JPWO2008093647A1 - Microarray, method for producing the same, and method for detecting interaction between organic molecule and active substance - Google Patents

Abstract

The organic molecules immobilized on the solid substrate can exist in various directions, and it is possible to detect intricate interactions between organic molecules and active substances, and there is no restriction on the directionality. The structure-activity relationship can be analyzed from the above, and the new organic molecule-immobilized microarray that can be easily and inexpensively manufactured, its manufacturing method, and the interaction between the active substance and the organic molecule using the same. A detection method for the above is provided. A microarray in which an organic molecule for detecting an interaction with an agent is immobilized, wherein A: a substrate has B: an organic carrier immobilized by an intermolecular force interaction, and B: the organic carrier. C: A microarray on which organic molecules are immobilized. In a preferred embodiment, the microarray is used to analyze by measuring at least one of transmission, reflection and interference of terahertz (THz) waves.

Description

  The present invention provides a novel organic molecule immobilized on a solid substrate for detecting an interaction with an active substance such as a biological component, natural product, allergic component, cell, antibody, sugar chain, protein, inorganic substance, or organic compound. The present invention relates to a microarray and a method for producing the same, and a method for detecting an interaction between an organic molecule and an active substance using the microarray.

  The present invention also provides a novel method for analyzing an interaction between an organic molecule and an agent capable of analyzing an interaction between the agent that interacts with an organic molecule that binds to the surface of the solid substrate by interaction, for example, an agent such as a protein, nucleic acid, or antibody. And an analysis apparatus and an analysis image apparatus therefor.

  Biological research developed using organic molecules is called chemical genetics or (in a broad sense) chemical biology. Such research is often carried out using specific and highly active organic molecules that can be used as drugs, and basically the goal is to find unknown molecular targets that can be used as seeds for drug discovery. However, in the process of finding a combination of interacting organic molecules and various biological components such as proteins, it may be directly developed into drug discovery. What is necessary for advancing such research is an organic molecule that specifically interacts with a biological component whose function is unknown. To find this, a method of screening organic molecules in a 96-well microplate, for example, is required. Has been adopted. In order to make this more efficient, technologies for immobilizing organic molecules on the substrate have been developed. Organic molecular microarrays using glass substrates, surface plasmon resonance (SPR) using gold substrates, and macros using filter paper as substrates. Arrays have been reported.

However, a problem common to these conventional immobilization techniques is that the orientation of organic molecules on the substrate is fixed in one direction. This is because all of these conventional techniques covalently bond the functional group of the organic molecule to the functional group on the substrate. In order to solve this, as a method for immobilizing organic molecules independent of the functional group on the substrate, a method of creating an organic molecule array on glass by covalent bond formation by photoreaction has been developed (for example, non-patent literature) 1) However, even in that case, it is not proved that the organic molecules on the substrate are actually arranged in a random direction. In addition, it is necessary to use a specific photoreactive group, and there is a problem that the versatility of the microarray is limited and its production is simple and not easy.

  In addition, various sugar chains are conjugated (covalently bonded) by forming a Schiff base with a hydrophobic amine and spotted on a membrane to create a sugar array, which can be used to interact with antibodies and lectins. Although it has been reported that this is detected (Non-Patent Document 2), in this case, the formation of a conjugate (covalent bond) due to the formation of a Schiff base that is chemically weak and has a low yield is a major problem. In addition, since the conjugated (covalent bond) must be purified before use for array preparation, there is a disadvantage that it is troublesome and burdensome.

  In SPR, there is a problem that it takes a cost to manufacture a substrate.

  In addition, the conventional glass array method and SPR often require an expensive array device. Further, the filter paper array has a weak carrier.

  In addition, in the array using such a conventional hard substrate, when the interaction point of the biopolymer is in a tangled position, the small molecule on the array cannot reach the interaction point. It was.

  Conventionally, analysis of active substances such as proteins, nucleic acids, antibodies, etc. has been performed from the state of binding due to the specific interaction with the surface of the solid substrate or substances fixed on the solid substrate. In the analysis, a labeling analysis method is known, in which a labeling substance for fluorescence, luminescence, etc. is used to label and analyze an agent bound on a solid substrate with a specific interaction. ing.

  However, in the case of analysis methods using fluorescence, luminescence, etc. by this labeling, it is necessary to select and prepare reagents for labeling, and to perform operations for labeling such as staining and color development. There were significant restrictions in terms of efficiency and cost reduction by shortening. In addition, because of labeling, the range of active substances to be analyzed is also limited.

  In order to solve the problem of the analysis method using labeling, an SPR method for performing analysis without labeling without labeling is also known. However, in this SPR as well, it is necessary to immobilize substances such as organic molecules that interact with the active substance on the surface of the solid substrate as in the above labeling method. The surface needs to be configured as a metal thin film, and it is not easy to fix organic molecules or the like to the metal thin film, and the solid substrate having the metal thin film on the surface is expensive.

  Conventionally, for example, in the above-described labeling analysis method using a microarray using a glass substrate, and also in the SPR method, an expensive array device is separately required for high-throughput analysis, and a solid substrate is used. In addition, there are still problems to be solved when fixing substances such as organic molecules. For example, it is an improvement measure for preventing a flow-down during cleaning and enabling a quantitative analysis. In addition, since substances such as organic molecules are usually fixed in a fixed direction with respect to the solid substrate, there is a restriction on the reactivity with the active substance, which may hinder high sensitivity analysis. Improvement of this etc. was also demanded.

  On the other hand, in recent years, the use of terahertz waves (THz waves) as technically undeveloped electromagnetic waves left between visible light and radio waves for sensing and imaging of biological molecules has been attracting attention. Actually, the biotin-avidin interaction was analyzed on the glass slide using the transmitted light of the terahertz (THz) wave (Non-patent Document 3), and the DNA sample was formed on the Ti / Au wiring produced on the silicon substrate. (Non-patent Document 4) has been reported. However, in the former report, since analysis is performed using a phase difference by a method using a THz system (THz-TDS) using a very expensive laser called a femtosecond laser, it is a large-scale system, It lacks practical generality. In the latter case, it is possible to analyze by using a wiring as a waveguide and forming a resonator. However, in practice, it is difficult to reuse the substrate, and advanced technology is required for each analysis. There is a problem that it is necessary to create a necessary substrate.

  In addition, a method of generating a transmission image by irradiating a freeze-dried specimen with a terahertz (THz) wave in a frequency band of 0.1 to 10 THz has been proposed (Patent Document 1). However, this method has a major limitation of freeze-drying.

  Furthermore, it has also been reported that RNA and pellets and films were prepared for RNA and analyzed by THz-TDS (Non-patent Document 5). However, this is only a result of dropping and drying on a substrate, and it is not practical. In addition, when drying on a substrate, the liquid sample is dried in a ring shape, so that only very inhomogeneous and unreliable data can be obtained, making quantitative measurement difficult. Therefore, as a measure for improving this problem, it is attempted to analyze a biomolecule sample dropped on a well provided on a filter membrane by measuring transmitted light of a terahertz (THz) wave with a TDS system after drying. (Non-Patent Document 6). However, even in this case, the sample placed on the membrane is only measured. For example, reaction processing that is indispensable when analyzing interactions with biopolymers, and operations such as washing. The process is not considered at all. For this reason, in the actual analysis, the loaded sample flows due to the cleaning process or the like, and the reaction process for analyzing the interaction is difficult.

Analysis using terahertz (THz) waves eliminates the problems associated with conventional labeling analysis, and the interaction between an organic molecule as an unknown component immobilized on a substrate and a known agent or vice versa. Open up new possibilities to enable analysis without labeling, and even with labeling, it can overcome the problems of the prior art and perform qualitative-quantitative analysis with higher sensitivity. Although imaging is expected to be possible, in order to actually analyze the active substance with high sensitivity, as in the case of the above-described conventional labeling analysis method and SPR method, the biological sample as a test sample is highly The binding state due to the interaction between the active substance such as a molecule and the organic molecule is formed so as to enable high-throughput analysis stably and easily at low cost, and these are formed by terahertz (THz) waves. It has been a major problem to be solved is that to be suitable for analysis. In addition to solving such problems, it has become an important issue to establish a suitable technique for terahertz (THz) wave analysis as a technique that enables imaging (image formation).
Angew. Chem. Int. Ed., 42, 5584-5587 (2003) Nature Biotechnology, 20, 1011-1017 (2002) Phys. Med. Biol., 47 (2002) 3789-3795 Appl. Phys. Lett., 80 (2002) 154-156 Optocs Express, 13 (2005) 5250-5215 Japan Society of Spectroscopy 2006 “The Symposium of the Terahertz Spectroscopy: The Cutting Edge of Terahertz Spectroscopy—Things Seen by Terahertz Spectroscopy—Abstracts of Lectures” (November 2, 2006), pp. 46-51 JP 2006-84422 A

  The present invention solves the conventional problems from the background as described above, and allows organic molecules immobilized on a solid substrate to exist in various directions, and the inclusion of organic molecules and active substances. It is possible to detect the interaction, and since there is no restriction on the directionality, it is possible to analyze the structure-activity relationship, and the new organic molecule immobilization that can be realized easily and inexpensively. It is an object of the present invention to provide a microarray and a method for producing the same, and a method for detecting an interaction between an agent and an organic molecule using the microarray.

