JP2004191368A - Target molecule detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simultaneously isolating and detecting all the pluralities of target molecules (e.g. genes) and simultaneously isolating and detecting genes through the use of a relatively small amount of probe molecules (e.g. a DNA conjugate). <P>SOLUTION: In the target molecule detection method, one type of probe molecules or more (e.g. a DNA conjugate) and two types of target molecules or more (e.g. genes) are placed under conditions which enable interaction. Target molecules which have interacted with any probe molecule and formed a complex and target molecules which have not formed any complex are isolated from each other through the use of the difference of mobility between the target molecules which have formed the complex and the target molecules which have not formed any complex. The target molecule detection method includes the detection of the target molecules which have formed the complex and/or the target molecules which have not formed any complex. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a method for detecting a target molecule. When the detection method of the present invention is used as a nucleic acid detection method, it is a gene separation detection method based on an unprecedented principle, and specific simultaneous separation detection of all genes including SNPs is possible. Further, the method of the present invention is also useful as a method for detecting various target molecules other than nucleic acids.

  According to the Ministry of Health, Labor and Welfare's statistics (http://www.mhlw.go.jp/topics/syokuchu/index.html), about 2000 food poisonings occur every year (about 30,000 victims). Food poisoning is caused by various factors such as poor air permeability of houses with improved airtightness, overconfidence in refrigerators with improved performance, distribution of contaminated foods due to the activation of the import industry, and contamination of domestic water due to thawing of contaminated foods. Is likely to occur all year round. A major feature of food poisoning in recent years is that food poisoning caused by pathogenic Escherichia coli O157 (O157: H7) has increased in place of Vibrio parahaemolyticus transmitted from fish and shellfish. O157 is Escherichia coli belonging to enterohemorrhagic Escherichia coli, and causes diarrhea due to produced toxin. Currently, identification of the causative bacteria of food poisoning requires an inspection process that takes several days, such as culturing the stool of a patient on an agar medium supplemented with sorbitol. However, in order to more quickly identify the causative bacterium, it is sufficient if the genotype of verotoxin can be identified quickly and easily.

  On the other hand, even if the same drug is administered to patients who show the same symptoms, there are cases where the `` individual difference in sensitivity to drugs '' has been a problem, in that high effects may be exhibited while side effects may occur. Has become. This is due to the sensitivity of the drug to different genotypes. However, if a doctor can easily determine the genotype of a patient, administration of a drug with strong side effects should be avoided. In order to realize such “tailor-made medicine” that provides optimal treatment for each individual, the individual differences in genotypes represented by single nucleotide polymorphisms (SNPs) must be rapidly determined. It is essential to provide a medical environment that can be easily identified.

  As described above, in the medical field such as genetic testing and genetic diagnosis, what is most demanded at present is "a technique for quickly and easily detecting a target gene".

  At present, the most prominent gene detection method is a gene detection technique using a substrate of several cm square called a DNA chip or a DNA microarray. The former is a method of synthesizing DNA (DNA: the body of a gene) as a probe on a glass substrate one base at a time by combining photolithography technology and DNA solid-phase synthesis technology. Is a technique of immobilizing different probe DNAs on spots on a substrate using a DNA spotter. When the fluorescent-labeled target DNA is added onto this substrate, it forms a double strand with the immobilized probe DNA. Since only the portion where the double strand is formed emits fluorescence, the expression of many genes is detected as a spot pattern. However, at present, there is a problem that non-specific adsorption of the sample onto the spot is likely to occur. In addition, the reaction between the target DNA (dissolved in a solution) and the probe DNA (immobilized on a solid) becomes a solid-liquid heterogeneous reaction, and a long-term hybridization (duplex formation reaction) is required. These are major principles of DNA chips or DNA microarrays.

  In addition, as a method for detecting gene mutation using capillary electrophoresis, an SSCP (single-chain higher-order structure polymorphism) method and an RFLP (restriction enzyme fragment length polymorphism) method are known. The former is based on the fact that the higher-order structure of the normal DNA and the mutant DNA in the single-stranded state are different, and the latter is based on the fact that the restriction enzyme cleavage sites of the normal DNA and the mutant DNA are different. Is a method of detecting However, in both cases, there is a limit to use as a general SNP detection method unless there is a single nucleotide mutation in a site where a higher-order structure is generated or a site where a restriction enzyme acts.

  The present inventors have previously proposed a gene diagnosis method using a DNA conjugate and using capillary electrophoresis (Non-Patent Document 1).

  In this method, a capillary whose inner wall is coated with polyacrylamide is filled with a DNA conjugate composed of polyacrylamide and DNA (DNA is complementary to a target gene), and the target gene is electrophoresed there. is there. As a result, when the normal type and the mutant type (single nucleotide polymorphism) coexist in the target gene, it is possible to detect both types separately.

However, this method could only detect and separate into two main groups: genes that easily hybridize to the DNA conjugate that filled the capillary and other genes. In addition, the capillary had to be filled with the DNA conjugate, which required a large amount of the DNA conjugate.
Electrophoresis, 23, 2267-2273 (2002)

  Therefore, an object of the present invention is to provide a new simple and accurate detection method which can be applied to analysis systems for various biological components, including genetic diagnosis and genetic testing. In particular, it is an object of the present invention to provide a method capable of separating and detecting all kinds of genes at the same time and simultaneously separating and detecting genes using a relatively small amount of a DNA conjugate.

