JP2009109452A - Orientation control material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an orientation control material for immobilizing a target material on a solid phase material by avoiding or suppressing direct interaction with the solid phase material including an immobilization element for immobilizing the target material. <P>SOLUTION: The target material is immobilized on the solid phase material by utilizing as the orientation control material, a material having following characteristics: (a) including organic molecules having a chain structure; (b) having the first element capable of interacting with the target material on at least one end of the chain structure; (c) and having the second element capable of interacting with the solid phase material on at least the other end of the chain structure. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an orientation control material for controlling the orientation of a substance.

  Attempts have been made to apply functional substances such as polypeptides to solid phase materials and use them in diagnostic applications, analytical applications, reaction reactor applications, and sensor applications. Here, since the polypeptide or the like exerts its function, the substantial catalytic efficiency is lowered unless the catalytic site is immobilized in an exposed state. Therefore, it has been attempted to immobilize a polypeptide or the like on the surface of a solid phase material with a certain direction, that is, orientation.

  For example, as a technique for orientation fixation using physical adsorption, a plurality of amino acids containing a carboxyl group are introduced by genetic recombination into the C-terminal side of a polypeptide as an orientation target, while polylysine is introduced on the surface of a solid phase material. There is a method of orienting and immobilizing a polypeptide by treatment with the like (Patent Document 1). There is also a method in which a hydrophobic peptide chain is introduced into the N-terminal or C-terminal of a polypeptide to be oriented by gene recombination, and such a polypeptide is immobilized on the substrate surface (Patent Document 2). Furthermore, as a result of chemical bonding to the surface of the solid phase material, an unnatural amino acid is introduced into the target polypeptide, and a reactive group that reacts with the non-natural amino acid is introduced into the solid phase material to orient the polypeptide. Then, there is a method of fixing (Patent Document 3).

JP 2004-347317 A JP-A-2-79975 JP-T-2006-511797

  In any of the methods disclosed in the above-mentioned patent documents, a part of the immobilizing element such as a functional group or a compound for immobilizing the substance while being oriented is contained in the solid phase material. That is, in any of the methods disclosed in the above-mentioned patent documents, in order to align and immobilize a substance with respect to a solid phase material, a bond that simultaneously realizes the alignment and immobilization of the substance with the solid phase material. Is used. For this reason, it has been difficult to achieve good orientation and fixation.

  In addition, as shown in Patent Documents 1 to 3, it is reasonable to use a method such as gene recombination to introduce a specific amino acid or the like into a part of the substance in order to orient and fix the substance. It is. However, in order to obtain a modified polypeptide that ensures its original function and can be oriented and fixed, many technical problems such as acquisition of the gene of the polypeptide, gene transfer operation, and activation of the obtained polypeptide are solved. It has to be done with great effort, but it was not always successful.

  As described above, in the present situation, when a substance is oriented and fixed to a solid phase material, there is only an attempt to orient the substance in accordance with the immobilization to the solid phase material. Attempts to align without being limited are limited to some special cases, and the operation is complicated and not practical. Further, no attempt has been made to align and fix the substance without introducing a special element for realizing the orientation of the substance to the substance side or the solid phase material side.

  Accordingly, it is an object of the present invention to provide an orientation control material that realizes orientation control of a substance with respect to the solid phase material and use thereof when the substance is oriented and fixed to the solid phase material.

  The present inventors have separated the function of orienting a substance with respect to the solid phase material and the function of immobilizing the substance with respect to the solid phase material when the substance is oriented and fixed with respect to the solid phase material. Focused on making it. Then, by using a material containing a polypeptide chain having a predetermined N-terminal amino acid residue as an orientation control material that realizes the function of orienting the substance with respect to the solid phase material, the target substance is oriented with respect to the solid phase material. It was found that the target substance can be immobilized on the solid phase material by other immobilization principles while adsorbing and maintaining the orientation state. The present inventors have completed the invention based on these findings. According to the present invention, the following means are provided.

  According to the present invention, the target substance contains a polypeptide chain and the N-terminal amino acid residue is any one selected from the group consisting of alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, proline and glutamine. An orientation control material for controlling the orientation of the material is provided. The N-terminal amino acid residue of this polypeptide chain can be either alanine or isoleucine. In addition, the N-terminal amino acid sequence of the polypeptide chain can be any selected from Table 1.

  Further, the second or third amino acid residue from the N-terminal amino acid of the polypeptide chain preferably has a hydroxyl group, and preferably the second or third amino acid residue is Either serine or threonine.

  Moreover, the orientation control material of the present invention can also comprise two or more polypeptide chains. Furthermore, the orientation control material of the present invention can also have a polymer chain in which the polypeptide chains are linked. In this embodiment, a plurality of the polypeptide chains may be provided with respect to the polymer chain.

  The orientation control material of the present invention can have a hydrophilic region or a hydrophobic region. In addition, the orientation control material of the present invention can also include an element that interacts with a solid phase material that is intended to orient and fix the target substance. Furthermore, the orientation control material of the present invention may further comprise an immobilizing element for immobilizing the target substance on a solid phase material to be oriented and fixed.

  In the orientation control material of the present invention, the target substance can include a biomolecular material, preferably a polypeptide chain, and more preferably an antibody.

  According to the present invention, there is provided a solid phase carrier for immobilizing a target substance, comprising: a solid phase material; and any one of the above orientation control materials held on the surface of the solid phase material. Provided. The solid phase material may be a molded body that includes at least a part of the orientation control material. The orientation control material may be fixed to the surface of the solid phase material.

  According to the present invention, the step of preparing any one of the above solid-phase carriers, the target substance is supplied to the surface of the solid-phase material on which the orientation control material is held, and the target substance is supplied to the solid-phase material. An immobilizing step of immobilizing the solid phase body, wherein the target substance is oriented and immobilized.

  According to the present invention, there is provided a solid phase body comprising a solid phase material, any one of the above orientation control materials held on the surface of the solid phase material, and a target substance oriented and fixed by the orientation control material. Provided.

  The present invention relates to an orientation of a target substance comprising a polypeptide chain, wherein the N-terminal amino acid residue is any one selected from the group consisting of alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, proline and glutamine The present invention relates to an orientation control material that controls According to the alignment control material of the present invention, the target substance can be oriented and adsorbed on the surface of the solid phase material held on the surface, and can be immobilized on the solid phase material. The orientation control material of the present invention can realize the orientation of the target substance with respect to the solid phase material separately from the fixation. For this reason, the freedom degree of the means which can be employ | adopted for orientation is increasing rather than implement | achieving orientation and fixation by one interaction and coupling | bonding. In addition, since the orientation controlling material of the present invention can orient the target substance relative to the solid phase material, it is not necessary to introduce elements for orientation into the target substance or solid phase material. Modification of the phase material can be avoided.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate. First, the orientation control material of the present invention will be described, and then, with reference to FIGS. 1 to 5 as appropriate, the orientation and immobilization of the target substance on the solid phase material using the solid phase carrier of the present invention, the orientation control material, and The solid phase body of the present invention will be described. FIG. 1 shows an example of a solid phase carrier that holds the orientation control material of the present invention, FIG. 2 shows another example of the solid phase carrier of the present invention, and FIG. 3 includes the orientation control material. 4 shows an example of a wheel model having an α-helix structure. FIG. 4 illustrates a process of orientation and immobilization of a target substance (a process for producing a solid phase), and FIG. 5 illustrates a solid phase of the present invention. Illustrate the body.

(Orientation control material)
The orientation control material of the present invention is a material that orients the target substance to be immobilized on the solid phase material. Here, the target substance is not particularly limited. Examples of target substances include (1) inorganic materials such as metals, metal oxides, semiconductors, ceramics, and glass, (2) organic materials such as so-called plastics, and (3) biomolecular materials such as proteins, nucleic acids, saccharides, and lipids. (4) One or two or more materials selected from composite materials obtained by combining two or more materials selected from the various materials (1) to (3) described above can be used. The target substance is preferably a biomolecular material.

