JP2007279028A - Biological substance structure with pore and manufacturing method therefor, biological substance carrier using the same, refining method for biological substance, container for affinity chromatography, chip for separation, analytical method for the biological substance, separator for analyzing objective substance, and sensor chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological substance structure, capable of containing a biological substance of a large amount than that in conventional one, under the condition where reactivity of the biological substance is maintained, and having micropores. <P>SOLUTION: This biological substance structure formed with the pores, having 300nm or more to 100μm or smaller for diameter is constituted of the biological substance and a principal chain comprising a compound to be bonded with the biological substance. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biological material structure having pores and a method for producing the same, and a biological material 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 target The present invention relates to a separation device for analyzing substances and a sensor chip.

  In the fields of medicine / diagnosis, genetic analysis, proteomics, microelectronics, membrane separation, etc., diagnosis, testing, and inspection using the interaction between biological substances and substances that specifically interact with the biological substances (target substances) Analysis, purification, etc. may be performed. Therefore, in order to appropriately perform the diagnosis, inspection, analysis, purification, and the like, various techniques have been developed for structures containing biological materials.

  For example, a technique has been proposed in which a biological material is immobilized or supported on a substrate at a high concentration to form a biological material immobilized product. In this case, if a structure holding a space capable of effectively performing interaction between the biological material and the target substance can be applied to the biological material immobilized product, a more sensitive biological material immobilized product can be realized. .

  In recent years, as a method for solving these problems, attempts have been made to immobilize biological materials on a substrate having a porous structure. A substrate having a porous structure has a very large surface area compared to a substrate having no porous structure. For this reason, the sensitivity of the interaction with the target substance can be increased by immobilizing or supporting a large number of biological substances on the surface of the base material. As an example, as described in Patent Document 1, it has been proposed to use a porous material as a base material on which a protein is immobilized.

Patent Document 2 and Patent Document 3 propose a method for crosslinking a biological material using a foaming agent. This method has an advantage that a foam-like structure can be formed in a crosslinked structure of a biological material simply by using an aqueous solution by incorporating a foaming agent into the system.
In addition, as described in Non-Patent Document 1, a technique for producing a structure made of a protein having a porous structure using an electrospray method has been proposed.
As a method for manufacturing a porous structure for the purpose of manufacturing a three-dimensional wiring structure, Patent Document 4 proposes a method using phase separation. In this method, after forming the phase separation structure, the porous structure is formed by removing at least one phase forming the phase separation structure.

JP 2004-191341 A International Publication No. 02/085284 A2 Pamphlet JP-T-2004-537136 JP 2001-83347 A J. et al. Colloid and Interface Science 269 (2004) 336-340

  However, there is still room for improvement in the conventional technology, and further technical improvements are made regarding the creation of a micro space containing biological material (that is, a space where the interaction between the biological material and the target material can be effectively performed). Development was required. For example, in the technique described in Patent Document 1 and the like described above, the production is multistage or the microstructure cannot be produced by a simple method.

Further, with the technique described in Patent Document 2, it is difficult to control the microstructure.
Furthermore, in the electrospray method described in Non-Patent Document 1 or the like, fiber production often depends on conditions such as viscosity, and biological materials may be denatured by application of a high electric field.
Further, in the technique described in Patent Document 3 and the like, in order to remove at least one phase after forming the phase separation structure, the organic solvent having an affinity only for the phase to be removed is selectively used. The phase had to be dissolved and removed. For this reason, the solvent to be used is limited, and when the method is performed using a biological material, the biological material may be denatured by the selective solvent to be washed.

  The present invention was devised in view of the above problems, and a biological material structure that can contain a larger amount of biological material than the prior art while maintaining the reactivity of the biological material, and a method for producing the same , And a biological material carrier having the same, whereby a highly efficient and easy separation method of the target substance and a method of analyzing the target substance, and an affinity chromatography container and a separation chip used therefor, An object of the present invention is to provide a separation device for analyzing a target substance and a sensor chip.

  As a result of intensive studies to solve the above-described problems, the inventors of the present invention have a predetermined diameter with respect to a biological material structure formed by including a biological material and a compound capable of binding to the biological material. By forming a hole, the diffusion efficiency of the target substance into the biomaterial structure can be increased, and a biomaterial structure capable of promptly interacting with the target substance is realized. I got the point of being able to do it. Further, in this biological material structure, the biological material and the compound, and the biological material and a non-binding substance that does not bind to the compound coexist in the medium, and then the non-binding material is removed as necessary. I got the point that it can be manufactured. The present invention has been developed based on the above viewpoint.

  That is, the gist of the present invention is characterized by having a main chain composed of a biological material and a compound capable of binding to the biological material, and a hole having a diameter of 300 nm to 100 μm is formed by the main chain. In a biological material structure (claim 1).

  Another gist of the present invention is a biological substance, a compound that can bind to the biological substance, and a non-binding substance that does not bind to the biological substance and the compound and can form a region having a diameter of 300 nm to 100 μm. A biological material structure obtained through a coexistence step in a predetermined medium, wherein a main chain composed of the biological material and the compound is formed, and the main chain has a diameter of 300 nm to 100 μm. The biological material structure is characterized in that pores are formed (Claim 2).

At this time, it is preferable that the biological material structure has a particulate lump composed of the biological material and the compound (claim 3).
In addition, the ratio of the weight of the biological material to the weight of the biological material structure is preferably 0.1 or more (claim 4).
Furthermore, the biological material structure preferably has a diameter of 1 μm or more in a dry state (Claim 5).

Moreover, it is preferable that the molecular weight of this compound is 1000 or more (Claim 6).
Furthermore, it is preferable that at least one of the compounds has two or more functional groups capable of binding to a biological substance (claim 7).
The compound is preferably uncharged (claim 8).
Furthermore, the compound is preferably miscible with water and miscible with at least one organic solvent (claim 9).

Moreover, it is preferable that the diameter of this compound in the state mixed in the liquid is 1 nm or more (claim 10).
Furthermore, the biological material is preferably a biomolecule (Claim 11), more preferably a protein (Claim 12).

  Still another subject matter of the present invention is a method of producing a biological material and a biological material structure having a main chain composed of a compound that can bind to the biological material, the biological material, the compound, and the biological material. Production of a biological material structure characterized by having a mixing step in which a non-binding substance capable of forming a region having a diameter of 300 nm to 100 μm that does not bind to the substance and the compound coexists in a predetermined medium. It resides in a method (claim 13).

At this time, it is preferable that the manufacturing method of the biological material structure of this invention has a removal process which removes the said non-binding substance after this mixing process.
Further, the non-binding substance is preferably a water-miscible substance (Claim 15), and more preferably a water-soluble polymer compound (Claim 16).

Still another subject matter of the present invention lies in a biological material carrier characterized in that the biological material structure is fixed to a solid phase carrier (claim 17).
At this time, the thickness of the biological material structure is preferably 50 nm or more (claim 18).

  According to still another aspect of the present invention, the biological material structure is brought into contact with a sample liquid containing a target substance that can be specifically adsorbed on the biological material, and the biological material structure and the sample liquid are separated. Then, the present invention resides in a method for purifying a target substance, wherein the target substance adsorbed on the biological material structure is released (claim 19).

  Still another gist of the present invention is that an eluent that flows out of the flow channel by flowing a sample solution containing a target substance that specifically interacts with the biological material through a flow channel holding the biological material structure. Among them, the present invention resides in a method for purifying a target substance, wherein a fraction containing the target substance is collected (claim 20).

  Still another subject matter of the present invention lies in an affinity chromatography container comprising a container main body capable of containing a fluid and the biological material structure held in the container main body ( Claim 21).

  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 structure held in the flow channel. ).

  Still another subject matter of the present invention is the above-described biological material structure, which is a biological material structure using a biological material capable of specifically adsorbing a substance having a specific structure, and a sample liquid containing the target substance The biological substance structure and the sample liquid are separated, the amount of the target substance adsorbed on the biological substance is measured, and the structure of the target substance is analyzed. It exists in the analysis method of a substance (Claim 23).

  Still another subject matter of the present invention is the above-described biological material structure, in a flow path holding the biological material structure using a biological material 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 24).

  Still another subject matter of the present invention is the above-mentioned biological material structure using a substrate in which a channel is formed and a biological material that is held in the channel and can specifically interact 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.

  Still another subject matter of the present invention is the above-described biological material structure using a substrate on which a flow path is formed and a biological material that is held in the flow path 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.

  Still another subject matter of the present invention lies in a sensor chip comprising the biological material structure (claim 27).

ADVANTAGE OF THE INVENTION According to this invention, the biological material structure which can contain a larger amount of biological material than before, and has a micropore, and its simple manufacturing method can be provided, maintaining the reactivity of a biological material.
Further, according to the present invention, a biological material carrier having the biological material structure can be provided. Since the biological material structure itself contains a large amount of biological material and has pores, a substrate having a special porous structure as in the prior art is usually unnecessary. Furthermore, it is possible to provide a target substance purification method and target substance analysis method that are highly efficient and easily separated, and an affinity chromatography container, a separation chip, and a target substance analysis separation apparatus used therefor. .
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 and implemented without departing from the gist of the present invention. .

[I. Structure of biological material structure]
The biological material structure of the present invention is a structure formed by including a biological material and a compound capable of binding to the biological material (hereinafter, referred to as “binding compound” as appropriate). Specifically, it is a structure having a main chain composed of a biological substance and a binding compound. In the biological material structure of the present invention, a hole having a predetermined diameter (hereinafter referred to as “specific hole” as appropriate) is formed by the main chain.

[I-1. Biological material]
The biological material is an element constituting the biological material structure of the present invention, and any material can be used depending on the purpose as long as the effects of the present invention are not significantly impaired.
In particular, when the biological material structure of the present invention is used for purification or analysis of a target substance, the biological substance is usually a predetermined substance (hereinafter, a substance that interacts with a biological substance is referred to as an “active substance” as appropriate) ) Should be used.

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

  In addition, for example, when the biological material structure of the present invention is used for analyzing a target material, a biological material that can interact with the above-mentioned active substance is used. Then, it is tested whether or not the target substance interacts with the biological substance. If the target substance and the biological substance interact with each other, the target substance corresponds to one of the active substances. That is, it can be analyzed that the target substance has a specific structure that causes interaction with the biological 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 biological 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. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction.

Furthermore, when the biological material structure of the present invention is used for purification or analysis, the above interaction is optional 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 avidin protein and biotin, biologically active substance (medicine or drug candidate compound) and living body such as protein Binding and dissociation with substances, 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 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, for example, a substance other than a biomolecule such as a cell can be used as the biological substance.
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 structure 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, another biological material or other compound that has been biotinylated can be immobilized easily and in large quantities. In yet another aspect, when protein A is used as the biological material, when an antibody is used as the other biological 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.
Furthermore, among the examples of the biological material, the low molecular compound is not particularly limited as long as it satisfies the requirements for the biological material (for example, has the ability to interact as described above). Although those with unknown functions or those with already known ability to bind to proteins can be used, drug candidate compounds and the like are preferably used.

In addition, when the biological material structure of the present invention is used for separation and purification using affinity separation techniques such as affinity purification and analysis, the biological material described above becomes a target substance of a target substance that is a target for separation and purification. Furthermore, when the biological material structure of the present invention is used for analysis, these biological materials become target materials for measuring the interaction (binding property, etc.) between the target substance and the biological material in the specimen.
Moreover, a biological material may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

[I-2. Compound for binding]
As the binding compound, any compound can be used as long as it 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. Usually, it is preferable to select an appropriate material according to the kind of the biological material and the use of the biological material structure 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 addition, when the biological material is a nucleic acid, usually, a group such as an amino group, a hydroxyl group, or a thiol group introduced into the end of the nucleic acid is bound to a binding functional group of the binding compound. Further, when the biological substance 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 bonding functional group, specific examples of the bonding functional group include a succinimide group, an epoxy group, an aldehyde group, and a p-nitrophenyl 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 which is the higher order structure. In particular, when two or more binding functional groups are contained in one molecule, a biological material structure can be easily formed when concentration is performed.

  However, in order for the biological material structure of the present invention to easily form a particulate lump described later, 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. In addition, when there is a binding between the binding compounds, the biological material cannot be bound efficiently, and further, when the specimen is reacted with the biological material structure of the present invention, the desired reaction efficiency may not be obtained. Because.

  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 this way, in order to form a biological material structure in which the binding compounds are not bonded to each other, a binding functional group that does not bond the binding compounds to each other is selected, and the situation where the binding compounds are bonded to each other is eliminated. 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.

