JP2007046975A - Labelled probe bonded matter, its manufacturing method and its utilizing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a labeling method of matter effective, when a large number of kinds of labels are applied, as required, even to minute matter, such as a fine granular substrate or the like used for immobilizing a probe molecule used in bioassay, and labeled matter. <P>SOLUTION: A large number of kinds of labels can be applied even to the minute matter, as necessary, by using a labeling material prepared according to the compositional condition of atoms contained, at least corresponding to the numerical value data of the (n) figure of the binary system using the number (n) of kinds of a plurality of kinds of pre-selected atoms and the presence or the level of the contents of the individual selected atoms, as a part of the material which constitutes the minute matter. Furthermore, bioassay becomes possible by the matter labeled by the labeling method. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a plurality of objects obtained by individually combining a plurality of types of probes, and a probe coupling provided with a label for identification in order to determine the type of each object, that is, the type of each of these probes. The present invention relates to an object and a manufacturing method thereof. The present invention further relates to a method for discriminating a probe of the object, a method for detecting, identifying and quantifying a probe bound to the object, and a method for detecting, identifying and quantifying a target substance bound to the probe.

  When handling multiple types of objects, it is often necessary to accurately determine the type of each object. In particular, when multiple types of objects to be handled themselves cannot be easily distinguished from each other, identification signs corresponding to the types of the objects are attached and used for discrimination of the types of the objects. is doing.

  There are various methods for identifying the identification, but in essence, a distinguishable encoded identifier (code) that is more than the number of types to be distinguished is assigned to each type. When the object size to be marked is large, a character string type identifier is often used as a discriminable encoded identifier (code) to be used. For example, the bar code corresponds to a character string type identifier and converted into bar line width and interval information according to a predetermined rule. In order to make it easier to assign identifiers that can be represented by a series of serial numbers to character string type identifiers, for example, country codes (country codes), area codes (area codes) ), And a combination of a plurality of hierarchical marker parts such as individual numbers in the area (intra-area station number + individual line number) are combined and integrated into a single character string type identifier in many cases.

  On the other hand, when the object size to be marked is small, it is technically difficult to attach a visually distinguishable character string type identifier as described above. For example, an IC tag employs a method of identifying information electronically described on an IC chip as a character string type identifier through an electromagnetic wave digitized with an external detector. . Note that the size of the IC tag itself is on the order of 100 μm, and it is technically more difficult to attach a character string type identifier to an object size of 10 or less μm or less, in some cases, several μm or less. It becomes.

  For example, an example in which the object size is a few tens of μm or less, and in some cases, a few μm or less, is one in which probe molecules are immobilized on the surface of microscopic particles used for detection of biologically relevant substances.

  For example, for the classification of various microorganisms, a method for investigating the close relationship between taxonomics by evaluating the cross-reactivity of antibody molecules specific for related microorganisms to the wells on the surface of the target microorganism has long been used. It's being used. At that time, antibody molecules used as probes are immobilized on the surface of the gold fine particles, and the accumulation of the gold fine particles around the microorganism is observed to determine the presence or absence of a cross-reaction. In addition, as a method for identifying the epitope sequences identified by various antibody molecules, for example, a peptide that reacts with an antigen molecule and an antibody molecule immobilized on the surface of micro ferrite particles from a random peptide library displayed by E. coli A technique for separating E. coli strains expressing the protein by applying a panning method is also widely known. In this method, when recovering magnetically micro ferrite particles, only E. coli strains expressing peptides having amino acid sequences that can react with antibody molecules are separated in a form linked to ferrite particles.

  The main advantage of microparticles that immobilize probe molecules that are used to detect these biomaterials is that they can be used by probe molecules by using a means to separate solid microparticles from the liquid phase. Separation of the bound substance to be detected and other contaminants present in the liquid phase is easy. That is, after a substance to be detected existing in a liquid phase is bound to a probe molecule, a conjugate of the substance to be detected and the probe molecule and an unreacted probe molecule once fix the probe molecule. It is separated and recovered from the liquid phase by using microscopic particles. After that, whether or not the conjugate of the detection target substance and the probe molecule actually exists in the collected mixture of the detection target substance and the probe molecule and the unreacted probe molecule is further quantified. Is advanced.

  The advantage that the separation from the liquid phase is simplified by immobilizing the probe molecule on the solid phase surface can also be achieved by immobilizing the probe molecule on the surface of the solid phase substrate having a macro size. For example, when detecting the presence or absence of a specific IgG antibody present in a specimen sample, an antigen peptide having an epitope sequence for the Ig antibody to be detected is immobilized on the surface of the solid phase substrate at a predetermined density, The specimen sample is brought into contact, and the IgG antibody to be detected is fixed to the surface of the solid phase substrate through the reaction with the antigenic peptide. Then, after removing the specimen sample from the surface of the solid phase substrate, the IgG antibody molecule fixed on the surface of the solid phase substrate is quantified with the anti-IgG antibody with the detection label attached to its Fc region (constant region). To react. Unreacted “labeled anti-IgG antibody” is washed and removed from the surface of the solid-phase substrate, and then reacted with the IgG antibody molecules fixed on the surface of the solid-phase substrate quantitatively. IgG antibodies are detected and quantified using the detection label.

In the method of immobilizing probe molecules on the surface of a macroscopic solid-phase substrate, the solid-phase substrate itself is large, so after dividing the same substrate surface into multiple regions, different types of probe molecules are immobilized in each region. You can also Specifically, when dividing into a plurality of areas, each section is arranged in an array or matrix, and an address for identifying each section is assigned (addressed), and each section has a different type. A probe array in which probe molecules are immobilized can be used. For example, the hybridization reaction with a nucleic acid probe, is utilized for immobilization of the target nucleic acid molecule comprising a nucleotide sequence complementary portion and the base sequence of nucleic acid probes, DNA probe array to more secure the DNA nucleic acid probe; antigen・ Peptide antigens and arrays that immobilize multiple types of antigen peptides with known amino acid sequences, which are used to immobilize target antibody molecules that have specificity for antigen peptides by antibody reactions; conversely, antigen-antibody reactions An antibody array for immobilizing a plurality of known specific antibody molecules, which is used for immobilization of a target antigen molecule using a specific antibody; binding of a substrate molecule on the receptor protein to the receptor protein Used for immobilization of target substrate molecules, such as receptor protein arrays that immobilize multiple types of known receptor molecules There is a state.

  The probe array is used when simultaneously immobilizing multiple types of target molecules contained in a specimen sample by using the corresponding multiple types of probe molecules. Since the solid phase substrate itself used has a macro size, separation from the liquid phase and subsequent cleaning operation are extremely simple. In addition, when detecting a target substance immobilized on a substrate as a conjugate with each probe molecule on the probe array, the fixing position (address) of each probe molecule is determined in advance, and each target substance is determined according to the address. Advance detection.

  Probe arrays, which immobilize probe molecules on the surface of a macroscopic solid phase substrate, have the advantage of being easy to handle when separating from the liquid phase and detecting target substances, and can be used in various fields. Is progressing. However, the reaction yield is relatively low due to the reaction between the probe molecule fixed in the specific region and the target molecule contained in the sample sample on the surface of the solid substrate of macro size. (The apparent reaction rate is slow). Specifically, the probe molecules are immobilized in a limited area on the surface of the solid phase substrate, but the target molecules contained in the specimen sample are uniformly distributed throughout the liquid phase. The target molecules that can react with the probe molecules are limited to those existing in a limited region and in a range close to the surface of the solid phase substrate. When a target molecule present in this limited area forms a conjugate with an immobilized probe molecule, the concentration of “free target molecule” present in the liquid phase in this area rapidly increases. As a result of the reduction, a decrease in the reaction rate is relatively reduced. As a result, the total number of target molecules (reaction yield) that forms a conjugate with the probe molecules is limited per total number of immobilized probe molecules. The disadvantage that the reaction yield is relatively low (the apparent reaction rate is slow) becomes more conspicuous as the occupied area ratio of the fixed region of each probe molecule on the surface of the solid phase substrate becomes smaller. . In other words, this defect becomes more prominent as the number of types of probe molecules constituting the probe array increases.

  Ideally, this defect can be eliminated by stirring the reaction solution quickly and making the concentration distribution in the liquid phase uniform, but in reality, the surface of a solid substrate with a macro size is used. When used, the reaction is often carried out in a situation where the reaction solution is gently stirred or left to stand. Even if the reaction yield is relatively low (the apparent reaction rate is slow), the total number of target molecules that form a conjugate with the probe molecule (reaction yield) per total number of immobilized probe molecules Reflects the initial concentration of the target molecule in the sample solution. However, the ratio of the area occupied by each probe molecule's immobilization region decreases, and the total number of target molecules (reaction yield) that form a conjugate with the probe molecule and the initial concentration of the target molecule in the sample solution. The relationship in does not show high linearity. That is, when using a high-density probe array that fixes various types of probe molecules in a high-density matrix on the surface of a macroscopic solid-phase substrate, target molecules that are immobilized as conjugates with each probe molecule This quantification is one of the factors that lower the quantification accuracy when quantifying the target molecule concentration in the sample solution.

  In addition, when configuring the probe array, a lattice-like frame region for partitioning may be provided around the immobilization region of each probe molecule. Alternatively, there may be a case where a probe molecule non-immobilization region exists around the probe molecule immobilization region even when the lattice-shaped frame region is not provided. Since the surface of the solid phase substrate is exposed in this lattice-like frame region or the non-immobilized region of the probe molecule, various target molecules often cause non-selective adsorption. This non-selectively adsorbed target molecule raises the background signal level during detection, and binds the difference between the signal level detected in the fixed region of the probe molecule and the background signal level to the probe molecule. When determining the signal due to the target molecule forming the body, it becomes a systematic error factor. In addition, since the reaction yield is relatively low (the apparent reaction rate is slow), it is one of the factors that lower the quantitative accuracy of the target molecule concentration range in the sample solution.