  The biomolecular material which is the subject of the present invention does not mean only one molecule, but may be an aggregate of homologous molecules composed of two or more molecules or a complex with different molecules. Further, it may be an organization such as a self-organization composed of a large number of same or different molecules.

  The biomolecular material is not particularly limited, and is a naturally occurring molecule that is present in an organism such as an animal, plant, microorganism, or virus, is produced by the organism, or is metabolized by the organism, or a molecule obtained by artificially modifying these. It may be an artificially designed molecule that does not depend on natural molecules. In addition to molecules collected from living organisms, they may be molecules produced artificially in organisms other than the organism in which the molecules originally exist, or may be artificially synthesized outside the organism, chemically synthesized, enzymes, etc. It may be a molecule synthesized by

  The biomolecular material typically includes biological materials such as polypeptides, nucleic acids, saccharides, lipids, osteogenic materials, various biological cells and parts thereof, tissues, and living organisms themselves. In addition, when the biomolecular material is immobilized on the solid phase, it may be combined with other organic materials and / or inorganic materials. Of these, the biomolecular material to be immobilized on the solid phase material preferably contains a polypeptide chain.

  In the present specification, the polypeptide means a polypeptide having any size, structure or function. Accordingly, oligopeptides having about 30 or less amino acid residues are also included. Examples of the target substance containing a polypeptide chain include various proteins, enzymes, antigens, antibodies, lectins, and cell membrane receptors. Of these, antibodies can be preferably used. The antibody means a naturally occurring or wholly or partially synthetically produced immunoglobulin. Also included are all derivatives thereof that retain specific binding ability.

  The nucleic acid may be single-stranded or double-stranded. It may contain DNA, RNA, DNA / RNA hybrids, DNA-RNA chimeras, bases and other modifications, whether artificial or natural. Further, the chain length is not particularly limited.

(Polypeptide chain)
The orientation control material includes a polypeptide chain. In addition to natural amino acids such as L-amino acids, polypeptide chains can be composed of non-natural amino acids such as modified amino acids and D-amino acids. The polypeptide chain is preferably free at the N-terminus. It is considered that the free N-terminal amino group contributes to the oriented adsorption of the target substance in the orientation control material.

  The N-terminal amino acid residue of the polypeptide chain is preferably not an acidic amino acid residue having an excess carboxyl group. Further, it is preferably a hydrophobic amino acid residue. Specifically, any one selected from the group consisting of alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, proline and glutamine is preferable. With these amino acid residues, good alignment efficiency can be obtained. In particular, it can be oriented with the antigen recognition site exposed in a protein such as an antibody. The N-terminal amino acid residue is preferably either alanine or isoleucine. With these amino acid residues, proteins such as antibodies can be more efficiently oriented.

  It is preferred that the second or third amino acid residue from the N-terminal amino acid residue of the polypeptide chain has a hydroxyl group. According to the study by the present inventors, good alignment efficiency can be obtained by having such an N-terminal amino acid sequence. Such an amino acid residue can be either serine or threonine. More preferred is threonine.

The amino acid sequence at the N-terminal of the polypeptide chain can be any one selected from the sequences shown in the upper column of Table 2. These amino acid sequences can be preferably used for orientation and immobilization on a solid phase material so as to expose an antigen recognition site of a polypeptide, typically an antibody. Among them, the amino acid sequence that can be oriented so as to expose the antigen recognition site with high orientation can be selected from the sequences shown in the column of Table 2. Furthermore, the amino acid sequence from which a high adsorption amount can be obtained can be selected from the sequences shown in the lower column of Table 2.

  The polypeptide chain of the orientation control material may have a secondary structure such as an α-helix structure or a β-sheet structure due to the polypeptide chain. Such a rigid secondary structure makes it possible to stably hold the N-terminus of the polypeptide chain at a certain site. In addition, a dipolar interaction, etc. is expressed based on the secondary structure, and an interaction for orientation fixation can be expressed between the target substance and the solid phase material. It can be expected to increase. Such secondary structure may be formed by the whole polypeptide chain, or may be formed in part.

  The α-helix structure of the polypeptide chain may be an amino acid sequence in which the number of hydrophobic amino acid residues (isoleucine, valine, leucine, phenylalanine, methionine, alanine, tryptophan) is dominant in the composition of constituent amino acid residues. it can. That is, the α-helix structure preferably has an amino acid sequence in which 50% or more of the total number of constituent amino acid residues is the hydrophobic amino acid residue. More preferably, the ratio of hydrophobic amino acid residues is 60% or more, more preferably 70% or more, still more preferably 80% or more, and most preferably 90% or more. Considering the formation of the α-helix structure, it is preferable that the hydrophobic amino acid residues contain more alanine, leucine, and isoleucine. More preferably, the hydrophobic amino acid residue is selected from alanine and isoleucine.

  The amino acid residues other than the hydrophobic amino acid residues preferably contain more amino acid residues selected from neutral amino acid residues such as cysteine, threonine, serine, tyrosine, glutamine and asparagine. .

  Considering the stability of the α-helix structure, the number of amino acid residues is preferably 9 residues or 9 residues or more. Further, when the constituent amino acid residues are converted as amino acids, the total molecular weight (amino acid equivalent total molecular weight) is preferably about 700 or more. The number of amino acid residues constituting the α-helix structure is not particularly limited, but is preferably about 20 or less considering the molecular weight.

  The hydrophobic amino acid residues in the α-helix structure are preferably rich on one side. Since the hydrophobic amino acid residue is predominantly coordinated on one side of the α-helix structure, stable hydrophobic interaction is exhibited and the orientation property of the orientation control material 20 with respect to the solid phase material 10 is improved. Can do.

  Here, in the α-helix structure, when the two rotations of the α-helix are regarded as one wheel, the Wheel model shown in FIG. 3 can be constructed. For example, in this model, it is hydrophobic in the composition of amino acid residues coordinated to one side of the α-helix, in the figure the position of a, b, e, f on the lower side or c, d, g on the upper side It is preferable that the amino acid residue (isoleucine, valine, leucine, phenylalanine, methionine, alanine, tryptophan) has an amino acid sequence that is 80% or more, more preferably 90% or more. As the hydrophobic amino acid residue, it is more preferable to use alanine, leucine and isoleucine preferentially, which can be selected from the same amino acid residues as already described. More preferably, alanine and isoleucine are used predominantly, and most preferably only alanine and isoleucine are used.

The α-helix structure is preferably a helix structure composed of only 7 or more, preferably 8 or more, more preferably 9 or 9 or more alanine residues and isoleucine residues in succession. Prepare. A preferred hydrophobic region in the case where the orientation control material 20 has a polypeptide chain includes the amino acid sequences shown in the following table consisting of 8 or 9 amino acids. Most preferably, it is AIAAAIAAI.

  The orientation control material may include one such polypeptide chain or two or more.

  The orientation control material can include organic molecules and inorganic molecules in addition to the polypeptide chain. Examples of organic molecules include various nucleic acid derivatives including nucleic acids such as DNA and RNA, modified bases, lipids, polysaccharides, surfactants, organic polymers, and various organic compounds. Examples of inorganic molecules include simple metals, metal clusters, and metal compounds.

  In the orientation control material, it is preferable that one or two or more polypeptide chains are linked to a chain-like organic molecule. A large number of polypeptide chains may be linked. The connection place is not particularly limited. The structure obtained by linking a polypeptide chain to an organic molecule may be linear as a whole, or may be branched such that the polypeptide chain is branched. The orientation control material can also take a form in which the polypeptide chain is linked as a side chain or a terminal of an organic polymer chain whose polypeptide chain is longer than the polypeptide chain. Such an orientation control material can be obtained by introducing a polypeptide chain into a partial functional group of the organic polymer chain. In introducing a polypeptide chain into a polymer chain, a carboxyl group or an amino group in the polypeptide chain can be used. For the polymer chain, the amount of polypeptide chain introduced can be adjusted by selecting the binding form or reaction form.