  Further, the method for examining the binding between the binding compounds contained in the biological material structure is determined by the formation of insoluble matter when the biological material in the biological material structure is decomposed by the method described later. Or it is judged by detecting the compound which suggests the coupling | bonding of the compound for coupling | bonding by analyzing a biological material structure by a pyrolytic gas chromatography. Specifically, for example, in the step of binding a binding compound having a biological substance and 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. Can be inferred from the bonds involving photoreactive groups or the presence of residual photoreactive groups (affinity chromatography: Tokyo Chemical Doujin, author: Kasai Kenichi) Matsumoto Istake, Beppu Masatoshi P238 ~).

  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 described above so that the biological material is not deactivated. For example, when immobilizing a protein, if 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.

  Furthermore, it is usually desirable to use a compound that is miscible with water as the binding compound. This is because water is used as a medium such as a solvent or a dispersion medium, although a medium used in manufacturing the biological material structure is arbitrary. Specifically, when manufacturing a biological material structure, 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 structure 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. That is, it is more preferable that it is miscible with water and miscible with at least one 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 structure 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 (charge having the same sign) as that of the biological material, 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. This may prevent the biological substance from effectively binding to the binding functional group of the compound for use. In addition, when the biological material structure of the present invention is used for separation and purification, the binding compound and the biological material can be easily bonded by an additive such as pH or salt of the solution used at the time of use. It is expected to break.

  Furthermore, when the target substance is separated using the biological substance structure of the present invention and the target substance has the same charge as the binding compound, the biological substance contained in the biological substance structure In the case where the target substance and the binding compound have opposite charges, the target substance and the binding compound are not attracted by electric attraction. This is because it is estimated that nonspecific interaction such as specific adsorption occurs.

  Similarly, when the biological substance structure of the present invention is used to detect an interaction between selective biological substances, the active substance as an analyte has the same charge as the binding compound. May interfere with the specific interaction with the biological substance that is the ligand, and if the analyte and the binding compound have opposite charges, the analyte and the binding compound This is because it is estimated that nonspecific interactions such as nonspecific adsorption occur.

  It should be noted that the binding compound is non-charged, at least in terms of structural formula, is nonionic or zwitterionic (has both positive and negative charges, and the mutual charges substantially cancel each other) If so, the binding compound is uncharged. However, in the production process of the biological material structure of the present invention, even if the binding compound has a charge due to hydrolysis of the binding functional group or the like, 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.
Specific examples of low molecular weight compounds that can be used as binding compounds include glutaraldehyde, diepoxybutane, diepoxyhexane, diepoxyoctane, bismaleimidohexane, bis (sulfosuccimidyl) suberate, disuccimidyl glutarate, ethylene Glycol bis (succimidyl succinate), ethylene glycol bis (sulfosuccimidyl succinate), succimidyl 4-N-maleimidomethylcyclohexane 1-carboxylate, sulfosuccimidyl 4- (p-maleimidophenyl) butyrate, succimidyl 4- (p-maleimidophenyl) butyrate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester and the like can be mentioned.

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.

  That is, for the synthesis of a synthetic polymer compound to be used as a binding compound, at least a monomer species that can be combined with a biological substance to form a particulate mass (this is a conjugate formed by reacting with a biological substance). A type of monomer that can form a gate) (partial), and particle masses are linked together to form a chain-like and / or network-like structure (ie, a structure of biological material structure) A monomer having a binding functional group (this is a part of a monomer having a binding functional group for building a chain-like and / or network-like structure that is bound between conjugates) It is preferable to use a monomer having a hydrophilic or amphiphilic functional group. 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. The monomers mentioned here may be different monomers, but one monomer may have two or more of the above functions.

  When the specific example of the monomer which comprises the said synthetic polymer compound is given, as a monomer used in radical polymerization, polymerizable unsaturated aromatics, such as styrene, chlorostyrene, (alpha) -methylstyrene, divinylbenzene, vinyltoluene, etc. Polymerizable unsaturated carboxylic acids such as (meth) acrylic acid, itaconic acid, maleic acid, and phthalic acid; polymerizable unsaturated sulfonic acids such as styrenesulfonic acid and sodium styrenesulfonate; methyl (meth) acrylate, (meta ) Ethyl acrylate, (meth) acrylic acid-n-butyl, (meth) acrylic acid-2-hydroxyethyl, 2- (meth) acrylic acid hydroxypropyl, (meth) acrylic acid glycidyl, N- (meth) acryloyloxy Succinimide, ethylene glycol-di- (meth) acrylate, ( T) Polymerizable carboxylic acid esters such as tribromophenyl acrylate, glycosyloxyethyl (meth) acrylate, 2-methacryloyloxyethyl phosphorylcholine; (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N, N -Dimethylacrylamide, N-isopropyl (meth) acrylamide, N-vinylformamide, 3-acrylamidophenylboronic acid, N-acryloyl-N'-biotinyl-3,6-dioxaoctane-1,9-diamine, butadiene, isoprene , Vinyl acetate, vinyl pyridine, N-vinyl pyrrolidone, N- (meth) acryloylmorpholine, vinyl chloride, vinylidene chloride, vinyl bromide, and the like; polymerizable unsaturated nitriles; vinyl halides; Conjugated die 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, a functional compound that can serve as a crosslinking agent may coexist in addition to the monomer described above. Examples of the functional 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, vinyl Examples thereof include N-alkylolamides of aromatic acids (for example, N-methylol-p-vinylbenzamide), N- (isobutoxymethyl) acrylamide and the like.

Further, 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, 1,3 -Polyfunctional monomers such as butylene glycol, 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-described biological substance include N- (meth) acryloyl 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 thereof include oxysuccinimide, 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 (meth) acrylate.

  Furthermore, specific examples of the hydrophilic monomer include (meth) acrylic acid, itaconic acid, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, maleic acid, styrene sulfonic acid, p-styrene. Sodium sulfonate, (meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropylacrylamide, N-vinylformamide, (meth) acrylonitrile, N- (meth) acryloylmorpholine, N-vinylpyrrolidone, N- Examples thereof include vinylacetamide, N-vinyl-N-methylacetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, 2-methacryloyloxyethyl 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- ( And (meth) acryloylmorpholine, N-vinylpyrrolidone, N-vinylacetamide, N-vinyl-N-methylacetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, 2-methacryloyloxyethyl phosphorylcholine, and the like. .

  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. Any radical polymerization initiator can be used as long as the effects of the present invention are not significantly impaired. 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- Methoxyvaleronitrile), 2,2′-azobis- (2,4-dimethylvaleronitrile), 2,2′-azobis- (2-amidinopropane) hydrochloride and other azobis type initiators, benzoyl peroxide, cumene hydro Peroxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfate (eg ammonium persulfate), perester (eg t-butyl) Ruokuteto and 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 SUNBRIGHT 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, it is crosslinked within one biological substance to be immobilized, and the main chain cannot be formed efficiently, and the present invention. It may become impossible to form a biological material structure. In addition, when crosslinked in a biological material, the activity of the biological material may not be retained. 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, there is a possibility that a biological material structure in which the particulate lumps gather effectively cannot 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 preparing a binding compound before the production of the biological material structure 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 structure. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 5 micrometers or less.

  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, still more preferably 5% or more, and usually 90% or less, preferably 80% or less, more preferably Is 70% or less. This is because if it is below this range, the binding compound may not be able to bind efficiently to the biological material, and if it exceeds this range, it may not be miscible with the solvent or dispersion medium.

[I-3. Structure of biological material structure]
[I-3-1. Main chain constituting biological material structure]
When viewed microscopically, the biological material structure of the present invention has a main chain composed of the biological material and a binding compound. The matrix structure (matrix) of the biological material structure of the present invention is configured by, for example, aggregation or bonding of the main chains. That is, the biological material structure of the present invention is a structure containing a biological material and a binding compound, as schematically shown in FIGS. 1 (a) to 1 (c), and its skeleton is a biological body. It is formed by a main chain formed by bonding a substance and a binding compound. Thus, the biological material structure of the present invention is configured as a matrix having a structure in which the main chain is bound in a chain and / or network.

  1 (a) to 1 (c) show the vicinity of the surface of an example of the biological material structure of the present invention immobilized on a solid support in order to explain the matrix structure of the biological material structure of the present invention. It is a schematic diagram which expands and shows. Further, in FIGS. 1A to 1C, a circular portion represents a biological material, and a linear portion represents a binding compound. However, FIG. 1 (a) to FIG. 1 (c) show the structure of the main chain regardless of whether or not it forms a particulate lump, which will be described later, in order to explain the structure of the main chain composed of the biological material and the binding compound. A structure is drawn two-dimensionally. Therefore, when the biological material structure has a particulate lump, FIGS. 1A to 1C recognize that the main chain of the granular lump of the biological material structure is two-dimensionally expanded. Should.

Hereinafter, the main chain of the biological material structure of the present invention and the matrix structure composed of the main chain will be described.
As described above, the biological material structure of the present invention is a structure having a main chain composed of a biological material and a binding compound. Further, the main chain of the biological material structure of the present invention constitutes a skeleton having a matrix structure, and specifically, a biological material and a binding compound are bonded to each other. Specifically, a binding compound is bound to a biological substance by a binding functional group, and a chain-like and / or network-like structure is formed by repeating the structure.

Therefore, the biological material structure of the present invention usually has two or more partial structures represented by the following 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 of the present invention 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 of the present invention may include, for example, a partial structure in which biological materials R ¹ or 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 of the present invention has at least a part of a bridge structure in which a biological material exists between binding compounds and a binding compound exists between biological materials. ing. The main chain of the matrix structure is constituted by both the biological material and the binding compound. Therefore, the biological material structure of the present invention is formed when both the biological material and the binding compound are present in the system at the time of production.

It can be confirmed, for example, by the following method that the biological material structure has a main chain composed of the biological material and the binding compound.
Since the biological material structure of the present invention has a main chain composed of the biological material and the binding compound as described above, the structure collapses by decomposing the binding of the biological material that is a constituent element thereof. If only the biological material of the biological material structure is decomposed using this, the main chain composed of the biological material and the binding compound can be confirmed. For example, when the biological material is decomposed while preventing the binding compound from being decomposed, in the biological material structure of the present invention, the biological material structure is largely collapsed. Further, by investigating the collapsed one, it is possible to specify the compound constituting the biological material structure other than the biological material component. Furthermore, when the biological material structure of the present invention is bound to a solid phase carrier, for example, when the biological material structure is immobilized to some solid phase carrier, at least a part of the binding compound is a biological material. In the biological material structure according to the present invention, when the biological material is decomposed while preventing the binding compound from being decomposed, the biological material structure according to the present invention can Thus, the portion immobilized via the biological material is detached from the solid phase carrier.

  Therefore, specifically, the biological compound is decomposed by an enzyme or other chemical that does not decompose the binding compound but only the biological substance is decomposed, and the substance detached from the solid support by this treatment is examined, or By investigating the substance remaining on the surface of the solid phase carrier, the compound constituting the biological material structure other than the biological material component can be specified. If the biological material structure has a main chain composed of the biological material and the binding compound, the binding compound is detected in the detached substance. In addition, the binding compound is not detected on the surface of the solid phase carrier, or the amount is decreased even if it is detected.

  On the other hand, if the biological material structure uses a polymer membrane according to a conventional method, the main chain is a polymer chain, so that even if the biological material is decomposed, all the polymer membranes (ie, polymer chains) Among the substances that remain on the solid phase carrier and have been desorbed, biological substance constituent elements are detected, but polymer chains corresponding to the binding compounds are not detected. Based on this difference, the presence or absence of the main chain composed of the biological material and the binding compound can be confirmed, and it can be determined whether or not the biological material structure of the present invention is present.

Any enzyme or chemical for decomposing biological material used in the above method may be used appropriately depending on the type of biological material or binding compound used. 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.
Further, when the biological substance is a protein, for example, proteolytic enzymes such as microbial protease, trypsin, chymotrypsin, papain, rennet, V8 protease, cyanogen bromide, 2-nitro-5-thiocyanobenzoic acid, hydrochloric acid, sulfuric acid, Examples thereof include chemical substances having a protein resolution such as sodium hydroxide.

Furthermore, when the biological substance is a lipid, for example, lipolytic enzymes such as lipase and phospholipase A2 can be mentioned.
In addition, when the biological substance is sugar, examples thereof include glycolytic enzymes such as α-amylase, β-amylase, glucoamylase, pullulanase, and cellulase.
In addition, the enzyme and chemical | medical agent for decomposing | disassembling a biological material may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.
However, among the examples, those that may decompose not only the biological substance but also the binding compound should be avoided because the above main chain may not be confirmed accurately.