  In addition, the present invention eliminates the problems of conventional labeling analysis methods and label-free spectroscopy, and enables high-throughput analysis of biologically relevant molecules as a highly sensitive spectroscopic analysis with ease and low cost. It is an object of the present invention to provide a new method for analyzing an interaction between an organic molecule and an active substance, which can be performed by using waves, and further to imaging (image formation), and an apparatus therefor.

  The present invention is characterized by the following in order to solve the above problems.

First: a microarray in which organic molecules for detecting an interaction with an agent are immobilized,
A: A microarray in which B: an organic carrier is immobilized on the substrate by an intermolecular force interaction, and C: an organic molecule is immobilized on the B: organic carrier.

  Second: The intermolecular force interaction is a microarray that is at least one of a hydrophobic interaction, an electrostatic interaction, and an ionic interaction.

  Third: B: A microarray in which C: organic molecules are immobilized by a conjugate (covalent bond) on an organic carrier.

  Fourth: B: A microarray in which C: an organic molecule is immobilized on an organic carrier by a conjugate (covalent bond) of at least one of an ester bond, an amide bond, a urea bond, a urethane bond, and an acetal bond; To do.

  Fifth: B: The organic carrier is a microarray that is a polymer or oligomer, or a cyclic compound.

  6th: B: Organic carriers are polyalkylene glycols, polyethers, fatty acids, peptides or polypeptides, oligomers or polymers of amino acids, steroids, terpenes, polycyclic aromatic compounds, porphyrins, and sugars A microarray that is at least one of the chains.

  Seventh: B: The organic carrier is a microarray of polyethylene glycol (PEG) having an average molecular weight of 450 or more, polyethylene oxide, or a long chain fatty acid having 8 or more carbon atoms.

  Eighth: A: The substrate is a microarray that is an organic membrane.

  Ninth: A: The organic membrane is a microarray that is a cellulose polymer, polyolefin polymer, diene polymer, fluorine polymer, polyester polymer, or polyarylene polymer.

  Tenth: A: The organic membrane is a microarray made of nitrocellulose (NC) or polyvinylidene fluoride (PVDF).

  Eleventh: A method for manufacturing any of the above microarrays, comprising the following steps.

<1.1> B: C: Organic molecules are immobilized on an organic carrier,
<1.2> A: Immobilize on the substrate.

  Twelfth: A method of manufacturing any one of the above microarrays, which includes the following steps.

<2.1> A: Immobilize B: organic carrier on the substrate,
<2.2> B: C: Organic molecules are immobilized on the organic carrier.

  13th: Using any one of the above microarrays, C: detecting an interaction between an organic molecule and an agent.

  14th: A method for analyzing a state in which an organic molecule is bound to an active substance by its interaction, wherein the organic molecule is immobilized on a substrate surface by reactive bonding or through bonding with a carrier, THz) An analysis method of interaction between an organic molecule and an active substance is characterized in that analysis is performed by measuring at least one of transmission, reflection and interference of waves.

  Fifteenth: A method for analyzing an interaction between an organic molecule and an agent is characterized in that the organic molecule is immobilized on the substrate surface via a carrier that is conjugated (covalently bonded) to the organic molecule.

  Sixteenth: A method for analyzing an interaction between an organic molecule and an active substance is characterized in that the carrier is an organic carrier and is fixed to the surface of the substrate by an intermolecular force interaction.

  Seventeenth: A method for analyzing an interaction between an organic molecule and an active substance is characterized in that a binding state is analyzed by measuring transmission absorption of a terahertz (THz) wave.

  18th: A method for analyzing an interaction between an organic molecule and an active substance, characterized in that a binding state is analyzed by measuring a spectrum change of transmitted light of a terahertz (THz) wave.

  Nineteenth: A method for analyzing an interaction between an organic molecule and an active substance is characterized in that a binding state is analyzed by measuring transmission of terahertz (THz) waves, and then absorption of reflection and re-transmission.

  20th: A method for analyzing an interaction between an organic molecule and an active substance, characterized in that near-field light is used and an angle change of total reflected light is measured to analyze a binding state.

  21st: The above terahertz (THz) wave has a frequency within a range of 0.01 to 500 THz, and is an interaction analysis method between an organic molecule and an active substance.

  Twenty-second: a terahertz irradiation unit that irradiates a terahertz (THz) wave to an active substance bonded by interaction and an organic molecule fixed to the substrate surface through a reaction bond or a bond with a carrier; And a detection unit that measures at least one of transmission, reflection, and interference of the terahertz wave thus generated.

  23rd: A terahertz irradiating unit irradiates a terahertz (THz) wave with a frequency in the range of 0.01 to 500 THz, and is an organic molecule-active substance interaction analyzer.

  24th: An interaction analysis between an organic molecule and an active substance, comprising: an image composing unit that generates an image based on a detection result in the detecting unit in the above apparatus; and an image display unit that displays the image An image device is assumed.

  According to the microarray of the present invention as described above, since the organic molecules exist in various directions by the fixation with respect to the organic carrier fixed to the substrate with intermolecular force interaction, the interaction with the active substance is not caused. The process proceeds more efficiently and the detection sensitivity is greatly improved. In addition, since the directionality is not limited, the structure-activity relationship can be analyzed. It is also possible to detect intricate interactions between organic molecules and agents.

  Further, according to the manufacturing method of the present invention, spot fixation is possible, and it is realized as simple and inexpensive.

  It is also possible to visualize the interaction with any labeled agent.

  Furthermore, according to the microarray of the present invention, it can be applied to the tracking of a chemical reaction. In this case, the reaction does not need to proceed in a high yield, and the used reagent is removed when the substrate is washed, It is not necessary to purify in advance.

  Further, according to the present invention, organic molecules whose binding state with an active substance such as a biopolymer due to interaction is to be measured and detected are immobilized on the surface of the solid substrate by reactive binding or through binding with a carrier. Because it is irradiated with terahertz (THz) waves, high-throughput analysis of biologically relevant molecules, etc. can be realized stably, highly reliable, simple, and low-cost, with high-sensitivity qualitative or quantitative analysis, or Qualitative and quantitative analysis is possible. That is, according to the present invention, the problems in the conventional labeling analysis are solved, and the problems of the conventional terahertz (THz) analysis are solved, and the organic molecule as an unknown component fixed on the substrate and the known action are solved. Analyze interactions with substances or vice versa with high sensitivity without labeling, and even with labeling, analyze conventional problems with high sensitivity It is possible to do. The analysis is also realized as imaging (image formation).

It is the figure which showed the form of the detection of the transmission absorption by a terahertz (THz) wave. It is the figure which showed the detection form of the spectrum change of the transmitted light by a terahertz (THz) wave. It is the figure which showed the form of imaging of (DELTA) f. It is the figure which showed the form of imaging of intensity change (DELTA) l. It is the figure which showed the detection form of the transmission, reflection, and re-transmission by a terahertz (THz) wave. It is the figure which showed the detection form which uses a near field light. It is the figure which showed the result of the visualization by the fluorescence detection of Cy3-O-PEG-O-Cy3 (4) and MPEG-O-Cy3 (5). It is the figure which showed the result of the visualization by the fluorescence detection of interaction with biotin-O-PEG-O-biotin (7) and MPEG-O-biotin (8) with streptavidin. It is the figure which showed the transmission image in the case of Example 2 (Example 2-2). It is the figure which showed the result of the Smoothing process of the light-absorbency image in the case of FIG. It is the figure which showed the result of visualization by the alkaline phosphatase-BCIP detection of biotin-O-PEG-O-biotin (7) and MPEG-O-biotin (8). It is the figure which showed the result of visualization by the alkaline phosphatase-chemiluminescence detection of biotin-O-PEG-O-biotin (7) and MPEG-O-biotin (8). It is the figure which showed the result of visualization by alkaline phosphatase-chemiluminescence detection of MPEG-O-biotin (8). It is the figure which showed the result of having analyzed the peak using the image data of FIG. It is the figure which showed the result of visualization by alkaline phosphatase-chemiluminescence detection of digoxins (9) (10) (11). It is the figure which showed the transmission image (absorbance) and the transmission reflection image (absorbance) in the case of Example 2 (Example 2-5). It is the figure which showed the result of visualization by the fluorescence detection of biotin fix | immobilized on the PEG sheet. It is the figure which showed the result of tracking biotin esterification reaction. It is the figure which showed the result of having fluorescence-detected the interaction of the immobilized sugar derivative and lectin. It is the figure which showed the result of having detected the terahertz (THz) wave about the interaction of the immobilized sugar derivative and lectin as a transmission image and a transmission reflection image. It is the figure which showed the result of having fluorescence-detected the interaction of the immobilized sugar derivative and lectin. It is the figure which showed the result of having detected the interaction of the immobilized sugar derivative and lectin by the terahertz (THz) wave.