  When producing a probe array, a spotting method in which probe molecules prepared separately in advance are applied to a predetermined immobilization region and immobilized is widely used. In this spotting method, the density of probe molecules immobilized per unit surface area is the same as in the case where microparticles are introduced into a liquid containing probe molecules separately prepared in advance and the probe molecules are immobilized on the surface. It will have high reproducibility. On the other hand, when immobilizing synthesizable DNA probe (oligonucleotide) molecules in an array by a solid phase reaction, each DNA probe is placed on the surface of a macro size solid phase substrate using a photolithographic method. There is also a method in which oligonucleotides synthesized sequentially according to the base sequence of (oligonucleotide) are used as immobilized probe molecules. Regarding the oligonucleotide synthesized directly on the surface of the solid phase substrate, it is difficult to confirm the base sequence after the synthesis, and an oligonucleotide partially lacking the base may be mixed. Alternatively, in principle, all (oligo) nucleotides that are short by one nucleotide are all present together in a certain ratio. When such a DNA molecule different from the target base sequence is mixed, it causes a relative decrease in the reaction yield in the probe hybridization reaction. In some cases, if a DNA molecule that is different from the target base sequence happens to have a common base sequence with a DNA probe molecule that is immobilized on another address, another type of nucleic acid molecule that differs from the original target nucleic acid molecule Will also be combined on such addresses. Therefore, the undesired contamination of the oligonucleotide described above becomes one of the causes of the decrease in the quantitative accuracy.

  On the other hand, probe molecule-immobilized fine particles, in which probe molecules are immobilized on the surface of microparticles, are prepared by injecting microparticles into a liquid containing probe molecules separately prepared in advance and immobilizing the probe molecules on the surface. Since it is prepared, the phenomenon of non-selective adsorption of various target molecules on the surface of microscopic particles is substantially suppressed. The probe molecule to be immobilized is prepared separately and then sufficiently purified. As will be described later, solid-phase sequential synthesis on the surface of microscopic particles can avoid the drawbacks of the above-described photolithography method, so that this method can also be used suitably.

  The probe molecule-fixed microparticles can usually maintain a uniformly dispersed state in the liquid phase, and the reaction between the target molecules uniformly distributed throughout the liquid phase and the solid phase / liquid phase is not However, macroscopically, it is possible to carry out a uniform reaction throughout the liquid phase. Therefore, the reaction yield is relatively low, which is remarkable when using a high-density probe array in which various probe molecules are immobilized on a macro-sized solid phase substrate surface in a high-density matrix. The disadvantage that the apparent reaction rate is slow) is substantially eliminated when this probe molecule fixed fine particle is used. Therefore, when probe molecule-immobilized microparticles are used, when the target molecule concentration in a sample solution is quantified by quantifying the target molecule immobilized as a conjugate with the probe molecule, the factors that decrease the quantitative accuracy listed in the probe array Most of them can be avoided.

  Further, the probe molecule-fixed fine particles usually retain the advantage that they can be easily separated from the liquid phase after the reaction is finished in a state of being uniformly dispersed in the liquid phase. For example, a solid-liquid separation method using filtration or centrifugal separation can be used, and when particles themselves are mainly composed of a magnetic material, they can be separated from the liquid phase using magnetic force. .

  Of course, the probe molecule-immobilized microparticles immobilize one type of probe molecule on the surface of each microparticle. Therefore, when multiple types of probe molecules are used, a large number of corresponding probe molecule-immobilized microparticles are prepared in advance. There is a need to. These probe molecule-fixed fine particles are different from each other in the probe molecules constituting the individual probe molecule-fixed fine particles, but cannot be easily distinguished from each other in appearance. In other words, it is necessary to identify the individual probe molecule-immobilized fine particles by making it possible to distinguish the micro-particles used for many types of probe molecule-immobilized fine particles. That is, it is necessary to attach a label for mutual identification to the micro particles used.

  Several proposals have been made regarding techniques for attaching microscopic particles constituting the probe molecule-fixed fine particles to each other for mutual identification. For example, Patent Document 1 (Japanese Patent Laid-Open No. 61-225656) discloses a technique for identifying by the difference in particle size of micro particles used; Patent Document 2 (Japanese Patent Laid-Open No. 62-81566) includes a microscopic technique. A technique for distinguishing by combining a difference in particle diameter of particles and a fluorescent label (total n types); Patent Document 3 (Japanese Patent Laid-Open No. 62-195556) includes coloring a micro particle, A method for distinguishing; Patent Document 4 (Japanese Patent Laid-Open No. 1-95800) discloses a technique for forming micro particles with different metal elements and distinguishes them; Patent Document 5 (Japanese Patent Laid-Open No. 2-299698) Patent Document 6 (Japanese Patent Application Laid-Open No. 7-83927) discloses a technique for distinguishing according to the fluorescence wavelength of a micro particle as an inorganic phosphor, and Patent Document 7 (US). Patent No. 6 No. 02671) is a technique for distinguishing by using semiconductor nanocrystals as micro particles; Patent Document 8 (U.S. Pat. No. 6,440,667) discloses a microscopic material having different magnetism, color, and shape. Method for making distinction using particles; Patent Document 9 (US Pat. No. 6,600,562) discloses a method for making a distinction by using semiconductor nanofluorescent particles for microscopic particles. These relate to a technique for giving individual addresses (labels) to particles.

In these conventional techniques, the number of types of microscopic particles that can be distinguished, that is, the number of types of labels is at most about a dozen, and when the number of types of labels is further increased, this is a technique with poor expandability. In particular, the particle size, shape, and main constituent materials of the micro particles are a technique with poor expandability when the number of types of labels is further increased while maintaining commonality.
Japanese Patent Laid-Open No. 61-225656 Japanese Patent Application Laid-Open No. 62-81566 Japanese Patent Laid-Open No. 62-195556 JP-A-1-95800 JP-A-2-299698 Japanese Patent Laid-Open No. 7-83927 US Pat. No. 6,602,671 US Pat. No. 6,440,667 US Pat. No. 6,600,562

  As described above, when determining the presence / absence and measuring the concentration of various biological substances contained in the liquid phase, such as nucleic acid molecules, proteins, sugar chain molecules, etc., target substances for inspection (target substances) A method of separating from a liquid phase once as a conjugate by causing a substance (usually referred to as a probe molecule) that specifically binds to) to act is widely used. Specifically, a technique is widely used in which a probe molecule is immobilized on the surface of a solid phase, a target substance existing in the liquid phase and a conjugate are formed, and the solid phase and the liquid phase are separated. . After separation from the liquid phase, the detection is performed using detection means that matches the presence / absence and the amount of the target substance constituting the conjugate with the probe molecule.

  In this case, probe molecule-immobilized fine particles in which each probe molecule is individually immobilized on a fine particle surface compared to a probe array in which multiple types of probe molecules are regularly immobilized on a solid surface substrate with a large surface area Is significantly superior in terms of the reactivity with the target substance in the liquid phase and the quantitativeness thereof. However, in the probe array, identification of each probe molecule type can be easily performed based on the immobilization position (address). However, in the probe molecule-immobilized microparticle, the microparticles used for immobilization are labeled in advance. Then, it is necessary to identify the label and specify the type of probe molecule.

  As described above, several means for labeling microparticles have been proposed, but the types that can be used are limited, and when there are many types of probe molecules to be used, all of them are individually labeled. It was not possible to satisfy the purpose. In particular, as in the case of microparticles used for immobilizing probe molecules, the size, shape, and main constituent materials can be increased as needed, while maintaining commonality. The development of a labeling method with high expandability is awaited.

  The present invention solves the above-mentioned problems, and the object of the present invention is to provide a sign that can be used while the object to be marked is a small size, and the shape and main constituent materials are common. A labeling method with a high expandability, a probe binding object labeled by the labeling method, a probe binding object, and a method for discriminating a probe bound to the probe binding object, enabling the number of types to be increased as necessary. An object of the present invention is to provide a method for detecting, identifying and quantifying the probe, and a method for detecting, identifying and quantifying a target substance bound to the probe.

  As a result of diligent research to solve the above problems, the present inventor first came up with the following. For example, the size of microscopic particles used for immobilizing probe molecules is selected to be in the submicron region in some cases, in order to disperse the particles uniformly in the liquid phase. . At that time, in order to make the amount of probe molecules immobilized on the surface of the microparticles uniform, it is necessary to make the particle diameter of the fine particles used constant and to make the surface area of each fine particle constant. Of course, it is necessary to unify the outer shape of the fine particles into, for example, a spherical shape. Furthermore, when immobilizing separately prepared probe molecules on the surface of the fine particles, if the material of the surface of the fine particles is completely different, it is necessary to use different immobilization means. Specifically, even if it is a metal fine particle, if the metal element which comprises it is completely different, it is necessary to utilize a different fixing means. On the contrary, if the means for immobilizing the probe molecules on the surface is different, it is difficult to make the density of the probe molecules immobilized per unit area the same.

Therefore, it is desirable to use a labeling method that is highly extensible so that the particle size, shape, and main constituent materials of the microparticles can be increased as needed, while maintaining commonality. . This particle size, shape, and main constituent materials also satisfy the requirement of maintaining commonality, and there is no ambiguity in identifying the labels attached, and labels that can be applied as needed Development of a highly scalable labeling method that can arbitrarily increase or decrease the type of the above. As a result, for example, in addition to the main constituent components, materials that can be added as small amounts can be added as a material used when producing microscopic particles. It was found that it is possible to add information by utilizing the composition of this optionally added secondary component. Specifically, as the composition of such optionally added secondary constituents, a plurality of types of atoms are selected in advance, and regarding the number of types (n) of atoms, the presence or absence of each is selected. In a two-level state, the number of types of atoms selected in advance (n) and the presence / absence of the content of each of the selected atoms correspond to binary n-digit numerical information. The inventors have conceived that the composition conditions of contained atoms can be set. Furthermore, when this method is generalized, a plurality of types of atoms are selected in advance, and regarding the number of types (n) of atoms, when a plurality of content levels, for example, M levels are set, for each atom, It becomes a state of M levels, and by using the number of types (n) of a plurality of types of atoms selected in advance and the level of the content of each of the selected atoms, numerical information of M-digit n-digits is obtained. The inventors have conceived that it is possible to set the composition conditions of the corresponding atoms. In addition to these ideas, the present inventor has determined that the number of types (n) of a plurality of types of atoms selected in advance and the presence or absence of the content of each of the selected atoms, or the difference in the level of content, For example, even if the particle size is a few μm or less, by applying various analysis means, the content level can be specified for a plurality of types of atoms selected in advance. It was also confirmed that the labeling information attached as the composition was sufficiently distinguishable, and the present invention was completed.