  The orientation control material can take a form having a large number of polypeptide chains in a polymer chain. With such a form, the adsorptivity of the target substance can be increased, and as a result, the efficiency of alignment can be increased and the amount of alignment fixation can be increased.

  When the polymer chain is a polymer of a thermoplastic or thermosetting resin and is itself a molding material, the orientation control for molding can be performed by introducing a polypeptide chain into the polymer chain constituting the molding material. It can also be a material. Similarly, when the polymer chain is a thermoplastic or thermosetting resin polymer and is already molded into a predetermined three-dimensional shape as a carrier for immobilizing the target substance, the polymer chain constituting this molded body It is also possible to obtain an orientation control material that adopts a carrier form in which a polypeptide chain is introduced on the surface of the molded body and the polypeptide chain is present on the surface of the molded body.

  The kind of monomer which comprises an organic polymer chain is not specifically limited. It may be the same as that constituting a resin material preferable as a solid phase material to be described later, or may be different.

  The orientation control material can comprise a hydrophilic region. By providing the hydrophilic region, the target substance can be stabilized when the target substance surface is hydrophilic, such as when the target substance has a polypeptide chain. In some cases, hydrophilic interaction can contribute to the oriented adsorption of the target substance. The hydrophilic region may be a part of the polypeptide chain, or may be a part other than the polypeptide chain described above, for example, an organic molecule. When the polypeptide chain has a hydrophilic region, the polypeptide chain can have an amino acid sequence including a polar amino acid residue having an additional amino group, carboxyl group or the like. Moreover, when an organic molecule is provided with a hydrophilic region, it can have a polymer chain or an organic functional group having a hydrophilic functional group such as an amino group, a carboxyl group, or a hydroxyl group.

  The orientation control material can comprise a hydrophobic region. By providing the hydrophobic region, the target substance can be stabilized, for example, when the target substance surface is hydrophobic, and it can contribute to the oriented adsorption of the target substance by hydrophobic interaction. The hydrophobic region may be a part of the polypeptide chain, or may be in a part other than the polypeptide chain described above, such as an organic molecule. When the polypeptide chain has a hydrophobic region, the polypeptide chain can have an amino acid sequence including the above-described hydrophobic amino acid residues having an additional alkyl group and the like. The hydrophobic region may form the α-helix structure already described. When the organic molecule has a hydrophilic region, the organic molecule can have a hydrocarbon group such as an alkyl group.

(Solid material interaction element)
The orientation control material can include an element (solid phase material interaction element) that interacts with a solid phase material to be oriented and fixed to the target substance. By providing the solid phase material interaction element, the orientation control material can be easily fixed to the solid phase material, and the orientation control material itself can be oriented with respect to the solid phase material. The N-terminus of the peptide chain can be easily exposed. As a result, the amount and orientation of the target substance immobilized on the solid phase material can be increased. In particular, when the principle for immobilizing the orientation control material 20 on the solid phase material 10 is light immobilization, coating, or the like, and the solid phase material 10 is not fixed at a specific site, By providing the second element, the orientation control material 20 can be easily oriented and fixed on the surface of the solid phase material 10.

  The interaction in which the solid phase material interaction element can be expressed with the solid phase material is not particularly limited. For example, non-covalent interactions such as electrostatic interaction, ionic bond, hydrogen bond, dipole interaction, hydrophobic interaction, and hydrophilic interaction are preferable. The orientation control material can comprise one or more such solid phase material interaction elements. For example, the electrostatic interaction may be an action based on a positive charge or an action based on a negative charge. Although it does not specifically limit as an acidic functional group which generate | occur | produces a negative charge, A carboxyl group, a sulfone group, a phosphoric acid group etc. are mentioned. Preferably, it is a carboxyl group. Examples of the basic functional group that generates a positive charge include amino groups such as primary amino groups and secondary amino groups, and quaternary ammonium groups. An amino group such as a primary amino group is preferable.

(Fixed element)
The orientation control material can also include an immobilization element that immobilizes the target substance on the solid phase material. The immobilization principle for immobilizing the target substance and the solid phase material is not particularly limited. Examples of the immobilization principle include non-covalent interactions such as hydrogen bonding, hydrophobic interaction, hydrophilic interaction, and electrostatic interaction, light immobilization based on covalent bond and light deformation caused by light irradiation.

  When electrostatic interaction is used as the immobilization principle, the orientation control material is an element capable of developing a charge that is paired with the charge of the solid phase material surface (such as a functional group capable of developing a positive or negative charge). ). When a covalent bond is used as an immobilization principle, the orientation control material solid phase material has an organic functional group that can be covalently bonded to the surface of the solid phase material or a compound that can bind to the surface of the solid phase material. it can. Those organic functional groups can be appropriately selected by those skilled in the art depending on the type of solid phase material, for example, the type of resin of the solid phase material and the type of functional group provided in the constituent resin.

  Note that the orientation control material is not necessarily provided with such an immobilization element. In order to realize the immobilization to the solid phase material, it is only necessary to have a functional group that is applicable to the solid phase material side, and an appropriate functional group can be provided by supplying a polyvalent crosslinking compound to the solid phase material. A group may be added. For example, a functional group such as an amino group or a carboxyl group included in a polypeptide chain or an organic molecule can be covalently bonded to a crosslinkable functional group provided on the surface of a solid phase material under a certain condition, or a separate polyvalent crosslink If a functional functional group is supplied, it can be easily covalently bonded to a solid phase material.

(Molecular weight)
The molecular weight of the orientation control material is preferably 5000 or less. When the molecular weight is 5000 or less, the target substance can be oriented and adsorbed on the surface of the solid phase material without hindering the immobilization of the solid phase material and the target substance. More preferably, it is 3000 or less. Note that this limitation cannot always be applied when the orientation control material is a resin material.

  Further, in relation to the molecular weight of the target substance, the molecular weight of the orientation control material is preferably 1/10 or less of the molecular weight of the target substance. This is because if the molecular weight of the orientation control material greatly exceeds one-tenth of the molecular weight of the target substance, immobilization of the target substance on the solid phase material tends to be hindered. Although it depends on the molecular weight of the target substance, the molecular weight of the orientation control material is more preferably 1/20 or less of the molecular weight of the target substance. Note that this limitation cannot always be applied when the orientation control material is a resin material.

When the orientation control material is composed of a polypeptide chain, the total number of amino acid residues is preferably 12 or more. In addition, the upper limit is not particularly limited, but in terms of synthesis efficiency in the case of chemical synthesis and in the case of light fixation, in order to suppress steric hindrance, it is preferable to have a molecular weight of 5000 or less, and 20 residues or less. Preferably there is. As such an orientation control material 20, a polypeptide having the following amino acid sequence can be preferably used.

(Solid phase material)
A solid phase material that aligns and fixes a target substance using the alignment control material of the present invention will be described. The solid phase material may be any material as long as the target substance can be immobilized using any of the above-described immobilization principles using an orientation control material, and the material and form thereof are not particularly limited.

  The solid phase material can include an element (an immobilization element) used for an immobilization principle for immobilizing a target substance on the solid phase material. When electrostatic interaction is used as the immobilization principle, for example, the surface of the solid phase material can be provided with a positive charge derived from an amino acid such as polylysine or lysine on the surface of the solid phase material.

  It is preferable that the immobilization principle does not interfere with non-covalent alignment adsorption between the target substance and the alignment control material. For example, as an immobilization principle, covalent bond or optical immobilization can be preferably used. As a principle of immobilization, when a covalent bond is used, the solid phase material can be provided with a functional group that can be covalently bonded to a functional group of the target substance on the surface thereof. For example, the solid phase material may have a crosslinkable functional group capable of undergoing a crosslink reaction with a functional group included in the target substance. The type of the crosslinkable functional group can be selected depending on the functional group provided in the target substance to be immobilized and the technique used. For example, when the target substance has an amino group, various crosslinkable functional groups such as an NHS group, an aldehyde group, and an epoxy group can be used. Moreover, when a target substance has a thiol group, crosslinkable functional groups, such as a maleimide group, are mentioned. Furthermore, when the target substance has an aromatic group, a diazonium group and the like can be mentioned.