In addition, when a biological substance is decomposed and a substance remaining on the solid phase carrier after the decomposition is confirmed, a specific confirmation method is arbitrary. For example, surface plasmon resonance (SPR), crystal resonator This can be confirmed by measurement using a microbalance (QCM), electron microscope, ellipsometry, or the like.
Furthermore, when analyzing a substance detached from a solid phase carrier after decomposition of a biological material, the specific analysis method is arbitrary. For example, liquid chromatography, gas chromatography, mass spectrometry (MS), infrared Examples include spectroscopy, nuclear magnetic resonance (NMR), 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.

[I-3-2. Particulate mass of biological material structure]
When viewed macroscopically than the above description, the biological material structure of the present invention usually has a particulate mass (particulate mass) composed of the biological material and the binding compound. This particulate lump is presumed to be formed by combining the main chain after the above-mentioned main chain is rounded or two or more gather. And this particulate lump couple | bonds together and the three-dimensional structure of the biological material structure of this invention is comprised.
Hereinafter, this particulate lump will be described.

  The biological material structure of the present invention is usually a structure in which a particulate lump is bound to each other in a chain and / or network form as schematically shown in FIGS. 2 (a) and 2 (b). It has become. In addition, this particulate lump is usually formed by binding a plurality of biological substances and binding compounds as shown in FIG. 3 (a) into particles as shown in FIG. Although it does not necessarily have a round shape, in FIGS. 2 (a) and 2 (b), the particulate lump is schematically shown as a circle.

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. Here, the bond usually refers to a bond consisting of one or more of a covalent bond, ionic bond, chelate bond, coordination bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic bond, Among these, a covalent bond is preferable. In this case, it may be in a state where only a part of the particulate lump is bound, but it is preferable that as many particulate lumps are bound as possible, and all the particulate lumps are bound together and the biological material structure More preferably, the body is constructed.
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.

Moreover, there is no restriction | limiting in the particle size of the particulate lump which comprises a biological material structure body, However, Usually, it is 10 micrometers or less, Preferably it is 5 micrometers or less, More preferably, it is 1 micrometer 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. In addition, although a minimum is arbitrary, it is 1.5 nm or more normally.
Furthermore, when measuring the particle size of the particulate mass individually, it is sufficient that at least a part of the particulate mass in the biological material structure has 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). When observed with a microscope, the shape of the particulate mass may be observed as a shape protruding from the biological material structure into a tuft shape in addition to the particulate shape. ) Is presumed to be formed by protruding a particulate lump from the biological material structure, the diameter (usually the short diameter) of this tuft-like lump portion may be within the above range.

  One method for examining the presence of particulate lumps is to examine Power Spectral Density (power spectral density) from images measured by AFM. 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.

For example, when measuring in a 1 μm × 1 μm field of view, Power Spectral Density (PSD) analysis is performed from the obtained AFM image, and the value of 2D isotropic PSD is 100 (nm ⁴ ) or more and the wavelength is less than 100 nm. The ratio Ic / It between the sum of the power spectra (Ic) and the total power spectrum (It) is usually 30% or more, preferably 50% or more, particularly preferably 60% or more, more preferably 70% or more. Thus, the presence of a submicron-order particulate lump can be shown. This is because the power spectrum represents irregularities on the surface of the object, and if the ratio Ic / It is in the above range, irregularities resulting from the structure of the particulate mass can be confirmed on the surface of the biological material structure.

  At this time, although a commercially available AFM apparatus can be used, NanoScope IIIa manufactured by Digital Instruments is preferable. 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 the analysis of the surface shape, and particularly preferably Power Algorithm attached to NanoScopeIIIa manufactured by Digital Instruments. The Density function is used (reference document: NanoScope command reference manual).

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, an example of the method of confirming the particulate lump by the light scattering measurement method is shown. This example is an example of a method for confirming a particulate lump by measuring the forward scattered light intensity of a biological material structure. By measuring the forward scattered light intensity of the biological material structure, the correlation length can be obtained to evaluate the particulate mass. This method measures the scattering angle dependence of the scattering intensity of the light transmitted through the biological material structure through an analyzer, and based on the Debye-Bueche theory (light intensity) ^−1/2 Is obtained with respect to the square of the wave number (= {(4πn / wavelength of light) × sin (scattering angle / 2)} ² ), 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 biological material structure may be swelled in a liquid or in a dry state, but is preferably measured in a liquid.

[I-3-3. Gel-like structure of biological material structure]
From the viewpoint of the production process, the biological material structure of the present invention is usually composed of many conjugates in which a biological material and a binding compound are bound, and binds to the biological material. It is a gel-like structure in which the compound for use is bound in a chain and / or network form (see FIGS. 1 and 4). This conjugate is a combination of a biological substance and a binding compound, and it can be prepared simply by mixing the biological substance and the binding compound in a medium such as a solvent or dispersion medium and bringing the molecules into contact with each other. it can.

[I-3-4. Advantages of main chain, particulate mass and gel structure of biological material structure]
The biological material structure of the present invention has a matrix skeleton formed by both the biological material and the compound, or a particulate mass formed by both the biological material and the binding compound is assembled and / or Since it has a structure formed by bonding, the ratio of biological substances can be increased. Therefore, the biological material structure of the present invention can hold a larger amount of biological material than before. 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.

  In addition, since a large amount of biological material can be retained, the biological material structure of the present invention can suppress nonspecific interactions that occur in the binding compound (and a solid phase carrier and the like 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, analysis and affinity separation using the interaction between the biological substance and the active substance can be performed while eliminating the influence of the nonspecific interaction.

  Furthermore, a space (void) is formed inside the biological material structure of the present invention. This void is usually formed between the particulate lumps. That is, the particulate lumps are not completely in close contact with each other, and spaces (voids) are formed between the particulate lumps. Even when the biological material structure of the present invention does not have a particulate lump, a space is formed between the main chains.

  This space is formed in such a size that the target substance that interacts with the biological substance can enter when affinity separation or the like is performed using the biological substance structure. Therefore, when affinity separation or the like is performed, an interaction between a biological substance and a target substance usually occurs in this space. Therefore, although the biological material structure of the present invention has a three-dimensional structure, the biological material contained therein (inside) can interact with the target material and the like.

  That is, although the biological material structure of the present invention can be provided with many biological materials in three dimensions, the biological material can interact without losing activity. For this reason, the biological material structure of the present invention can contain a larger amount of biological material than before while maintaining the reactivity of the biological material.

  There is no limitation on the method for examining whether the biological material structure of the present invention has a space between the particulate masses. For example, the biological material structure is confirmed 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.

  Further, when the volume of the biological material structure of the present invention in a dry state is compared with the volume when the liquid is included, the volume increases due to the penetration of the liquid into the space. It may be recognized that the biological material structure of the present invention has a space. These volume changes may be confirmed by any method. For example, when the biological material structure 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.

[I-3-5. Holes formed in the biological material structure]
In the biological material structure of the present invention, a specific hole having a predetermined size diameter is formed. This specific hole is formed by the main chain. That is, when the main chain exists while avoiding a predetermined region, the avoided space eventually forms a space region (that is, a specific hole) where the main chain does not exist.
The diameter of the specific pore is larger than the particulate lump and smaller than the biological material structure, but may be any as long as the structure of the biological material structure can be maintained. It is usually 300 nm or more, preferably 500 nm or more, more preferably 1000 nm or more, and usually 100 μm or less, preferably 50 μm or less, more preferably 30 μm or less.

Due to the specific pores, the biological material structure of the present invention can be more efficiently separated when affinity separation or the like is performed. The reason is guessed as follows.
That is, when affinity separation or the like is performed using a biological material structure, a target substance that interacts with the biological material enters the space formed in the biological material structure. At this time, the entry speed of the target substance is relatively low. This is because the invasion of the target substance into the space within the biological material structure is a phenomenon in which molecules proceed slowly like diffusion.
On the other hand, the specific hole is usually larger than the space. Therefore, a fluid substance can enter the specific hole. That is, a fluid such as a sample solution can flow into the specific hole. For this reason, since the entry of the target substance into the interior of the biological material structure is performed more quickly than the entry into the space, the interaction between the biological substance and the target substance can be performed more quickly. It is guessed.
In addition, since the specific hole is formed, the specific surface area of the biological material structure is increased.

  The diameter of the specific hole can be measured by observing the dried biological material structure using a microscope such as TEM, SEM, AFM, or laser microscope. Moreover, the diameter of the specific hole here refers to the longest diameter in the opening of the specific hole.

  The specific hole is not limited in shape, depth, number, or the like. For example, the specific hole may be formed only near the surface of the biological material structure of the present invention, or may extend to the inside of the biological material structure. Moreover, many holes may be formed and one hole may be formed. There is no restriction | limiting in the number of holes, The structure of this biological material structure should just be maintained.

[I-3-5. Other features of the structure of the biological material structure]
Further, the size of the biological material structure of the present invention is not limited and is arbitrary. However, when the biological material structure is not bonded to any solid phase carrier, the diameter of the biological material structure is usually 1 μm or more, preferably 5 μm or more, more preferably 10 μm or more in a dry state. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 10 cm or less. Here, the diameter of the biological material structure 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 structure is too small, it may be difficult to recover the biological material structure by centrifugal separation or the like in purification separation when used for affinity separation or the like.

[I-4. Composition of biological material structure]
[I-4-1. Biomaterial content ratio]
In the biological material structure 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 structure)” is usually 0.1 or more, preferably 0. .3 or more, more preferably 0.5 or more, still more preferably 0.7 or more, and particularly preferably 0.9 or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 0.999 or less. When the ratio of the biological material is below this range, the binding compound in the biological material structure to be configured cannot be sufficiently covered with the biological material, and nonspecific adsorption to the binding compound may occur. Further, when separation and purification is performed using this biological material structure, the efficiency of the separation and purification may be reduced.

[I-4-2. Method for measuring the content ratio of biological substances]
The method for measuring the ratio of the biological material is not particularly limited. For example, the biological material contained in the biological material structure of the present invention is decomposed using an enzyme, a drug, or the like, and the material derived from the biological material and the binding compound is used. Each may be quantified 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. Specific examples thereof include those exemplified as enzymes and chemicals for degrading the biological material used in the method for confirming the presence or absence of the main chain composed of the biological material and the binding compound.
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 and substance derived from binding compound After decomposing biological material structure, there is no limitation on the method for quantifying biological substance and binding compound-derived substance. Are, for example, liquid chromatography, gas chromatography, mass spectrometry (MS), infrared spectroscopy, nuclear magnetic resonance ( ¹ H-NMR, ¹³ C-NMR, ²⁹ Si-NMR), high-performance liquid chromatography (HPLC ), Gel electrophoresis, capillary electrophoresis, absorbance measurement, fluorescence measurement, elemental analysis, amino acid quantification and the like. In the analysis, each measurement method may be used alone, or two or more kinds may be arbitrarily combined.

[I-4-3. Biomaterial reaction number ratio]
In the biological material structure of the present invention, most of the biological material in the biological material structure has not lost its activity. Specifically, when the biological material structure is brought into contact with a solution containing an active substance capable of specifically interacting with the biological material, the biological material with respect to the number of biological materials in the biological material structure is determined. The ratio of the number of the above-mentioned active substances interacting with the substance (hereinafter referred to as “reaction number ratio” as appropriate) is usually 0.05 or more, preferably 0.1 or more, more preferably 0.2 or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 1.0 or less. As described above, since the biological material structure of the present invention can contain many biological materials while having voids, the reaction number ratio can be increased as in the above range. Thereby, a biological substance can be used effectively. Note that the reaction number ratio is not always obtained, and may be out of the above range depending on the types and amounts of biological substances and active substances, conditions at the time of measurement, and the like. However, it is one of the excellent advantages of the biological material structure of the present invention that an excellent reaction number ratio can be obtained as described above when conditions and the like are appropriately set.
This reaction number ratio can be obtained as a ratio of the number of molecules when the biological substance and the active substance are molecules such as a biomolecule and an active molecule, respectively. The reaction number ratio can be measured by, for example, the SPR (surface plasmon resonance) method or the QCM (quartz crystal microbalance) method.
This advantage can be similarly obtained even when the biological material structure is immobilized on a solid phase carrier to form a biological material carrier.

[II. Manufacturing method of biological material structure]
The biological material structure of the present invention is usually produced through a step of coexisting a biological material, a binding compound, and a non-binding substance in a predetermined medium (reaction medium) (hereinafter referred to as “mixing step” as appropriate). Is done. In this mixing step, the biological material and the binding compound are mixed in a medium and combined by coexisting in the same system, whereby the biological material structure of the present invention is formed. Furthermore, in the mixing step, the specific holes are formed by the non-binding substance functioning as a template.
In addition, after the mixing step, a concentration step for removing the medium, a drying step, a step for removing unbound substances, or the like may be performed.
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. Mixing process]
In the mixing step, the biological substance, the binding compound, and the non-binding substance are allowed to coexist in a predetermined medium.