The present invention for solving the above problems is as follows.
[Claim 1] One or more probe molecules and two or more target molecules are placed under conditions capable of interacting with each other,
A target molecule that has formed a complex by interacting with any of the probe molecules, and a target molecule that has not formed a complex are compared with a target molecule that has formed a complex and a target molecule that has not formed a complex. Isolate using the difference in mobility,
A method for detecting a target molecule, comprising detecting a target molecule that has formed a complex and / or a target molecule that has not formed a complex.
[2] The detection method according to [1], wherein the probe molecule is a conjugate of a polymer and a functional moiety capable of interacting with a target molecule.
[Claim 3] The detection method according to claim 2, wherein the functional portion is a nucleic acid, the target molecule is a nucleic acid, and the interaction is hybridization.
[4] The detection method according to [3], wherein the nucleic acid constituting the functional portion is DNA, RNA, or an artificial nucleic acid.
[5] The detection method according to [4], wherein the target molecule is at least one nucleic acid selected from the group consisting of a disease-related gene and a gene of a pathogenic bacterium.
[6] The detection method according to any one of [3] to [5], wherein the hybridized double-stranded nucleic acid is detected using an intercalator.
[Claim 7] The detection method according to claim 2, wherein the functional portion is an antibody, the target molecule is an antigen, and the interaction is an antigen-antibody reaction.
[8] The detection method according to [2], wherein the functional moiety is a ligand, the target molecule is a metal, and the interaction is a coordination bond.
[Claim 9] The detection method according to claim 2, wherein the functional moiety is a receptor, the target molecule is a hormone, and the interaction is hormone-receptor binding.
[10] The detection method according to any one of [2] to [9], wherein the polymer constituting the conjugate is a polymer of one or more vinyl monomers.
[Claim 11] The detection method according to claim 10, wherein the vinyl monomer is at least one member selected from the group consisting of acrylamide, N-isopropylacrylamide, N, N'-dimethylacrylamide, and N-vinylpyrrolidone.
[12] The detection method according to any one of [2] to [11], wherein at least two types of probe molecules having different molecular weights of polymers constituting the conjugate are used.
[Claim 13] The detection method according to claim 12, wherein the functional portion constituting the conjugate varies depending on the type of the polymer.
[14] The detection according to any one of [1] to [13], wherein by moving at least one of the probe molecule and the target molecule, the probe molecule and the target molecule are placed under a condition capable of interacting with each other. Method.
[15] The detection method according to any one of [1] to [13], wherein the target molecule that has formed the complex and the target molecule that has not formed the complex are separated by electrophoresis.
[16] The method according to [15], wherein the probe molecule is subjected to an electrophoresis method after interacting with the target molecule.
[Claim 17] The interaction between the probe molecule and the target molecule, and the separation between the target molecule that forms a complex with the probe molecule and the target molecule that does not form a complex, are performed by holding the probe molecule. The detection method according to any one of claims 1 to 13, which is performed by causing a target molecule to migrate on an electrophoresis gel.
[18] The detection method according to [17], wherein the holding of the probe molecule on the electrophoresis gel is performed by causing the probe molecule to electrophorese on the electrophoresis gel.
[Claim 19] A fluorescence detector, a visible / ultraviolet spectroscopic analyzer, an organic mass spectrometer, a Fourier transform infrared spectrometer is used for detecting the target molecule forming the complex and / or the target molecule not forming the complex. The method according to any one of claims 1 to 18, which is performed by an apparatus, a time-of-flight mass spectrometer, a nuclear magnetic resonance spectrometer, a gas chromatograph analyzer, a gas chromatograph-mass spectrometer, and / or a liquid chromatograph analyzer. Detection method.

  The present inventors have conducted various studies in order to achieve the above object, and have found a new detection principle called the PRESS (probe-regulated simultaneous separation) method, which is a method for simultaneous control of probe separation, and have completed the present invention.

In particular, in the present invention, rapid and simple simultaneous gene separation and detection was successfully developed by applying capillary electrophoresis, which requires a small amount of sample and requires a short analysis time.
Prior to the present invention, a DNA conjugate, which is a biocomposite material obtained by graft-polymerizing DNA and a monomer, was developed as a DNA separation detection probe used in a capillary tube. This material is easily obtained by mixing and polymerizing DNA and monomers. Then, it was found that DNA conjugates having different DNA graft ratios obtained by changing the mixing ratio of the two migrate at different speeds in the capillary tube. By using this DNA conjugate as a probe having a narrow bandwidth (probe band), a method for simultaneously separating and detecting a specific gene from a gene mixture was successfully developed. The present invention synthesizes a plurality of probe DNA conjugates for detecting a plurality of target genes, specifically captures target genes migrating in a capillary tube to which an electric field is applied by probes having different migration speeds, and each target gene -Based on an unprecedented novel approach where probe complexes are detected at different times.

  Further, the present invention makes it possible to prepare a conjugate for a molecule other than DNA in the same manner as described above, use this as a probe, and detect a molecule that interacts with the molecule as a target.

Gene diagnosis / detection technology with higher generality As a method for detecting gene mutation using capillary electrophoresis, the SSCP method and the RFLP method are mainstream, but there is a limit to use as a general SNP detection method. However, the method shown in the present invention is to realize gene identification in a base sequence-specific manner, and it is possible to separate and detect the entire gene more accurately and easily than any method reported in Japan and overseas. Its high potential application in the fields of gene isolation, gene diagnosis, and gene analysis is expected in the future.

  The problem of the gene detection method using a DNA chip or a DNA microarray mainly arises from the fact that DNA as a probe is immobilized on a solid substrate. On the other hand, the problem of the gene detection method by the capillary electrophoresis is caused by the analysis principle itself. In a gene detection method applying the principle proposed in the present invention, a DNA conjugate performs the same function as individual probe spots of an array-type DNA chip. In addition, by incorporating a different principle of the conventional method of “migrating the probe itself” into the capillary tube, it is not only possible to solve the problems of various gene detection methods using capillary electrophoresis, It also solves the problem of the array type DNA chip that is necessary. In other words, mounting a gene detection method based on the principle of the PRESS method on a chip leads to the development of a “DNA chip” with a concept completely different from the conventional one.

Possibility of speeding up and simplifying gene diagnosis by chipping Conventional reports on DNA molecule recognition are poor in generality and insufficient in information level. However, in the present invention, extremely high-order information recognition is performed in that genes are accurately identified based on DNA complementarity (clear distinction of a base sequence composed of four nucleic acid bases). This completely new information-recognition-type detection system enables the development of a nanodiagnostic device that can accurately identify the base sequence of a gene at the level of one base and output the result in a clear form. It has already been reported recently that capillary electrophoresis is a technique that requires a very short analysis time and can be easily mounted on a chip. Therefore, the results of the present invention are expected to greatly contribute to speeding up and simplifying gene separation, gene diagnosis, and gene analysis.

Prospects as flow-based gene detection technology In connection with gene diagnosis, microfabrication technologies such as microchips and microarrays are rapidly developing. As the problems with DNA chips have come to light, systems that can be handled in a flow (flow path) system are likely to cause a breakthrough in diagnostic technology. The method of the present invention is considered to be a flow-based gene separation / detection technology with extremely high potential compared to conventional methods.

"Generality as an analytical method"
By controlling the order of injection of the target substance and the probe and the direction of the positive and negative electrodes for each separation target, the present principle can be applied to all analytical systems that can detect the target substance by some kind of interaction. . For example, it is conceivable to use not only DNA-DNA but also an interaction between an antigen and an antibody, which can be applied to various medical fields such as genetic diagnosis and immunodiagnosis. In addition, by utilizing the interaction between metal and ligand or between hormone and receptor, it can be applied to the field of environmental analysis.

The method for detecting a target molecule of the present invention comprises:
(1) placing one or more probe molecules and two or more target molecules under conditions capable of interacting with each other;
(2) a target molecule that forms a complex by interacting with any of the probe molecules, and a target molecule that does not form a complex, and a target that does not form a complex with a target molecule that forms a complex. Separation using the difference in mobility with molecules,
(3) detecting a target molecule that has formed the complex and / or a target molecule that has not formed the complex.