Even when a covalent bond is used as an immobilization principle for immobilizing a target substance on a solid phase material, the solid phase material itself must always have a functional group capable of covalently binding to the target substance as described above. There is no. If a functional group that reacts with a polyvalent crosslinkable compound such as a polyvalent crosslinkable polymer that crosslinks between the target substance and the solid phase material is provided, it is shared with the functional group included in the target substance 4 via such a compound. Bonding is possible. Examples of the crosslinkable functional group provided in such a polyvalent crosslinkable compound include an amino group, a carboxyl group, an aldehyde group, and an epoxy group. As the polyvalent crosslinkable compound corresponding to these crosslink sites, Examples include arm PEG (manufactured by NOF Corporation), EMCS, SPDP (above, manufactured by Dojin Kagaku), BS ³ , DMS, SMCC (above, manufactured by Pierce).

  From the viewpoint of considering the influence on the target substance, particularly from the viewpoint of ensuring the activity when the target substance is a biomolecular material, it is preferable to use light immobilization as the principle of immobilization of the target substance. It is preferable to use light fixation as an immobilization principle because it is not necessary to provide an immobilization element to the orientation control material.

  Here, light fixation refers to photoirradiation in a state where a photoresponsive material containing a photoresponsive component that is deformed by light irradiation is used as the solid phase material 10 and the target substance is disposed on or near the surface of the solid phase material. By doing so, it is immobilized on the solid phase material. The relationship between light irradiation, light-responsive component, and target substance fixation in light fixation is not necessarily clear, but light fixation can be performed at least by applying light to the solid phase material of the target substance. It can be defined that this is a fixing method capable of increasing the amount of adsorption.

  A solid phase material for utilizing light fixation has a photoresponsive component in a matrix constituting the solid phase material. The matrix (matrix) of the solid phase material is not particularly limited as long as the photoresponsive component can be held so as to be photofixable. For example, various organic materials including low molecular materials or high molecular materials, inorganic materials such as glass, organic-inorganic composite materials, and the like can be used. Considering the dispersibility and retention ability of the photoresponsive component in the matrix, a polymer material or a composite material containing a polymer material is preferable.

  The polymer material constituting the matrix is not particularly limited, and various thermoplastic or thermosetting polymers can be used. For example, (1) polymers of carbon multiple bond monomers such as olefin polymers, vinyl polymers, acrylic polymers, methacrylic polymers, styrene polymers, diene polymers, and (2) cyclic monomers such as cyclic ether polymers. (3) polymers of bifunctional monomers such as ester polymers, urethane polymers, urea polymers, and the like. Among these, in consideration of the convenience of copolymerization, a polymer of a monomer having a double bond (hereinafter, also referred to as a double bond monomer) such as an olefin polymer, an acrylic polymer, or a methacrylic polymer, or a urethane type Polymers are preferred. More preferably, it is a double bond monomer. In particular, acrylic polymers, methacrylic polymers, and acrylic-methacrylic copolymers have less non-specific adsorption when immobilizing proteins such as antibodies, so they effectively suppress background signals in antibody chips and microreactors. Can do. In addition, urethane polymer and urethane- (meth) acrylic polymer can be preferably used in the present invention in that the amount of light deformation can be increased, and when a surface richer in polarity than acrylic polymer is required. Can also be preferably used.

  It is preferable that the matrix is configured to cause shape deformation (hereinafter also referred to as light deformation) due to a change in the molecular structure of the photoresponsive component. In this specification, the optical deformation includes deformation due to entanglement between the target substance and the surface of the solid phase material due to movement at a molecular level, in addition to a shape change in a normal macro sense. Some of these deformations cannot be clearly observed by ordinary observation means due to problems of deformation amount and deformation form. Photodeformation is caused by the presence of a light-responsive component in the matrix material and, for example, induced by a change in the volume, density, free volume, etc. of the matrix material or the light-responsive component upon light irradiation. it is conceivable that.

(Photo-responsive component)
The photoresponsive component is a component that causes a change in molecular structure or molecular arrangement by light. The phenomenon in which molecular structure changes due to light is generally referred to as photochromism. As the photoresponsive component used in the present invention, a compound generally referred to as a photochromic compound can be used. Among them, a compound that causes photoisomerization is preferably used. In addition, there are compounds that cause changes in molecular arrangement (especially anisotropic changes) such as photo-induced orientation and photoassociation with or without molecular structure changes such as photoisomerization. As long as light immobilization on the surface of the solid phase material is possible, it can be used as the photoresponsive component of the present invention.

  As the light-responsive component, various components that have been used for such light fixation can be used. Examples of the photoresponsive component include a photoisomerized compound such as a component that causes trans-cis photoisomerization. For example, an organic compound such as an azo compound, a spiropyran compound, a spirooxazine compound, a diarylethene compound, or a chalcogenite. Examples thereof include inorganic materials generically called glass.

The photoresponsive component is preferably a compound (azo compound) having a dye structure having an azo group (—N═N—). The azo compound undergoes cis-trans isomerization by light irradiation or the like, and the movement at the molecular level by this isomerization plasticizes the matrix material to facilitate deformation. Of these, amino-type azobenzene compounds having a structure of aminoazobenzene or a derivative thereof are preferable. The amino-type azobenzene compound can typically be represented by the formula (1).

In formula (1), R ₁ and R ₂ each independently represent a hydrogen atom or a substituent, and X represents a hydrogen atom, an electron-withdrawing substituent or an electron-donating substituent.

Examples of the substituent in R ₁ and R ₂ include alkyl group, hydroxyalkyl group, halogen atom, nitro group, amino group, carboxyl group, aryl group, allyl group, alkyl ester group, alkyl ether group, alkylamino group, alkylamide Examples include a group, an isocyanate group, and an epoxy group. In particular, when _{one of the} substituents in R ₁ and R ₂ is an acrylic compound such as acrylic acid or (meth) acrylic acid having a polymerizable double bond at the terminal, or other double bond component Formula (1) represents a photoresponsive polymer having a polymerizable group derived from the double bond component, as well as a double bond monomer. When _{one of the} substituents in R ₁ and R ₂ is a polycondensation or polyaddition polymerizable component such as an isocyanate group, an amino group, a carboxyl group, or a hydroxyl group, the formula (1) is a polymerizable monomer. And a photoresponsive polymer (polymer having an azo dye-containing unit) having a polymerizable group derived from the polymerizable component. Considering the deformability of the photoresponsive material, it is preferable that the polymer skeleton of the photoresponsive material is a (meth) acrylic polymer, a urethane polymer, or a urethane-acrylic polymer. Is preferably a (meth) acrylic monomer, a urethane monomer or a polymer containing these ((meth) acrylic polymer, urethane polymer or urethane-acrylic polymer).

  Examples of the electron-withdrawing substituent in X include a cyano group, a nitro group, and a sulfo group. Examples of the electron donating substituent include an amino group, a dimethylamino group, and an alkyl group. Those in which such substituents are bonded are preferable because the matrix material is considered to have a large plasticizing action by repeating cis-trans isomerization during light irradiation. In addition, the photoresponsive component which is an azo compound can be used 1 type or in combination of 2 or more types.

  Such a photoresponsive component may be added to the matrix as a component separate from the matrix material, but is present in a part (main chain or side chain) of the matrix material through a chemical bond. It is preferable. The photoresponsive component is preferably present uniformly in the matrix. By doing so, the target substance can be photo-immobilized at a desired site on the surface of the solid phase material.

  In the case where hydrogen bonding, hydrophobic interaction, hydrophilic interaction, or the like is used as the principle of immobilization of the target substance, appropriate functional groups and treatments can be applied to the solid phase material 10 respectively. Techniques for this are well known to those skilled in the art. The immobilization element in the solid phase material may be at least on the surface layer side of the solid phase material. Such a layer for immobilization may be supported on a suitable carrier.