  In addition, in the present invention, the label includes at least two types of atoms selected from a plurality of types of atoms selected in advance. At least one of mass spectrometry, X-ray photoelectron spectroscopy, and Auger electron spectroscopy, which is a means suitable for measurement, is used.

That is, the probe coupling object according to the present invention is:
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The sign is
Including at least two types of atoms selected from a plurality of types of preselected atoms;
The label is a probe-binding object characterized by being identified by at least one of mass spectrometry, X-ray photoelectron spectroscopy, and Auger electron spectroscopy.

  In this case, it is preferable that the plurality of types of atoms selected in advance are not selected from atoms constituting the probe, but selected from various atoms used when constituting the surface to which the probe is bound or the surface thereof. That is, mass spectrometry, X-ray photoelectron spectroscopy, or, from the remaining atomic groups excluding the group of atoms that make up the probe from the various groups of atoms that can be used in constructing the object or its surface, What can be detected by any of Auger electron spectroscopy can be suitably used as the plurality of types of preselected atoms. The label may be identified by a “detection result” that any one of the “selected at least two types of atoms” is not detected, that is, the content is zero. In other words, in the present invention, “including at least two selected atoms” means “a state in which the label can be identified by paying attention to the content of the atoms”. It means that.

One aspect of the probe coupling object of the present invention is:
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types (n) of a plurality of types of atoms selected in advance and the presence / absence of the content of each of the selected atoms, the composition conditions of contained atoms corresponding to numerical information of binary n digits are configured. And then
Using a indicator having a composition that satisfies the composition conditions of the contained atoms,
The probe coupled object is characterized by being attached with the discrete identification information expressed as the binary n-digit numerical information.

Other aspects of the probe coupling object of the present invention include:
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types (n) of a plurality of types of atoms selected in advance and the content level of each of the selected atoms, the composition conditions of the contained atoms corresponding to at least binary n-digit numerical information are configured And then
Using a indicator having a composition that satisfies the composition conditions of the contained atoms,
The probe coupled object, wherein the discrete identification information expressed as numerical information of at least binary n digits is attached.

One aspect of the method for producing a probe-coupled object of the present invention is as follows.
A method for producing a probe binding object according to any of the above aspects,
Binding of the probe to the labeled object surface is
According to the probe binding object, characterized by sequentially supplying corresponding monomers to the surface of the object to which the probe is bound according to the base sequence of the probe, and synthesizing a nucleic acid chain by a solid phase synthesis method. This is a manufacturing method.

Another aspect of the method for producing the probe-coupled object of the present invention is as follows.
A method for producing a probe binding object according to any of the above aspects,
Binding of the probe to the labeled object surface is
A method for producing a probe-binding object, characterized in that a nucleic acid chain synthesized in advance and purified according to the base sequence of the probe is applied to the surface of the object.

The method of determining the type of probe that the probe binding object of the present invention has is,
A method of discriminating the type of probe-bound object that is labeled according to any of the above aspects, that is, the type of probe that is bound to the object,
Corresponding to at least binary n-digit numerical information by using the number of types of atoms selected in advance (n) and the presence or absence of the content of each selected atom, or the content level, After configuring the composition conditions of the contained atoms,
A label attached to each object using a labeler having a composition that satisfies the composition conditions of the contained atoms,
For each of the plurality of types of preselected atoms, the plurality of types of preselected atoms in the composition of the labeler using means capable of detecting the presence or absence of the content or the level of the content. The presence or absence of the individual content of the atom, or the content level,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
It is a method for discriminating probe coupled objects, wherein the type of each probe coupled object is identified based on the discrete identification information expressed as numerical information of at least binary n digits.

The method for quantifying the probe of the probe binding object of the present invention is as follows.
A method for identifying, detecting or quantifying a probe bound to a probe binding object according to any of the above aspects,
The type of object, i.e. the type of probe coupled to the object,
The label of the object, which is attached using the indicator, is used to detect the presence or absence of the content or the level of the content of each of the plurality of preselected atoms. With respect to the plurality of preselected atoms in the composition of the indicator, the presence / absence of the content of each of the atoms, or the content level is identified,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
Discriminating by identifying based on the discrete identification information expressed as numerical information of at least binary n digits,
Detection is performed by using, as an indicator derived from the probe, an atom, a molecule, or a substance that is included in the probe and not included in the object. Alternatively, a method for identifying, detecting, or quantifying a probe bound to an object characterized by quantification.

The target nucleic acid detection, identification, or quantification method of the present invention is a target nucleic acid detection, identification, or quantification method using a probe-binding object,
At least the following steps (1) and (2):
(1) A step of obtaining a hybrid by reacting the probe-binding object according to any one of the above aspects and a target substance;
(2) Using at least binary n-digit numerical information by using the number of types (n) of a plurality of types of atoms selected in advance and the presence / absence or level of content of each of the selected atoms. After configuring the composition conditions of the corresponding atoms,
The label attached to the binding object on which the hybrid is formed using a labeling composition that satisfies the composition conditions of the contained atoms, the content of each of the plurality of types of preselected atoms. Using the means capable of detecting the presence or absence, or the level of the content, the presence or absence of the content of the individual atoms or the content of the plurality of types of the preselected atoms in the composition of the labeler Identify the level,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
If necessary, detect, identify and quantify the probe bound to the binding object on which the hybrid is formed by the method described above,
At the same time or subsequently, the target substance forming the hybrid is detected and specified by using as an index the atoms, molecules or substances contained in the target substance and not included in the probe binding object and the probe. Quantifying;
A method for detecting, identifying and quantifying a target substance characterized by comprising:

  By using the object labeling method according to the present invention, for example, even a minute object such as a fine particulate substrate used for immobilizing a probe molecule used for detection of a biological substance can be used. As a part of the constituent material, at least a binary system using the number of types (n) of a plurality of atoms selected in advance and the presence or absence of the content of each of the selected atoms or the content level By using a labeling material prepared in accordance with the composition conditions of the contained atoms, which corresponds to n-digit numerical information, it is possible to attach many types of labels as necessary.

  As described above, in the object labeling method used in the present invention, for example, as a material used when producing microparticles, in addition to the main constituent components, a component that can be mixed in a small amount As described above, a material capable of having a secondary constituent component that can be arbitrarily added is selected, and labeling information is added using the composition of the secondary constituent component that can be optionally added. Specifically, as the composition of such optionally added secondary constituents, a plurality of types of atoms are selected in advance, and regarding the number of types (n) of atoms, the presence or absence of each is selected. It will be in a two-level state. Then, the composition conditions of the contained atoms corresponding to the binary n-digit numerical information using the number of kinds (n) of the plurality of kinds of atoms selected in advance and the presence / absence of the content of each of the selected atoms This is a method of giving labeling information depending on the composition of the set contained atoms. Furthermore, when this method is generalized, a plurality of types of atoms are selected in advance, and regarding the number of types (n) of atoms, when a plurality of content levels, for example, M levels are set, for each atom, There are M levels. By utilizing this, the number of types of the plurality of types of atoms selected in advance (n) and the content level of each of the selected atoms are used to correspond to numerical information of M-ary n digits. By setting the composition conditions of the contained atoms, it is possible to increase the number of types of labels as necessary, and this is a scalable method.

That is, in the object labeling method used in the present invention, first, when preparing an object, a plurality of types of atoms are selected in advance as secondary constituents that can be arbitrarily added, and the number of types (n ), The vector type having the contents (C _i ) of these n kinds of atoms as elements, using the individual contents (C _i ; i = 1,..., N) of the selected atoms. information _{{C 1, ··, C i} , ··, C n} to set the. The information in this Vector _{{C 1, ··, C i} , ··, C n} , the corresponding n-dimensional space _{(C 1, ··, C i} , ··, C n) on respective discrete It corresponds to the point that exists. On the other hand, the object is composed of main constituents and the optionally added secondary constituents. When the content of the main constituents is expressed as C ₀ , the overall composition is a vector. the type of information can be represented _{{C 0, C 1, ··} , C i, ··, C n} as. In other words, the overall _{composition: {C 0, C 1,} ··, C i, ··, C n} composition conditions containing atoms to _{achieve: g (C 1, ··,} C i, ··, C _n), the composition of arbitrarily adding a minor component _{{C 1, ··, C i} , ··, C n} is determined by. As the composition of the object, selecting the total number N _TOTAL Road, the overall _{composition: {C 0, C 1,} ··, C i, ··, C n} containing atoms for achieving the composition condition: g (C ₁ ,..., C _i ,..., C _n ) are N _TOTAL in total.

Vector type information indicating the total composition of the total number N _TOTAL : {C ₀ , C ₁ ,..., C _i ,..., C _n } is the corresponding (n + 1) -dimensional space (C ₀ , C ₁ , ···, C _i , ···, C _n ), corresponding to a set of N _TOTAL discrete points in the vicinity of the point (C ₀ , 0, ···, 0, ···, 0) To do. Composition conditions containing _{atoms: g (C 1, ··,} C i, ··, C n) is a vector type indicating the overall composition _{information: {C 0, C 1,} ··, C i, ··, and C _n}, information {C ₁ vector type indicating the content of arbitrarily adding a minor component, ··, C _i, ··, and C _n}, associates each function (operator) It corresponds to. _{That, g (C 1, ··,} C i, ··, C n) ⇒ can be carried out _{{C 0, C 1, ··} , C i, ··, C n} association that can be expressed as. In the present invention, the content of arbitrarily adding a minor component _{{C 1, ··, C i} , ··, C n} For, when preset composition conditions of the total number N _TOTAL street, suitable By selecting the detection means, the detection accuracy of the detection means to be used is taken into consideration so that the respective compositions can be distinguished from each other, and the content of secondary constituents that can be arbitrarily added {C ₁ , .., C _i ,..., C _n } are selected “discretely”.