The probe molecule used in the method of the present invention is not particularly limited as long as it is a molecule capable of interacting with the target molecule when placed under conditions capable of interacting with the target molecule. Such a probe molecule can be, for example, a conjugate of a polymer with a functional moiety capable of interacting with the target molecule.
Further, if the molecular weight of the molecule conjugated with the functional moiety capable of interacting with the target molecule is different, a probe molecule having a different detection behavior can be obtained, and therefore, the molecular weight of the molecule forming the conjugate does not matter at all. Examples include a conjugate of a probe moiety capable of interacting with a target molecule and a high molecular weight substance such as a protein, and a conjugate of a low molecular weight substance such as an oligopeptide.

FIG. 1 shows a conceptual diagram of the method of the present invention.
For example, the figure shows a case where a target substance (ab) is detected from a mixture (abc). Probe molecules (A and B) that capture each target substance by some interaction are synthesized in advance. Here, A and B are molecularly designed so that “the movements in the flow path are different”. Then, when a probe molecule is injected into the flow channel in advance and a target substance is injected later, a target substance-probe molecule complex (aA / bB) is formed. For example, by dissolving a reagent that binds only to the complex in a solution in the flow channel, only the complex (that is, the target substances a and b captured by the probe) is detected (for example, the DNA is In the case of detection, only the complex is detected by previously dissolving a fluorescent reagent such as ethidium bromide that binds only to double-stranded DNA. When the reagent that binds only to the complex is not dissolved, a, b, and c are all separated and detected (for example, when detecting DNA, only the DNA captured by the probe molecule is detected at different times). Detected, but other uncaptured DNA is also detected). This principle can be fused with any detection method using a flow channel, and examples of the method include capillary electrophoresis, liquid chromatography, and gas chromatography.

When the probe molecule is a conjugate of a polymer and a functional moiety capable of interacting with the target molecule, the functional moiety, the target molecule, and the interaction can be as follows.
(1) The functional part is a nucleic acid, the target molecule is a nucleic acid, and the interaction is hybridization. (Target molecule: nucleic acid)
(2) The functional part is an antibody, the target molecule is an antigen, and the interaction is an antigen-antibody reaction. (Target molecule: antigen)
(3) The functional moiety is a ligand, the target molecule is a metal, and the interaction is a coordination bond. (Target molecule: metal)
(4) The functional moiety is a receptor, the target molecule is a hormone, and the interaction is hormone-receptor binding. (Target molecule: hormone)

Embodiment (1) (target molecule: nucleic acid)
The mode of the above (A) in which the functional portion is a nucleic acid, the target molecule is a nucleic acid, and the interaction is hybridization, is specifically a method for detecting a target nucleic acid. This method
(1) placing a probe molecule (probe nucleic acid) composed of a conjugate of a nucleic acid and a polymer and a sample containing a target nucleic acid under hybridizing conditions;
(2) separating the target nucleic acid hybridized with the probe nucleic acid from the nucleic acid not hybridized; and (3) separating the target nucleic acid hybridized with the probe nucleic acid and / or the target nucleic acid not hybridized with the probe nucleic acid. Detecting.

[Conjugate (probe nucleic acid)]
In the conjugate used in the method of the present invention, the constituent nucleic acid can be, for example, DNA, RNA, or artificial nucleic acid. Here, the artificial nucleic acid means all nucleic acid analogs in which a sugar-phosphate diester skeleton in a nucleic acid molecule is converted into a more stable skeleton such as an ester or an amide, and examples thereof include PNA.

  Further, the polymer constituting the conjugate can be a polymer of one or more vinyl monomers. The vinyl monomer can be, for example, acrylamide, N-isopropylacrylamide, N, N'-dimethylacrylamide, N-vinylpyrrolidone, and the like. However, if it is a vinyl monomer, it is possible to constitute a polymer, and any monomer may be used.

  Various methods are applied as a method for synthesizing a DNA conjugate. For example, a method may be mentioned in which a terminal aminated single-stranded DNA is coupled with methacryloyloxysuccinimide to obtain a terminal vinylated single-stranded DNA, which is then radically copolymerized with an acrylamide monomer. At this time, the catalyst in the coupling reaction or the copolymerization reaction may be any substance, and the reaction conditions are not particularly limited. For example, terminally aminated single-stranded DNA and methacryloyloxysuccinimide are coupled in a buffer, and the product and acrylamide monomer are started with N, N, N'N'-tetramethylethylenediamine in a Tris-HCl buffer. Radical polymerization may be performed as an agent. Of course, a catalyst other than these may be used, or a co-catalyst or an additive may be used. Further, the solvent, buffer, reaction temperature and the like are not limited.

  It is appropriate that the molecular weight of the polymer constituting the conjugate is, for example, in the range of tens of thousands to hundreds of thousands for each of the different conjugates serving as probes from the viewpoint that the migration speed in the capillary tube is different. The molecular weight of the polymer constituting the conjugate is preferably in the range of 30,000 to 300,000.

  The number of nucleic acids contained in one molecule of the conjugate is suitably, for example, in the range of 1 to several tens from the viewpoint that the migration speed in the capillary tube is different. The number of nucleic acids contained in one molecule of the conjugate preferably ranges from 1 to 20.

In the method of the present invention, at least two types of probe molecules having different molecular weights of the polymers constituting the conjugate can be used. In that case, the nucleic acid constituting the conjugate may be different depending on the type of the polymer.
By doing so, the following becomes possible. For example, when there are a plurality of target genes, by synthesizing a conjugate having a different molecular weight and binding a complementary gene to each of the conjugates and using the conjugate as a probe, the migration time of each target gene-probe conjugate can be reduced. It becomes possible to control. This makes it possible to construct a system in which target genes are separated and detected at different times.

  As the target nucleic acid, for example, any nucleic acid such as any disease-related gene such as cancer or diabetes, and any gene of a pathogenic bacterium such as O157 can be considered, and this method is applicable regardless of its chain length and base sequence.

In step (1), a probe molecule and a nucleic acid sample containing a target nucleic acid are placed under hybridization conditions.
Specifically, (a) a probe molecule and a nucleic acid sample containing a target nucleic acid are allowed to coexist in the same solution to allow hybridization, or (b) the target nucleic acid is placed on an electrophoresis gel holding probe molecules in advance. Hybridization can also be performed by causing the nucleic acid sample to migrate. The retention of the probe molecules in the electrophoresis gel can be performed, for example, by collecting the probe molecules in a loop and then introducing them into a capillary tube for analysis using a rotary valve, aspirating, pressurizing, or performing electrophoresis. Can be done by

The hybridization of (a) can be specifically performed under the following conditions.
For example, the hybridization may be promoted by previously mixing the probe molecule and the target nucleic acid, and the mixed solution may be introduced into the capillary tube by a pressurization method. At this time, the inside of the capillary tube is desirably at a low temperature (about 0 to 30 ° C.) or in a state where a suitable double-strand formation promoter (such as magnesium chloride) is added.