  The solid phase material may include an element for immobilizing the orientation control material on the solid phase material in addition to the immobilization element for immobilizing the target substance. Such an element can be appropriately selected from elements that can be employed as an immobilizing element for immobilizing the target substance already described in the solid phase material. The element for fixing the orientation control material varies depending on the type of the orientation control material, but may be the same as or different from that of the inherent immobilization principle for immobilizing the target substance.

  Therefore, the solid phase material can include two different elements, an immobilizing element for immobilizing the target substance and an element for immobilizing the orientation control material. For example, the solid phase material can include a certain type of crosslinkable functional group for immobilizing the target substance and another type of crosslinkable functional group for immobilizing the orientation control material. If the target substance and the orientation control material can be immobilized by different immobilization principles, the target substance is immobilized on the solid phase material after the target substance is oriented and adsorbed by the orientation control material. Can hardly affect the fixed state of the orientation control material, and the intended orientation state of the target substance can be obtained easily and reliably.

  The solid phase material can also include an element for fixing the orientation control material on the same fixing principle as that of the target substance. For example, when both the target substance and the orientation control material are immobilized on the solid phase material by light fixation, the solid phase material only needs to have a single type of photoresponsive component for these immobilization elements. In addition, by including two or more kinds of photoresponsive components in the solid phase material, the target substance and the alignment control material can be immobilized by light irradiation in different wavelength regions.

  The solid phase material may not necessarily include an element for immobilizing the orientation control material. For example, the orientation control material can be fixed to the solid phase material by simple coating or the like, or the orientation control material can be fixed to the solid phase material by using heat, pressure, or the like.

  The three-dimensional form of the solid phase material is not particularly limited. In addition to a film-like body, a sheet-like body, and a plate-like body, various shapes such as a spherical body, an indefinitely shaped body, a needle-like body, a rod-like body, and a flake-like body can be adopted. Also, the size is not particularly limited. The solid phase material may be self-supporting or may be applied in the form of a film on the surface of another carrier. The solid phase material can be applied to the surface of the carrier by a known method such as spin coating, dip coating, or ink jet.

(Solid phase carrier with orientation control material on the surface)
The solid phase carrier of the present invention can comprise a solid phase material and an orientation control material held on the surface of the solid phase material. In a preferred embodiment of the solid phase carrier of the present invention, the solid phase material has a predetermined three-dimensional shape such as a substrate shape or a particulate shape that can be used as it is as various devices or diagnostic / analytical materials.

  A first embodiment of the solid phase carrier of the present invention is shown in FIG. As shown in FIG. 1A and FIG. 1B, a form in which the orientation control material 20 is immobilized on the surface of the solid phase material 10 prepared separately from the orientation control material 20 can be mentioned. The solid phase carrier 32 of this form can be obtained by supplying the orientation control material 20 to the surface of the solid phase material 10 and immobilizing the orientation control material 20 on a preselected immobilization principle. The solid phase carrier 32 shown in FIG. 1 (a) is provided with the alignment control material 20 in the form of dispersed dots in the surface of the solid phase material 10, and the solid phase carrier 32 shown in FIG. An orientation control material 20 is provided in the form of a film on almost the entire surface of the solid phase material 10.

  A second embodiment of the solid phase carrier of the present invention is shown in FIG. As shown in FIG. 2, a form in which the solid phase material 10 is formed from at least a part of the orientation control material 20 can be mentioned. For example, a solid phase material 10 in which organic molecules and inorganic molecules constituting a part of the orientation control material 20 are polymer materials, and a material containing these polymer materials has a predetermined three-dimensional shape by molding or the like. The form which forms is mentioned.

  One aspect of the solid phase carrier 32 of the second embodiment is shown in FIG. As shown in FIG. 2A, a solid phase is formed on the surface of a solid phase material 10 having a predetermined three-dimensional shape formed by using a material containing an organic polymer or an inorganic polymer that is a part of the orientation control material 20. As a result of the polypeptide chain being introduced into the polymer chain on the surface of the solid phase material 10 from the outside of the material 10, a form of the solid phase carrier 32 having the orientation control material 20 on the surface of the solid phase material 10 can be mentioned.

  Another embodiment is shown in FIG. As shown in FIG. 2 (b), as a result of plasticizing and molding a material including an orientation control material 20 having one or more polypeptide chains linked to an organic polymer chain or the like, the material is oriented on the surface of the solid phase material 10. The form of the solid-phase support | carrier 32 which has the control material 20 is mentioned. In this embodiment, the orientation control material 20 can be held not only on the surface of the solid phase material 10 but also inside the solid phase material 10.

(Method for producing a solid phase in which the target substance is oriented and fixed)
Next, the process of immobilizing the target substance on the solid phase material or solid phase carrier will be described. In the following description, these series of steps will be described separately as a solid phase material preparation step, an alignment control material immobilization step on the solid phase material, and a target substance immobilization step.

(Preparation process of solid phase material)
In order to immobilize the target substance on the solid phase material using the orientation control material, first, the solid phase material is prepared. For example, as shown in FIG. 4A, the solid phase material can be formed as a layered immobilization layer on the surface layer of a substrate-like carrier.

(Fixing process of orientation control material)
Next, as shown in FIG. 4B, the orientation control material is supplied to the surface of the solid phase material, and the orientation control material is fixed to the surface of the solid phase material. The orientation control material may be dispersed and immobilized on the surface of the solid phase material, or may be immobilized on a non-specific region on the surface of the solid phase material, in other words, the entire surface of the solid phase material. Or you may fix according to a specific pattern. As a method of supplying the orientation control material to the surface of the solid phase material, various conventionally known coating methods and printing methods (including inkjet) can be used. Also, a conventionally known method can be employed for patterning the orientation control material. As an example of patterning, droplet spots of the orientation control material can be supplied in an array.

  In order to immobilize the orientation control material on the surface of the solid phase material, an appropriate method should be used depending on the type of solid phase material and the type of element for immobilization to the solid phase material that the orientation control material may have. You can choose. It may be simply fixed due to drying or evaporation of the solvent, or may be accompanied by pressure. Moreover, you may use the element for these fixation | immobilization with which an orientation control material and / or a solid-phase material are equipped. Further, an appropriate binder component may be used separately.

  When the orientation control material includes an element capable of interacting with the solid phase material interaction element, the orientation control material is easily fixed to the solid phase material. Moreover, by providing such an interaction element, the orientation control material itself can be easily oriented and fixed.

  The orientation control material may be immobilized on the solid phase material by the same immobilization principle as the target substance is immobilized on the solid phase material. By doing so, the orientation fixing step can be simplified, and elements for fixing the orientation control material to be provided in the solid phase material can be omitted. As such an immobilization principle, light immobilization is preferable. In the case of light fixation, the alignment control material and the target substance can be fixed only by light irradiation at the time of fixing, and adverse effects caused when the alignment control material and the target substance are fixed to the alignment control material and the target substance can be reduced.

(Fixing process of alignment control material by light fixation)
When using a photoresponsive material as the solid phase material, it is preferable to photofix the orientation control material. In light immobilization, an orientation control material is supplied to or near the surface of a solid phase material that is a photoresponsive material, and the orientation control material is immobilized on the surface of the solid phase material by light irradiation.

  In the light fixation of the alignment control material, the supply of the alignment control material to the surface of the solid phase material is not particularly limited, but it can be applied in a state dissolved or suspended in the liquid medium via the liquid medium. preferable. When the liquid medium is used, the orientation control material can be easily developed on the surface of the solid phase material, and the orientation control material has its structure (for example, its secondary structure in the case of the orientation control material 2 polypeptide). ) Can be maintained and fixed.

  The liquid medium is particularly preferably water or a composition liquid containing water as a main medium. The composition liquid containing water as the main medium is appropriately selected according to the type of the orientation control material, and examples thereof include water, a buffer solution, and a buffer solution adjusted in pH. The liquid medium can also be selected to enhance the interaction between the orientation control material and the solid phase material. For example, the interaction can be enhanced by adjusting the pH, electrolyte concentration, polarity, etc. of the liquid medium. As such a liquid medium, water, an aqueous solvent which is an organic solvent compatible with water, a non-aqueous solvent may be used alone or in combination. Moreover, you may add a component required in order to provide the said liquid property for improving the interaction of the solid-phase material 10 and orientation control material. Furthermore, for example, a surfactant may be added to the liquid medium.