Capable discrimination of each composition mutual respect article showing the entire composition of the total number N _TOTAL street, as a code indicating the type of differences in each article (difference in composition) (seed number), discrete of the total number N _TOTAL Street Numerical information: A set {f (N)} of f (N) is separately selected. Then, the selected "discretely", the content of arbitrarily adding a minor component _{{C 1, ··, C i} , ··, C n} is determined by the total number N _TOTAL Street containing atoms composition _{condition: g (C 1, ··,} C i, ··, C n) the set _{{g (C 1, ··,} C i, ··, C n)} of each element g of ( C ₁ ,..., C _i ,..., C _n ), one element f (N) in a set {f (N)} of a total number N _TOTAL discrete numerical information: f (N) ) Is “one-to-one correspondence” as indicated by g (C ₁ ,..., C _i ,..., C _n ) ⇔f (N). That is, the “discrete identification information” expressed as the numerical information f (N) corresponding to a code (species number) indicating a difference in type (composition difference) of each article is referred to as “discretely”. "have been set _{composition: {C 1, ··, C} it, ··, C n} is given as labels utilized.

When the labeled probe-binding object according to the present invention uses this object labeling method, the total number N _TOTAL of the types of articles showing the overall composition that can be distinguished from each other is N _TOTAL ≧ 2 ⁿ −. so as to satisfy a composition constituting a _{"label": {C 1, ··, C} i, ··, C n} "discretely" newly discovered method of setting, an application of it is there.

In this case, in the present invention, the label includes at least two types of atoms selected from a plurality of types of atoms selected in advance. At least one of mass spectrometry, X-ray photoelectron spectroscopy, and Auger electron spectroscopy, which is a means suitable for measurement, is used. Therefore, the probe coupling object of the present invention is
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The sign is
Including at least two types of atoms selected from a plurality of types of preselected atoms;
The label is a probe-binding object characterized by being identified by at least one of mass spectrometry, X-ray photoelectron spectroscopy, and Auger electron spectroscopy.

The labeled probe binding object according to the present invention described above is, for example,
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types of atoms selected in advance (n) and the content of each selected atom (C _i ; i = 1,..., N), the total number of atoms included in N _TOTAL composition _{condition: g (C 1, ··,} C i, ··, C n) the set of _{{g (C 1, ··,} C i, ··, C n)} which can constitute the composition according to the setting procedure of conditions, the total number n _tOTAL street containing atomic composition _{condition: g (C 1, ··,} C i, ··, C n) the set of _{{g (C 1, ··,} C i, · , C _n )} corresponding to a subset of elements N _LABEL (where N _LABEL is an integer satisfying 1 ≦ N _LABEL ≦ N _TOTAL ) containing atoms: g _j (C ₁ ,. , C _i ,..., C _n ) {g _j (C ₁ ,..., C _i ,..., C _n ): j = 1 _,.
Number of elements N _LABEL (where, N _LABEL is an integer satisfying 1 ≦ N _LABEL ≦ N _TOTAL) numerical information: the set of _{f j (N) {f j} (N): j = 1, ···, N LABEL },
Numerical information of the number of elements N _LABEL: f _j set of _{(N) {f j (N} ): j = 1, ···, N LABEL} for each element f _j of (N), one-to-one Corresponding compositional composition of atoms with N _LABEL elements: g _j (C ₁ ,..., C _i ,..., C _n ) set {g _j (C ₁ ,..., C _i ,. C _n ): j = 1,..., N _LABEL } elements g _j (C ₁ ,..., C _i ,..., C _n )
Said containing atomic composition _{condition: g j (C 1, ··} , C i, ··, C n) satisfying the _{composition: {C 1, ··, C} i, ··, C n} -indicator having a Using,
This g _j (C ₁ ,..., C _i ,..., C _n ) and a one-to-one correspondence (g _j (C ₁ ,..., C _i ,..., C _n ) ⇔f _j ( N)), the discrete identification information represented as the numerical information f _j (N) is attached,
Said containing atomic composition _{condition: g (C 1, ··,} C i, ··, C n) the set of _{{g (C 1, ··,} C i, ··, C n)} the number of elements in the summation : N _TOTAL is selected as N _TOTAL ≧ 2 ⁿ −1
The numerical information: the set of _{f j (N) {f j} (N): j = 1, ···, N LABEL} element number N _LABEL as well as compositions condition of the containing atoms: g _j (C _1, ·· , C _i, · ·, set _{{g j (C 1, ··} , C i, ··, C n): j = 1, ···, n LABEL} of C _n) number of elements n _LABEL of, N _TOTAL ≧ N _LABEL ≧ n · (n−1) +1 is selected, and the probe coupled object may be formed. Moreover,
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
By using the number of types of atoms selected in advance (n) and the content of each selected atom (C _i ; i = 1,..., N), numerical information of the total number N _TOTAL : f set of (N) with respect to {f (N)} each element f in (N), a one-to-one correspondence, the total number N _tOTAL street containing atomic composition condition: g (C _1, · ·, C _i, · ·, set of _{C n) {g (C 1} , ··, C i, ··, C n)} on which constitute,
Said containing atomic composition _{condition: g (C 1, ··,} C i, ··, C n) composition _{satisfies: {C 1, ··, C} i, ··, C n} a-indicator having a make use of,
The g (C ₁ ,..., C _i ,..., C _n ) and the one-to-one correspondence (g (C ₁ ,..., C _i ,..., C _n ) ⇔f (N)) The discrete identification information expressed as the numerical information f (N) is attached,
The numerical information: the sum of the number of elements of the set {f (N)} of f (N): N _TOTAL may be a probe coupled object characterized in that N _TOTAL ≧ 2 ⁿ −1 is selected. Is possible.

At this time, numeric information: as f (N), the _{composition: {C 1, ··, C} i, ··, C n} with respect to, atomic individual content _{(C i; i = 1,} ··, n) Is converted into two levels, in particular, a value corresponding to “binary n-digit numerical information” generated by converting the value into two levels of “presence / absence of content”. One form. Further, as numerical information: f (N), the content of each atom (C _i ; i = 1,..., N) is converted into a value divided into two or more levels, for example, M levels. The second form of the present invention is a form in which the technique of the first form is generalized and expandability is given, such as using the “M-ary n-digit numerical information” generated in step (1).

The labeled probe binding object according to the first aspect of the present invention is an object to which a probe is bound,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types (n) of a plurality of types of atoms selected in advance and the presence / absence of the content of each of the selected atoms, the composition conditions of contained atoms corresponding to numerical information of binary n digits are configured. And then
Using a indicator having a composition that satisfies the composition conditions of the contained atoms,
The probe coupled object is characterized by being attached with the discrete identification information expressed as the binary n-digit numerical information.

Further, the labeled probe binding object according to the second embodiment of the present invention, which has further expandability with respect to the first embodiment,
An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types (n) of a plurality of types of atoms selected in advance and the content level of each of the selected atoms, the composition conditions of the contained atoms corresponding to at least binary n-digit numerical information are configured And then
Using a indicator having a composition that satisfies the composition conditions of the contained atoms,
The probe coupled object characterized by being attached with the discrete identification information expressed as numerical information of at least binary n digits.

  Furthermore, as preferable forms thereof, the following forms can be exemplified.

For example, in the first aspect of the present invention described above and the object according to the second aspect,
In the sign,
The number of types (n) of plural types of atoms selected in advance is 5 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are (Positive integer) can be 31 or more.

In the above sign,
The number of types (n) of plural types of atoms selected in advance is 8 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are It is also possible to set a positive integer) to 255 or more.

Furthermore, as an aspect showing high expandability,
In the sign,
The number of types (n) of plural types of atoms selected in advance is 12 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are The positive integer) can be 4095 or more. Alternatively, in the label,
The number of types (n) of plural types of atoms selected in advance is 16 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are The positive integer) can be 65535 or more.

  In the labeled probe binding object according to the first embodiment and the second embodiment of the present invention described above, the shape of the object itself is not used for the label information and can be arbitrarily selected. Is possible. Therefore, the shape of the object can be selected from irregular fine particles, regular fine particles, fibers, or sheets.

  On the other hand, in the labeled probe binding object according to the present invention, the label includes the number of types of atoms selected in advance (n), the presence / absence of the content of each of the selected atoms, or the level thereof. For example, it can be applied to a magnetic material having a plurality of types of atoms as constituent components. Therefore, it is possible to select an aspect in which the object is a magnetic body.

  In addition to the shape of the object, the size of the object is not used for the sign information. Therefore, in the labeled object according to the present invention, the size of the object can be arbitrarily selected according to the application. Is possible. For example, as the size of the object, when the object is in the form of fine particles, the size corresponding to the particle size is in the range of 1 nm to 10 μm, and when the object is in the shape of a fiber or one piece, the vertical and horizontal It is also possible to select at least two of the heights in the range of 1 nm to 10 μm.

  The probe bound to the probe binding object according to the present invention is a substance having a function as a so-called probe, that is, a function as one of a pair of substances capable of recognizing and binding to each other. Furthermore, any substance may be used as long as it has such a function and can be bound to the object according to the present invention. Examples of such substances include nucleic acids, DNA, RNA, cDNA, cRNA, oligodeoxyribonucleotides, polydeoxyribonucleotides, oligoribonucleotides, polyribonucleotides, peptide nucleic acids (PNA), oligopeptide nucleic acids, peptides, oligopeptides, Examples thereof include polypeptides, proteins, antigens, antibodies, enzymes, ligands, ligand receptors, sugars, and sugar chains.

  The method for binding the probe to the object according to the present invention may be any method as long as it can form a state in which the probe can function as a probe and can be bonded to the object. For example, covalent bonding, ionic bonding, and physical adsorption can be performed. Can be used.

  In the case of an oligomer or polymer in which the probe is composed of a plurality of monomers, the basic configuration of the present invention is an embodiment in which the probe is bound to the object, so the probe is synthesized on the object by so-called solid-phase sequential synthesis. A combination method can be adopted. The solid phase sequential synthesis method is a method that is already commonly used in nucleic acid synthesis, peptide nucleic acid synthesis, peptide synthesis, and the like, and these methods can be employed as they are in the present invention. In the production of a so-called nucleic acid chip, for example, US Pat. No. 5,744,305 describes a method of synthesizing and binding a nucleic acid probe on a solid phase substrate using photolithography and deprotection reaction by light. However, since this method is a reaction efficiency of the photodeprotection reaction or all the reactions are heterogeneous reactions on the substrate, the reaction yield, the reaction rate, and an undesired short on the substrate. The problem that many strand nucleic acids are formed remains.