The hybridization of (b) can be specifically performed under the following conditions.
For example, a probe molecule may be introduced into a capillary tube by a pressurization method, and a solution containing a target nucleic acid may be introduced later by the same pressurization method. At this time, the inside of the capillary tube is desirably at a low temperature (about 0 to 30 ° C.) or in a state where a suitable double-strand formation promoter (eg, magnesium chloride) is added.

In the step (2), the target nucleic acid hybridized with the probe molecule is separated from the nucleic acid not hybridized. This separation method is not particularly limited, but it is preferable to use electrophoresis.
When the target nucleic acid hybridized with the probe molecule is separated from the non-hybridized nucleic acid by the electrophoresis method, when the hybridization is performed by the method (a), the target nucleic acid contains the probe molecule and the target nucleic acid. After hybridization with the nucleic acid sample, the sample is subjected to electrophoresis.
When the hybridization is performed by the method (b), the hybridization between the probe molecule and the nucleic acid sample containing the target nucleic acid, and the hybridization between the probe molecule and the non-hybridized nucleic acid of the target nucleic acid. Can be performed by causing a nucleic acid sample containing a target nucleic acid to migrate on an electrophoresis gel holding probe molecules.

  Regarding electrophoresis, since it is necessary to migrate a negatively charged target nucleic acid and a probe molecule, a sample is introduced from the cathode side and migrated to the anode side. The electrophoresis is preferably capillary electrophoresis, but may be slab electrophoresis. At the time of electrophoresis, for example, the inside of the capillary tube is desirably filled with a Tris-borate buffer or the like. However, other buffers may be used, and pure water or pure water (or buffer) to which a small amount of an organic solvent is added has no problem. These electrophoresis solutions are desirably in a state to which a suitable double-strand formation promoter (such as magnesium chloride) has been added. The electrophoresis temperature is preferably as low as possible (about 0 to 30 ° C.).

In the step (3), the target nucleic acid hybridized with the probe molecule is detected.
The detection of the target nucleic acid hybridized with the probe molecule can be performed, for example, by performing hybridization and / or separation in the presence of an intercalator and detecting a double-stranded nucleic acid to which the intercalator is attached.
The intercalator used in the method of the present invention is not particularly limited, and examples thereof include ethidium bromide and various nucleic acid detection reagents (SYBR Green I, SYBR Green II, SYBR Gold) manufactured by Molecular Probe.

The double-stranded nucleic acid to which the intercalator has been attached can be detected, for example, as follows.
For example, if a fluorescent intercalator such as ethidium bromide is dissolved in a buffer in advance, only the complex formed by the probe molecule and the target nucleic acid is fluorescently labeled, and only the target nucleic acid is separated by using a fluorescence detector. Is detected.

One embodiment of the method of the present invention will be described more specifically with reference to FIG.
In (A), probe DNA conjugates having different migration speeds are held in a capillary tube in advance. Then, the target DNA mixture is injected from the cathode side. At this time, the fluorescent intercalator that binds to the double-stranded DNA is dissolved in the electrophoresis solution.
In (B), unlike the DNA conjugate, the target DNA has a high electrophoresis speed because the polymer is not bonded thereto, so that the target DNA catches up with the DNA conjugate, and the DNA conjugate and the target DNA can be hybridized. .
In (C), the target DNA migrates first, and then the target DNA hybridized (sequence-specific binding) with the DNA conjugate is detected by the action of the fluorescent intercalator.

  The detection of a target molecule that has formed a complex and / or a target molecule that has not formed a complex can be performed using a fluorescence detector, as described above, when only the complex is fluorescently labeled. In contrast, when a fluorescent label is not used, instead of a fluorescence detector, for example, a visible / ultraviolet spectrometer, an organic mass spectrometer, a Fourier transform infrared spectrometer, a time-of-flight mass spectrometer, a nuclear magnetic resonance Using an analyzer, a gas chromatograph analyzer, a gas chromatograph-mass spectrometer, and / or a liquid chromatograph analyzer, etc., detection of a target molecule forming a complex and / or a target molecule not forming a complex is performed. It can be carried out. The detection of a target molecule that has formed a complex and / or a target molecule that has not formed a complex can be performed using any device that can detect the difference between the two.

Embodiment (2) (target molecule: antigen)
The functional portion is an antibody, the target molecule is an antigen, the interaction is an antigen-antibody reaction (2), specifically, a target antigen detection method,
(1) a step of placing a probe molecule comprising a conjugate of an antibody and a polymer and a sample containing a target antigen under conditions that allow an antigen-antibody reaction;
(2) separating the target antigen that has reacted with the probe molecule from the target antigen that has not reacted, and (3) detecting the target antigen that has reacted with the probe molecule and / or the target antigen that has not reacted with the probe molecule. including.

In the present invention, any substance can be used as the antigen, and examples thereof include high molecular weight substances such as proteins and polysaccharides having a repeating structure.
Further, the antibody may be any antibody against the antigen of the other party, and the antibody may be a monoclonal antibody or a polyclonal antibody.
Examples of antigens and antibodies include, for example, collagen and anti-collagen monoclonal antibody, HCV core protein and anti-HCV core protein monoclonal antibody, HBV (hepatitis B virus) and HBV antibody, HIV (human immunodeficiency virus) and HIV antibody , HCV (hepatitis C virus) and HCV antibodies, but are not limited thereto.

  A conjugate having an antibody as a functional moiety can be prepared, for example, by a method of converting an amino group having an antibody into a vinyl group, and then subjecting this to radical copolymerization with an acrylamide monomer. At this time, the catalyst for the reaction for converting the amino group to a vinyl group or the copolymerization reaction may be any substance, and the reaction conditions are not particularly limited. For example, a product obtained by converting an amino group of an antibody into a vinyl group and an acrylamide monomer in a Tris-HCl buffer solution may be subjected to radical polymerization using N, N, N'N'-tetramethylethylenediamine as an initiator. good. Of course, a catalyst other than these may be used, or a co-catalyst or an additive may be used. Further, the solvent, buffer, reaction temperature and the like are not limited.

  It is appropriate that the molecular weight of the polymer constituting the conjugate is, for example, in the range of tens of thousands to hundreds of thousands for each of the different conjugates serving as probes from the viewpoint that the migration speed in the capillary tube is different. The molecular weight of the polymer constituting the conjugate is preferably in the range of 30,000 to 300,000.

  The number of antibodies contained in one molecule of the conjugate is suitably, for example, in the range of 1 to 50, from the viewpoint that the migration speed in the capillary tube is different. The number of antibodies contained in one molecule of the conjugate is preferably in the range of 1-20.

  In the method of the present invention, at least two types of probe molecules having different molecular weights of the polymers constituting the conjugate can be used. In that case, the antibodies that make up the conjugate can be different depending on the type of polymer.