  Embodiments of the present invention will be described below.

In the microarray of the present invention, as described above,
A: a substrate, B: an organic carrier, C: an organic molecule, and A and B, that is, an organic carrier is immobilized on the substrate by an intermolecular force interaction. Further, organic molecules are immobilized on B and C, that is, on the organic carrier.

  Usually, C: the organic molecule itself does not interact with the solid A: substrate at all or has only a very weak interaction. For this reason, organic molecules are easily separated by washing. However, in the present invention, B: an organic carrier is immobilized on the A: substrate by the intermolecular force interaction, and C: the organic molecule is immobilized on the B: organic carrier. Is securely and stably fixed to the substrate. In this case, the organic molecules can be immobilized in various directions.

  Here, the intermolecular force interaction for fixing A: organic carrier to A: substrate is essentially different from chemical bonding action such as so-called conjugate (covalent bond), Considered as any of hydrophobic interactions, electrostatic interactions, ionic interactions. It may be called physical bond formation.

  Hydrophobic interaction is called hydrophobic bonding, which is a substance that has a weak interaction with water and a weak affinity for water, or a functional group with that property. This means that substances having (hydrophobic groups) are aggregated and stabilized. This means that the existence of a set of each other is a thermodynamically stable state, not of the nature of actively quoting and joining each other. (For example, see “Chemical Dictionary” published by Tokyo Chemical Co., Ltd. (1989 1st edition), page 1313).

  The electrostatic interaction is a so-called electrostatic interaction. An ionic interaction is an interaction derived from the presence of ions.

  B: Such intermolecular force interaction is formed with the organic carrier. A: The substrate may be selected as a material that is easy to obtain and manufacture at a low cost and easy to handle, and is light transmissive. By making it, it can be made favorable for visualization.

  Such a substrate may be an inorganic material such as an organic polymer, silicon, metal, or ceramics. The organic polymer may be in the form of, for example, a membrane (film-like body), a film, a sheet, a bulk, or a woven fabric or a nonwoven fabric.

  In any case, the substrate of the present invention has an organic solvent resistance.

  In the present invention, for example, an organic polymer membrane having a pore size of 0.5 μm or less is considered suitable as the substrate as described above.

  Examples of such organic membranes include cellulose polymers, polyolefin polymers, diene polymers, fluorine polymers, polyester polymers, polycarbonate polymers, polyamide polymers such as nylon, polyether polymers, and polyethersulfone polymers. And various homopolymers such as polyarylene polymers or copolymers. More specifically, for example, nitrocellulose (NC), polyvinylidene fluoride (PVDF) and the like can be exemplified as suitable ones.

  When such a membrane, such as a nitrocellulose membrane or PVDF, is used, it penetrates when the active substance is dropped and penetrates into the inside of the membrane. Therefore, the internal state is also measured by terahertz (THz) wave measurement. Since this is reflected in the detection result, it is possible to perform analysis with higher accuracy.

  With respect to the intermolecular interaction with the organic carrier in the substrate of the present invention, it can be determined whether or not the hydrophobic interaction is dominant by determining the degree of hydrophobicity. As a means for the determination, the target substance is dissolved in an organic solvent that does not mix with water and mixed with water, and when the equilibrium is reached (concentration difference in organic solvent / water) Or log P in which this is expressed in common logarithm. As the organic solvent, n-octanol is often used. There is a ClogP method as a method of predictive calculation from a molecular structure on a computer. Alternatively, the degree of hydrophobicity can also be determined by reverse phase chromatography.

  For example, when calculated as a molecular weight of 2400, ClogP has the following value.

PVDF: 51.63
Nylon: 10.38
PTFE: 47.94
Polyethylene: 105.24
Polyester (PET): 16.26
Polyethersulfone (PES): 24.30
Polycarbonate: 52.14
For example, in light of experimental findings, when a polymer is used as a substrate, if the value of ClogP with a molecular weight of 2400 is 10 or more, it may be determined that the hydrophobicity is effective in the present invention. . On the other hand, the ClogP value is as follows:
Cellulose: -29.76
Nitrocellulose: -3.24
It can be considered that what is obtained as a negative numerical value like this mainly brings about electrostatic interaction, not hydrophobic interaction.

For the measurement of hydrophobicity,
You can refer to http://en.wikipedia.org/wiki/%E7%96%SE%E6%B0%B4%E6%80%A7.

  In the present invention, in order to obtain a hydrophobic substrate, the surface of various materials may be subjected to a hydrophobic surface treatment by a hydrophobizing agent such as PDMS (polydimethylsiloxane) or other means. Good.

  On the other hand, when the surface of a polymer or the like is modified with a polyanion or a polycation, an ionic interaction and further a hydrophobic interaction can be imparted thereto.

  In the present invention, those in which the substrate and the organic carrier are chemically covalently bonded and those that cause extremely strong bonding due to the presence of a hydrophilic group or the like are not preferable. This is because it is difficult to realize the intended purpose and effect of the present invention.

  The organic carrier of the present invention is also selected so that the substrate realizes intermolecular force interaction such as hydrophobicity as described above.

  In the present invention, B: an organic carrier is capable of fixing by A: binding by interaction with a substrate, fixing, etc., but in general, a polymer or oligomer, an aromatic compound or a heterocyclic compound, A cyclic compound such as a saccharide is preferred. Here, the distinction between an oligomer and a polymer is not strict, and an oligomer having a repeating unit of about 10 or less can be used as an oligomer, and a polymer having more than that can be used as a polymer. More specifically, in the present invention, B: as an organic carrier, polyalkylene glycols, polyethers, fatty acids, peptides or polypeptides, amino acids, in particular oligomers or polymers of β-amino acids, steroids, terpenes , Polycyclic aromatic compounds, porphyrins, sugar chains and the like. More preferably, for example, polyethylene glycol (PEG) or polyethylene oxide having an average molecular weight of 450 or more and 70000 or less, an ether or esterified hydroxyl group at one end thereof, or a long chain fatty acid having 8 or more carbon atoms can be considered. . Here, for example, when the molecular weight of a polyether such as polyethylene glycol or polyethylene oxide is 450 or more, the experimental value of logP exceeds 10, and it is determined as “hydrophobic”, which is effective for forming a hydrophobic interaction. It turns out that it is a thing. Even in fatty acids, if the alkyl chain has 8 carbons (C), ClogP is calculated to be 2.68, and if it is 8 or more, a hydrophobic interaction is sufficiently formed.

  On the other hand, PEG (polyethylene glycol) and fatty acids are considered to contribute to the electrostatic interaction between the cellulose and nitrocellulose.

A: Bonded to a substrate by an intermolecular force interaction such as hydrophobicity, fixed by fixing, etc. B: Organic carrier, C: Fixes an organic molecule. This immobilization is chemical or physical. Bonding, adhesion, fixing, etc. may be used. For example, it may be a bond by formation of a chemical conjugate (covalent bond), an ionic bond, a hydrogen bond, an electrostatic bond, or the like. Among them, it is preferably considered that it can be conjugated (covalently bonded) as one that enables stronger immobilization. This conjugate (covalent bond) may be various, but more convenient and versatile ones include ester bond, amide bond, urea bond, urethane bond, bond with phenol group, acetal bond, etc. Are preferably considered. Functional groups for forming these conjugates (covalent bonds), such as hydroxy group (OH), carboxyl group (COOH), amino group (NH ₂ ) carbonyl group (—CO—), etc. C: It is desirable that each organic molecule has.

  On the other hand, the type of C: organic molecule is not particularly limited. It may be various low molecular or high molecular organic molecules necessary for examining the interaction with the active substance. For example, various bioactive natural products such as biotin, digoxin, digoxigenin and the like shown in the examples may be used. Various types such as alcohols, phenols, carboxylic acids, amines, saccharides, amino acids, peptides, proteins, polyethers and the like are also considered. These organic molecules are preferably considered to be compounds having no repetitive structure and having a molecular weight of about 4000 or less. In any case, B is preferably a functional group that can be conjugated (covalently bonded) to an organic carrier, or a derivative into which the functional group is introduced.

  Various agents for detecting the interaction with C: organic molecules may be used. For example, it may be a nucleic acid (DNA, RNA), amino acid, peptide, protein, lectin, antibody, vitamin, hormone, environmental hormone, cell, sugar chain, natural or synthetic organic compound, inorganic compound or metal, allergic component, etc. .

And the microarray of the present invention, as described above,
<1.1> B: C: Organic molecules are immobilized on an organic carrier,
<1.2> A: A method of fixing to a substrate with a required pattern, or
<2.1> A: Immobilize B: organic carrier on the substrate,
<2.2> B: It can be produced easily and inexpensively by a method of immobilizing C: organic molecules on an organic carrier.