  On the other hand, in the present invention, if the object to which the probe is bound is appropriately selected from particles, fine particles, etc., it is convenient that it can be applied to the solid phase automatic synthesizer used in the above-mentioned nucleic acid synthesis, peptide nucleic acid synthesis, peptide synthesis, etc. There is sex. Furthermore, although it is a solid phase synthesis, that is, a heterogeneous system, particles are used, a high reaction yield and a high reaction rate can be obtained as compared with using a heavy solid substrate. In addition, since a general scheme such as the deprotection of a nucleic acid sequential reaction using an acid can also be used as a synthesis scheme, unnecessary short-chain nucleic acid compared to a method using photodeprotection on a substrate. There is an advantage that the abundance of is also small.

  When using the above solid-phase synthesis, for example, if the probe is a nucleic acid, it is possible to perform automatic synthesis after introducing a hydroxyl group onto the surface of the object to which the probe is to be bound. In the case of a peptide, an automatic synthesis can be performed after introducing an amino group on the surface of an object. In addition, you may use the silane coupling agent which has an amino group for the introduction | transduction of the amino group to the object surface.

  In addition, as a method for binding a probe to an object according to the present invention, a method of providing a probe synthesized and purified in advance can be employed. Conventionally used methods can be applied to the method of applying the probe synthesized and purified in advance to the object. For example, the object is treated with a silane coupling agent having various functional groups (amino group, thiol group, hydroxyl group, epoxy machine, succinimide ester group, maleimide group, halide group, etc.), and the desired functional group is introduced to the object surface. Then, a method of reacting with a probe having a functional group capable of reacting with these functional groups can be mentioned. In the case where the functional groups do not match, the bond may be imparted through a linker substance having two different functional groups, for example, N- (6-maleimidocaproyloxy) succinimide (Dojindo Laboratories). . Examples of the functional group on the probe side include a functional group that reacts with the functional group introduced into the object as described above, or a functional group that reacts via the linker substance. For example, if the probe is a nucleic acid, amino groups, thiol groups, and succinimide groups can be mentioned as examples. On the other hand, if the probe is a peptide, protein, enzyme, or antibody, either a thiol group is introduced synthetically into the probe using a cysteine residue or a thiol group of an existing cysteine residue is used, and the substrate surface is In addition, for example, a maleimide group may be formed on the surface of an object using an aminosilane or a pulling agent and the N- (6-maleimidocaproyloxy) succinimide and bonded to the thiol group of the probe.

  Examples of the ionic bond include an ionic bond between an amino group and a thiol group. When using an ionic bond, it goes without saying that it is necessary to control the ionization of the functional group to react by controlling the pH during the binding reaction.

  The present invention further includes, in addition to the invention of the probe-binding object according to the present invention having the above-described configuration, for the probe-binding object to which a label is attached, by identifying the attached label, The present invention also provides an invention of a method for discriminating, and thus discriminating between probes bound to the object.

That is, the object and probe discrimination method according to the present invention are:
A method of discriminating the type of probe-bound object to which the above-mentioned label is attached, that is, the type of probe bound to the object,
Corresponding to at least binary n-digit numerical information by using the number of types of atoms selected in advance (n) and the presence or absence of the content of each selected atom, or the content level, After configuring the composition conditions of the contained atoms,
A label attached to each object using a labeler having a composition that satisfies the composition conditions of the contained atoms,
For each of the plurality of types of preselected atoms, the plurality of types of preselected atoms in the composition of the labeler using means capable of detecting the presence or absence of the content or the level of the content. The presence or absence of the individual content of the atom, or the content level,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
It is a method for discriminating probe coupled objects, wherein the type of each probe coupled object is identified based on the discrete identification information expressed as numerical information of at least binary n digits.

  Furthermore, the present invention provides a method for detecting, identifying, or quantifying a probe-bound object that has been labeled.

That is, the probe detection, identification, or quantification method according to the present invention includes:
The type of object, i.e. the type of probe coupled to the object,
The label of the object, which is attached using the indicator, is used to detect the presence or absence of the content or the level of the content of each of the plurality of preselected atoms. With respect to the plurality of preselected atoms in the composition of the indicator, the presence / absence of the content of each of the atoms, or the content level is identified,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
Discriminating by identifying based on the discrete identification information expressed as numerical information of at least binary n digits,
Detection is performed by using, as an indicator derived from the probe, an atom, a molecule, or a substance that is included in the probe and not included in the object. Alternatively, a method for identifying, detecting, or quantifying a probe bound to an object characterized by quantification.

  An atom, molecule, or substance that is included in the probe of the probe binding object but not included in the object can be selected as appropriate according to the type of the probe. For example, if the probe is a nucleic acid, it can be selected from carbon atoms, phosphorus atoms, nitrogen atoms, nucleobases, sugars, and phosphoric acids. Moreover, if a probe is a peptide nucleic acid, it can select from a carbon atom, a nitrogen atom, and a nucleobase, for example. Furthermore, if the probe is a peptide, protein, enzyme, or antibody, it can be selected from, for example, a carbon atom, a nitrogen atom, and a sulfur atom.

  In addition, the present invention also provides a so-called target substance detection, identification, or quantification method that specifically binds to a probe of a probe-bound object that has been labeled.

That is, the target substance detection, identification, or quantification method according to the present invention includes:
At least the following steps (1) and (2):
(1) a step of reacting the probe-binding object according to any one of claims 1 to 11 with a target substance to obtain a hybrid,
(2) Using at least binary n-digit numerical information by using the number of types (n) of a plurality of types of atoms selected in advance and the presence / absence or level of content of each of the selected atoms. After configuring the composition conditions of the corresponding atoms,
The label attached to the binding object on which the hybrid is formed using a labeling composition that satisfies the composition conditions of the contained atoms, the content of each of the plurality of types of preselected atoms. Using the means capable of detecting the presence or absence, or the level of the content, the presence or absence of the content of the individual atoms or the content of the plurality of types of the preselected atoms in the composition of the labeler Identify the level,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
If necessary, the probe bound to the binding object on which the hybrid is formed is detected, identified, and quantified by the method described above,
At the same time or subsequently, the target substance forming the hybrid is detected and specified by using as an index the atoms, molecules or substances contained in the target substance and not included in the probe binding object and the probe. Quantifying;
A method for detecting, identifying and quantifying a target substance characterized by comprising:

  In each of the above methods, when the object is positioned on a plane, the presence or absence of the content of each of the atoms or the level of the content is identified with respect to the plurality of types of preselected atoms in the composition of the indicator. In addition, when detecting the labeling substance, it is preferable to further provide a step of performing two-dimensional imaging on the plane on which the object is located in order to specify at least the position of the object on the plane.

  For example, mass spectrometry can be used as a means for detecting the presence or absence of the content of each of the plurality of types of preselected atoms, or the content level, and particularly suitable mass spectrometry. The method is time-of-flight secondary ion mass spectrometry (TOF-SIMS). Further, X-ray photoelectron spectroscopy (XPS) or Auger electron spectroscopy (AES) is used as a means capable of detecting the presence or absence or the level of the content of each of the plurality of preselected atoms. It is also possible.

  As the atoms included in the target substance and not included in the probe binding object and the probe, a halogen atom or a metal atom can be preferably used. In some cases, the halogen atom or metal atom may be attached to the target substance as a so-called label. When a metal atom is introduced as a label, introduction may be facilitated by using an organometallic complex or a substance containing an organometallic complex.

As an example of the organometallic complex, if the following compound (Sigma Aldrich) is used, ruthenium can be introduced into the target substance using the amino group of the target substance.
Bis (2,2'-bipyridine) -4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluorophosphate
When the target substance is a nucleic acid, when the nucleic acid is enzymatically synthesized based on a template nucleic acid by a primer extension reaction, a halogen atom or a metal atom is used for the primer or primer extension reaction, dATP, A mode of binding to dGTP, dCTP, dTTP (or dUTP), ATP, GTP, CTP, UTP is a preferable mode of the present invention. In addition, when the probe is a peptide nucleic acid and the target substance is a nucleic acid, examples of the atom specific to the target substance include a phosphorus atom and phosphoric acid. It should be noted that the substances used for probe binding, the probe itself, or the target substance, which have been described so far, can be detected separately by the labeling of the object according to the present invention and the detection means used in the present invention. It is.

  Next, the method for detecting a marker in the present invention will be described in more detail.

  As described above, it is possible to adopt a mode in which mass spectrometry is used as a means for detecting the presence or absence of the content of each of a plurality of types of atoms selected in advance or the level of the content. At that time, the mass spectrometry is more preferably time-of-flight secondary ion mass spectrometry (TOF-SIMS). Alternatively, X-ray photoelectron spectroscopy (XPS) or Auger electron spectroscopy (AES) is used as means for detecting the presence / absence or level of the content of each of the plurality of preselected atoms. It is also possible to adopt an aspect. The same method can be used for detection, identification, and quantification of the probe bound to the probe binding object.

  In addition, when using TOF-SIMS, XPS, or AES, these analysis methods are applicable also to the analysis of the measuring object which has a two-dimensional extension, for example, when an object is located on a plane, When identifying the presence / absence of each individual atom or the level of the content of the plurality of types of preselected atoms in the composition of the label, the probe or the target bound to the probe Also in the detection, identification, and quantification of a substance, it is possible to provide a mode in which a step of two-dimensional imaging is further provided on the plane on which the object is located. This is also true when a plurality of objects are arranged in a plane.

  The performance of the detection apparatus necessary for the present invention will be described by taking TOF-SIMS as an example of the detection apparatus. The label used in the present invention is composed of atoms, and its mass is at most 100, and the mass resolution is also as follows. Basically, 1 ms (mass unit) is sufficient. For detection, identification, and quantification of the probe and the target substance, the upper limit of mass is several hundreds at most, and a resolution of 1 ms is sufficient. In TOF-SIMS, measurement of high-mass materials and high-resolution measurement require a longer flight tube, high-speed primary ion velocity, high voltage for secondary ion extraction, etc., and the equipment is large and expensive. The running cost will be high.

  In comparison, the apparatus required for the present invention can be small and inexpensive.