  After supplying the orientation control material to the solid phase material, the orientation control material on the surface of the solid phase material can be irradiated with light to immobilize the orientation control material on the surface of the solid phase material. The light irradiation method for fixing the light is not particularly limited. What is necessary is just to irradiate so that arbitrary lights, such as various propagation light, near field light, or evanescent light, may arrive at the surface of solid-phase material, or its vicinity. Further, the light irradiation can be selectively performed on a part of the solid phase material using a known method.

  The wavelength range used for light fixation may be a wavelength range that causes a change in molecular structure or molecular arrangement in the photoresponsive component. Information regarding such wavelength ranges can be easily obtained for various available photoresponsive components or can be confirmed upon use.

  In addition, about the light irradiation for light fixation, employ | adopt the irradiation light and the light irradiation method already described in Unexamined-Japanese-Patent No. 2003-329682, Unexamined-Japanese-Patent No. 2004-93996, and Unexamined-Japanese-Patent No. 2004-251801. Can do. The light immobilization is disclosed by the present applicant in Japanese Patent Application Laid-Open No. 2003-329682, Japanese Patent Application Laid-Open No. 2004-93996, and Japanese Patent Application Laid-Open No. 2004-251801. Can also be applied.

  In addition, it is preferable to wash the surface of the solid phase material after the optical control of the orientation control material and remove the orientation control material that is not fixed.

  In the above description, the case where the solid phase carrier in which the orientation control material is immobilized on the surface of the solid phase material (the first embodiment of the solid phase carrier of the present invention) is used has been described. However, instead of preparing such a solid phase carrier, the solid phase carrier according to the second embodiment can be prepared and used.

(Target substance immobilization process)
Next, as shown in FIG. 4C, the target substance is supplied to the region where the orientation control material on the surface of the solid phase material is disposed, and the target substance and the solid phase material are fixed based on the principle of immobilization. Substance 4 is immobilized on a solid phase material. At this time, since the orientation control material has a predetermined polypeptide chain, the target substance can be oriented and adsorbed. That is, the target substance can easily approach the orientation control material. In addition, the target substance has a certain orientation with respect to the solid phase material due to the interaction with the polypeptide chain of the orientation control material. In this state, the target substance is immobilized on the surface of the solid phase material by the principle of immobilization between the target substance and the solid phase material, for example, covalent bond, electrostatic interaction, light immobilization and the like. By doing so, as shown in FIG. 4D, the target substance is immobilized on the solid phase material in a state where the orientation is controlled by the orientation control material. That is, the solid phase body 2 in which the target substance is immobilized on the solid phase material via the orientation control material can be obtained.

  In the case where the orientation control material includes a solid phase material interaction element, the orientation control material is oriented and adsorbed to the solid phase material with a certain orientation and is fixed to the solid phase carrier (the solid phase of the first embodiment). Carrier) can be formed. As a result, the orientation of the target substance with respect to the solid phase material can be further improved.

  In addition, according to the method of the present invention, since the orientation control material is used, it is avoided to add an element for controlling the orientation to the target substance and the solid phase material. For this reason, the target substance 4 can be easily oriented, and the influence of these additional elements on the activity and stability of the target substance is reduced.

  The orientation control material is held on the surface of the solid phase material in the solid phase carrier to the extent that it does not interfere with the fixation of the target substance and the solid phase material. This is realized by the relationship between the molecular weight of the orientation control material itself and the molecular weight of the target substance, and the amount of the orientation control material immobilized on the solid phase material, for example, the thickness of the layer at the time of immobilization is passed. It can also be realized by making it sufficiently smaller than the diameter. If the thickness of the immobilization layer of the orientation control material is sufficiently smaller than the diameter of the target substance, such as the diameter of the target substance, even if the direct contact between the target substance and the solid phase material is avoided or suppressed by the orientation control material, the specific fixation is achieved. Immobilization can be ensured according to the principle of optimization. For example, it is preferable that the layer thickness of the fixed orientation control material is ½ or less of the diameter of the target substance. More preferably, the layer thickness of the orientation control material is about 1 nm or less.

  In this immobilization process, when the solid phase material 10 is a light-responsive material and the inherent immobilization principle is light immobilization, the alignment control material 20 is optically immobilized, and further, the alignment control material 20 interacts with it. Together with the target substance 4, it can be optically immobilized on the solid phase material 10. In particular, when light fixation is used in such two stages, the orientation of the target substance 4 can be enhanced by the presence of the alignment control material 20 as compared with the case where the alignment control material 20 is not used. Further, according to light fixation, no special element for immobilization is originally required on the target substance 4 side, and the immobilization conditions are gentle, so that the target substance 4 can be easily oriented. In addition, inconveniences such as a decrease in the activity and stability of the target substance 4 due to the introduction of elements for orientation into the target substance 4 can be eliminated.

  The light fixation of the target substance 4 can be performed in the same manner as the light fixation of the orientation control material 20. That is, the target substance 4 may be supplied to the alignment control material 20 or the vicinity thereof and then irradiated with light. Even when the alignment control material 20 is optically fixed, the target substance 4 can be optically fixed using the alignment control material 20. This means that even when the alignment control material 20 is present on the surface of the photoresponsive material, the target substance 4 can be optically fixed through the alignment control material 20 and / or with the alignment control material 20. It means you can do it. It has never been known and expected that two or more target substances (here, corresponding to the alignment control material 20 and the target substance 4) can be light-immobilized in two or more stages by repeating such light irradiation. It wasn't.

  As described above, according to the manufacturing method of the present invention, when the target substance 4 is oriented and immobilized on the solid phase material 10, the orientation control material 20 held on the surface of the solid phase material 10 has a predetermined value. Since it has a polypeptide chain, the target substance 4 can be easily oriented and fixed to the solid phase material 4 while maintaining the activity of the target substance 4 without separately introducing an orientation element into the target substance 4. Can do. In addition, it is possible to avoid or suppress a direct interaction for alignment between the target substance 4 and the solid phase material 10 and to fix the target substance 4 to the solid phase material 10. From the above, the adverse effect on the target substance 4 due to the orientation of the target substance 4 with respect to the solid phase material 10 can be reduced. Furthermore, the amount of the target substance 4 immobilized can be increased by the orientation control material 20.

  In addition, by immobilizing the target substance 4 via the alignment control material 20, the target substance 4 is not affected by the interaction for alignment with the solid phase material 10. For this reason, it is suppressed or avoided that the solid phase material 10 reduces its activity, storage stability, and the like. In other words, it is possible to immobilize the target substance around the active part of the target substance without being adversely affected by immobilization (disappearance of the active site due to immobilization or interference with the three-dimensional structure necessary for realizing the function by immobilization). Fixed.

  Furthermore, when the target substance 4 is immobilized by light immobilization, the target substance 4 is immobilized by photodeformation of the solid phase material 10, so that the target substance 4 may be greatly affected by the solid phase material 10. However, it is possible to fix the target substance 4 to the solid phase material 10 in a state in which the stability and activity of the target substance 4 are further improved by interposing the orientation control material 20 and fixing the orientation.

(Solid phase on which target substance is immobilized)
The solid phase body of the present invention can include a solid phase material, an orientation control material held on the surface of the solid phase material, and a target substance that is oriented and fixed by the orientation control material. According to the solid phase body of the present invention, since the target substance is orientation-controlled by the orientation control material and is fixed to the solid phase material, the target substance is oriented while ensuring the activity and stability of the target substance. .

  The solid phase body of the present invention will be described with reference to FIG. For example, as shown in FIG. 5A, the solid phase body 2 is provided with an alignment control material 20 in the form of dots on the surface of the solid phase material 10. The substance 4 may be immobilized, or, as shown in FIG. 5B, different target substances 4 are immobilized on the surface of the orientation control material 20 that is entirely immobilized on the surface of the solid phase material 10. May be. Further, as shown in FIG. 5C, in the solid phase body 2, the solid phase material 10 includes at least a part of the orientation control material 20, and the polypeptide chain of the orientation control material 20 is placed on the surface of the solid phase material 10. The form which has.