  These fixations can be spot-blotting by a required pattern. In this case, the spot fixing means may be any of various methods, and conventionally known means are appropriately employed. A: As a method for immobilization on an organic membrane or the like as a hydrophobic substrate, various methods such as the spotting method, an ink jet method, a micro contact printing method, an electrospray deposition method (ESD), and a photolithography method are used. The method is considered.

  In addition, a method by transfer of C: organic molecule migrated or C: organic molecule immobilized on B: hydrophobic organic carrier is also considered. Transfer by thin layer chromatography (TLC) blotting, transfer from gel by electrophoresis, transfer from paper chromatography, and the like. Any of these means are contemplated in the present invention.

  In the method <2.1> <2.2>, C: organic molecules may be spot-fixed in a required pattern in <2.2>, or B: organic carrier in <2.1> in advance. The pattern may be fixed.

  In the present invention, also in the production of the above microarray, as described above, B: polyethylene glycol (PEG) is considered as a suitable form as an organic carrier. In this case, PEG has an average molecular weight (MW). ) Is preferably in the range of, for example, 450 to 35000. C: When immobilization with an organic molecule is carried out by formation of a conjugate (covalent bond), this conjugate (covalent bond) is, for example, esterified , Acetalization, amidation, ureaization, or urethanization. For example, it is desirable that the terminal functional group PEG for this purpose is provided. Examples of reaction conditions include the following as the condensing agent.

Ester bond: DCC (dicyclohexylcarbodiimide), EDC (1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide), EDC Hydrochloride, DIC (diisopropylcarbodiimide), HfCl ₄ (hafnium tetrachloride), Bu ₃ P ( Tributylphosphine), lanthanide triflate, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline)
Acetal formation: iodine, NIS (N-iodosuccinimide), ICl (iodine chloride)
Reaction solvents include methylene chloride, chloroform, DMF, DMSO, DMF, DMA, methyl alcohol, water, ether, THF, ethanol, isopropanol, butanol, trifluoroethanol, benzene, toluene, pyridine, hexane, or two of these The above mixed solvent is considered. The reaction temperature is usually about room temperature (10 ° C. to 25 ° C.), but may be further heated or cooled.

  For example, various means including a conventionally known method may be employed for detecting the interaction between the organic molecule and the active substance using the microarray of the present invention as described above. For example, fluorescence detection, alkaline phosphatase activity detection, peroxidase activity detection, chemiluminescence detection, detection by radioisotope (RI) method or colloidal gold staining, detection by spectroscopic analysis, etc. may be considered. And in these detections, the visualization is also possible.

In particular, in the present invention, an embodiment in which terahertz (THz) waves are used for detection of an interaction between an organic molecule and an active substance using the microarray is preferable. In addition, in the method for analyzing the interaction between the organic molecule and the active substance according to the present invention using the terahertz (THz) wave and the apparatus therefor, for example, the following various label-free or labeling forms are organic. It is possible to analyze an interaction between a molecule and an active substance such as a biopolymer.
<1> Measurement of Transmitted Absorption For example, as illustrated in FIG. 1, the sample (102) is irradiated with a terahertz (THz) wave from the terahertz light source (101), and the transmitted absorption is detected by the detector (103). The result is displayed as an image on the display device (104). Terahertz (THz) wave irradiation light and transmitted light are guided through a parabolic mirror (105). By using a multi-element detector (103), the imaging can be of higher resolution.
<2> Spectral Change of Transmitted Light Further, as illustrated in FIG. 2, the change in the spectrum accompanying the transmission of the terahertz (THz) wave from the terahertz light source (101) is detected by the detector (103), the sensor (106), etc. By measuring and detecting, the presence or absence of the interaction between the organic molecule and the active substance can be detected by the change in refractive index, and the amount thereof can be analyzed.

  In this case, it is possible to form an image by condensing light and detecting by a single element.

  As for this imaging, for example, the spectral change information is used to image the frequency change Δf as shown in FIG. 3, or the transmission intensity change Δl of the monochromatic light source is imaged as shown in FIG. You can also

That is, analysis with a monochromatic light source is possible.
<3> Transmission, reflection, and re-transmission As illustrated in FIG. 5, for example, when using a carrier membrane or the like, after the terahertz (THz) wave is absorbed through the membrane while being absorbed through the membrane and reflected by the reflecting member It is possible to receive absorption of proteins and the like again. In this case, since the optical path length is effectively propagated twice, a minute difference can be measured with high sensitivity. Either a condensing system or a parallel light optical system is possible. Of course, the front and back of the membrane or the like can be reversed from FIG. (The sample can be on the reflective member side).
<4> Method using near-field light As shown in FIG. 6, the presence / absence or quantification of the interaction between the organic molecule and the active substance by the change in the angle of the total reflection light due to the change in the intensity or the change in the refractive index using the near-field light. Measurement is possible. The sample (sample) on the membrane in the figure can be measured even on the prism side. For imaging, it is considered to move the sample on the prism or to form an imaging system with a multi-element detector.

  Since the terahertz (THz) wave is an electromagnetic wave having a long wavelength, the amount of the near-field light oozing out is large and analysis with high sensitivity becomes possible. In addition, since the wavelength is long, it is suitable for analyzing the interaction with the active substance by the organic molecules that are transmitted through the thin substrate and immobilized thereon.

  And there is also a great feature that analysis when moisture is contained is also possible.

  For example, in any of the above forms, the analyzer of the present invention requires an irradiation unit including a terahertz light source and a detection unit, and for imaging, an image is generated from the detection result of the detection unit. It is essential to include an image configuration unit and an image display unit. As the terahertz light source, for example, a terahertz (THz) wave parametric transmitter, a light injection type terahertz (THz) wave parametric transmitter, a quantum cascade laser, an optical mixing type THz wave generator, a differential frequency mixed terahertz (THz) wave generation Various means such as an apparatus, a free electron laser, and a backward wave tube may be used. In the analysis method of the present invention and the analysis apparatus therefor, the frequency of the terahertz (THz) wave is generally within the range of 0.01 to 500 THz, and more practically 0. .1-10 THz frequency band can be suitably considered.

Various detectors may also be used. For example, a current collecting element, a porometer, a substrate absorption superconducting terahertz wave detecting element, or a quantum dot terahertz wave detecting element can be used. Among these, as a current collecting element, in addition to DTGS, for example, LiTaO ₃ (lithium tantalate), PZT (piezoelectric element), TGS (triglycine sulfate), PbTiO ₃ magnetic alignment film (lead titanate alignment film), PVF ₂ (Polyvinylidene fluoride) can be used. As the bolometer, for example, a silicon bolometer or an InSb bolometer (indium antimony bolometer) can be used. Further, instead of the terahertz light source and the wave detector, a terahertz wave time domain spectroscopy system using a femtosecond pulse laser semiconductor antenna may be used as a system in which a terahertz (THz) wave generator and detector are integrated. it can. This system includes two semiconductor antennas. A terahertz wave is emitted from one semiconductor antenna, and the terahertz wave is detected by the other semiconductor antenna.

The image construction unit is, for example, a computer, and calculates the transmission intensity of the terahertz wave at each point of the sample based on, for example, electronic signals sequentially input from a minute signal detector. On the other hand, the image construction unit is connected to, for example, the secondary scanning stage, controls the movement operation of the two-dimensional scanning stage, and detects the amount of movement from the initial position of the two-dimensional scanning stage to calculate the position of the two-dimensional scanning stage. To do. The image construction unit associates the transmission intensity of the terahertz wave with the position of the two-dimensional scanning stage at predetermined time intervals and develops the transmission image, and outputs the result to an image display unit (for example, a CRT display or a liquid crystal display). In this image display unit, a transmission image of the sample in which the transmission intensity is replaced with light and shade is displayed.

  The above example explanation is for transmission measurement, but other cases are similarly configured with various technical means.

  The analysis using such terahertz (THz) waves is based on the microarray of the present invention described above, that is, A: the substrate is fixed with B: organic carrier by intermolecular force interaction, and B: organic carrier. C: A microarray in which organic molecules are immobilized is preferably used. However, in the analysis using the terahertz (THz) wave according to the present invention, an active substance such as a biopolymer as a sample interacts. Organic molecules that are bound to each other can be fixed to the substrate by reactive binding or by binding via a carrier.

  Here, for example, reactive bonding refers to bonding by a reaction using a compound having a thiol group (SH group) to a gold (Au) substrate, or self-organization to an inorganic substrate such as silicon or zinc oxide. Known methods such as immobilization of organic molecules by forming a film (SAM film), binding using peptides, proteins, etc. that specifically bind to inorganic materials such as gold, silver, silicon, zinc, titanium, or oxides thereof Although it may be based on means, of course, it is not limited to this. In addition, immobilization via a bond with a carrier means that the organic molecule and the carrier are chemically or physically bound.