[II-1-1. Biological material]
The biological material is as described above. When preparing a biological material, the biological material is usually prepared as a solution or dispersion in which the biological material is dissolved or dispersed in some solvent. In this case, the solvent or dispersion medium for diluting the biological material is preferably prepared in consideration of the activity of the biological material, the stability of the structure, and the like.

[II-1-2. Compound for binding]
The binding compound is as described above.

[II-1-3. Unbound material]
As the non-binding substance, a substance that does not bind to the biological substance and the binding compound and can form a region having a diameter of 300 nm to 100 μm is used.

  By using a non-binding substance that does not bind to the biological substance and the binding compound, the matrix structure or particulate mass of the biological substance structure can be more reliably produced. That is, if a non-binding substance that binds to a biological substance and a binding compound is used, the formation of the main chain or particulate mass is inhibited, or the non-binding substance cannot be removed later, and specific pores are formed. It may not be possible. In addition, when a non-binding substance binds to a biological substance or a binding compound, the non-binding substance is incorporated into the biological substance structure, so that non-specific mutuals resulting from the non-binding substance are used when performing affinity separation or the like. There is a possibility that the separation performance deteriorates due to the action.

  In addition, the non-binding substance has a diameter similar to that of the specific hole (usually 300 nm or more and 100 μm or less) inside or on the surface of the biological material structure of the present invention when the biological material structure of the present invention is manufactured. It is possible to form a region (phase) having the same. This region (phase) is to form a specific hole later in or on the surface of the biological material structure.

Here, the “region (phase)” means a phase composed of non-binding substances separated by a structure in which main chains or particulate lumps formed by a biological substance and a binding compound are connected. ) Is removed, the specific hole according to the present invention is formed.
For the formation of the region (phase), any method can be used as long as a region having the same size as that of the specific hole (usually, 300 nm or more and 100 μm or less) can be formed. For example, in the process of mixing a biological material, a binding compound and an unbound material in a medium and then concentrating or evaporating the medium to form a biological material structure, the unbound material aggregates, precipitates, precipitates, A phenomenon in which an aggregate region (phase) of non-binding substances is formed by phase separation or the like can be used. In the present specification, the formation of the aggregate region of these non-binding substances on or inside the biological material structure is referred to as “phase separation”. This phase may be formed in the presence of a predetermined medium, and may be performed in a state where the medium is completely dried. However, a phase of an unbound substance is formed by a process such as evaporation to remove the medium. It is desirable (phase separation).

Alternatively, pores may be formed by forming fine particle-containing biological material structures using fine particles such as polymer spheres as non-binding substances and then removing the fine particles. In this case, the fine particles can be considered as a region (phase) formed of a non-binding substance.
Further, the biological material and the binding compound are excluded from the binding, and after the biological material structure is formed, the biological material and the binding compound that could not be macroscopically bonded, and the non-binding material (these are the phases) ) Can also be removed to form specific holes.

  Among these, it is particularly preferable that the formation of pores in the biological material structure of the present invention is performed by a method of removing a region (phase) formed by phase separation. That is, it is preferable to use, as a non-binding substance, a substance that can be phase-separated from the biological substance and the binding compound in the process of forming the biological substance structure by concentration or evaporation of a medium to be used during the production of the biological substance structure. According to this method, unbound substances can be easily removed, and the process is very simple. Furthermore, since there are many choices of the non-binding substance that can be used in such a method, the non-binding substance can be selected according to the properties of the biological substance. Biological substances tend to be denatured and the like, and a method of selectively dissolving a conventionally used compound in an organic solvent cannot be used. For this reason, it is very useful to be able to arbitrarily select a non-binding substance that can use a medium that does not affect biological substances.

  As described above, the non-binding substance is preferably a substance that can be phase-separated from the biological substance and the binding compound in the step of forming the biological substance structure. By using as the non-binding substance what can be phase-separated from the biological substance and the binding compound in the process of forming the biological material structure, the specific hole is formed in the region (phase) of the non-binding substance formed by phase separation. It can function as a template to do. That is, if the non-binding substance is phase-separated from the biological substance and the binding compound, the main chain of the biological substance structure is not formed in the portion where the non-binding substance exists. For this reason, if the non-binding substance is removed after the main chain is formed, a specific hole is formed in the portion where the non-binding substance was present.

  However, the extent to which the non-binding substance does not bind to the biological substance and the binding compound and the degree to which the non-binding substance is phase-separated from the biological substance and the binding compound are within the range where the biological substance structure of the present invention can be obtained. I just need it. Thus, for example, if there is no substance that does not bind to the biological substance and the binding compound at all, and the non-binding substance completely phase-separates from the biological substance and the binding compound, or is difficult to obtain, the desired A biological material or a binding compound to such an extent that it can bind to the biological material and the binding compound to such an extent that a biological material structure having a structure can be obtained, or a biological material structure having a desired structure can be obtained. It is possible to use compatible materials as non-binding materials.

  The non-binding substance is preferably miscible with the medium, and may be liquid at room temperature. Accordingly, when the biological substance and the binding compound are mixed in the medium, the non-binding substance can coexist in the medium. Moreover, the dimension of a specific hole can be changed by changing the molecular weight and usage-amount of a non-binding substance. Therefore, the specific hole having a desired diameter can be formed more stably.

  When the non-binding substance is dissolved in the medium, the solubility is arbitrary, but the solubility of the non-binding substance in the medium is usually 0.001% by weight or more, preferably 0.01% by weight or more, more preferably Is 0.1% by weight or more. Moreover, there is no upper limit to the solubility of the non-binding substance in the medium, and it can be mixed at an arbitrary ratio.

  Further, as the non-binding substance, it is preferable to use a substance that can be removed after the biological substance and the binding compound are bonded to form a main chain. The unbound material may be removed by any means. However, since a non-binding substance is usually removed by washing with a washing solvent (described later), it is desirable to use a substance that is soluble in a predetermined washing solvent.

Specific types of non-binding substances may be arbitrarily selected according to biological substances, binding compounds, media, washing solvents, and the like. However, since water is usually used as a medium, a water-miscible substance is used as the non-binding substance. Among them, a water-soluble substance is preferably used, and a water-soluble polymer compound is particularly preferably used. The non-binding substance is preferably uncharged. It should be noted that the non-binding substance is non-charged at least in terms of the structural formula, non-ionic or zwitterionic (having both positive and negative charges, and the mutual charges substantially cancel each other) If so, the unbound material is uncharged.
When a phase is formed by a phase separation mechanism, when a water-soluble polymer compound is used as a non-binding substance, a synthetic polymer compound or a natural polymer compound may be used. As the monomer of the water-soluble polymer compound, a hydrophilic monomer having no binding functional group used in the synthesis of the above-described binding compound can be used, and preferably an uncharged monomer can be used. More preferably, the non-binding substance is polyethylene glycol. Moreover, when using a natural polymer, the natural polymer compound used with the above-mentioned compound for a coupling | bonding can be used. In addition, for example, salts such as glycerin and sodium chloride, water-soluble low-molecular compounds such as sucrose and lipids, and the like can be used for phase separation.

  In addition, as described above, in a method for forming a region using a mechanism other than phase separation, aggregates such as polymer spheres and micelles, metal nanoparticles such as gold and silver, and the like can be used. These substances may be any substances that can be removed from the biological material structure after the biological material structure is formed. For example, after the biological material structure is formed, the biological material structure may be removed by outflow or the like by swelling the biological material structure with a medium and widening the interval between the main chains forming the biological material structure. It may be removed from the inside of the biological material structure by decomposing.

In addition, as other non-binding substances, for example, salts such as glycerin and sodium chloride, water-soluble low molecular compounds such as sucrose and lipids, and the like can be used.
In addition, a non-bonding substance may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios.

  Further, when a polymer compound is used as the non-binding substance, the molecular weight is not limited, but is usually 100 or more, preferably 300 or more, more preferably 500 or more, and usually 1 million or less, preferably 500,000 or less, More preferably, it is 100,000 or less. If the molecular weight is too small, it may be difficult to form a pore having a predetermined size (usually 300 nm) by being compatible with the biological substance and the binding compound, and if it is too large, it may be difficult to mix with water.

[II-1-4. medium]
In the mixing step, the biological substance, the binding compound, and the non-binding substance are allowed to coexist in a predetermined 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 biological material structure of the present invention can be produced. Usually, a biological material, a binding compound, a non-binding material, and an additive used as appropriate can be mixed. It is desirable to use one. At this time, the mixing state of the biological substance, the binding compound, the non-binding substance and the additive is arbitrary, and it may be in a dissolved state or a dispersed state. In order to bind, it is preferable that the biological substance and the binding compound exist 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.

[II-1-5. Additive]
In any step of the manufacturing process of the biological material structure, the biological material, the binding compound, the non-binding material and the medium, and the mixture thereof are not significantly disturbed by the present invention. Arbitrary additives may coexist. Examples of additives include the above-mentioned salts, moisturizers such as acids, bases, buffers, glycerin, polyethylene glycol, saccharides, metal ions such as zinc as stabilizers for biological materials, antifoaming agents, denaturing agents, Examples thereof include preservatives such as sodium azide.
Moreover, an additive may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

[II-1-6. Operation of mixing process]
When the biological material structure of the present invention is produced, the biological material, the binding compound and the non-binding material are mixed in the presence of a medium. At this time, a stirring operation is usually performed. Thereby, a mixture in which at least a biological substance, a binding compound and a non-binding substance coexist in the medium is obtained. In this mixture, the biological substance and the binding compound are bonded to form a main chain, but the main chain is not formed in the portion where the non-binding substance exists, so the biological material structure in which the specific hole is formed Is obtained.

  In mixing, the biological material, the binding compound, the non-binding material, the medium, the additive and the like may be prepared in any state as long as the biological material structure of the present invention 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 arbitrary. Moreover, the order which mixes these is also arbitrary. Furthermore, it is also possible to prepare this mixture on a solid phase carrier for the purpose of immobilizing the biological material structure of the present invention on the solid phase carrier described later.

[II-1-7. Composition of mixing process]
The mixing ratio of the biological material, binding compound, non-binding material, medium, additive, etc. used in the mixing step is arbitrary as long as the biological material structure 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, still more preferably 0.7 or more, and particularly preferably 0.9 or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 0.999 or less. 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.

  That is, when the mixing ratio of the biological material is high, a biological material structure having a main chain having the binding compound as a bonding point as shown in FIG. 1 (a) is formed. When the ratio is high, as shown in FIG. 4, a biological material structure having a main chain in which the biological material becomes a bonding point is formed. Therefore, at this time, it is possible to suppress non-specific adsorption to the binding compound by increasing the ratio of the biological substance as in the above range. FIG. 4 is an enlarged schematic view showing the vicinity of the surface of an example of the biological material structure of the present invention immobilized on a solid phase carrier in order to explain the structure of the biological material structure of the present invention. In FIG. 4, the circular portion represents the biological material, and the linear portion represents the binding compound. However, FIG. 4 is a two-dimensional drawing of the structure of the main chain, regardless of whether or not it forms a particulate mass, in order to explain the structure of the main chain composed of the biological substance and the binding compound. . Accordingly, when the biological material structure has a particulate mass, FIG. 4 should be recognized as a two-dimensionally expanded main chain of the particulate mass of the biological material structure.

  Further, the ratio (concentration) of the biological material and the binding compound in the medium is arbitrary as long as the biological material structure of the present invention can be obtained, but the total concentration of the biological material 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 may not easily be generated.

  Furthermore, the value of the mixing ratio represented by “(weight of non-binding substance) / {(weight of biological substance) + (weight of binding compound) + (weight of non-binding substance)}” is usually 0.001. Above, preferably 0.01 or more, more preferably 0.1 or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, 0.95 or less, Preferably it is 0.7 or less, More preferably, it is 0.5 or less. If the mixing ratio is too small, it may be difficult to form specific pores, and if it is too large, it may be difficult to form a biological material structure.

[II-1-8. Formation mechanism of biological material structure]
The formation process of the biological material structure of the present invention is not clear, but can be estimated as follows. That is, when a mixture is prepared, a biological substance and a binding compound are bound in the mixture, and a conjugate is generated. This conjugate has a main chain in which a biological substance and a binding compound are bound, and can be prepared by simply mixing the biological substance and the binding compound in a solvent and bringing the molecules into contact with each other. Therefore, a conjugate is usually present in the mixture.

Conjugates form a matrix having a structure in which the conjugates are assembled and the biological materials and binding compounds of the conjugates are bonded to each other, and bonded in a chain and / or network form.
When the biological substance and the binding compound form a particulate lump, the biological substance and the binding compound are bound in the mixture (see FIG. 3A), and FIG. A particulate lump as shown in FIG. Confirmation of the particulate mass in the process of forming such a biological material structure can be confirmed 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. 2 (a) and 2 (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 addition, regarding the relationship between the conjugate and the particulate lump, it is assumed that the particulate lump is a form of the conjugate. Therefore, a large matrix structure of biological material structure having a chain-like and / or network-like structure is formed by aggregation of particulate masses and binding of biological materials and binding compounds of each other. can do. At this time, the biological material structure of the present invention is usually a matrix in which particulate lumps are bound.