Further, in the object labeling method according to the present invention, a plurality of types of atoms are selected in advance as the label attached to the target object, and the presence / absence or content of each of the number of types (n) of atoms is selected. It uses the information inherent in the composition, indicated at the level of Specifically, as a plurality of types (n types) of atoms selected in advance, for example, one type of atom is selected from n types of elements different from each other, and the presence or absence (two levels) of each atom is determined. In other words, the total number N _{TOTAL of the} compositions in which at least one of the plurality of types (n types) of atoms selected is included is N _TOTAL = ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n Is a positive integer). Alternatively, when the content of each atom is changed in, for example, two steps (two levels) of “small amount, small amount”, at least a plurality of preselected atoms (n types) are included, and the content The total number of composition types N _TOTAL having a difference in amount is N _TOTAL = 2 ⁿ .

On the other hand, if the content of a plurality of types (n types) of atoms selected in advance is changed in, for example, three levels (three levels) of “no inclusion, very small amount, small amount”, at least a plurality of types selected in advance. The total number N _{TOTAL of the} compositions included in one of the seed (n species) atoms is N _TOTAL = 3 ⁿ −1.
Furthermore, among a plurality of types (n types) of atoms selected in advance, the content of one specific atom is set in, for example, three levels (three levels) of “no inclusion, very small amount, small amount”. For the remaining (n-1) types of atoms, if the presence or absence (two levels) is changed, at least one of the plurality of types (n types) of atoms selected in advance is included in the total number N of composition types included. _TOTAL is N _TOTAL = 3 × (2 ⁿ −1) −1. In the above aspect, for example, for all the plural types (n types) of atoms selected in advance, for example, three levels (three levels) of “no inclusion, very small amount, small amount” are set. The content of one particular atom is changed in accordance with the three-level (three levels) index, but the three-level (three levels) index for the remaining (n-1) types of atoms. Among them, it corresponds to selecting only two kinds of indicators “not contained, small amount”. That is, this corresponds to an aspect of selecting and using {3 × (2 ⁿ −1) −1} types of the subset out of the total number of composition types N _TOTAL = 3 ⁿ −1 that can be set. In this way, by dividing the content of multiple types (n types) of atoms selected in advance into three or more levels (three levels) and diversifying the options, for example, the content can be divided into M levels. When classifying into (M level), the range of the total number of composition types N _TOTAL can be expanded until reaching {M ⁿ −1} types.

As described above, a plurality of types of atoms are selected in advance as the indicator attached to the target object, and the number of types (n) of atoms is indicated by the presence or absence or the level of content. By using the information inherent in the composition, at least a plurality of types (n types) of atoms selected in advance, the content of N _TOTAL = 2 ⁿ −1 can be reached only by changing the content. It is possible to give a sign. For example, the label of the kind reaching N _TOTAL = 2 ⁿ −1 corresponds to a numerical value of at least binary n digits when a plurality of types (n types) of atoms selected in advance are associated with each digit. It has become.

In addition, since the label attached to the target object uses information inherent in the composition, the number of types of atoms (n) to be selected in advance is appropriately selected according to the total number of target types of labels. It is desirable to do. For example, if the number (n) of atoms selected in advance is 5 or more, the total number of composition types N _TOTAL is at least N _TOTAL = {2 ⁵ −1 with the presence or absence or the level of content as an index. }, That is, it is possible to apply to 31 or more types as the total number of target marker types. For example, in identifying various bacteria, gene information unique to each bacterium, for example, a base sequence of rRNA can be used. In order to select the coding region on the genomic gene corresponding to the base sequence of rRNA, it is necessary to perform a DNA hybridization assay using DNA probes having various base sequences. More specifically, in the identification of infectious disease-causing bacteria using a DNA hybridization assay, the types of DNA probes used often exceed 31 types. In that case, by applying the labeling method according to the present invention to a fine-particle base material on which each DNA probe is immobilized, the number of types (n) of the atoms selected in advance is 8 or more, so that there are more than 255 types. Can be attached. In addition, single nucleotide polymorphisms (SNPs) found in human genome genes have been reported to greatly exceed 255 types. Regarding DNA probes used for detection of individual single nucleotide polymorphisms It is desirable to make a distinction according to the label attached to the particulate base material used for the immobilization. For example, if the number of types of atoms (n) selected in advance is 12 or more, it is possible to attach 4095 or more types of labels, and single nucleotide polymorphisms (SNPs) found in human genome genes Signs corresponding to a substantial portion of can be provided. Furthermore, if the number of types of atoms (n) to be selected in advance is 16 or more, it is possible to attach 65535 or more types of labels, for example, a huge amount corresponding to all genes existing in the human genome. Each number of DNA probes can be provided with a unique label. As described above, in the labeling method according to the present invention, the number of types (n) of atoms to be selected in advance, which is used for expressing the label information, is appropriately increased or decreased in accordance with the total number of target label types. It can be used for various assays, and various probe molecules can be distinguished from each other by a label attached to a particulate substrate used for immobilization. Extensibility to support various applications.

  For example, in the technique disclosed in Japanese Patent Laid-Open No. 1-95800, the microbeads that immobilize each DNA probe are used as fluorescent particles by using individual metal elements to form labeled particles. Thus, a technique for analyzing one kind of metal element and specifying the kind of each DNA probe is disclosed. This labeling method is a form in which one kind of metal element such as Cr, Fe, Zn, Ba, Ti or the like is used as one-character labeling information as a labeling device. Accordingly, as in the labeling method according to the present invention, for example, label information corresponding to binary n-digit numerical information using a plurality of types (n types) of atoms selected in advance and using the content level as an index. Is different from the technique of notation in the technical idea. Further, in the technique disclosed in Japanese Patent Laid-Open No. 7-83927, ultrafine particles for immobilizing probe molecules are prepared using a plurality of types of inorganic fluorescent materials, and the characteristic fluorescence of each inorganic fluorescent material is produced. A technique for observing and identifying the type of each probe molecule is disclosed. In this labeling method, the characteristic fluorescence of each of a plurality of types of inorganic fluorescent materials is used as labeling information for one character. Accordingly, as in the labeling method according to the present invention, for example, label information corresponding to binary n-digit numerical information using a plurality of types (n types) of atoms selected in advance and using the content level as an index. Is different from the technique of notation in the technical idea.

In more detail, if the features of the technical idea are described, in the labeling method according to the present invention, it is apparent that even if the five types of atoms are selected in advance and used for labeling. The essence is that, for example, 16 types are selected as a plurality of types (n types) of atoms selected in advance, and each atom is assigned to a corresponding digit, for example, corresponding to numerical information of binary 16 digits. In addition, among them, only five types of atoms are arbitrarily selected, and {2 ⁵ -1} types are used as a subset with the presence or absence of the selection as an index. That is, it is a form that can be expanded to a sufficiently wide application range in advance, but uses a partial subset thereof depending on the application range.

  On the other hand, in the object labeling method according to the present invention, the shape and size of the object itself can be arbitrarily selected as long as the information on the content regarding a specific atom can be used as a label. For example, when targeting fine particles used for immobilizing various probe molecules, it is preferable to use irregular fine particles such as needles, rods, and irregularities, and regular fine particles such as spheres and squares. In addition, the labeling method for an object according to the present invention can also be suitably applied to a fibrous or sheet-like object as a substrate used for immobilizing various probe molecules. In addition, the material of the fine particles used for immobilization of various probe molecules can be a magnetic material. When the form of such magnetic fine particles is selected, the liquid phase is in a state where the various probe molecules are immobilized. Separation operation using magnetic force can be applied from the inside.

  The base material used for immobilizing various probe molecules is desirably a size that can be uniformly dispersed in the liquid phase. That is, a fine size that is advantageous for dispersibility in the liquid phase is usually selected. For example, in the case of fine particles, it is desirable to select a size corresponding to the particle size in the range of 1 nm to 10 μm. In addition, when the base material is in the form of a fiber or each piece, it is desirable to select the size in at least two directions of the vertical, horizontal, and height in the range of 1 nm to 10 μm. In addition, it is preferable that the size lower limit is selected to be 100 nm or more in detecting the application of the cytometry method.

  For example, when the method for labeling an object according to the present invention is applied to fine particles that can be used for immobilizing various probe molecules, the composition of the materials constituting the fine particles themselves is changed variously, and a plurality of preselected materials are selected. With regard to the species (n types) of atoms, the presence or absence of the species or the level of the content may be different, and a form of labeling may be employed. Dry method of mixing, firing and grinding raw materials adjusted to the desired composition ratio, coprecipitation method of obtaining coprecipitates containing multiple types of atoms from solutions adjusted to the desired composition ratio, dissolving raw materials, spray pyrolysis A spray cracking method for breaking up is available. For example, a plurality of types (n types) of metal atoms selected in advance using an oxide superconducting material fine particle manufacturing method disclosed in JP-A-64-51222 are contained in the form of an oxide. It can be formed as oxide fine particles. Further, using a method for producing ferrite particles using a coprecipitation method disclosed in Japanese Patent Application Laid-Open No. 2002-128523, a plurality of metal elements selected in advance for a plurality of types (n types) of metal atoms. It is possible to form as ferrite particles containing.

  Furthermore, coating fine particles obtained by adding a coating layer to the core fine particles formed of various materials are configured, and the composition of the material of the coating layer is changed variously to select a plurality of types (n With regard to the atoms of the species, the presence or absence of the atoms or the level of the contents may be different, and a form of labeling may be employed. For example, a coating layer having a desired composition can be formed by using the following means by using particles such as glass, plastic, silicon, and metal as the core fine particles. For example, as a dry process, a PVD (Physical Vapor Deposition) method such as vacuum evaporation, ion plating, sputtering, or a CVD (Chemical Vapor Deposition) method is disclosed in Japanese Patent Application Laid-Open No. 07-053271. Coating layer formation using a medium dispersion method can be used. Furthermore, instead of the coating layer, a desired element can be introduced from the surface of the core fine particle using an ion implantation method, and the surface layer composition can be changed to form the surface layer.

  In addition, in the method for labeling an object according to the present invention, a plurality of types (n types) of atoms selected in advance included in the composition component constituting the labeling device can be identified with high accuracy, and There is no particular limitation as long as the content can be measured with good reproducibility. However, there are certain restrictions on the elements that can be used from the viewpoint of maintaining the performance required for the target object itself, for example, the magnetic properties. In addition, some elements cannot be used in principle depending on the manufacturing method. Therefore, it is preferable to select a plurality of types (n types) of atoms to be used as appropriate in consideration of the performance required for the target object itself and the manufacturing method. At that time, for example, it is preferable to select a typical metal element or a transition metal element that satisfies the above conditions.