  By doing so, the following becomes possible. For example, when there are a plurality of target antigens, it is possible to control the electrophoresis time of each target antigen-probe conjugate by synthesizing a conjugate having a different molecular weight to which an antibody for each is bound and using it as a probe. This makes it possible to construct a system in which the target antigen is separated and detected at different times.

  In step (1), the probe antibody and a sample containing the target antigen are placed under conditions that allow an antigen-antibody reaction. Specifically, (a) an antigen-antibody reaction is carried out by coexisting a probe antibody and a sample containing a target antigen in the same solution, or (b) the target antigen is placed on an electrophoresis gel holding the probe antibody in advance. An antigen-antibody reaction can also be performed by electrophoresing the containing sample. The retention of the probe antibody on the electrophoresis gel can be performed, for example, by collecting the probe antibody into a loop and then introducing the probe antibody into a capillary tube for analysis using a rotary valve, aspirating, pressurizing, or performing electrophoresis. Can be done by

The antigen-antibody reaction of (a) can be specifically performed under the following conditions.
For example, the antigen-antibody reaction may be promoted by previously mixing the probe antibody and the target antigen, and the mixed solution may be introduced into the capillary tube by a pressurization method. At this time, it is desirable that the temperature inside the capillary tube is low (about 0 to 30 ° C.).

The antigen-antibody reaction of (b) can be specifically performed under the following conditions.
For example, the probe antibody may be introduced into the capillary tube by a pressurization method, and a solution containing the target antigen may be introduced later by the pressurization method. At this time, it is desirable that the temperature inside the capillary tube is low (about 0 to 30 ° C.).

In the step (2), the target antigen that has reacted with the probe molecule is separated from the target antigen that has not reacted. This separation method is not particularly limited, but it is preferable to use electrophoresis.
When the target antigen that has reacted with the probe molecule is separated from the target antigen that has not reacted by electrophoresis, when the reaction is performed by the method (a), the probe molecule and the sample containing the target antigen are separated. After the reaction, the sample is subjected to an electrophoresis method.
When the reaction is carried out by the method (b), the reaction between the probe molecule and the sample containing the target antigen, and the separation of the target antigen that has reacted with the probe molecule from the unreacted target antigen are performed by the probe. This can be performed by causing a sample containing a target antigen to migrate on an electrophoresis gel holding molecules.

  Regarding electrophoresis, since it is necessary to migrate a negatively charged target and probe molecules, a sample is introduced from the cathode side and the target antigen is migrated to the anode side. The electrophoresis is preferably capillary electrophoresis, but may be slab electrophoresis. At the time of electrophoresis, for example, the inside of the capillary tube is desirably filled with a Tris-borate buffer or the like. However, other buffers may be used, and pure water or pure water (or buffer) to which a small amount of an organic solvent is added has no problem. The electrophoresis temperature is preferably as low as possible (about 0 to 30 ° C.).

In step (3), target antigens that have reacted with the probe molecules and / or target antigens that have not reacted with the probe molecules are detected.
The detection of the target antigen that has reacted with the probe molecule can be performed, for example, by using a secondary antibody that binds to the antibody as a fluorescent label and detecting an antigen-antibody complex to which the fluorescent label is attached.

  The fluorescent label used in the method of the present invention is not particularly limited, and examples thereof include 5′-carboxyfluorescein (FAM), 5′-hexachlorofluorescein (HEX), and isothiocyanate fluorescein (FITC).

  In the case where the detection of the complex-formed target molecule and / or the non-complex-formed target molecule is performed using a fluorescent label, the detection can be performed using a fluorescence detector as described above. On the other hand, when a fluorescent label is not used, a visible / ultraviolet spectrometer, an organic mass spectrometer, a Fourier transform infrared spectrometer, a time-of-flight mass spectrometer, a nuclear magnetic resonance spectrometer instead of the fluorescence detector Using a gas chromatograph analyzer, a gas chromatograph-mass spectrometer, and / or a liquid chromatograph analyzer to detect a target molecule forming a complex and / or a target molecule not forming a complex. Can be. The detection of a target molecule that has formed a complex and / or a target molecule that has not formed a complex can be performed using any device that can detect the difference between the two.

Embodiment (3) (target molecule: metal)
The functional moiety is a ligand, the target molecule is a metal, the interaction is a coordination bond (3), specifically, a target metal detection method,
(1) a step of placing a probe molecule comprising a conjugate of a ligand and a polymer and a sample containing a target metal under conditions capable of coordinating and binding;
(2) separating the target metal bound to the probe molecule from the unbound target metal; and (3) detecting the target metal bound to the probe molecule and / or the target metal not bound to the probe molecule. including.

In the present invention, as the metal, any metal that coordinates can be used, and examples thereof include Cu, Cr, Mo, Rh, Ru, and Zn.
In addition, the ligand may be any substance that can coordinate with the metal of the other party, and examples thereof include (methylsulfinyl) methanide and skeletal molecules of an organometallic compound such as dihydronaphthalenidyl. .

  Examples of coordinated metal-ligands include potassium and (methylsulfinyl) methanide, sodium and dihydronaphthalenidyl, sodium and naphthalene radical ions, metal halides and 2,2'-bipyridine-based ligands, ruthenium And a trinuclear ruthenium cluster, and various metals and a pyridinium β-diketone ligand, but are not limited thereto.

  A conjugate having a ligand as a functional moiety is, for example, a vinyl group-introducing reagent such as methacryloyloxysuccinimide acting on an amino group to convert the amino group of the ligand into a vinyl group, and then converting this to acrylamide. It can be prepared by a method of radical copolymerization with a monomer. At this time, the catalyst for the reaction for converting the amino group to a vinyl group or the copolymerization reaction may be any substance, and the reaction conditions are not particularly limited. For example, the product obtained by converting the amino group of the ligand into a vinyl group and an acrylamide monomer in a Tris-HCl buffer solution are subjected to radical polymerization using N, N, N'N'-tetramethylethylenediamine as an initiator. May be. Of course, a catalyst other than these may be used, or a co-catalyst or an additive may be used. Further, the solvent, buffer, reaction temperature and the like are not limited.

  It is appropriate that the molecular weight of the polymer constituting the conjugate is, for example, in the range of tens of thousands to hundreds of thousands for each of the different conjugates serving as probes from the viewpoint that the migration speed in the capillary tube is different. The molecular weight of the polymer constituting the conjugate is preferably in the range of 30,000 to 300,000.

  The number of ligands contained in one molecule of the conjugate is, for example, in the range of 1 to 50, from the viewpoint that the migration speed in the capillary tube differs. The number of ligands contained in one molecule of the conjugate is preferably in the range of 1-20.

  In the method of the present invention, at least two types of probe molecules having different molecular weights of the polymers constituting the conjugate can be used. In that case, the ligand constituting the conjugate may be different depending on the type of the polymer.