  In the present invention, a method using this carrier is considered as a more preferable one. In this case, the carrier may be either an organic substance or an inorganic substance, but it is considered that it is more preferable to form a microarray by fixing it to a substrate as an organic carrier. More practically, as described above, A: the substrate has B: an organic carrier immobilized by an intermolecular force interaction, and B: the organic carrier has C: an organic molecule immobilized. The microarray is characterized by this.

  For example, the THz wave of the present invention is used to detect the interaction between the organic molecule and the active substance using the microarray, but in the case of the microarray, there are various kinds of immobilized organic molecules. Since it exists in the direction, the interaction with the active substance proceeds more efficiently, and the sensitivity of detection is greatly improved. In addition, since the directionality is not limited, the structure-activity relationship can be analyzed. It is also possible to detect intricate interactions between organic molecules and working substances.

  Therefore, an example will be shown below and will be described in more detail. Of course, the invention is not limited by the following examples.

<Example 1>
Organic molecules were introduced into polyethylene glycol (PEG) as a hydrophobic organic carrier by formation of a conjugate (covalent bond).
(Example 1-1)
The fluorescent dye Cy3 was introduced along the following formula to synthesize Cy3-O-PEG-O-Cy3 (4).

First, HO-PEG-OH (1) with average molecular weight (MW) 3400 (1) (13.9 mg, 0.00770 mmol) and Cy3 (3) (8.8 mg, 0.016 mmol) in CH ₂ Cl ₂ (0.1 mL) To a stirred solution of was added DCC (2.4 mg, 0.012 mmol) and N, N-dimethyl-4-aminopyridine: DMAP (0.2 mg, 0.002 mmol) at room temperature.

After 19 hours, the mixture was filtered through silica gel (0.5 g, MeOH / CHCl ₃ = 10: 90), and the PEG-containing fraction was concentrated under reduced pressure. Furthermore, it refine | purified by the gel filtration column chromatography (Sephadex G-25, MeOH), and obtained Cy3-O-PEG-O-Cy3 (4) (14.9 mg, 29% weight yield, red solid). .

(Example 1-2)
Next formula

In this way, MPEG-O-Cy3 (5) was synthesized.

That is, first, MPEG-OH (2) (54.0 mg, 0.0108 mmol) having an average molecular weight (MW) of 5000 and CH ₂ Cl ₂ (0.1 mL) of Cy3 (3) (12.3 mg, 0.0216 mmol). To the stirred solution was added DCC (3.3 mg, 0.016 mmol) and DMAP (0.3 mg, 0.002 mmol) at room temperature.

After 22 hours, the same treatment as in Example 1-1 was performed to obtain MPEG-O-Cy3 (5) (61.1 mg, 100% weight yield) (red solid). The effective yield from ¹ HNMR spectrum was 98%.

(Example 1-3)
Next formula

The biotin-O-PEG-O-biotin (7) was synthesized from PEG and biotin.

  First, in a DMF (0.3 mL) stirred solution of HO-PEG-OH (1) (50.0 mg, 0.0147 mmol) and biotin (6) (7.2 mg, 0.029 mmol) having an average molecular weight (MW) of 3400 At room temperature, DCC (4.6 mg, 0.022 mmol) and DMAP (0.4 mg, 0.003 mmol) were added.

  After 4 days, it was concentrated by azeotropy with toluene under reduced pressure and purified by gel filtration chromatography (Sephadex G-25, MeOH) to give biotin-O-PEG-O-biotin (52.7 mg, 41% weight). Yield) (colorless solid).

(Example 1-4)
Next formula

In this way, MPEG-O-biotin (8) was synthesized.

  To a stirred solution of MPEG-OH (2) (51.6 mg, 0.0100 mmol) and biotin (6) (4.9 mg, 0.020 mmol) with an average molecular weight (MW) of 5000 in DMF (0.1 mL) was added DCC at room temperature. (3.1 mg, 0.015 mmol) and DMAP (0.4 mg, 0.003 mmol) were added.

After 12 hours, the same treatment as in Example 1-3 was performed to obtain MPEG-O-biotin (8) (50.0 mg, 96%) (colorless solid). The effective yield from ¹ HNMR spectrum was 65%.

(Example 1-5)
Next formula

Then, MPEG-O-AAc-O-digoxin (9) was synthesized from MPEG and digoxin.

First, in a stirred solution of MPEG-O-AAc-OH (12.8 mg, 0.00256 mg) and digoxin (2.00 mg, 0.00256 mmol) with an average molecular weight (MW) of 5000 in CH ₂ Cl ₂ (0.25 mL), DCC (2.6 mg, 0.013 mmol) and DMAP (0.6 mg, 0.0005 mmol) were added at room temperature.

Then, after 41 hours, the solution was concentrated under reduced pressure and purified by gel filtration column chromatography (Sephadex G25, MeOH) to obtain MPEG-O-AAc-O-digoxin (9) (14.5 mg, 98%) (colorless solid) Got. ¹ HNMR spectrum confirmed that the conjugate (covalent bond) was randomly formed with the hydroxyl group of digoxin and that the effective yield was 51% (0.094 mmol / g).
(Example 1-6)
Next formula

Then, MPEG-O-AAc-digoxigenin (10) was synthesized from MPEG and digoxigenin.

Average molecular weight (MW) 5000 in MPEG-O-AAc-OH ( 12.8mg, 0.00256mmol) and digoxigenin (1.00mg, 0.00256mmol) in CH ₂ Cl ₂ (0.25mL) stirred solution of at room temperature DCC (2.6 mg, 0.013 mmol) and DMAP (0.6 mg, 0.0005 mmol) were added.

After 20 hours, the same treatment as in Example 1-5 was performed to obtain MPEG-O-AAc-O-digoxigenin (10) (13.8 mg, 100% by weight) (colorless solid). According to ¹ HNMR spectrum, the effective yield was confirmed to be 30% for 3-O-conjugate (10a) and 70% for 12-O-conjugate (10b) (total 0.186 mmol / g). It was.
(Example 1-7)
Next formula

MPEG-O-AAc-O-digitoxigenin (11) was synthesized from MPEG and digitoxigenin as described above.

First, a stirred solution of MPEG-O-AAc-OH (13.4 mg, 0.00267 mmol) and digitoxigenin (1.00 mg, 0.00267 mmol) with an average molecular weight (MW) of 5000 in CH ₂ Cl ₂ (0.20 mL) DIC (0.0021 mL, 0.013 mmol) and DMAP (0.06 mg, 0.0005 mmol) were added at room temperature.

After 20 hours, the same treatment as in Example 1-6 was carried out to obtain MPEG-O-AAc-O-digitoxigenin (11) (14.5 mg, 100% by weight) (colorless solid). According to the ¹ HNMR spectrum, the effective yield was 100% (0.186 mmol / g).
(Example 1-8)
Next formula

As a result, MPEG-O-MTM (12) and various ROHs (alcohols and phenols) were used to obtain MPEG-O-CH ₂ -OR (13) in a yield of 90% or more.
<Example 2>
An organic molecule conjugated to a hydrophobic organic carrier was immobilized on a hydrophobic organic membrane. As a spot fixing means by dot blotting, dot blotting is performed in a state where a grid sheet is placed under a hydrophobic organic membrane and illuminated from a light source below.

  The immobilization procedure was based on the following process.

That is, a solution of organic molecules CHCl ₃ (1 × 10 ⁻² , 2 × 10 ⁻³ , 4 × 10 ⁻⁴ , 8 × 10 ⁻⁵ , 1.6 × 10 ⁻⁵ M) conjugated to PEG or MPEG. (0.1 × 10 ⁻³ mL) was dot blotted onto NC (nitrocellulose) or PVDF (polyvinylidene fluoride) membranes (75 mm × 75 mm).

  Then, it was immersed in a fixing solution (10 mL) for 10 minutes, and then washed with water (10 mL, 2 × 1 min, then 2 × 5 min).

  The immobilized membrane was immersed in a skim milk solution (10 mL) for 60 minutes to stir the solution.

Here, nitrocellulose (NC, GE Healthcare Bio-Sciences, Hybond-ECL, 100% nitrocellulose, pore size: 0.20 × 10 ⁻³ mL or 0.45 × 10 ⁻³ mL) is used for the membrane as described above. Alternatively, polyvinylidene fluoride (PVDF, GE Healthcare Bio-Sciences, Hybond-P, 100% polyvinylidene difluoride, pore size: 0.20 × 10 ⁻³ mL or 0.45 × 10 ⁻³ mL) is used. The following solutions are prepared and used.

(Example 2-1)
Using the Cy3-O-PEG-O-Cy3 (4) and MPEG-O-Cy3 (5) prepared in Example 1-1 and Example 1-2 in Example 1, dot blotting was performed on the NC membrane by the following procedure. And fixed.

  After the treatment of the skim milk solution, washing with DBB (10 mL, 6 × 5 min) and washing with water (10 mL, 1 h) were performed, and drying was performed for 120 minutes between Whatman papers.