Further, as schematically shown in FIG. 5, the non-binding substance forms a region (phase) in the biological material structure.
In the medium, in the process of forming the biological material structure from the biological material and the binding compound, the unbound material does not bind to these. For this reason, single or two or more non-binding substances are aggregated to form a region (phase) in the biological material structure. Since the biological material structure is not formed in this region (phase), this region (phase) can function as a template. Therefore, it is considered that the region (phase) will form a specific hole later.
Here, when a phase separation mechanism is used to form the region (phase), it is preferable to perform a concentration step and / or a drying step described later.
FIG. 5 is a diagram schematically showing a state in a mixture in which a biological substance, a binding compound, and a non-binding substance coexist. However, the dimensions and shapes of the respective parts in FIG. 5 are schematic and do not necessarily match the actual state.

[II-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 mixture is large, a conjugate or a biological material structure may be difficult to generate or may not be generated in the mixture. In these cases, by concentrating the mixture, the conjugate can be formed efficiently, and the biological material structure can be produced efficiently.
Furthermore, even when the above mixture contains a conjugate or a fragment of a biological material structure, the conjugate or the biological material structure can be further formed by concentration. Therefore, concentration may be performed for the growth of such conjugates or biological material structures.

However, in order to form a uniform biological material structure, it is preferable to uniformly mix the biological material and the binding compound in the medium in the initial stage of preparation of the mixture. 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.
In addition, when a phase separation mechanism is used for forming the region (phase) by the above-described non-binding substance, it is preferable to use a concentration step in order to promote the phase separation efficiently. Usually, in the mixing process, the biological substance, the binding compound, and the non-binding substance are mixed in the medium. However, the concentration process promotes the formation of the biological substance structure, and the medium acting as a co-solvent is lost. As a result, a non-binding substance that does not bind to the biological material structure forms a region (phase).

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.

  Further, the concentration of the mixture increases the probability that the biological substance and the binding compound are brought into contact with each other to facilitate the formation of a conjugate, or increases the probability that the conjugates or the particulate lumps are brought into contact with each other. An object of the present invention is to facilitate the construction of the biological material structure. Therefore, before or during concentration, the conjugate and biological material structure may be precipitated by precipitating the mixture by centrifugation, mixing with a poor solvent, or adding an additive such as ammonium sulfate. It may be.

As described above, when the concentration step or the drying step is performed, a region (phase) due to a non-binding substance is more easily formed. Since a biological material structure is not formed in this region (phase), as schematically shown in FIG. 6, the biological material structure is formed so as to avoid a region (phase) made of a non-binding substance (template). It is thought that
In addition, FIG. 6 is a figure which represents typically the mode of the biological material structure after a concentration process or a drying process. However, the dimensions and shapes of the respective parts in FIG. 6 are schematic and do not necessarily match the actual state.

[II-3. Removal process]
After the mixing step, preferably, a removal step for removing unbound substances from the biological material structure is usually performed through a concentration step and a drying step. As a result, as shown in FIG. 7, the portion where the region (phase) made of the non-binding substance was present becomes the specific hole.
FIG. 7 is a diagram schematically showing the state of the biological material structure after the removing step. However, the dimensions and shapes of the respective parts in FIG. 7 are schematic and do not necessarily match the actual state.

There is no limitation on the method for removing unbound material. For example, a biological material structure containing an unbound substance may be washed with a washing solvent to remove the unbound substance.
Here, as the cleaning solvent, a solvent in which the biological material structure is not dissolved or decomposed or the biological material structure is hardly soluble is used. Specifically, a solvent having a solubility of the biological material and the binding compound in the washing solvent of usually 60% by weight or less, preferably 40% by weight or less, more preferably 20% by weight or less is used. The lower limit is theoretically 0% by weight.

  The solubility of the non-binding substance in the washing solvent is arbitrary as long as the non-binding substance can be removed from the biological material structure, but is usually 1% by weight or more, preferably 3% by weight or more, more preferably 5% by weight or more. is there. However, even when the solubility of the unbound substance is low, the unbound substance can be removed by using a large amount of a washing solvent.

Furthermore, the washing solvent is preferably one that does not alter the biological material and the binding compound. This is to maintain the activity of the biological material and the binding compound.
Specific examples of the washing solvent include water-soluble organic solvents such as water, ethanol, 2-propanol, DMSO, and DMF. Of these, water is preferred.
In addition, a washing | cleaning solvent may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

  Further, the cleaning solvent may contain substances such as a buffer substance and a surfactant. For example, for the purpose of preventing the denaturation of biological substances, a substance having a buffering action such as sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, sodium hydrogen carbonate, sodium acetate, citric acid, sodium citrate, HEPES and the like is included. May be. Further, for example, salts such as sodium chloride, potassium chloride, sodium hydrogen carbonate, sodium carbonate may be contained for the purpose of shielding electrostatic interaction between biological substances and washing unbound substances. Moreover, you may contain surfactant, such as tween20 and SDS, for the purpose of improving a cleaning effect.

  Moreover, when manufacturing the biological material structure in which the non-binding substance is present in the specific hole, the removing step is not an essential step. For example, when a manufacturer manufactures a biological material structure in which unbound substances exist in specific holes, and the user removes unbound substances before use, the manufacturer does not necessarily perform the removal process. It doesn't matter. However, it is possible for the manufacturer to perform a removal process to produce a biological material structure in which the non-binding substance is removed from the specific hole (that is, the specific hole is empty). This is preferable because it saves labor.

[II-4. 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. By utilizing this, after the biological material carrier is manufactured, another biological material or other compound modified to specifically bind to the biological material in the biological material structure is bonded later, and as a result, the solid material is fixed. The above-mentioned another biological substance or other compound can be immobilized at a high density on the phase carrier. As a specific example, using avidin as a biological material, this avidin and a binding compound are bonded to form a biological material structure, and a biological material carrier is manufactured. Thereafter, another biological substance modified with biotin or another compound modified with biotin can be used to immobilize the other biological substance or other compound described above by an avidin-biotin interaction. Similarly, a biological substance can be immobilized via a histidine tag or glutathione-S-transferase.

As a specific example, a case where FK506, which is a physiologically active substance (medicine), is used as a compound to be bound to the biological material structure of the present invention and is bound to the biological material contained in the biological material structure will be described. In the case of binding to a biological material using an avidin-biotin interaction, an avidin (eg, streptavidin) is used as the biological material. At this time, an ethylene oxide chain is bound to FK506 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 (streptavidin), the binding compound, and the biotinylated FK506 may coexist at the same time, but it is preferable to contact the biotinylated FK506 after producing the biological material structure. .
Further, for example, the biological material structure of the present invention may be fixed to some solid phase carrier to produce a biological material carrier (solid phase carrier to which the biological material is fixed).

[III. Biological material carrier]
The biological material structure of the present invention can be immobilized on a solid phase carrier and used as an affinity purification tool, a pharmaceutical function analysis tool, or a sensor chip. Further, a surface treatment of a drug for DDS (drug delivery system) It can be applied to surface treatment of regenerative medical carriers, surface treatment of artificial organs, surface treatment of catheters and the like. When the biological material structure of the present invention is used for these surface treatments, etc., a biological material carrier in which the biological material structure of the present invention is fixed to a desired solid phase carrier by an arbitrary method is used. become.

Hereinafter, the biological material carrier will be described.
The biological material carrier of the present invention is obtained by fixing the biological material structure of the present invention to a solid phase carrier. That is, it has the biological material structure of the present invention on the surface of the solid phase carrier.

[III-1. Biological material structure of biological material carrier]
The biological material structure possessed by the biological material carrier of the present invention, and the biological materials and binding compounds that are constituent elements thereof are the same as those described above.
As described above, since the biological material structure of the present invention has a matrix skeleton composed of a main chain formed of both the biological material and the binding compound, the ratio of the biological material can be increased. For this reason, even the biological material carrier of the present invention can immobilize a larger amount of biological material than before. In other words, the biological substance can be immobilized at a large concentration (solid amount of solid phase carrier per unit surface; surface concentration) relative to the solid phase carrier. The polymer membrane according to the prior art has a structure in which a main chain is formed in advance by a polymer chain formed on a solid phase carrier, and a biological substance is bonded to the main chain in a branch shape (graft shape). It has become. Therefore, conventionally, the amount of biological material immobilized is limited by a predetermined upper limit value, and a large amount of biological material cannot be immobilized.

  Further, the thickness (film thickness) of the biological material structure is arbitrary, but when the biological material structure is dried, it is usually 50 nm or more, preferably 100 nm or more, more preferably 500 nm or more, and further preferably 1 μm or more. is there. In addition, although there is no restriction | limiting in particular in an upper limit, Usually, it is 10 cm or less. If the film thickness is too small, the biological material structure may be easily peeled off, it is difficult to make the film thickness of the biological material structure uniform, and the biological material should be fixed with high reproducibility. May become difficult, and there is a possibility that a film cannot be formed sufficiently. The above thickness can be measured by SEM, TEM, AFM, or the like.

[III-2. Solid phase carrier possessed by biological material carrier]
The solid phase carrier serves as a substrate for forming the biological material structure 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 structure 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. As specific examples, chips (substrates) on which a large number of biological substances can be arranged, flat glass members such as glass slides, fiber slides, microtiter plates, beads as chromatography carriers and latex diagnostic agents, and separation membranes Examples thereof include hollow fiber fibers and porous membranes. In particular, when a chip having a flow path is used as a solid phase carrier, the shape and dimensions of the flow path can be arbitrarily formed according to the application. However, when the biological material structure is immobilized on a substrate or the like, it is not necessary. However, when the biological material structure is simply filled and held in the flow path, the biological material is drawn from the flow path. In order to prevent the structure from flowing out, it is preferable to provide an outflow prevention means such as a filter in the flow path.

Further, the solid phase carrier may be used as it is, but after performing some surface treatment, a biological material structure may be formed on the surface. For example, the biological material structure 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 structure. The functional group is arbitrary, but for example, chemical groups such as hydroxyl group, carboxyl group, thiol group, aldehyde group, hydrazide group, carbonyl group, epoxy group, vinyl group, amino group, succinimide group, p-nitrophenyl group, etc. Examples thereof include a functional group that binds the solid phase carrier and the biological material structure by bonding.

In addition, when water is used as a medium such as a solvent or a dispersion medium during the production of the biological material carrier of the present invention, the solid phase carrier and the biological material structure are formed by physical adsorption by lyophobic interaction such as alkyl groups and phenyl groups. It is also possible to use a functional group that binds to each other.
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.

[III-3. Manufacturing method of biological material carrier]
The manufacturing method of the biological material carrier of the present invention is arbitrary. For example, a biological material structure may be prepared in advance and bound to a solid phase carrier. Normally, however, the biological material carrier of the present invention does not contain a biological material, a binding compound, and a non-binding material in the presence of a medium. Produced through a step of supplying a mixture coexisting with a binding substance to a solid phase carrier and forming a biological substance structure having a main chain composed of the biological substance and the binding compound on the surface of the solid phase carrier. . Hereinafter, this method will be described.

[III-3-1. Biological substances, binding compounds and non-binding substances used in the production of biological substance carriers]
The biological material, the binding compound, the non-binding material, and the like are the same as those described above in the description of the manufacturing method of the biological material structure. In addition, when the biological material carrier of the present invention is produced, additives may be allowed to coexist in the biological material, the binding compound, and the non-binding material as appropriate.

[III-3-2. Medium used for manufacturing biological material carrier]
The medium used when manufacturing the biological material carrier of the present invention is the same as the medium described above in the description of the manufacturing method of the biological material structure. Therefore, the medium is preferably selected in consideration of the activity of the biological substance and the binding compound, the stability of the structure, the nature of the non-binding substance, and the like.
Similarly to the production of the biological material structure, water is usually used as the medium when producing the biological material carrier of the present invention. Here, the fact that water can be used as a medium, that is, that a biological material carrier can be produced by supplying a mixture of the above-described biological substance, binding compound and non-binding substance in water to a solid support. This is one of the excellent advantages of the invention.

[III-3-3. Mixture used for production of biological material carrier]
The mixture used for the production of the biological material carrier is a mixture in which at least a biological material, a binding compound and a non-binding substance coexist in the presence of a medium. This mixture is similar to the mixture prepared during the production of the biological material structure.