  By doing so, the following becomes possible. For example, when there are a plurality of target metals, controlling the migration time of each target metal-probe conjugate by synthesizing conjugates having different molecular weights binding ligands to each of the conjugates and using them as probes. This makes it possible to construct a system in which the target metal is separated and detected at different times.

In step (1), the probe ligand and the sample containing the target metal are placed under conditions that allow coordination bonding.
Specifically, (a) a probe ligand and a sample containing a target metal are co-located and coordinated in the same solution, or (b) an electrophoresis gel holding a probe ligand in advance. By coordinating a sample containing a target metal with the target metal, coordination bonding can also be performed. The retention of the probe ligand in the electrophoresis gel can be performed, for example, by collecting the probe ligand in a loop and then introducing the probe ligand into an analytical capillary tube using a rotary valve, aspirating, pressing, electrophoresis, and the like. This can be done in various ways.

The coordination bond of (a) can be specifically performed under the following conditions.
For example, coordination bonds may be promoted by previously mixing the probe ligand and the target metal, and the mixed solution may be introduced into the capillary tube by a pressurization method. At this time, it is desirable that the temperature inside the capillary tube is low (about 0 to 30 ° C.).

The coordination bond of (b) can be specifically performed under the following conditions.
For example, the probe ligand may be introduced into the capillary tube by a pressurization method, and the solution containing the target metal may be introduced by the same pressurization method later. At this time, it is desirable that the temperature inside the capillary tube is low (about 0 to 30 ° C.).

In step (2), the target metal bound to the probe molecule is separated from the unbound target metal. This separation method is not particularly limited, but it is preferable to use electrophoresis.
When the target metal bound to the probe molecule is separated from the unbound target metal by electrophoresis, when the binding is performed by the method (a), the probe molecule and the sample containing the target metal are separated. After binding, the sample is subjected to electrophoresis.
When the binding is performed by the method (b), the binding between the probe molecule and the sample containing the target metal and the separation of the target metal bound to the probe molecule from the unbound target metal are performed by the probe. This can be performed by causing a sample containing a target metal to migrate on an electrophoresis gel holding molecules.

  With regard to electrophoresis, it is necessary to cause ionized target and probe molecules to migrate. Therefore, a metal is introduced from the side of the electrode which shows the opposite polarity to the charge of the target molecule / probe molecule complex to be detected, and migrates to the opposite side of the electrode. The electrophoresis is preferably capillary electrophoresis, but may be slab electrophoresis. At the time of electrophoresis, for example, the inside of the capillary tube is desirably filled with a Tris-borate buffer or the like. However, other buffers may be used, and pure water or pure water (or buffer) to which a small amount of an organic solvent is added has no problem. The electrophoresis temperature is preferably as low as possible (about 0 to 30 ° C.).

In the step (3), the target metal bound to the probe molecule and / or the target metal not bound to the probe molecule are detected.
The detection of the target metal bound to the probe molecule can be performed, for example, by previously binding a fluorescent molecule to the probe molecule and detecting the probe molecule / target metal complex.
The fluorescent molecule used in the method of the present invention that binds to the probe molecule in advance is not particularly limited, and examples thereof include FAM, HEX, and FITC.

  When the detection of the complex-forming target molecule and / or the non-complex-forming target molecule is performed using a fluorescent label, the detection can be performed using a fluorescence detector as described above. On the other hand, when a fluorescent label is not used, a visible / ultraviolet spectrometer, an organic mass spectrometer, a Fourier transform infrared spectrometer, a time-of-flight mass spectrometer, a nuclear magnetic resonance spectrometer instead of the fluorescence detector Using a gas chromatograph analyzer, a gas chromatograph-mass spectrometer, and / or a liquid chromatograph analyzer to detect a target molecule forming a complex and / or a target molecule not forming a complex. Can be. The detection of a target molecule that has formed a complex and / or a target molecule that has not formed a complex can be performed using any device that can detect the difference between the two.

Embodiment (4) (target molecule: hormone)
The embodiment in which the functional moiety is a receptor, the target molecule is a hormone, and the interaction is hormone-receptor binding (4), specifically, is a method for detecting a target hormone,
(1) placing a probe molecule comprising a conjugate of a receptor and a polymer and a sample containing a target hormone under conditions capable of binding a receptor-hormone;
(2) separating the target hormone bound to the probe molecule from the unbound target hormone; and (3) detecting the target hormone bound to the probe molecule and / or the target hormone not bound to the probe molecule. including.

In the present invention, the receptor may be any substance that can bind to a hormone. For example, a protein that exists in a cell and recognizes a physical / chemical stimulus to induce a response in the cell ( Nuclear receptors and cell membrane (penetrating) receptors).
In addition, the hormone may be one that binds to a receptor serving as a partner.
Examples of hormones and receptors include, for example, estradiol and environmental hormone molecules such as estradiol receptor and its receptors testosterone and dihydrotestosterone (DHT) and androgen receptor, taste substances and its receptors, and the like. Not limited.

  A conjugate having a hormone as a functional moiety can be prepared, for example, by a method of converting an amino group of a hormone into a vinyl group, and then subjecting the amino group to radical copolymerization with an acrylamide monomer. At this time, the catalyst for the reaction for converting the amino group to a vinyl group or the copolymerization reaction may be any substance, and the reaction conditions are not particularly limited. For example, a product obtained by converting the amino group of the hormone into a vinyl group and an acrylamide monomer in a Tris-HCl buffer solution may be subjected to radical polymerization using N, N, N'N'-tetramethylethylenediamine as an initiator. good. Of course, a catalyst other than these may be used, or a co-catalyst or an additive may be used. Further, the solvent, buffer, reaction temperature and the like are not limited.

  It is appropriate that the molecular weight of the polymer constituting the conjugate is, for example, in the range of tens of thousands to hundreds of thousands for each of the different conjugates serving as probes from the viewpoint that the migration speed in the capillary tube is different. The molecular weight of the polymer constituting the conjugate is preferably in the range of 30,000 to 300,000.

  The number of receptors contained in one molecule of the conjugate is suitably, for example, in the range of 1 to 50, from the viewpoint that the migration speed in the capillary tube differs. The number of receptors contained in one molecule of the conjugate preferably ranges from 1 to 20.

  In the method of the present invention, at least two types of probe molecules having different molecular weights of the polymers constituting the conjugate can be used. In that case, the receptor constituting the conjugate may be different depending on the type of the polymer.

  By doing so, the following becomes possible. For example, when there are a plurality of target hormones, it is possible to control the migration time of each target hormone-probe conjugate by synthesizing conjugates having different molecular weights, each binding a receptor to each, and using them as probes. This makes it possible to construct a system in which the target hormone is separated and detected at different times.