  Attached FIG. 7 shows the result of observing fluorescence derived from Cy3 (FUJIFILM, FLA-2000, 473 nm). FIG. 7 shows the following.

As shown in FIG. 7, it can be seen that the immobilization by using the dye (Cy3) as a PEG / MPEG conjugate is immobilized in a concentration-dependent manner. In addition, detection by fluorescence is clearly more sensitive than viewing by visible light. This example shows the basis of small molecule immobilization technology with PEG / MPEG support.
(Example 2-2)
Using biotin-O-PEG-O-biotin (7) and MPEG-O-biotin (8) prepared in Example 1-3 and Example 1-4 in Example 1, dot blotting was performed on a PVDF membrane by the following procedure. And fixed.

  In this example, the interaction between immobilized biotin and streptavidin was detected by fluorescence detection and transmission measurement using THz waves.

That is, as shown in Table 9, following immersion of the skim milk solution, a skim milk solution (10 mL) containing streptavidin-Alexa 633 (2.0 mg / mL in Water, 3.3 × 10 ⁻³ mL) was added at room temperature. It was immersed for 6 hours and washed with DBB (10 mL, 6 × 5 min) and water (10 mL, 1 h). After drying using Whatman paper, fluorescence detection and transmission measurement with THz waves were performed. In the transmission measurement using the THz wave, the frequency band 2 THz was used and the measurement was performed by THz-TDS.

FIG. 8 shows the result of fluorescence detection. The detection results with 633 mm UV light are shown, and blots (0.1 × 10 ⁻³ mL) at various concentrations in the case of biotin-O-PEG-O-biotin (7) and MPEG-O-biotin (8) are shown. ) Is observed.

The detection by the fluorescence of the biotin-streptavidin interaction shown in FIG. 8 can be detected in the range of 8 × 10 ⁻⁵ −5 × 10 ⁻² M, and is clearly concentration-dependent.

  In the case of biotin (6), streptavidin-derived fluorescence is not detected at all. This can be attributed to the fact that the biotin (6) blotted there was washed away, and the PEG / MPEG conjugate should be used. This shows the effectiveness of immobilizing small molecules by.

FIG. 9 shows the result of transmission measurement using THz waves as a transmission image. In addition, the white line in a figure has shown the damage | wound. FIG. 10 shows the result of smoothing the 2 THz absorbance image. For example, as shown in the change in absorbance of the Y = 88 NO line in FIG. 10, the detection of biotin-streptavidin interaction is 8 It can be detected in the range of × 10 ⁻⁵ -5 × 10 ⁻² M, and it is apparent that it is concentration-dependent.

In addition, in the case of biotin (6), no streptavidin-derived transmitted image was detected because the blotted biotin (6) was washed away by the washing operation, and the PEG / MPEG conjugate This clearly shows the effectiveness of immobilizing small molecules.
(Example 2-3)
In the following procedure, the same biotin as in Example 2-2 was immobilized, and the interaction with streptavidin was detected by the alkaline phosphatase-BCIP detection method.

  In this case, after DBB washing, it is incubated for 20 minutes with an NBT / BCIP solution (1.0 mL), and then washed with water.

FIG. 11 shows the results of visualization with alkaline phosphatase-BCIP detection at 5 × 10 ⁻² M for blots at 0.1 × 10 ⁻³ mL.
(Example 2-4)
In the following procedure, the same biotin as in Example 2-2 was immobilized on a blot (0.1 × 10 ⁻³ mL), and the interaction with streptavidin was determined by the alkaline phosphatase-chemiluminescence detection method. The effect was detected.

That is, following immersion in a skim milk solution, a skim milk solution (1.0 mL) containing streptavidin and an alkaline phosphatase conjugate (1.0 mg / mL in water, 8.3 × 10 ⁻⁴ mL) was added to room temperature. And then DBB washed. And it incubated with the CDP-star solution (0.5 mL).

  FIG. 12 shows the result of chemiluminescence detection.

The detection of biotin-streptavidin interaction shown in FIG. 12 by alkaline phosphatase activity-chemiluminescence is much more sensitive than using BCIP (FIG. 11) as in the literature (1 × 10 ⁻⁵ M to 1 × 10 6). Detection was possible in a concentration dependent range of ^-2 M. This sensitivity is equivalent to or slightly higher than the detection by fluorescence (FIG. 8).

  For the results of FIG. 13 visualized by alkaline phosphatase activity-chemiluminescence detection of MPEG-O-biotin, this image data was converted to ImageJ (ver. 1.37v) (Rasband, WS, ImageJ, US National Institutes of The results of peak analysis using Health, Bethesda, Maryland, USA, http // rsb.info.nih.gov / iJ /, 1997-2006) are shown in FIG.

Clear concentration dependence can be confirmed.
(Example 2-5)
In the following procedure, MPEG-O-AAc-O-digoxin (9), MPEG-O-AAc-O-digoxigenin (10), MPEG-O- of Examples 1-5, 1-6, and 1-7 were used. Blot (0.1 × 10 ⁻³ mL) was immobilized using each of AAc-O-digitoxigenin (11), terahertz (THz) by alkaline phosphatase chemiluminescence detection method and in the same manner as in Example 2-2 above. ) Interaction was detected by wave.

  FIG. 15 shows the results of visualization by an alkaline phosphatase chemiluminescence detection method.

FIG. 16 shows a transmission image (absorbance) and a transmission reflection image (absorbance) by THz waves.
<Example 3>
The method of immobilizing organic molecules on the membrane using the properties of PEG involves the production of conjugates on the membrane, in addition to the method of preparing and blotting the organic molecule-PEG conjugates already mentioned. The method is also practical. Here, a “PEG sheet” whose surface is modified with PEGs is prepared by immersing the membrane in a solution of PEGs in advance. A mixed solution of the organic molecule and the condensing agent to be immobilized is blotted and incubated for a predetermined time. According to this method, if the organic molecule group to be immobilized is not conjugated with PEGs, a membrane macroarray can be quickly prepared with little effort.

  For example, biotin or the like as an organic molecule can be immobilized by the following procedure, and fluorescence can be detected by interaction with streptavidin, or can be detected by a terahertz (THz) wave interacting with streptavidin.

  More specifically, first, a PVDF membrane (15 mm × 15 mm) is immersed in a solution of PEG having an average molecular weight (MW) of 3400 (500 mg) in methanol / water (1: 1, 10 mL) at room temperature for 10 minutes. Then wash with water (10 mL, 2 × 1 min, then 2 × 5 min) and dry between whatman paper for 120 minutes. Thereby, a PEG sheet is obtained.

  Meanwhile, a blotting solution of DMF (0.4 mL) of biotin (9.78 mg, 0.040 mmol), EDC-HCl (8.24 mg, 0.040 mmol) and DMAP (0.96 mg, 0.008 mmol) is prepared.

Using 0.1 × 10 ⁻³ mL of this, blot on the above PEG sheet. It is then incubated for 40 hours at room temperature.

After drying, immerse in fixer (10 mL) for 10 minutes. Wash with water (10 mL, 2 × 1 min, then 2 × 5 min), then immerse in skim milk solution (10 mL) for 60 minutes, and further streptazibine-Alexa 633 (2.0 mg / mL in Water, 3.3 × 10 ^-3 mL) in a skim milk solution for 6 hours.

  Wash with DBB (10 mL, 6 × 5 min), wash with water (10 mL, 1 hour) and dry on Whatman paper for 120 minutes.

  FIG. 17 shows the result of visualization by fluorescence detection (633 nm).

Further, in the same manner as in Example 2-2 of Example 2, the interaction with streptazibine could be confirmed as a THz transmission image.
<Example 4>
The microarray of the present invention can also be used for tracking organic reactions. For example, the synthesis reaction mixture of biotin-O-PEG-O-biotin (7) and MPEG-O-biotin (8) is prepared in advance as a sample.

These samples (0.1 × 10 ⁻³ mL) are dot blotted onto a PVDF membrane (65 mm × 55 mm). Next, it is immersed in a fixing solution (10 mL) for 10 minutes and washed with water (10 mL, 2 × 1 min, then 2 × 5 min).

Immerse it in a skim milk solution (10 mL) at room temperature for 60 minutes, and then add it to a skim milk solution (1.0 mL) containing streptavidin-Alexa 633 (2.0 mg / mL in Water, 1.67 × 10 ⁻³ mL). Immerse for 6 hours at room temperature.

  Wash with DBB (10 mL, 6 × 5 min) and water (10 mL, 1 hour) and dry between Whatman paper (120 minutes).

  FIG. 18A shows the result of visualizing the result of fluorescence detection (633 nm) with time. FIG. 18B shows a dot blot map.

  The degree of biotin esterification reaction can be traced from FIGS. 18 (a) and 18 (b).