[III-3-4. Supply of mixture to solid phase carrier]
When manufacturing the biological material carrier of the present invention, the above-mentioned mixture is supplied to a solid phase carrier. That is, the above-mentioned mixture is brought into contact with the solid phase carrier. Although the specific operation is arbitrary, for example, a mixture may be prepared in advance, and the mixture may be brought into contact with a solid phase carrier, or each component of the mixture may be prepared separately, and they may be placed on the solid phase carrier. A mixture may be prepared by mixing, and the mixture may be brought into contact with a solid phase carrier. Specifically, for example, after each of a solution containing a biological substance (such as an aqueous solution), a solution containing a binding compound (such as an aqueous solution), and a solution containing an unbound substance (such as an aqueous solution) are supplied onto the solid phase carrier, It can be performed by mixing each solution on a solid support. In addition, when preparing a mixture in advance, the conjugate (including a particulate mass that is one form thereof) or a biological material structure is prepared in the mixture before supply, and then the mixture is solid-phased. You may make it supply to a support | carrier.

[III-3-5. Formation of biological material structure on solid support surface]
Next, a biological material structure having a main chain composed of the biological material and the binding compound is formed on the surface of the solid phase carrier. Since the mixture includes a conjugate, a particulate mass, a biological material structure, or the like as described above, by bringing the mixture into contact with a solid phase carrier, for example, the conjugate, the particulate mass, or the The biological material structure is accumulated on the surface of the solid phase carrier, the biological material structure formed by combining conjugates and particulate masses in the mixture is bound to the solid phase carrier, and the biological material and binding in the mixture A biological material structure can be formed on the surface of the solid phase carrier by generating a biological material structure by binding the compound for use to the surface of the solid phase carrier.

Conditions for binding the biological material structure 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 structure 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.
Moreover, you may make it perform a concentration process or a drying process suitably like the time of manufacture of a biological material structure. Furthermore, usually, after supplying the mixture to the solid phase carrier, a non-binding substance is removed from the biological material structure by performing a removing step.

[III-4. Advantages of biological material support compared to conventional technology]
As described above, the above method can be applied to a solid phase carrier surface only by bringing a mixture of a biological substance, a binding compound, and a non-binding substance into contact with the solid phase carrier in a medium (usually a solvent). A biological material structure having pores can be formed, whereby the biological material carrier of the present invention can be produced, that is, the biological material can be immobilized on a solid phase carrier. It is a simple method.

  If the biological material carrier of the present invention is used, the biological material can be immobilized in a larger amount than before. For example, conventionally, a biological substance is bound to a polymer film (polymer film) formed on the surface of a solid support. However, in this case, the biological material is bonded to the tip of the polymer chain or grafted to the polymer chain to the main chain formed by the polymer chain, so that the biological material is attached to the solid phase carrier. Since they were bonded, it was necessary to form a polymer chain as the main chain, and a certain amount of polymer had to be used for the biological material to be immobilized, and the amount of biological material immobilized was limited.

In contrast, in the present invention, a biological material structure having a biological material structure skeleton (main chain) formed by both a biological material and a binding compound (corresponding to a polymer) is formed, and this biological material is formed. Since the biological substance can be immobilized on the solid phase carrier using the structure, the ratio of the biological substance in the biological substance structure can be increased (that is, the ratio of the binding compound can be reduced). Therefore, when immobilizing a biological material, there is no conventional limitation, and it is possible to immobilize the biological material more than before, that is, with a high density on the solid phase carrier surface.
Furthermore, since it is possible to form specific holes, it is possible to perform separation more efficiently when affinity separation or the like is performed.

  Another advantage is that the biological material carrier of the present invention can be produced by a simple method. In the conventional method, much labor is required to immobilize the biological material at a high density. Specifically, in the past, a polymer film was previously formed on a solid phase carrier, a biological material was immobilized on the polymer membrane, and a biological material structure including a ligand on the solid phase carrier was constructed. . However, in these methods, when preparing a polymer film on the solid phase, it is necessary to appropriately control the molecular weight of the polymer and the density of introduction to the solid support, which is very complicated and reproducible. It was difficult to fix well.

  In contrast, the biological material carrier of the present invention can be obtained by contacting a solid phase carrier with a mixture in which a biological material, a binding compound and a non-binding substance coexist in a medium (usually a solvent). It is possible to form a biological material structure having specific pores, that is, to immobilize the biological material, which can be manufactured very easily. Further, the solvent used for the production of the biological material carrier is not limited to the organic solvent, and the usable biological material is not limited thereby, so that the selection range of the biological material to be immobilized can be expanded.

  Furthermore, in the biological material structure having specific holes formed in the biological material carrier of the present invention, the biological material can be fixed on the solid phase carrier in a three-dimensional substantially uniform state. Therefore, it is possible to construct an optimum reaction field for interaction between a biological substance and an agent that specifically interacts with the biological substance. Thereby, for example, when the biological material carrier of the present invention is used in a sensor using the above-described interaction, the detection sensitivity of the sensor can be increased.

  The reason why the above-mentioned optimum reaction field can be constructed is as follows as inferred from the inventors of the present invention. That is, in the conventional method of immobilizing a biological material on a solid support surface-treated with a conventional polymer membrane, the biological material is biased on the surface of the membrane, and there is no void in which the active substance can enter the polymer membrane. End up. Furthermore, in the conventional technique in which the main chain is constructed only with a hydrophilic polymer, it is speculated that the hydrophilic polymer chain hinders the access of the active substance to the biological substance due to the excluded volume effect and the movement of the polymer chain (biological substance). Editing method for real-time analysis of material interaction-BIACORE, editing Kazuhiro Nagata, Handahiro, publisher Springer Fairlark Tokyo, page 258, Development and application of medical polymer materials, CMC, 19th page).

  However, in the biological material structure formed on the biological material carrier of the present invention, the biological material can be fixed on the solid support in a three-dimensional substantially uniform state. Furthermore, by using the technique of the present invention, the structure of the biological material structure forms a void in which the active substance can sufficiently react in the polymer film. The biological material structure of the present invention is formed with a specific hole that facilitates the entry of the active substance into the biological material structure. From these, it is considered that an optimal reaction field can be constructed.

  The biological material structure formed on the biological material carrier of the present invention can arbitrarily control the film thickness. In the prior art, it is usually difficult to arbitrarily control the thickness of the biological material structure from the submicron level to several micron level. However, according to the present invention, the thickness of the biological material structure can be controlled at the precise level as described above, and the degree of freedom in designing the biological material structure can be increased.

  For example, when performing interaction observation by SPR, it is considered that the optimal film thickness used for fixing the observation target is about 200 nm to 300 nm. In addition, for example, when performing a surface treatment of a medical instrument or a regenerative medical carrier, a micro-order film thickness is required for a film used for the surface treatment in order to realize sufficient strength and coating. Further, for example, when performing surface treatment of a drug for DDS (drug delivery system), it is required to arbitrarily control the film thickness used for the surface treatment in order to control drug release. . As described above, when some kind of membrane is used for immobilization of a biological material, its film thickness control is one of the important points, but conventionally it has been difficult to control the film thickness. However, according to the present invention, the film thickness according to the application can be arbitrarily controlled by adjusting the concentration, amount, reaction conditions (temperature, time, etc.) of the mixture.

  Further, in the biological material-carrying body of the present invention, when the biological material-carrying body of the present invention is used to allow the biological material and the active substance to interact with each other, non-specificity caused by a biological material structure component other than the biological material Interaction can be suppressed. This is because the ratio of biological substances in the biological substance structure can be increased, and the ratio of substances other than biological substances that cause non-specific interactions can be suppressed.

  In particular, when an uncharged compound is used as the binding compound, it is possible to further suppress the nonspecific interaction caused by the charge. That is, for example, according to the description of “Nanotechnology Basic Series Bionanotechnology Yasuhiro Horiike and Kazunori Kataoka (co-edition), page 186”, when a certain conventional technique is used, the polymer film has a charge. The pH and ionic strength of the buffer solution to be used have a great influence on the reaction of biological substances, and furthermore, nonspecific adsorption due to electrostatic interaction has been unavoidable for charged proteins. However, if an uncharged binding compound is used, this is not the case, and a specific interaction can be selectively generated.

[III-5. Use of biological material carrier]
The biological material carrier of the present invention can be suitably used as, for example, a biosensor that detects an agent that interacts with a biological material. The above biosensor analyzes the interaction using, for example, a so-called DNA array or DNA chip, or a sensor chip on which DNA or protein is immobilized, which is called a protein array or protein chip. For example, the biological material carrier of the present invention can be applied to this sensor chip. That is, when the biological material is immobilized on the sensor chip, the biological material structure can be formed on the sensor chip body by the method described above, and the sensor chip can be used as the biological material carrier of the present invention.

  Specific examples of such biosensors include fluorescence method, ELISA method, chemiluminescence method, RI method, SPR (surface plasmon resonance) method, QCM (quartz crystal microbalance) method, piezo method cantilever method, laser method cantilever Examples thereof include a method, a mass spectrometry method, an electrochemical method, an electrode method, a field effect transistor (FET) method, a FET using a carbon nanotube, and / or a sensor using a single electron transistor method. Among these, detection by the SPR method and the QCM method is preferably used because the sample can be easily analyzed without labeling.

  In order to induce surface plasmon waves in the SPR method, the surface of the sensor chip is preferably coated with a metal. The metal is not particularly limited as long as it can induce surface plasmon waves, and examples thereof include gold, silver, copper, aluminum, and alloys containing these. Of these, silver is preferable in terms of sensitivity and inexpensiveness, and gold is preferable in terms of stability. The metal layer is formed by vapor deposition, sputtering, plating, other coatings, etc., and its thickness is usually about 20 nm or more, preferably 30 nm or more, and usually about 300 nm or less, preferably about 160 nm or less.

When the biological material carrier of the present invention is applied to these SPR method and QCM method, the surface of the sensor chip body has a functional group in order to firmly bond the biological material structure to the surface of the sensor chip body. Preferably it is.
Furthermore, the present invention can be applied to a surface treatment of a drug for DDS (drug delivery system), a surface treatment of a regenerative medical carrier, a surface treatment of an artificial organ, a surface treatment of a catheter or the like.

[III-6. Biological substance immobilization kit]
What is used for immobilizing a biological material on a solid phase carrier to produce the biological material carrier described above, that is, the above-described binding compound and non-binding material, and the biological material and binding compound can be mixed. A biological material immobilization kit in which a medium such as a solvent or a dispersion medium is made into a kit may be used. That is, in order to manufacture a biological material carrier, a biological material immobilization kit including a binding compound, a non-binding material, and a medium in which the biological material and the binding compound can be mixed may be prepared. If the biological material immobilization kit is used, the biological material carrier can be easily manufactured, that is, the biological material structure can be easily prepared on the solid phase carrier. It can be fixed.

  The binding compound and non-binding substance provided in the biological material immobilization kit are the same as those described above. In the biological material immobilization kit, the binding compound and the non-binding substance may be provided in any state, for example, a solution dissolved in an arbitrary solvent, a dispersion liquid dispersed in an arbitrary dispersion medium, or a powder. The state of existence is arbitrary, such as a solid or massive solid.

  The medium provided in the biological material immobilization kit is the same as the medium described above as the medium used for manufacturing the biological material carrier. Further, in the biological material immobilization kit, this medium may be provided separately from the binding compound and the non-binding substance, and the binding compound and / or the binding compound as a solvent or dispersion medium of the non-binding substance and the like. It may be provided integrally with the non-binding substance.

Furthermore, the biological material immobilization kit may be provided with other elements as necessary.
For example, you may further provide the reagent etc. which accelerate | stimulate manufacture of a biological material structure. As a specific example, when polyacrylic acid is used as the binding compound, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: EDC) is used to activate the carboxyl group of polyacrylic acid. ) As a reagent.

[IV. Explanation of affinity separation]
The biological material structure and biological material carrier of the present invention are suitable for use in affinity separation. Therefore, the biological material structure and biological material carrier of the present invention can be used, for example, as affinity purification or a pharmaceutical function analysis tool.

  Specifically, for example, when a physiologically active substance is used as a biological substance for the purpose of affinity purification or a pharmaceutical 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 biological substance and the target substance. For example, when analyzing the action mechanism of a drug candidate compound on a biological substance contained in a biological substance structure, a plurality of drug candidate compounds can be analyzed by using a protein that contributes to a disease such as a receptor as the biological substance. By selecting the drug candidate compound or analyzing the mechanism of action, it becomes possible to select the drug candidate compound by bringing it into contact with the biological material structure and analyzing the drug candidate compound specifically adsorbed.

  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 biological substance contained in the biological substance structure and the target substance. At this time, first, a sample liquid (specimen) containing the target substance is brought into contact with the biological material structure to separate the target substance and other substances in the sample liquid.