In step (1), the probe receptor and the sample containing the target hormone are placed under conditions that allow receptor-hormone binding.
Specifically, (a) a probe receptor and a sample containing a target hormone are allowed to coexist in the same solution to form a receptor-hormone bond, or (b) a target hormone is added to an electrophoresis gel in which a probe receptor is previously held. Receptor-hormone binding can also be achieved by running a sample containing The retention of the probe receptor on the electrophoresis gel can be performed, for example, by collecting the probe receptor into a loop and then introducing the probe receptor into a capillary tube for analysis using a rotary valve, aspirating, pressurizing, or performing electrophoresis. It can be carried out.

Specifically, the receptor-hormone binding of (a) can be performed under the following conditions.
For example, receptor-hormone binding may be promoted by previously mixing the probe receptor and the target hormone, and the mixed solution may be introduced into a capillary tube by a pressurization method. At this time, it is desirable that the temperature inside the capillary tube is low (about 0 to 30 ° C.).

Specifically, the receptor-hormone binding of (b) can be performed under the following conditions.
For example, the probe receptor may be introduced into the capillary tube by a pressurization method, and a solution containing the target hormone may be introduced later by the same pressurization method. At this time, it is desirable that the temperature inside the capillary tube is low (about 0 to 30 ° C.).

In the step (2), the target hormone bound to the probe molecule is separated from non-hybridized unbound target hormone. This separation method is not particularly limited, but it is preferable to use electrophoresis.
When the target hormone bound to the probe molecule is separated from the unbound target hormone by electrophoresis, when the binding is performed by the method (a), the probe molecule and the sample containing the target hormone are separated. After binding, the sample is subjected to electrophoresis.
When the binding is performed by the method (b), the binding between the probe molecule and the sample containing the target hormone and the separation of the target hormone bound to the probe molecule from the unbound target hormone are determined by the probe. It can be performed by causing a sample containing a target hormone to migrate on an electrophoresis gel holding molecules.

  Regarding electrophoresis, since it is necessary to migrate a negatively charged target and a probe molecule, a sample is introduced from the cathode side and the target hormone is migrated to the anode side. The electrophoresis is preferably capillary electrophoresis, but may be slab electrophoresis. At the time of electrophoresis, for example, the inside of the capillary tube is desirably filled with a Tris-borate buffer or the like. However, other buffers may be used, and pure water or pure water (or buffer) to which a small amount of an organic solvent is added has no problem. The electrophoresis temperature is preferably as low as possible (about 0 to 30 ° C.).

In the step (3), a target hormone bound to the probe molecule and / or a target hormone not bound to the probe molecule are detected.
The detection of the target hormone bound to the probe molecule, for example, the binding and / or separation of the target hormone bound to the probe molecule is detected, for example, by preliminarily binding a fluorescent molecule to the probe molecule. This can be done by detecting the body. The fluorescent molecule used in the method of the present invention that binds to the probe molecule in advance is not particularly limited, and examples thereof include FAM, HEX, and FITC.

  In the case where the detection of the complex-formed target molecule and / or the non-complex-formed target molecule is performed using a fluorescent label, the detection can be performed using a fluorescence detector as described above. On the other hand, when a fluorescent label is not used, a visible / ultraviolet spectrometer, an organic mass spectrometer, a Fourier transform infrared spectrometer, a time-of-flight mass spectrometer, a nuclear magnetic resonance spectrometer instead of the fluorescence detector Using a gas chromatograph analyzer, a gas chromatograph-mass spectrometer, and / or a liquid chromatograph analyzer to detect a target molecule forming a complex and / or a target molecule not forming a complex. Can be. The detection of a target molecule that has formed a complex and / or a target molecule that has not formed a complex can be performed using any device that can detect the difference between the two.

Hereinafter, the present invention will be further described with reference to examples.
4-1) Using Reagent oligonucleotides (5'-GCTGGTGGC-3 ', 5'-ACACACACA-3', H 2 N-5'-GCCACCAGC-3 ', H 2 N-5'-TGTGTGTGT-3') is It was purchased from Sigma Genosis Japan Co., Ltd. (Hokkaido) as an HPLC purified grade product. The purchased oligonucleotide was first dissolved in sterile water, the absorbance at 260 nm was measured, and the concentration was calculated from the extinction coefficient calculated based on the nearest base pairing method. All other reagents used commercially available special grade reagents.

4-2) Synthesis of methacrylloyloxysuccinimide
A mixed solution (solution A) obtained by adding 1,3-dicyclohexylcarbodiimide (14.5 g) to dichloromethane (40 mL), methacrylic acid (4.03 g), N-hydroxysuccinimide (5.22 g), and 4-dimethylaminopyridine (2.80 g) ) And pyridine (10 mL) were dissolved in dichloromethane (40 mL) to prepare a mixed solution (solution B). The solution A was added dropwise to the solution B in an ice bath, and the obtained cloudy solution was filtered. Thereafter, dehydration of the filtrate, addition of ethyl acetate, filtration (collection of the filtrate), and evaporation were repeated three times to obtain methacryloyloxysuccinimide.

4-3) Synthesis of vinylated DNA Dimethyl sulfoxide (150 μL) in which methacryloyloxysuccinimide (32.5 μmol) obtained above was dissolved and disodium carbonate / sodium carbonate buffer in which aminated oligonucleotide (0.65 μmol) was dissolved The solution (pH 9.5, 650 μL) was mixed at 25 ° C. for 18 hours. The obtained mixed solution is separated by HPLC (reverse phase column, 0.1 M triethylamine-glacial acetic acid: 100 → 73%, acetonitrile: 0 → 27%, 30 minutes), and lyophilized to obtain a vinylated oligonucleotide. Was.

4-4) Synthesis of DNA conjugate A stock solution of a vinylated oligonucleotide and acrylamide (AAm) was mixed to a desired final concentration, and nitrogen was added for 10 minutes. The final molar fraction of the oligonucleotide is 5.52 × 10 ⁻² mol% (CGACCACCG-AAm) and 5.52 × 10 ⁻¹ mol% (TGTGTGTGT-AAm). Thereafter, ammonium persulfate (APS, final concentration: 3 mM) and N, N, N ', N'-tetramethylethylenediamine (TEMED, final concentration: 6 mM) were added to initiate a polymerization reaction. The reaction temperature was 25 ° C, the reaction time was 18 ° C, and the stirring speed was 300 rpm. After the completion of the reaction, the polymerization solution was subjected to gel filtration, and the fractionated fraction containing the DNA conjugate was dialyzed and lyophilized to obtain a DNA conjugate.