  The solid phase body of the present invention is not limited to the form shown in FIG. 5, and the solid phase material, the orientation control material and its immobilization, and the target substance in the solid phase body of the present invention have already been described. The solid phase material and the target substance, and the forms applicable to immobilization and preferred forms can all be applied.

  In such a solid phase, the target substance is preferably a polypeptide. This is because in the polypeptide, the active site exposure and the three-dimensional structure change for the activity expression are important for the activity expression, and these are ensured only by the orientation control. In addition, the activity and stability of the polypeptide are greatly affected by the solid phase material.

  Since the solid phase is fixed in a state advantageous for the activity and stability of the target substance, it is suitable for analysis and diagnostic use. Light immobilization is suitable for immobilizing biological materials and biological materials such as proteins including antibodies, sugar chains, nucleic acids and cells. Therefore, the solid phase body 2 of the present invention is suitable for analysis or diagnostic devices such as proteins, enzymes, antibody chips, sugar chain chips, nucleic acid chips such as DNA, and cell chips. It is also suitable for a bioreactor in which enzymes, cells, etc. are immobilized on the solid phase body 2. This is because the optical detection characteristics are excellent, so that the design and control of the reactor are easy, and the retention stability of the target substance 4 such as a biomolecular material is excellent. Therefore, a highly efficient bioreactor, particularly a microreactor can be produced.

(Detection method of interaction between target substance and other components)
The method for detecting the interaction between the target substance of the present invention and other components comprises the steps of supplying other components to the target substance immobilized on the solid phase of the present invention to exert the interaction, Detecting an interaction between the other component and the target substance. According to the detection method of the present invention, since the target substance is immobilized in an advantageous state for securing the activity and stability of the interaction and the like, it is possible to detect the interaction with good accuracy and sensitivity. . Examples of the interaction mentioned here include electrostatic binding interaction, ionic bonding interaction, hydrogen bonding interaction, hydrophobic interaction, hydrophilic interaction and the like. In addition, for example, an interaction between a ligand and a receptor for the ligand, an interaction between a protein having a specific amino acid sequence or structure and a substance such as a protein having an affinity for the protein, an enzyme and a substrate for the enzyme, Interaction between an antigen and an antibody against the antigen, a nucleic acid or modified nucleic acid having a specific base sequence, and a nucleic acid or modified nucleic acid having a base sequence complementary to the specific base sequence of the nucleic acid or modified nucleic acid Can interact with each other. In this detection method, the target substance is preferably a biomolecular material such as a biomaterial or a biomaterial, and it is preferable to detect such an interaction with an optical signal.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example, It can implement in various aspects in the scope of the present invention.

The azo film used in this example and the following examples uses an azo polymer (m: n = 15: 85) represented by the following formula. The azo film was prepared by dissolving 200 mg of this azo polymer in 16 ml of a pyridine solution, filtered through a 0.22 μm filter, drying a slide glass wiped with acetone-impregnated cotton, and then dripping 80 μl of a polymer solution with a spin cast machine. After rotating at 4000 rpm for 10 seconds, it was dried at 60 ° C. for 2 hours and then vacuum-dried under light shielding.

(Immobilized antibody ability and orientation when using N-terminal recognition peptide)
Synthetic peptides having various amino acid sequences of 100 μg / mL (FIG. 6) were dropped on an azo film in an amount of 0.1 μL, vacuum-dried, and irradiated with light (20 mW / cm ² ) at 25 ° C. for 0.5 hour. Thereafter, washing was performed 3 times with TPBS for 5 minutes to immobilize the synthetic peptide. 1 μL of 0-400 ng / mL Rabbit antibody IgG / TPB solution was added dropwise to each position where the peptide was dropped and fixed. This was incubated for 17 hours at 25 ° C. while being irradiated with light (20 mW / cm ² ). Thereafter, the plate was washed 3 times with TPBS for 1 minute.

(Evaluation of immobilization ability)
1 μg / mL Cy5-labeled Mouse anti Rabbit IgG (AP188S) / TPBS solution is dropped onto the azo film on which the peptide is immobilized, and reacted at 25 ° C for 30 minutes, and then 3 times for 1 minute with TPBS. Was washed. This slide glass was set with an array scanner, the fluorescence amount of Cy5 was measured, and the fluorescence amount of the spot where the peptide was dropped was quantified.

(Evaluation of immobilized antibody ability)
In addition, 1 μg / mL Cy5-labeled Mouse anti Goat IgG (AP127S) / TPPBS solution was dropped onto the peptide on the azo film on which the peptide was immobilized, reacted at 25 ° C. for 30 minutes, and then 1 mL with TPBS. Three washes were performed per minute. This slide glass was set with an array scanner, the fluorescence amount of Cy5 was measured, and the fluorescence amount of the spot where the peptide was dropped was quantified.

  In the conventional method, 0.1 μL of 0-400 ng / mL Rabbit anti-goat IgG / TPS solution was dropped into the synthetic peptide in the step of dropping the synthetic peptide, and light-fixed.

  The orientation was calculated based on the immobilized antibody activity per immobilization ability as an index, and the value obtained when each peptide was immobilized using the conventional method as 1. The result is shown in FIG.

  As shown in FIG. 6, by photofixing the peptide in two steps, a value that was 2.3 to 5 times higher than the conventional method was obtained. This supports that the two-step immobilization method may be able to control the orientation of the immobilized antibody. Moreover, it supports that the protective effect with respect to an antibody is working through the peptide.

(Evaluation of hydrophobic helix structure)
(Evaluation of the amount of peptide immobilized)
1 μL of a 100 μg / mL aqueous solution of various synthetic peptides shown in FIG. 7 was dropped onto an azo film, vacuum-dried, and then irradiated with light (20 mW / cm ² ) at 25 ° C. for 0.5 hour. Thereafter, the plate was further washed 3 times with TPBS for 5 minutes to immobilize the synthetic peptide. After immobilization, 50 μL of 1 μg / mL Cy5 mono-reactive-dye (GE Healthcare Biosciences PA25001) / PBS solution was added dropwise and reacted at room temperature for 30 minutes. Thereafter, the plate was washed 3 times for 1 minute with TPBS, and this slide glass was set with an array scanner, and the fluorescence amount of the spot where the peptide was dropped was measured.

(Evaluation of immobilization ability)
50 μL of 1 μg / mL Cy5-labeled Goat anti mouse IgG / TPBS solution was dropped onto the slide glass coated with the synthetic peptide, and this was irradiated with light (20 mW / cm2) at 25 ° C. for 17 hours. Immobilization]. Thereafter, the plate was washed 3 times with TPBS for 1 minute. The slide glass was set with an array scanner, and the fluorescence amount of the spot where the peptide was dropped was measured.

  The results of evaluating these peptides are shown in FIG. As shown in FIG. 8, it was found that in the peptides IAT, IAT (A9), and IAT (G), the peptide was immobilized on the azo film. On the other hand, it was found that peptides IAT (A7), IAT (A5), and IAT (I> G) were not immobilized on the azo film. Peptides IAT, IAT (A9), and IAT (G) have been confirmed to form a nearly 100% helical structure by CD spectrum measurement. FIG. 8 schematically shows the peptide structure on the left side of the peptide name. The zigzag of the peptide structure shown in FIG. 8 shows an α-helix structure, and the ellipsoid added downward from the zigzag structure of the peptide display of IAT (G) and IAT indicates the hydrophobic group of isoleucine. Represents. From the above, it was found that a hydrophobic region, particularly an α-helix structure rich in hydrophobic amino acid residues, is important for immobilizing peptides as orientation control materials. In addition, since the results of immobilization ability depend on the amount of peptide immobilized, it is well supported that the evaluation of the amount of peptide immobilized by Cy5 mono-reactive-dye reflects the actual amount of immobilization. it seems to do.