Further, in the same manner as in Example 2-2 of Example 2, the degree of biotin esterification reaction could be traced by observing permeation detection with time.
<Example 5>
A sugar chain as an organic molecule was introduced into a fatty acid compound having a long alkyl chain as a hydrophobic organic carrier by formation of a conjugate (covalent bond).
(Example 5-1)
Lactose (Lac) was introduced according to the following formula.

Lactose octanoylhydrazone (Lac-C8)
Lactose (20.0 mg, 0.0555 mmol) and octanoylhydrazide (17.6 mg, 0.111 mmol) are dissolved in 0.1 M sodium acetate-acetic acid (pH 2.5) / methanol / chloroform (2: 5: 3, 1 mL), and then at 50 ° C. for 4 days. Stir. After concentration under reduced pressure, the obtained residue was purified by silica gel column chromatography to obtain lactose octanoylhydrazone (21.0 mg, 0.00433 mmol, 78%) as a white solid. ¹ H NMR (300MHz, CD ₃ OD) δ 4.32 (d, J = 7.5Hz, 1H), 3.95-3.34 (m, 12H), 3.23 (dd, J = 9.0, 9.0Hz, 1H), 2.16 (dd, J = 8.1, 6.6Hz, 2H), 1.61 (br, 2H), 1.31 (br, 8H), 0.90-0.87 (m, 3H); ¹³ C NMR (125MHz, CDCl ₃ ) δ 195.7, 176.0, 149.1, 104.7 , 91.5, 80.1, 77.4, 76.7, 76.3, 74.6, 72.2, 71.7, 70.2, 62.6, 62.0, 35.4, 32.8, 30.3, 30.0, 26.8, 23.7, 15.0.
Lactose stearylhydrazone (Lac-C18)
Lactose (20.0 mg, 0.0555 mmol) and stearylhydrazide (66.7 mg, 0.222 mmol) were dissolved in 0.1 M sodium acetate-acetic acid (pH 2.5) / methanol / chloroform (6: 4: 0.4, 1 mL), and the mixture was stirred at 50 ° C. for 4 days. Stir. After concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography to obtain lactose stearylhydrazone (10.4 mg, 0.0017 mmol, 30%) as a white solid. ¹ H NMR (300 MHz, CD ₃ OD) δ 4.32 (d, J = 7.5 Hz, 1H), 3.96-3.44 (m, 12H), 3.23 (dd, J = 8.7, 8.7 Hz, 1H), 2.15 (ddd, J = 8.4, 8.4, 7.5Hz, 2H), 1.61-1.59 (m, 2H), 1.28 (br, 28H), 0.89 (t, J = 6.6Hz, 3H).
(Example 5-2)
Lacto-N-tetraose was introduced along the following formula.

Lacto-N-tetraose octanoylhydrazone (4-C8)
Lacto-N-tetraose (0.5 mg, 0.71 μmol) and octanoylhydrazide (1.12 mg, 7.06 μmol) are dissolved in 0.1 M sodium acetate-acetic acid (pH 2.5) / methanol / chloroform (6: 5: 2, 0.3 mL), Stir at room temperature for 2 weeks. The reaction mixture was concentrated under reduced pressure, distilled water (0.2 mL) was added, and the mixture was extracted with chloroform (0.3 mL × 3). After the organic layer was concentrated under reduced pressure, lacto-N-tetraose octanoylhydrazone (0.488 mg, 0.575 μmol, 81%, purity 76.7%) was obtained as a white solid. HRMS (ESI, positive) calcd for C _{3 4} H _{5 2} N ₃ O _{2 1} [(M + H) ⁺ ] 848.3870, found 848.3845.
Lacto-N-tetraose stearylhydrazone (4-C18)
Lacto-N-tetraose (0.50mg, 0.71μmol) and stearylhydrazide (2.04mg, 7.06μmol) were dissolved in 0.1M sodium acetate-acetic acid (pH 2.5) / methanol / chloroform (6: 5: 2, 0.3mL) Stir at room temperature for 2 weeks. The reaction mixture was concentrated under reduced pressure, distilled water (0.2 mL) was added, and the mixture was extracted with chloroform (0.3 mL × 3). After the organic layer was concentrated under reduced pressure, lacto-N-tetraose stearylhydrazone (0.702 mg, 0.71 μmol, quant, purity 22.7%) was obtained as a white solid. HRMS (ESI, positive) calcd for C _{4 4} H _{8 2} N ₃ O _{2 1} [(M + H) ⁺ ] 988.5435, found 988.5390.
(Example 5-3)
Lacto-N-fucopentaose was introduced according to the following formula.

Lacto-N-fucopentaose octanoylhydrazone (5-C8)
Lacto-N-fucopentaose (0.5 mg, 0.59 μmol) and octanoylhydrazide (0.890 mg, 5.86 μmol) are dissolved in 0.1 M sodium acetate-acetic acid (pH 2.5) / methanol / chloroform (6: 5: 2, 0.3 mL), Stir at room temperature for 2 weeks. The reaction mixture was concentrated under reduced pressure, distilled water (0.2 mL) was added, and the mixture was extracted with chloroform (0.3 mL × 3). After the organic layer was concentrated under reduced pressure, lacto-N-tetraose octanoylhydrazone (0.469 mg, 0.472 μmol, 80%, purity 83%) was obtained as a white solid. HRMS (ESI, positive) calcd for C _{4 0} H _{7 2} N ₃ O _{2 5} [(M + H) ⁺ ] 994.4449, found 994.4412.
Lacto-N-fucopentaose stearylhydrazone (5-C18)
Lacto-N-fucopentaose (0.50 mg, 0.59 μmol) and stearylhydrazide (1.75 mg, 5.86 μmol) were dissolved in 0.1 M sodium acetate-acetic acid (pH 2.5) / methanol / chloroform (6: 5: 2, 0.3 mL), Stir at room temperature for 2 weeks. The reaction mixture was concentrated under reduced pressure, distilled water (0.2 mL) was added, and the mixture was extracted with chloroform (0.3 mL × 3). After the organic layer was concentrated under reduced pressure, lacto-N-tetraose stearylhydrazone (0.388 mg, 0.342 μmol, 58%, purity 35.5%) was obtained as a white solid. HRMS (ESI, positive) calcd for C _{5 0} H _{9 0} N ₃ O _{2 5} [(M + H) ⁺ ] 1133.2548, found 1132.8397
<Example 6>
Table 5 in Example 2 shows sugar derivatives that are conjugates of sugar chains and long-chain alkyl chain fatty acid compounds obtained as a result of introduction from Example 5-1 to Example 5-3 in Example 5. It was immobilized on a hydrophobic organic membrane in the same manner as in the process.

The immobilized array of seven sugar derivatives was then allowed to interact with lectin (congerin I), which was detected with fluorescence at 473 nm. FIG. 19 shows the result. In any compound, the strength of the interaction changes depending on the concentration, and it can be said that a specific interaction is detected. For example, when compared at a concentration of 4 × 10 ^-4 M, it is clear that, as is known so far, tetrasaccharide has stronger interaction and pentasaccharide is stronger than disaccharide, lactose. Recognize. Even in the same lactose, the fluorescence is stronger when conjugated with an alkyl chain, and the fluorescence of carbon chain 18 (C18) is stronger than that conjugated with carbon chain 8 (C8). This is not because there is a difference in the strength of the interaction, but because there is a difference in the amount of the compound retained on the membrane, which means that a strongly hydrophobic compound is more strongly immobilized. This is due to the nature of the present invention.

  Thus, it is one of the features of the present invention that a weak interaction between a sugar and a lectin, which is generally difficult to detect with a glass array, can be detected.

Also, an immobilized array of seven sugar derivatives was allowed to interact with lectin (congerin I), and this was detected by THz wavelength as in Example 2. FIG. 20 shows the results as a transmission image (absorbance) and a transmission reflection image (absorbance). In any compound, the strength of the interaction changes depending on the concentration, and it can be said that a specific interaction is detected. It can be clearly seen that tetrasaccharide is stronger in interaction and lactose is stronger than the disaccharide lactose. Moreover, the fluorescence of carbon chain 18 (C18) is stronger than that conjugated with carbon chain 8 (C8). This is not because there is a difference in the strength of the interaction, but because there is a difference in the amount of the compound retained on the membrane, which means that a strongly hydrophobic compound is more strongly immobilized. This is due to the nature of the present invention.
<Example 7>
(Example 7-1)
According to the following schemes 1 to 3, a polyethylene glycol conjugate of a sugar chain was synthesized. Via a two-step click reaction, a triethylene glycol linker having an azide group was introduced into the reducing end of the sugar chain by an oxime formation reaction, and PEG was introduced into this azide group by a Huisgen reaction.

  The first step reaction can be performed in an acetonitrile-acetate buffer solution that dissolves sugar chains well, and linkers can be introduced in good yield regardless of the structure of the target sugar chain. The second stage reaction forms a triazole ring which has been shown to have little biological activity.