[IV-1. Substances subject to separation and purification]
The target substance to be separated and purified is an agent that specifically interacts (affinity binds) with a biological substance. As an example of such a target substance, the same biological substance as the biological substance structure described above can be used.
As a specific example, when separating a substance that can be a drug candidate (drug candidate substance), a substance that can cause a desired interaction that is desired to occur in the drug candidate substance is used as a biological substance, and contains a drug candidate substance. 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.

In addition, for diagnostic analysis tools, blood, serum, plasma, bone marrow, urine, feces, nasal discharge, saliva, and other body fluids, cells, tissues, and biological substances such as antibodies A substance that specifically binds becomes a target substance, and a disease can be diagnosed by analyzing the amount or presence of the target substance.
Further, when a nucleic acid such as protein, DNA, RNA or the like is used as a biological substance, a protein screening method and function modification method using a protein-nucleic acid linking molecule as an 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 structure of the present invention ( For example, see Yanagawa et al., “Protein Nucleic Acid Enzyme”, Vol. 48 No. 11 P1474 (2003)).

[IV-2. Sample solution containing the target substance]
The target substance usually coexists with other substances in the specimen which is 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 structure 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 liquid becomes too high, and there is a possibility that the target substance will sufficiently penetrate into the biological material structure and cannot come into contact with the biological material.

[IV-3. Method of contacting biological material structure with sample liquid]
The method for bringing the sample liquid containing the target substance into contact with the biological material structure of the present invention is not particularly limited. For example, the sample liquid containing the target substance is allowed to flow along with the mobile phase in a column filled with the biological material structure. The method of making it contact is mentioned. At this time, the biological material structure 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 placed in a container such as a microtube, and a biological material structure is added to the sample liquid, or the container is placed with a biological material structure. It is also possible to make contact by adding a sample solution containing the target substance to the liquid crystal.

  Furthermore, it is possible to further improve the efficiency of separation and purification by immobilizing the biological material structure 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 structure 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 structure, any conditions may be set as appropriate, preferably 25 ° C. or lower, more preferably 10 ° C. or lower. Is desirable.
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.

[IV-4. Separation of target substances]
How the target substance is separated after the contact between the biological material structure of the present invention and the sample liquid is arbitrary depending on the type of the biological substance and the target substance.
For example, if the biological substance and the target substance interact specifically, resulting in a difference in retention time (retention time) between the target substance and other substances, 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 a specific interaction with the biological substance, the target substance is adsorbed on the biological substance, and the sample liquid is separated from the biological substance structure in that state, and then The target substance can be separated from other substances by releasing the target substance from the biological substance and collecting it. The method for releasing the target substance from the biological substance is not particularly limited. For example, in addition to the added salt, pH, temperature rise, etc., the biological substance structure is a known chemical substance having a higher adsorption power than the separation / purification target or equivalent Alternatively, even if the adsorption power is weak, the target substance can be released from the biological material structure 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, it may be mixed with a solvent according to the purpose. 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 structure 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 on the biological material in the second embodiment to be described later, or when measuring the amount of the target substance in the fractionation by the measurement unit 21 in the fourth embodiment, A measuring instrument using the exemplified method can be used.

[IV-5. Advantages of affinity separation using biological material structures]
In the affinity separation according to the prior art, since the biological material is immobilized on the surface of the affinity chromatography carrier used for the separation, the introduction amount of the biological material is limited (usually, protein monolayer adsorption is 0.3 to 1.0 μg / cm ² ). Therefore, conventionally, in the affinity chromatography carrier, when the biological substance and the target substance are allowed to interact, the amount of the target substance that can interact in the unit volume is small.

  Furthermore, conventionally, the affinity chromatography carrier could not be completely covered with a biological material, 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.

  On the other hand, in the biological material structure of the present invention, the reactivity of the biological material introduced into the biological material structure can be maintained. Further, by increasing the ratio of the biological material in the biological material structure, it is possible to increase the amount of the target substance that specifically interacts with the biological material in the unit volume of the biological material structure. Furthermore, since the ratio of the binding compound can be kept low by increasing the ratio of the biological material in the biological material structure, nonspecific adsorption based on the binding compound can be suppressed. As a result, non-specific adsorption can be suppressed as compared with the conventional case, and the biological material structure has specific pores, so that the time required for separation and purification can be shortened.

  Moreover, the biological material structure 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 structure of the present invention.

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

[IV-6-1. First Embodiment]
In the present embodiment, the 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.
FIG. 8 is a cross-sectional view schematically showing the affinity chromatography container used in the present embodiment.

  In this embodiment, as shown in FIG. 8, an affinity chromatography container 3 (hereinafter referred to as “affinity container” as appropriate) 3 holding a biological material structure 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 structure 2 is fixed to the inner surface of the container body 1, thereby preventing the biological material structure 2 from coming out of the container body 1. It is held in the main body 1. However, the biological material structure 2 may be simply housed in the container body 1 without being fixed to the container body 1.
Furthermore, the biological material of the biological material structure 2 used in the present embodiment and the target material to be purified specifically interact, so that the target material can be specifically adsorbed to the biological material. 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 structure 2 comes into contact with the sample liquid, and the target substance is adsorbed on the biological material.
Next, the sample liquid is discharged out of the affinity container 3 in order to separate the sample liquid and the biological material structure 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 biological substance of the biological substance structure 2.
Then, the target substance is released from the biological material structure 2 by, for example, 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 biological substance. 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 biological substance that specifically interacts with the target substance is used, and the biological substance structure of the present invention contains a very large amount of the biological substance that maintains 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 substance causes a given interaction with the biological substance using this, it is screened what kind of substance interacts with the biological substance. 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 structure 2 is not fixed to the container body 1 in the configuration of the present embodiment, the biological material structure 2 is collected by using a centrifuge before discharging the sample liquid. It is preferable because efficiency and recovery efficiency are improved.

[IV-6-2. Second Embodiment]
In the present embodiment, the 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. 8 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 biological substance that the biological substance structure 2 has, thereby allowing the substance to be used. 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, the sample liquid is injected into the affinity container 3 to bring the biological material structure into contact with the sample liquid, as in the first embodiment. Subsequently, the sample liquid is discharged out of the affinity container 3 in order to separate the biological material structure 2 and the sample liquid. Thereby, when the target substance has a specific structure capable of specifically interacting with the biological substance, the target substance is adsorbed to the biological substance of the biological substance structure 2, whereby 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 specific structure described above by checking whether or not the target substance is adsorbed on the biological substance, that is, whether or not the target substance remains in the affinity container 3. It turns out whether or not.
In order to examine whether or not the target substance is adsorbed on the biological substance, specifically, the amount of the target substance adsorbed on the biological substance may be measured. For example, as in the first embodiment, the target substance is introduced into the biological material structure 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 biological 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.

In this way, it is possible to analyze whether or not the target substance in the sample solution has at least a specific structure corresponding to the biological substance.
Furthermore, the technique of the present embodiment can also be applied when examining the molecular structure that an active substance should have in order to interact with a biological substance by utilizing this. That is, for example, when a group of substances (target substances) is separated from other substances by interacting with a biological substance, if the group of substances have 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 biological substance.

Moreover, since the biological material structure 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.

[IV-6-3. Third Embodiment]
In the present embodiment, the target substance is separated and purified from a sample solution in which the target substance (substance to be purified) and other substances coexist in the liquid.
FIG. 9 is a diagram schematically showing the 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”) 10 as shown in FIG. 9 is used. The affinity chromatography apparatus 10 includes a tank 11, a pump 12, an auto-injector 13, an affinity separation chip 14 that is a biological material carrier, a flow path switching valve 15, recovery bottles 16 and 17, and a control unit 18. And has.

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 structure (not shown). Here, the biological material of the biological material structure used in the present embodiment and the target substance to be purified change the retention time of the target substance flowing through the flow path 14B by specifically interacting. It is assumed that Note that a filter (not shown) for preventing the biological material structure from flowing out is formed at the downstream end of the flow path 14B, whereby the biological material structure is securely held in the flow path 14B. It has become so.

  Therefore, the supplied carrier liquid (including the liquid into which the sample liquid has been injected) flows through the flow path 14B filled with the biological material structure. 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 stores information on the retention time due to the interaction between the target substance and the biological substance included in the biological substance structure 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. 9, 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 structure in the flow path 14B, the biological material 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 biological substance that specifically interacts with the target substance is used, and the biological substance structure of the present invention contains a very large amount of the biological substance that maintains its activity. Furthermore, it is possible to easily perform highly efficient separation and purification by suppressing non-specific interaction.

Furthermore, using this, as in the first embodiment, when an active substance that causes a predetermined interaction between the biological substance and the biological substance is unknown, what kind of substance is It can also be screened for interaction.
In the configuration of this embodiment, the biological material structure 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.

[IV-6-4. Fourth Embodiment]
In the present embodiment, the 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. 10 is a diagram schematically showing the outline of the affinity chromatography apparatus used in the present embodiment. However, in FIG. 10, the same parts as in FIG. 9 are denoted by the same reference numerals as in FIG.

  In this embodiment, an affinity chromatography apparatus 20 as shown in FIG. 10 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 biological substance of the biological substance structure. 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.

  Furthermore, the affinity chromatography device 20 includes a measurement unit 21 that measures the amount of the 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 structure 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 structure and the sample liquid are in 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 device 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 biological substance. . Moreover, since the biological material structure of the present invention is capable of easily performing 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 biological 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 structure, a specific structure corresponding to the structure, 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.

  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 structure using albumin]
(1) Synthesis of Compound for Binding (Polymer A) N-acryloylmorpholine (NAM, manufactured by KOHJIN) as a monomer 2.82 parts by weight and N-acryloyloxysuccinimide (N-ACRYLOXYSUCCINIMIDE (NAS)) 0 .84 parts by weight and 20.87 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. To prepare a monomer solution.

This monomer solution was heated to 70 ° C. in an oil bath, and a solution in which 0.0124 parts by weight of a polymerization initiator azobisisobutyronitrile (AIBN, manufactured by Kishida Chemical Co., Ltd.) was dissolved in 0.50 parts by weight of dehydrated dioxane. The polymerization was started by adding. The polymerization was carried out for 8 hours under a nitrogen atmosphere.
After the polymerization, the polymer-generated solution was reprecipitated by adding dropwise to 0.5 L of ethanol (manufactured by Junsei Co., Ltd.), and then pulverized by removing the solvent to obtain a binding compound polymer A. .

The obtained polymer A was subjected to SEC (Size Exclusion Chromatography) measurement calibrated with standard polystyrene. As a result, the weight average molecular weight (Mw) of the polymer A was estimated to be about 59,500.
The molar ratio (NAS / NAM) between NAS and NAM contained in the obtained polymer A was estimated to be NAS / NAM = 23/77 from ¹ H-NMR measurement.

(2) Formation of a biological material structure on the surface of a gold chip (2-1) Surface treatment of a gold chip A substrate surface made of a flat resin having a size of 2.5 cm long × 2.5 cm wide × 1.2 mm thick A chip on which gold was deposited at a thickness of about 80 nm was immersed in a 10 mM ethanol solution of 16-mercaptohexadecanoic acid (16-MERCAPTOCHEXADENOIC ACID: ALDRICH) and reacted at room temperature for 12 hours for surface treatment. After completion of the reaction, the gold chip was washed with ethanol. This surface treatment is a treatment for introducing a carboxyl group into the surface of the gold chip via a gold-sulfur bond.

  Next, 1 mL of an aqueous solution of 0.1 M N-hydroxysuccinimide (NHS, manufactured by Wako Pure Chemical Industries) and 0.4 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC, Dojindo) The substrate (gold chip) into which the carboxyl group was introduced was immersed in a solution mixed with 1 mL of an aqueous solution and further 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, 47.4 mg / mL albumin (manufactured by SIGMA) and 47.6 mg / mL polyethylene glycol 600 (manufactured by Wako Pure Chemical Industries, Ltd .; hereinafter referred to as “PEG” as appropriate) ) And an aqueous solution (carbonate buffer 110 mM, pH 9.4) and polymer A mixed at a weight mixing ratio of 10: 1 (biological substance: binding compound) were spotted on the surface of a gold chip by 5 μL. After drying this at room temperature, it was immersed in 3 mL of 1M ethanolamine-HCl aqueous solution (pH 8.5) for 30 minutes for the purpose of blocking unreacted active ester groups. As a result, a biological material structure including albumin, polymer A, and polyethylene glycol 600 as the biological material, the binding compound, and the non-binding material was formed on the surface of the gold chip.
Thereafter, the gold chip was thoroughly washed with demineralized water for the purpose of removing polyethylene glycol, and dried at room temperature to obtain a biomaterial structure gold chip carrying the biomaterial structure on the surface.