4-5) Examination of simultaneous separation and detection of multiple genes by capillary electrophoresis First, the migration behavior of oligonucleotides and DNA conjugates was examined. After injecting a solution in which each component alone was dissolved for 5 seconds, a voltage (-30 kV) was applied, and the migration time of each component was measured.
Next, simultaneous separation and detection of multiple genes by capillary electrophoresis was examined. A solution containing a DNA conjugate (one single solution or two mixed solutions) was injected into the capillary tube for 5 seconds. Then, after injecting the oligonucleotide mixed solution for 5 seconds, a voltage (-30 kV) was applied, and the migration time of the oligonucleotide-DNA conjugate complex was measured.
All samples were injected from the cathode side by a pressurization method (5 psi). For detection, a UV (in the case of studying the electrophoretic behavior of each component alone; 260 nm) and a fluorescence (in the case of studying simultaneous separation detection of multiple genes; an excitation wavelength of 488 nm and a fluorescence wavelength of 600 nm) were used. The electrophoresis solution was a 100 mM Tris-borate buffer (pH 7.4), and 5 mg / L ethidium bromide (fluorescent intercalator) was added only when fluorescence detection was performed. The capillary electrophoresis apparatus used was PACE MDQ manufactured by Beckman, and the capillary tube used was a commercially available tube (Agilent, CEP coated capillary) having an inner wall treatment (inner diameter: 75 μm, sample introduction to detector: 50cm). The oligonucleotide concentration and the DNA conjugate concentration are both 20 μM (each mixed at 10 μM).

4-6) Results of DNA conjugate synthesis In the present invention, two types of probe DNA conjugates (CGACCACCG-AAm and TGTGTGTGT-AAm) were synthesized. The gel filtration column was able to separate the DNA conjugate, unreacted vinylated oligonucleotide, and polymerization initiator from the polymerization solution. The fraction containing the DNA conjugate was collected and freeze-dried to obtain a DNA conjugate powder. The powder was dissolved in sterilized water and subjected to UV measurement to measure the oligonucleotide content contained in the DNA conjugate. The result was 5.14 × 10 ⁻² mol% (CGACCACCG-AAm) and 1.45 × 10 ⁻¹ mol% ( TGTGTGTGT-AAm).

4-7) Results of migration of various components in capillary electrophoresis Next, the migration behavior of each component (oligonucleotide and DNA conjugate) by capillary electrophoresis was examined. As a result, it was found that the migration times of the two kinds of oligonucleotides and the mixture thereof were almost the same (3.5 minutes), but the migration times of the DNA conjugates having different oligonucleotide graft amounts were different (CGACCACCG-AAm). : 29.5 minutes, TGTGTGTGT-AAm: 28.5 minutes). This is considered to be because the charge / mass ratio of the TGTGTGTGT-AAm conjugate, which has about three times more oligonucleotide grafts, was higher and migrated to the cathode side earlier than the CGACCACCG-AAm conjugate.

4-8) Results of simultaneous separation and detection of multiple genes by capillary electrophoresis Based on the above results, simultaneous separation and detection of multiple genes by capillary electrophoresis using a DNA conjugate as a probe band was examined. As a result, when one type of probe DNA conjugate was used to separate and detect a mixed solution of two types of oligonucleotides, each oligonucleotide was specifically separated and detected depending on the type of DNA conjugate (Fig. 3 (a, b)). This indicates that the double-stranded DNA-binding intercalator specifically binds to the complex formed by the oligonucleotide and the DNA conjugate, and the fluorescence is detected. Thus, when two types of probe DNA conjugates were used to separate and detect a mixed solution of two types of oligonucleotides, each oligonucleotide was successfully separated and detected specifically (FIG. 3 (c)). .

FIG. 2 is a schematic explanatory view of a method (general) of the present invention. FIG. 1 is a schematic explanatory diagram of the method (nucleic acid detection method) of the present invention. 7 shows the results of simultaneous separation detection in Examples.

Claims (19)

Placing one or more probe molecules and two or more target molecules under conditions capable of interacting,
A target molecule that has formed a complex by interacting with any of the probe molecules, and a target molecule that has not formed a complex are compared with a target molecule that has formed a complex and a target molecule that has not formed a complex. Separate using the difference in mobility,
A method for detecting a target molecule, comprising detecting a target molecule that has formed a complex and / or a target molecule that has not formed a complex. The detection method according to claim 1, wherein the probe molecule is a conjugate of a polymer and a functional moiety capable of interacting with a target molecule. The method according to claim 2, wherein the functional part is a nucleic acid, the target molecule is a nucleic acid, and the interaction is hybridization. The detection method according to claim 3, wherein the nucleic acid constituting the functional portion is DNA, RNA, or an artificial nucleic acid. The detection method according to claim 4, wherein the target molecule is at least one nucleic acid selected from the group consisting of a disease-related gene and a gene of a pathogenic bacterium. The detection method according to any one of claims 3 to 5, wherein the hybridized double-stranded nucleic acid is detected using an intercalator. The detection method according to claim 2, wherein the functional portion is an antibody, the target molecule is an antigen, and the interaction is an antigen-antibody reaction. The method according to claim 2, wherein the functional moiety is a ligand, the target molecule is a metal, and the interaction is a coordination bond. The method according to claim 2, wherein the functional moiety is a receptor, the target molecule is a hormone, and the interaction is hormone-receptor binding. The detection method according to any one of claims 2 to 9, wherein the polymer constituting the conjugate is a polymer of one or more vinyl monomers. The detection method according to claim 10, wherein the vinyl monomer is at least one selected from the group consisting of acrylamide, N-isopropylacrylamide, N, N'-dimethylacrylamide, and N-vinylpyrrolidone. The detection method according to any one of claims 2 to 11, wherein at least two types of probe molecules having different molecular weights of polymers constituting the conjugate are used. 13. The detection method according to claim 12, wherein the functional portion constituting the conjugate differs depending on the type of the polymer. The detection method according to any one of claims 1 to 13, wherein at least one of the probe molecule and the target molecule is moved so that the probe molecule and the target molecule are placed under a condition capable of interacting with each other. The detection method according to any one of claims 1 to 13, wherein the separation of the target molecule that has formed the complex and the target molecule that has not formed the complex is performed by an electrophoresis method. 16. The detection method according to claim 15, wherein the probe molecule is subjected to an electrophoresis method after interacting with the target molecule. The interaction between the probe molecule and the target molecule, and the separation of the target molecule that formed a complex with the probe molecule and the target molecule that did not form a complex, were performed on an electrophoresis gel holding the probe molecule. The detection method according to any one of claims 1 to 13, which is performed by causing a molecule to migrate. The detection method according to claim 17, wherein the holding of the probe molecule on the electrophoresis gel is performed by causing the probe molecule to electrophorese on the electrophoresis gel. Detection of the target molecule forming the complex and / or the target molecule not forming the complex is performed by a fluorescence detector, a visible / ultraviolet spectrometer, an organic mass spectrometer, a Fourier transform infrared spectrometer, a time-of-flight type The detection method according to any one of claims 1 to 18, wherein the detection is performed by a mass spectrometer, a nuclear magnetic resonance spectrometer, a gas chromatograph analyzer, a gas chromatograph-mass spectrometer, and / or a liquid chromatograph analyzer.