The linker 7 used in this synthesis was synthesized as shown in Scheme 1. Commercially available triethylene glycol 1 was monotritylated to give 2, and the remaining hydroxyl group was converted to 3 by Mitsunobu reaction with N-hydroxyphthalimide. The phthalimide moiety was removed by hydrazine treatment, and the resulting amino group was converted to Boc by a conventional method. The obtained trityl group of 4 was removed by catalytic hydrogenation to obtain 5, and then azide (DEAD, PPh ₃ ) using DPPA was performed to obtain 6. Finally, deprotection of the Boc group was performed with TFA, and linker 7 could be obtained.

  The obtained knowledge about the reaction between the linker 7 and lactose and the Huisgen reaction using the model compound 9 are shown in Scheme 2.

  When linker 7 was reacted with lactose at room temperature and pH 2.5, the oxime formation reaction was completed within 2 days, and 8 could be obtained almost quantitatively. Similar results were obtained when maltose was used as the sugar chain. Product 8 is highly polar and difficult to handle and also forms as an isomer mixture in the oxime functional moiety. Therefore, this reaction was evaluated only by LC-MS, after which the reaction mixture was concentrated and used for the next Huisgen reaction.

  Triazole ring formation by Huisgen reaction proceeds even without catalyst, but in this example using acetylene compound 9, it was revealed that the reaction proceeds rapidly when copper sulfate and sodium ascorbate coexist. The reaction was carried out at 60 ° C. for 12 hours and further at room temperature for 3 to 4 days. Also for this reaction, the product was analyzed by LC-MS, and it was confirmed that 8 was completely consumed and triazole compound 10 was clearly formed. Next, conjugate formation with PEG was performed using the above reaction conditions. Scheme 3 shows the conjugate synthesis of lactose and PEG.

The PEG used here is poly (ethylene glycol) monomethyl ether (MPEG) having an average molecular weight of 5000. MPEG acetylene 12 was quantitatively obtained by amide condensation of a known carboxylic acid 11 (Japanese Patent Application No. 2003-383694) and propargylamine. This was reacted with 8 under the above-mentioned conditions (8 → 10) to obtain 13. 13 can be obtained quickly and easily by gel filtration column chromatography or silica gel column chromatography taking advantage of the properties of MPEG. ^{The 1} H NMR spectrum was analyzed, and the reaction progress rate was determined to be 91.5% and the loading amount was 0.167 mol / g.

(Example 7-2)
Maltose MPEG conjugation was performed according to Scheme 4.

The procedure was exactly the same as that of lactose shown in Scheme 3, and conjugate 15 could be obtained. ^{The 1} H NMR spectrum was analyzed and the reaction progress was determined to be 75.0% and the loading amount was 0.136 mmol / g.

<Example 8>
An attempt was made in the same manner as in Example 6 to detect the interaction between the sugar chain array using lactose and its conjugates 10 and 13 synthesized in Example 7 and lectin, Congelin I.

  That is, 6 types of each of lactose and 10, 13 methanol solutions were prepared, and 0.2 mL each was dot-blotted in series on a PVDF membrane. Immobilization, washing, and blocking were sequentially performed, and incubated for 20 hours in a plastic bag with Congerin I labeled with FITC. The membrane was washed, air dried and analyzed by fluorescence and terahertz waves. The results are shown in FIGS. 21 and 22, respectively.

  From the results obtained by either of the detection methods of FIGS. 21 and 22, the interaction is concentration-dependent, and it can be determined that a specific interaction has been detected.

For the fluorescence detection of FIG. 21, only the lactose MPEG conjugate 13 showed interaction with Congerin I at a concentration of 4 × 10 ⁻⁴ M.

In the terahertz wave analysis of FIG. 22, it was possible to detect the interaction with higher sensitivity than in FIG. That is, it was revealed that the interaction could be detected with the MPEG conjugate 13 at a concentration of 8 × 10 ⁻⁵ M. Low molecular weight conjugate 10 was also detected at a concentration of 4 × 10 ⁻⁴ M, but unconjugated lactose did not detect its interaction even at a high concentration of 1 × 10 ⁻² M. This is consistent with the result in Example 6.

  The results in FIG. 22 show that conjugation is effective for immobilization of sugar chains, and in particular, MPEG with an average molecular weight of 5000 is superior, whereas low molecular weight conjugates have an immobilization yield. It shows that it falls a little.

Claims (24)

A microarray on which organic molecules for detecting an interaction with an agent are immobilized,
A: A microarray in which B: an organic carrier is immobilized on a substrate by an intermolecular force interaction, and B: an organic molecule is immobilized on the B: organic carrier.   The microarray according to claim 1, wherein the intermolecular force interaction is at least one of a hydrophobic interaction, an electrostatic interaction, and an ionic interaction.   The microarray according to claim 1 or 2, wherein C: an organic molecule is immobilized on the organic carrier by a conjugate (covalent bond).   B: The organic carrier has C: an organic molecule immobilized on the organic carrier by a conjugate (covalent bond) of at least one of an ester bond, an amide bond, a urea bond, a urethane bond, and an acetal bond. The microarray according to claim 3.   B: The microarray according to claim 1 or 2, wherein the organic carrier is a polymer, an oligomer, or a cyclic compound.   B: Organic carriers are polyalkylene glycols, polyethers, fatty acids, peptides or polypeptides, oligomers or polymers of amino acids, steroids, terpenes, polycyclic aromatic compounds, porphyrins, and sugar chains The microarray according to claim 1, wherein the microarray is at least one of them.   B: The microarray according to claim 6, wherein the organic carrier is polyethylene glycol (PEG) having an average molecular weight of 450 or more, polyethylene oxide, or long chain fatty acids having 8 or more carbon atoms.   A: The microarray according to claim 1 or 2, wherein the substrate is an organic membrane.   A: The microarray according to claim 8, wherein the organic membrane is a cellulose polymer, a polyolefin polymer, a diene polymer, a fluorine polymer, a polyester polymer, or a polyarylene polymer.   The microarray according to claim 9, wherein the organic membrane is nitrocellulose (NC) or polyvinylidene fluoride (PVDF). The method for producing a microarray according to any one of claims 1 to 10, comprising the following steps.
<1.1> B: C: Organic molecules are immobilized on an organic carrier,
<1.2> A: Immobilize on the substrate. The method for manufacturing a microarray according to any one of claims 1 to 10, comprising the following steps.
<2.1> A: Immobilize B: organic carrier on the substrate,
<2.2> B: C: Organic molecules are immobilized on the organic carrier.   A microarray detection method, comprising: detecting an interaction between C: an organic molecule and an agent using the microarray according to any one of claims 1 to 10.   A method for analyzing a state in which an organic molecule is bonded to an active substance by its interaction, wherein the organic molecule is immobilized on a substrate surface by a reactive bond or through a bond with a carrier, and a terahertz (THz) wave An analysis method for an interaction between an organic molecule and an active substance, wherein the analysis is performed by measuring at least one of transmission, reflection, and interference.   15. The method for analyzing an interaction between an organic molecule and an agent according to claim 14, wherein the organic molecule is immobilized on the substrate surface via a carrier that is conjugated (covalently bonded) to the organic molecule.   16. The method for analyzing an interaction between an organic molecule and an active substance according to claim 15, wherein the carrier is an organic carrier and is fixed to the surface of the substrate by intermolecular force interaction.   The interaction analysis method according to claim 14, wherein the coupling state is analyzed by measuring transmission absorption of a terahertz (THz) wave.   15. The method for analyzing an interaction between an organic molecule and an active substance according to claim 14, wherein the binding state is analyzed by measuring a spectral change of transmitted light of a terahertz (THz) wave.   15. The method for analyzing an interaction between an organic molecule and an active substance according to claim 14, wherein the binding state is analyzed by measuring transmission of terahertz (THz) waves, and then absorption of reflection and re-transmission.   15. The method for analyzing an interaction between an organic molecule and an active substance according to claim 14, wherein the near-field light is used to measure the angle change of the total reflected light to analyze the binding state.   The method for analyzing an interaction between an organic molecule and an active substance according to any one of claims 14 to 20, wherein the terahertz (THz) wave has a frequency within a range of 0.01 to 500 THz.   A terahertz irradiation unit that irradiates a terahertz (THz) wave to an active substance that is bound by interaction and an organic molecule that is fixed to the substrate surface through a reaction bond or a bond with a carrier; and the irradiated terahertz An apparatus for analyzing an interaction between an organic molecule and an active substance, comprising: a detection unit that measures at least one of wave transmission, reflection, and interference.   23. The apparatus for analyzing an interaction between an organic molecule and an active substance according to claim 22, wherein the terahertz irradiation unit irradiates a terahertz (THz) wave having a frequency in a range of 0.01 to 500 THz.   24. An organic molecule and an active substance, comprising: an image forming unit that generates an image based on a detection result of the detection unit in the apparatus according to claim 22; and an image display unit that displays the image. Interaction analysis imaging device.