(3) Observation of structure of biological material structure The surface of this chip was observed with an AFM (atomic force microscope, Nanoscope IIIa Multimode manufactured by Digital Instruments). A drawing-substituting photograph showing the observation result in a 10 μm × 10 μm field of view is shown in FIG. As can be seen from FIG. 11, a hole having a diameter of about 1 μm was confirmed on the chip surface. In addition, FIG. 12 shows a drawing-substituting photograph showing the observation result in a higher magnification field of view (500 nm × 500 nm field of view). In FIG. 12, a smaller space was observed, suggesting the formation of a particulate mass.

[Example 2: Biological material structure using streptavidin]
(1) Formation of a biological material structure on the surface of a 96-well plate (1-1) Surface treatment of a 96-well plate 1 mL of an aqueous solution of 0.1 M N-hydroxysuccinimide (NHS, manufactured by Wako Pure Chemical Industries) and 0.4 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC, manufactured by Dojindo Laboratories) aqueous solution 1 mL was mixed and further diluted with 18 g of demineralized water to introduce a carboxyl group. 300 μL / well was added to a 96-well plate (Sumilon (registered trademark) ELISA product / carbo type, manufactured by Sumitomo Bakelite Co., Ltd.), and allowed to react for 15 minutes. This is for introducing a succinimide group capable of binding the well and the biological material structure to the surface in the well.

(1-2) Formation of biological material structure Next, an aqueous solution (carbonate buffer 11 mM, pH 9.4) containing 30 mg / mL streptavidin (manufactured by SIGMA) and 30 mg / mL polyethylene glycol 600 and polymer A are weighed. 5 μL of the mixture mixed at a mixing ratio of 10: 1 (biological material: binding compound) was spotted into the wells of a 96-well plate. After drying this at room temperature, for the purpose of blocking unreacted active ester groups, 300 μL of 1M ethanolamine-HCl aqueous solution (pH 8.5) was immersed in 1 well at room temperature for 30 minutes. As a result, a biological material structure comprising streptavidin, polymer A, and polyethylene glycol 600 as the biological material, the binding compound, and the non-binding material was formed on the well surface of the plate.
Thereafter, the well was thoroughly washed with demineralized water for the purpose of removing polyethylene glycol 600 and dried at room temperature to obtain a biological material structure plate carrying the biological material structure on the surface.

(2) Evaluation by ELISA method The performance of the sample was analyzed by ELISA. The biological material structure plate was blocked with PBS (−) 200 μl / well containing 1 wt% BSA at 37 ° C. for 1 hour. The solution was removed, 100 μl of PBS (−) containing 1 μg / mL biotinylated horseradish peroxidase (PIERCE) and 1% BSA at room temperature was added to each well, and incubated at room temperature for 1 hour. After removing the solution, the plate was thoroughly washed with PBS (-) containing 0.05% Tween20. Add 0.4mg / mL orthophenylenediamine (OPD, Sigma) and citric acid-phosphate buffer solution (pH 5.0) containing about 0.014% hydrogen peroxide solution and react at room temperature to develop color Was done. After 5 minutes or 30 minutes, the reaction was stopped by adding a 1N H ₂ SO ₄ solution, and further diluted 2-fold with a 0.5N H ₂ SO ₄ solution, and then measured at a measurement wavelength of 490 nm and a reference wavelength of 650 nm.
The results are shown in FIG. In FIG. 13, this example showed higher absorbance (OD) than Comparative Examples 2 and 3 (described later). This suggests that the formation of pores in the biological material structure facilitated the diffusion of the substrate or product and facilitated the enzymatic reaction, suggesting that this method is effective. It was.

[Comparative Example 1]
Succinimide groups were introduced on the surface of the chip coated with gold in the same manner as in “(2-1) Surface treatment of gold chip” in Example 1.
Next, a mixture obtained by mixing a 50.4 mg / mL albumin (manufactured by SIGMA) aqueous solution (carbonate buffer 110 mM, pH 9.4) and polymer A at a weight mixing ratio of 10: 1 (biological substance: binding compound) 5 μL spotted on the surface of the chip. After drying this at room temperature, it was immersed in 3 mL of 1M ethanolamine-HCl 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 gold chip.

Thereafter, the gold chip was thoroughly washed with demineralized water and dried at room temperature to obtain a biomaterial structure gold chip carrying the biomaterial structure on the surface.
In the same manner as in Example 1, the AFM surface of the gold chip was observed. A drawing-substituting photograph showing the observation result at 10 μm × 10 μm is shown in FIG. 14, and a drawing-substituting photograph showing the observation result in the 500 nm × 500 nm field of view is shown in FIG. As can be seen from FIG. 15, the biological material structure produced in this comparative example has a space, but no specific hole is formed as shown in FIG.

[Comparative Example 2]
A succinimide group was introduced into the surface of the well of the 96-well plate in the same manner as in “(2-1) Surface treatment of plate” in Example 1.
Next, a mixture of 30 mg / mL streptavidin aqueous solution (manufactured by PIERCE) (carbonic acid buffer 11 mM, pH 9.4) and polymer A at a weight mixing ratio of 10: 1 (biological substance: binding compound) was added to 96 wells. 5 μL spotted on the surface of the plate. This was dried at room temperature, and then immersed in 300 μL / well of 1M ethanolamine-HCl aqueous solution (pH 8.5) for the purpose of blocking unreacted active ester groups. As a result, a biological material structure comprising streptavidin and polymer A as the biological material and the binding compound was formed on the well surface of the 96-well plate.

Thereafter, the inside of the well of the plate was thoroughly washed with demineralized water and dried at room temperature to obtain a biological material structure plate carrying the biological material structure on the surface.
Using this plate, ELISA analysis was performed in the same manner as in “(2) Evaluation by ELISA method” in Example 2. The results are shown in FIG.

[Comparative Example 3]
A succinimide group was introduced into the surface of the well of the 96-well plate in the same manner as in “(2-1) Surface treatment of plate” in Example 1.
Next, 5 μl of 27.2 mg / ml streptavidin (PIERCE) aqueous solution (carbonate buffer 10 mM, pH 9.4) was spotted on the surface of a 96-well plate. This was dried at room temperature, and then immersed in 300 μL / well of 1M ethanolamine-HCl aqueous solution (pH 8.5) for 30 minutes. Thus, streptavidin was two-dimensionally immobilized on the well surface of the 96-well plate.

Thereafter, the inside of the well of the plate was thoroughly washed with demineralized water and dried at room temperature to obtain a plate carrying the biological material structure on the surface.
Using this plate, ELISA analysis was performed in the same manner as in “(2) Evaluation by ELISA method” in Example 2. The results are shown in FIG.

The present invention can be used in a wide range of industries. There is no restriction on the specific application, and it can be used for any application. Usually, it is used for an application that uses "interaction" between a biological substance and an agent that specifically interacts with the biological substance. It is preferable.
For example, the present invention is suitably used for a sensor chip for analyzing an interaction between biological materials, a surface treatment of a medical material requiring biocompatibility, a biosensor, a diagnostic device, and the like. Moreover, it is suitable for use in fields such as medical care, 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. Furthermore, for example, by using a biological material carrier having a biological material structure formed on the surface of a flow channel, and the like, an automated affinity purification apparatus or pharmaceutical action can be obtained by flowing a mixed solution to be separated and purified through the flow channel. Etc. can be easily produced.

(A)-(c) is a schematic diagram which expands and shows the surface vicinity of an example of the biological material structure of this invention fixed to the solid-phase carrier. (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 binding | bonding typically, (b) is a model of a particulate lump. FIG. It is a schematic diagram which expands and shows the surface vicinity of an example of the biological material structure of this invention fixed to the solid-phase carrier. It is a figure which represents typically the mode in the mixture in which the biological material, the compound for binding, and the non-binding substance coexist in the mixing process of the manufacturing method of the biological material structure of the present invention. It is a figure which represents typically the mode of the biological material structure after the concentration process or the drying process of the manufacturing method of the biological material structure of this invention. It is a figure which represents typically the mode of the biological material structure after the removal process of the manufacturing method of the biological material structure of this invention. FIG. 8 is a cross-sectional view schematically showing a container for affinity chromatography as one embodiment of the present invention. FIG. 9 is a diagram schematically showing an outline of an affinity chromatography apparatus as one embodiment of the present invention. FIG. 10 is a diagram schematically showing an outline of an affinity chromatography apparatus as one embodiment of the present invention. It is a drawing substitute photograph showing the result of having observed the biological material structure in Example 1 of this invention. It is a drawing substitute photograph showing the result of having observed the biological material structure in Example 1 of this invention. It is a graph showing the result of the ELISA analysis in Example 2 and Comparative Examples 2 and 3 of the present invention. 6 is a drawing-substituting photograph showing a result of observing a biological material structure in Comparative Example 1. FIG. 6 is a drawing-substituting photograph showing a result of observing a biological material structure in Comparative Example 1. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container body 2 Biological material structure 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 21 Measurement unit

Claims (27)

A biological material structure having a main chain composed of a biological material and a compound capable of binding to the biological material, wherein pores having a diameter of 300 nm to 100 μm are formed by the main chain. A biological substance, a compound that can bind to the biological substance, and a non-binding substance that does not bind to the biological substance and the compound and can form a region having a diameter of 300 nm to 100 μm coexist in a predetermined medium. A biological material structure obtained through a process,
A main chain composed of the biological material and the compound is formed,
A biological material structure, wherein pores having a diameter of 300 nm to 100 μm are formed by the main chain. The biological material structure according to claim 1 or 2, wherein the biological material structure comprises a particulate mass comprising the biological material and the compound and having a particle size of 10 µm or less. The biological material structure according to any one of claims 1 to 3, wherein a ratio of the weight of the biological material to the weight of the biological material structure is 0.1 or more. The biological material structure according to any one of claims 1 to 4, wherein the biological material structure has a diameter of 1 µm or more in a dry state. The biological material structure according to any one of claims 1 to 5, wherein the molecular weight of the compound is 1000 or more. The biological material structure according to any one of claims 1 to 6, wherein at least one of the compounds has two or more functional groups capable of binding to the biological material. The biological material structure according to claim 1, wherein the compound is uncharged. The biological material structure according to claim 1, wherein the compound is miscible with water and miscible with at least one organic solvent. The biological material structure according to any one of claims 1 to 9, wherein a diameter of the compound in a state of being mixed in a liquid is 1 nm or more. The biological material structure according to any one of claims 1 to 10, wherein the biological material is a biomolecule. The biological material structure according to any one of claims 1 to 11, wherein the biological material is a protein. A method for producing a biological material structure having a main chain composed of a biological material and a compound that can bind to the biological material,
A mixing step in which a non-binding substance capable of forming a region having a diameter of 300 nm to 100 μm that does not bind to the biological substance, the compound, and the biological substance and the compound coexists in a predetermined medium. A method for producing a biological material structure, comprising: 14. The method of manufacturing a biological material structure according to claim 13, further comprising a removing step of removing the non-binding substance after the mixing step. The method for producing a biological material structure according to claim 13 or 14, wherein the non-binding substance is a water-miscible substance. The method for producing a biological material structure according to any one of claims 13 to 15, wherein the non-binding substance is a water-soluble polymer compound.   A biological material carrier, wherein the biological material structure according to any one of claims 1 to 12 is fixed to a solid phase carrier. The biological material carrier according to claim 17, wherein the biological material structure has a thickness of 50 nm or more. The biological material structure according to any one of claims 1 to 12 is brought into contact with a sample liquid containing a target substance that can be adsorbed specifically to the biological material,
Separating the biological material structure and the sample solution;
A method for purifying a target substance, wherein the target substance adsorbed on the biological material structure is released. A sample liquid containing a target substance that specifically interacts with the biological material is circulated through the flow path holding the biological material structure according to any one of claims 1 to 12.
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 structure according to any one of claims 1 to 12, which is held in the container body. A substrate formed with a flow path;
A separation chip comprising the biological material structure according to claim 1 held in the flow path. The biological material structure according to any one of claims 1 to 12, comprising a biological material structure using a biological material capable of specifically adsorbing a substance having a specific structure, and a target substance. Contact the sample solution,
Separating the biological material structure and the sample solution;
A method for analyzing a target substance, comprising measuring the amount of the target substance adsorbed on the biological substance and analyzing the structure of the target substance. The biological material structure according to any one of claims 1 to 12, wherein the biological material structure uses a biological material 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 12, wherein a substrate on which a flow path is formed and a biological material held in the flow path and capable of specifically interacting with a substance having a specific structure are used. A separation chip comprising a material structure;
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 12, wherein a substrate on which a flow path is formed and a biological material held in the flow path and capable of specifically interacting with a substance having a specific structure are used. A chip mounting part for mounting a separation chip comprising a substance structure;
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;
A separation device for analyzing 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 structure according to claim 1.