  As an example, as an example of a combination of plural kinds of atoms to be used, for example, in the case of 8 kinds, Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, in the case of 12 kinds, Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag, Pd, and in the case of 16 types, Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag , Pd, V, Cr, Ru, and Rh.

As a method for analyzing and detecting the labeled object according to the present invention, the object labeling method according to the present invention, that is, presence or absence of a plurality of types of preselected atoms used as a labeling device. Alternatively, any analysis method can be used as long as it can identify the content level. For example, mass spectrometry such as time-of-flight secondary ion mass spectrometry (TOF-SIMS), X-ray photoelectron spectroscopy (XPS), or Auger electron spectroscopy (AES) can be suitably used. Furthermore, if these methods capable of two-dimensional imaging are used, it can be used as a detection method when a plurality of different particles exist two-dimensionally.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, although these Examples are examples of the best embodiment concerning this invention, this invention is not limited to the form shown in this Example.

Example 1
Based on the method for producing fine ferrite particles described in JP-A-2002-128523, a solution containing divalent iron ions as an essential component and other various metal element ions constituting the target ferrite is added. Utilizing and oxidizing the divalent iron ions to trivalent iron ions using an oxidizing agent, ferrite fine particles are prepared. In addition to the main component iron ions, the ferrite fine particles to be prepared can be selected from various metal elements in addition to the main component metal element iron by selecting the type and concentration of metal element ions added to the raw material solution. In a desired content ratio.

In this embodiment, in addition to iron as a main component metal element constituting ferrite, at least eight kinds of metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti are selected as a trace component metal element. Ferrite fine particles to which one or more metal elements are added and ferrite fine particles to which none of these eight metal elements are added are prepared. That is, at least the ferrite fine particles to which one or more metal elements selected from eight metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti are added are among the eight metal elements. Depending on the type of metal element contained, it is classified as (2 ⁸ −1) type = 255 type. Furthermore, ferrite fine particles to which none of these eight kinds of metal elements are added are added, and the total number of ferrite fine particles is 256 based on the presence or absence of each of the eight kinds of metal elements in the composition. being classified.

  In the present example, the concentration of each metal element ion added as a trace component was the same in the solution containing a plurality of metal element ions used when producing the ferrite fine particles. Further, the particle diameter of the finally produced ferrite fine particles was approximately 500 nm.

  The prepared ferrite fine particles are separated from each reaction solution and washed with ultrapure water. In the recovery / separation operation of the ferrite fine particles such as recovery / separation from the reaction solution and recovery after washing, a technique of collecting ferrite particles using a magnet is used. A total of 256 types of washed ferrite fine particles are suspended in ultrapure water, respectively, to give suspensions having a dispersion concentration of about 5.5 mg / mL (about 500 particles / μL).

  About 1 μl of each of the obtained ferrite fine particle suspensions, total 256 species, is collected and mixed, and then the ferrite fine particles contained therein are collected once and resuspended in 100 μl of pure water. 1 μl of this resuspension is placed on a copy paper. Spot and dry. Next, the copy paper is appropriately cut out, and the metal element composition contained in the existing ferrite fine particles is measured for each spotting portion. In this example, a TOF-SIMS device (manufactured by ION-TOF: TOF-SIMS IV), an XPS device (manufactured by JEOL: JPS-9200), an AES device (manufactured by JEOL: JAMP-9500F) are used. Measurement of spectra derived from nine types of metal elements such as Fe, Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti, while two-dimensional imaging of each spotting region is performed using three types of measurement methods. I do.

As a result of the two-dimensional imaging, an image of each ferrite fine particle existing region (spot region) is obtained from each spotting site based on the spectrum derived from the main component metal element in any of the three types of measurement methods. At the same time, the spectrum derived from the trace component metal element contained in each ferrite fine particle is not subject to interference caused by other component metal elements, and at least the trace component metal elements: Co, Ni, Mn, Zn, It was confirmed that individual characteristic spectra of Cu, Mg, Al, and Ti can be measured. That is, eight metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti are selected as traceable metal elements that can be added to the main component metal element Fe contained in the ferrite fine particles. , the presence or absence of the content of the eight metallic element, by subjecting a label corresponding to binary 8-digit numerical information, it was confirmed to be possible determine the ferrite particles of the total of 256 species ⁽²⁸ species).

Next, among the 256 types of ferrite fine particles, fine particles labeled with “Ni, Mn, Zn, Cu, Mg, Al, Ti” and labeled with “Co, Mn, Zn, Cu, Mg, Al, Ti”. For two types of fine particles, oligonucleotide probes were bound by the following steps.
(1) An appropriate amount of fine particles was placed in a 0.5 ml microtube, 100 μl of ethanol was added and shaken and stirred, and then ethanol was removed using a separator using the magnet described above, and the fine particles were collected.
(2) A 1 wt% aqueous solution of an amino group-bonded silane coupling agent, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane KBM603 (Shin-Etsu Chemical Co., Ltd.) is stirred at room temperature for 2 hours, and the silane The methoxy group in the molecule of the compound was hydrolyzed. Subsequently, the fine particles obtained in (1) above were immersed in 100 μl of this solution at room temperature for 1 hour, washed with pure water, collected, and dried. Next, it was baked in an oven heated to 120 ° C. for 1 hour to finally introduce amino groups onto the surface of the fine particles.
(3) 2.7 mg of N- (6-maleimidocaproyloxy) succinimide (EMCS) was dissolved in a 1: 1 mixed solution of dimethyl sulfoxide (DMSO) / ethanol to a concentration of 0.3 mg / ml. The fine particles subjected to the silane coupling treatment were immersed in 100 μl of the EMCS solution at room temperature for 2 hours to react the amino group supported on the substrate surface by the silane coupling treatment with the succinimide group of the EMCS solution. At this stage, an EMCS-derived maleimide group is present on the surface of the fine particles. The substrate pulled up from the EMCS solution was sequentially washed with a mixed solvent of DMSO and ethanol and ethanol, and then dried under reduced pressure.
(4) Requesting a DNA synthesizer (Vex Co., Ltd.) to synthesize two types of nucleic acid, a single-stranded nucleic acid having the base sequence of SEQ ID NO: 1 and a single-stranded nucleic acid having the base sequence of SEQ ID NO: 2. did. A thiol (SH) group was introduced into the 5 ′ end of each single-stranded DNA by using a thiol modifier (Glen Research) at the time of synthesis. Deprotection and DNA recovery were performed by conventional methods, and purification was performed using HPLC. All the series of steps from synthesis to purification were performed by a synthesizer.
SEQ ID NO: 1
5 'HS- (CH2) ₆ -O-PO ₂ -O-ACTGGCCGTCGTTTTACA 3'
SEQ ID NO: 2
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-CGTACGATCGATGTAGCTAGCATGC 3'
(5) The single-stranded DNA is dissolved in 50 mM phosphate buffer (pH 7.0) at a concentration of 8 μM, and the microparticles obtained in (3) are immersed in 100 μl of each solution for 1 hour at room temperature. The maleimide group on the microparticles and the thiol group of the nucleic acid were reacted to bind the nucleic acid as a probe on the microparticle. Thereafter, it was appropriately washed with the above-mentioned phosphate buffer and stored as it was at 4 ° C. in the buffer until it was used next time.

The nucleic acid-binding fine particles were appropriately washed with pure water, recovered and dried under reduced pressure, and then observed with a TOF-SIMS apparatus based on the above-described method. As a result, for the two types of ferrite fine particles, in addition to the atom as the label, each of the ion ions of PO ⁻ , PO ₂ ⁻ , PO ₃ ⁻ and the fragment ions of the phosphoric acid moiety of the nucleic acid Strongly observed. Among the observed ions, PO ₂ ⁻ and PO ₃ ⁻ were particularly strongly observed. In addition, when the nucleic acid concentration at the time of binding the nucleic acid to the microparticles was changed and reacted, and the PO ₂ ⁻ and PO ₃ ⁻ ions were similarly observed with TOF-SIMS, the nucleic acid concentration was linear within a certain range of the nucleic acid concentration. Proportional ionic strength was observed.

  From the above, according to the present invention, the probe-bound microparticles provided with atomic labels and the probes bound to the microparticles, and the detection and identification of the probes on the microparticles are relative, but quantitative. It was shown to be possible.

  Next, as a model target nucleic acid, a single-stranded nucleic acid having a base sequence complementary to each of the base sequences of the above-mentioned nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2 as SEQ ID NO: 3 and SEQ ID NO: 4 Synthesized.

A (Br) represents adenylic acid having a bromine atom bonded thereto, and this part is an automatic synthesizer using 8-bromo-3′-deoxyadenosine phosphoramidite (Glen Research) whose structure is shown in the figure below. It was introduced at the time of synthesis. Deprotection and DNA recovery were performed by conventional methods, and purification was performed using HPLC. A series of steps from synthesis to purification was similarly performed by a synthesizer.
SEQ ID NO: 3
5 'TGTA (Br) A (Br) A (Br) A (Br) CGA (Br) CGGCCA (Br) GT 3'
SEQ ID NO: 4
5 'GCA (Br) TGCTA (Br) GCTA (Br) CA (Br) TCGA (Br) TCGTA (Br) CG 3'

  These nucleic acids were dissolved in 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl at a concentration of 50 nM, and 100 μl thereof was dissolved in the probes of SEQ ID NO: 1 and SEQ ID NO: 2. An appropriate amount of particles to which nucleic acid was bound was mixed and dispersed, and allowed to stand at 40 ° C. for 16 hours, and hybridization between the probe nucleic acid on the microparticles and the target nucleic acid was attempted. Thereafter, after washing with the above buffer solution and then with pure water at room temperature, the fine particles were collected and dried under reduced pressure.