(1) Evaluation of antibody immobilization ability of synthetic peptide 1 μL of 100 μ / mL synthetic peptide shown in FIG. 9 was dropped on an azo film, vacuum-dried, and then irradiated with light at 25 ° C. for 2 hours (20 mW / cm ² )did. Thereafter, the plate was washed 3 times with TPBS for 5 minutes to immobilize the synthetic peptide. 1 μg / mL Cy5-labeled Goat anti mouse IgG / TPS solution was added dropwise thereto, and this was incubated for 17 hours at 25 ° C. while being irradiated with light (20 mW / cm ² ). Thereafter, the plate was washed 3 times with TPBS for 1 minute to photofix the antibody. This slide glass was set with an array scanner, the fluorescence amount of Cy5 was measured, and the fluorescence amount of the spot where the peptide was dropped was quantified. The results are shown in FIG.

(2) Evaluation of immobilized antibody activity (1) Various synthetic peptides shown in FIG. 9 were photofixed in the same manner as in the evaluation of the antibody immobilization ability of synthetic peptides. 1 μg / mL Rabbit anti goat IgG / TPS solution was added dropwise thereto, and this was incubated for 17 hours at 25 ° C. while being irradiated with light (20 mW / cm ² ). Subsequently, the antibody was immobilized by washing 3 times with TPBS for 1 minute. 1 μg / mL of Cy5-labeled Goat anti mouse IgG / TPS solution was added dropwise, reacted at 25 ° C. for 30 minutes, and then washed 3 times with TPBS for 1 minute. This slide glass was set with an array scanner, the fluorescence amount of Cy5 was measured, and the fluorescence amount of the spot where the peptide was dropped was quantified. The results are shown in FIG.

  In Comparative Example 1, only water was dropped in place of the synthetic peptide solution in (1), and in Comparative Example 2, 10 μg / mL Rabbit anti goat IgG / TPPBS solution was used in place of the synthetic peptide solution in (2). As a comparative example 3, the sequence EATAAIAAIAAAI was used as a synthetic peptide in (1) and (2), and as a comparative example 4, the sequence EAAAIAAIAAAI was used as a synthetic peptide in (1) and (2).

  The orientation of each synthetic peptide was determined using the ratio of the immobilized antibody activity obtained in (2) to the immobilization ability obtained in (1), and 10 μg / mL Rabbit anti goat IgG in Comparative Example 2 was used. The ratio of the immobilized antibody activity to the immobilization ability in the case of dry light fixation was calculated as 1. The results are also shown in FIG.

  As shown in FIG. 9, the immobilization ability of the synthetic peptides 1 to 33 increased 10 times or more compared to the case of Comparative Example 1. This is thought to be because the peptides listed in FIG. On the other hand, in Comparative Examples 3 and 4, the immobilization ability was significantly reduced. This indicates that the N-terminal amino group interacts with the molecular surface of the antibody, and this phenomenon indicates that the carboxyl group of glutamic acid (E) located at the N-terminal is interlinked between the N-terminal amino group and the antibody molecule. This was thought to be because the action was inhibited.

  The activity of the antibody adsorbed on the carrier via the synthetic peptide (immobilized antibody activity) increased 3-20 times compared to the case of the directly photo-immobilized antibody (Comparative Example 2). The value (orientation) indicating the activity per immobilized antibody was 3-6 times that of the directly immobilized antibody (Comparative Example 2). This is because (1) the antigen recognition site of the immobilized antibody does not interact with the carrier (or peptide) and is oriented to the aqueous solution side, and (2) compared to the case of direct immobilization. It supports that the antibody is in a state of maintaining the original three-dimensional structure.

  It is important that the amino group at the N-terminal side of the synthetic peptides 1-33 screened using the immobilization ability and orientation as an index is not modified at the N-terminal amino group. It was found that threonine or serine is preferably contained therein.

  N-terminal 3 residues with high immobilized antibody ability and orientation more than 3 times are IAA, VAA, FAA, PAA, AAA, LAA, QAA, IAT, ATA, FAT, WAT, VAT, LAT, AAT , PAT, IHT, IPT, IIT, IMT, IST, ITT, IQT, IAS, IGS, IVS, ISS, ITS, IQS, INS, IAY, IAE, IAI. Among them, the N-terminal 3 residues having orientation of 5 times or more are IAT, ITT, ITS, IAS, and the N-terminal 3 residues having particularly high immobilized antibody ability and orientation of 3 times or more are IPT, IMT, IST, IQT, IQS, INS.

SEQ ID NOs: 1-45: polypeptides for immobilization of the target substance

It is a figure which shows an example of the solid-phase carrier of this invention. It is a figure which shows the other example of the solid-phase carrier of this invention. It is a figure which shows the wheel model in alpha-helix structure. It is a figure which shows an example of the manufacturing process of the solid-phase body of this invention. It is a figure which shows the example of the solid-phase body of this invention. It is a figure which shows the amino acid sequence of the synthetic peptide used in Example 1, and the evaluation result of orientation. FIG. 3 is a view showing an amino acid sequence of a synthetic peptide used in Example 2. It is a figure which shows the evaluation result of the synthetic peptide in Example 2, the upper figure is a figure which shows the amount of peptide fixation, and the lower figure is a figure which shows antibody immobilization ability. The three-dimensional structure of the peptide is schematically displayed on the left side of the peptide name in the upper diagram. It is a figure which shows the amino acid sequence of the synthetic peptide used in Example 3, and the evaluation result of the immobilization ability of each of these synthetic peptides, immobilization antibody activity, and orientation.

Explanation of symbols

  2 solid phase body, 4 target substance, 10 solid phase material, 12 carrier, 20 orientation control material, 32 solid phase carrier

Claims (18)

  An orientation that controls the orientation of the target substance, including a polypeptide chain, and whose N-terminal amino acid residue is selected from the group consisting of alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, proline, and glutamine Control material.   The orientation control material according to claim 1, wherein the N-terminal amino acid residue is one of alanine and isoleucine. The orientation control material according to claim 1 or 2, wherein the N-terminal amino acid sequence of the polypeptide chain is any one selected from the following table.

  The orientation control material according to any one of claims 1 to 3, wherein the second or third amino acid residue from the N-terminal amino acid of the polypeptide chain has a hydroxyl group.   The orientation control material according to claim 4, wherein the second or third amino acid residue is one of serine and threonine.   The orientation control material according to claim 1, comprising two or more polypeptide chains.   The orientation control material according to claim 1, which has a polymer chain in which the polypeptide chains are linked.   The orientation control material according to claim 7, comprising a plurality of the polypeptide chains with respect to the polymer chain.   The orientation control material according to claim 1, comprising an element that interacts with a solid phase material to be oriented and fixed to the target substance.   The orientation control material according to claim 1, further comprising an immobilization element that immobilizes the target substance on a solid phase material to be oriented and fixed to the target substance.   The orientation control material according to claim 1, wherein the target substance includes a biomolecular material.   The orientation control material according to claim 11, wherein the biomolecular material includes a polypeptide chain.   The orientation control material according to claim 12, wherein the biomolecular material is an antibody. A solid phase carrier for immobilizing a target substance,
A solid phase material;
The orientation control material according to any one of claims 1 to 13, which is held on the surface of the solid phase material,
A solid phase carrier comprising:   The solid phase carrier according to claim 14, wherein the solid phase material is a molded body molded including at least a part of the orientation control material.   The solid phase carrier according to claim 14, wherein the orientation control material is fixed to the surface of the solid phase material. Preparing a solid phase carrier according to any one of claims 14 to 16,
An immobilization step of supplying a target substance to the surface of the solid phase material on which the orientation control material is held, and immobilizing the target substance on the solid phase material;
A method for producing a solid phase in which a target substance is oriented and fixed. A solid phase material;
The orientation control material according to any one of claims 1 to 13, which is held on the surface of the solid phase material,
A target substance oriented and fixed by the orientation control material;
A solid phase body.