Next, the microparticles subjected to hybridization were observed with a TOF-SIMS apparatus based on the above-described method. As a result, the atom as a label attached to the fine particle, the fragment ion derived from the nucleic acid probe and the target nucleic acid, and the Br ⁻ ion derived from the target nucleic acid are observed from the ferrite fine particle hybridized with the complementary strand. It was done. Incidentally, Br ^- ions as isotopic ions present in nature, ⁷⁹ Br ^{-, 81} Br ^- ions both was observed at approximately the same ionic strength. In addition, from ferrite particles hybridized with non-complementary nucleic acids, atoms as a label and fragment ions derived from nucleic acid probes were observed, but Br ⁻ ions were not observed. Moreover, when the target nucleic acid concentration at the time of hybridization was changed and reacted, and observed with the TOF-SIMS apparatus, Br ⁻ ion intensity linearly proportional to the target nucleic acid concentration was observed within a certain range of the target nucleic acid concentration. It was.

  Thus, the target nucleic acid of the target nucleic acid that specifically binds (hybridizes) to the nucleic acid probe bound to the probe-bound microparticle provided with the label of the atom of the present invention is contained in the target nucleic acid, It was shown that detection, identification, and relative quantification were possible using atoms not contained in the probe bound to the microparticles.

  In addition, arbitrary probes can be couple | bonded with 256 types of ferrite fine particles referred to in the present Example, and it can use for the detection of the target substance couple | bonded specifically with this probe.

(Example 2)
In Example 2, in addition to iron as a main component metal element constituting ferrite, as trace component metal elements, eight kinds of metal elements such as Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti are used. Further, ferrite fine particles to which one or more metal elements selected from a total of 12 kinds of metal elements, including four kinds of metal elements of Ga, Ge, Ag, and Pd, are added, and these 12 kinds of metal elements are: Ferrite fine particles to which none of them are added are prepared. That is, ferrite fine particles to which at least one metal element selected from 12 metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag, and Pd is added. Among the 12 types of metal elements, (2 ¹² −1) types = 4095 types are classified according to the types of metal elements contained. Further, ferrite fine particles to which none of these 12 kinds of metal elements are added are added, and the ferrite fine particles to be prepared have a total of 4096 kinds based on the presence or absence of each of the 12 kinds of metal elements in the composition. being classified.

  Also in this example, the concentration of each metal element ion added as a trace component was the same in the solution containing a plurality of metal element ions used when producing the ferrite fine particles. However, the content of each of the 12 metal elements with respect to the Fe content of the main component metal element is reduced to 8/12 of the ratio in Example 1. Further, the particle diameter of the finally produced ferrite fine particles was approximately 500 nm.

  The prepared ferrite fine particles are separated from each reaction solution and washed with ultrapure water. In the recovery / separation operation of the ferrite fine particles such as recovery / separation from the reaction solution and recovery after washing, a technique of collecting ferrite particles using a magnet is used. A total of 4096 kinds of washed ferrite fine particles are suspended in ultrapure water, respectively, to give suspensions having a dispersion concentration of about 5.5 mg / mL (about 500 particles / μL).

  About 1 μl of each of the resulting ferrite fine particle suspensions, totaling 4096 types, was collected and mixed, and then the ferrite fine particles contained therein were collected once and resuspended in 100 μl of pure water. Spot and dry. Next, in the same measurement procedure and conditions as in Example 1, the copy paper is appropriately cut out and the metal element composition contained in the existing ferrite fine particles is measured for each spotting portion.

As for the ferrite fine particles of Example 2, as a result of two-dimensional imaging, in any of the three types of measurement methods, each of the ferrite fine particle existing regions (spot regions) is based on the spectrum derived from the main component metal element from each spotting site. An image is obtained. At the same time, the spectrum derived from the trace component metal elements contained in each ferrite fine particle is not affected by interference from other component metal elements, and at least the trace component metal elements:
It was confirmed that characteristic spectra of each of Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag, and Pd can be measured. That is, for the main component metal element Fe contained in the ferrite fine particles, the trace component metal elements that can be blended are Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag, and Pd. select the 12 metal elements, the presence or absence of the content of the 12 bimetallic element, by subjecting a label corresponding to binary 8-digit numerical information, it can determine the ferrite particles of the total 4096 species (2 ¹² kinds) It was confirmed that there was.

  From the above results, in addition to the main component metal element iron constituting the ferrite, as a trace component metal element, when further increasing the number of types of metal elements that can be selected, for any of these multiple types of metal elements, Various types of detection means can be applied to measure the characteristic spectrum of each metal element, and as long as its content can be evaluated, it is possible to increase the types of labels that can be attached to ferrite fine particles. It is verified that

  Next, among the 4096 types of ferrite fine particles, fine particles labeled with “Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag”, and “Ni, Mn, Zn, Cu, For two types of microparticles labeled with “Mg, Al, Ti, Ga, Ge, Ag, Pd”, oligopeptide nucleic acids as probes were bound in the same manner as in Example 1. However, the maleimide group on the fine particle of the probe used for binding and the thiol group involved in binding bind a cysteine residue having no nucleobase to the amino terminus of the oligopeptide nucleic acid (the 5 ′ end in the nucleic acid probe). Introduced by. The base sequence is identical to SEQ ID NO: 1 and SEQ ID NO: 2. The oligopeptide nucleic acid was also synthesized by Bex Corporation.

  When the microparticles bound with the oligopeptide nucleic acid probe were observed with a TOF-SIMS apparatus, nucleobase fragments derived from the oligopeptide nucleic acid probe were observed in addition to the atoms as the label of the microparticle. Other fragment ions of the oligopeptide nucleic acid probe were not observed or could not be distinguished from aminosilane coupling agents or fragment ions derived from EMCS.

  Next, hybridization was performed in the same manner as in Example 1 on the two types of ferrite particles to which the oligopeptide nucleic acid probe was bound. However, the prepared complementary strand has the same base sequence as SEQ ID NOs: 3 and 4, but has no bromine atom bonded thereto.

When the ferrite fine particles after hybridization were observed with a TOF-SIMS apparatus, only from the ferrite fine particles hybridized with the complementary strand, in addition to the atoms as the fine particle labels, the target nucleic acid derived fine particles themselves, In addition, PO ⁻ , PO ₂ ⁻ and PO ₃ ⁻ ions which were not contained in the oligopeptide nucleic acid were observed.

Further, as in Example 1, the reaction was carried out by changing the target nucleic acid concentration at the time of hybridization, and observed with a TOF-SIMS device, and was linearly proportional to the target nucleic acid concentration within a certain range of the target nucleic acid concentration. PO ₂ ⁻ and PO ₃ ⁻ intensities were observed.

  From these results, in the case of the oligopeptide nucleic acid probe and the unlabeled target nucleic acid, the usefulness of the present invention was shown as in Example 1.

  An arbitrary probe can be bound to the 4096 types of ferrite fine particles referred to in the present embodiment and used for detection of a target substance that specifically binds to the probe.

(Example 3)
Also in Example 3, in addition to iron as a main component metal element constituting ferrite, a trace component metal element is selected from eight metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti. Ferrite fine particles to which one or more metal elements are added and ferrite fine particles to which none of these eight metal elements are added are prepared. In Example 3, in the solution containing a plurality of metal element ions used when producing the ferrite fine particles, regarding the concentration of each metal element ion added as a trace component, two types of Ni and Zn are used. Two levels of the same concentration as in Example 1 and 1/10 of the concentration were used.

That is, at least ferrite fine particles to which one or more metal elements selected from eight metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti are added are Co, Mn, Cu, Mg, Among the six kinds of metal elements of Al and Ti, the kind of metal element contained (number of cases 2 ⁶ ), and the content level concerning two kinds of Ni and Zn (each three levels: 0, 1/10, According to the difference of 1) (number of cases 3 ² ), (2 ⁶ × 3 ² −1) types = 575 types are classified. Further, ferrite fine particles to which none of these eight kinds of metal elements are added are added, and the total number of 576 kinds of ferrite fine particles is based on the content level of each of the eight kinds of metal elements in the composition. It is classified as (2 ⁶ × 3 ² types). Also in this example, the particle diameter of the finally produced ferrite fine particles was approximately 500 nm.

  About 1 μl is collected from each of the obtained ferrite fine particle suspensions (total 575 types), spotted on a copy paper and dried. Next, in the same measurement procedure and conditions as in Example 1, the copy paper is appropriately cut out and the metal element composition contained in the existing ferrite fine particles is measured for each spotting portion.

As for the ferrite fine particles of Example 3, as a result of two-dimensional imaging, in any of the three kinds of measurement methods, each of the ferrite fine particle existing regions (spot regions) is based on the spectrum derived from the main component metal element from each spotting site. An image is obtained. At the same time, the spectrum derived from the trace component metal element contained in each ferrite fine particle is not subject to interference caused by other component metal elements, and at least the trace component metal elements: Co, Ni, Mn, Zn, Cu, Mg, Al, Ti, Ga, Ge, Ag, Pd can be measured individually characteristic spectrum, in addition, the difference in content level of Ni and Zn can be clearly distinguished It was confirmed that there was. That is, eight metal elements of Co, Ni, Mn, Zn, Cu, Mg, Al, and Ti are selected as traceable metal elements that can be added to the main component metal element Fe contained in the ferrite fine particles. , Due to the difference in the content level of the eight metal elements, it exceeds the binary 8-digit numerical information, for example, by attaching labels corresponding to the numerical information consisting of 6-digit binary and 2-digit binary, a total of 576 types it was confirmed to be possible determine the ferrite particles of ^{(2 6} × 3 2 kinds).

From the above results, in addition to iron as a main component metal element constituting ferrite, as a trace component metal element, any of the n types of metal elements can be selected with respect to the number of selectable metal elements (n). As long as it is possible to apply various detection means to measure a characteristic spectrum of each metal element and evaluate not only the presence / absence of the metal element but also multiple levels of content (number of levels M), for example, It is verified that it is possible to attach a label corresponding to numerical information of M-digit n-digit numbers to the ferrite fine particles. Next, among the 576 types of ferrite particles, the eight Ni atoms of “Co, Ni, Mn, Zn, Cu, Mg, Al, Ti” are not contained, and the same concentration as in Example 1 and the 1 Nucleic acid probes were bound by the following method for two levels of / 10 concentration, a total of three types of microparticles.
(1) In the same manner as in Example 1, treatment with an aminosilane coupling agent and EMCS was performed to introduce maleimide groups on the surface of the fine particles.
(2) A 0.5 mg / ml 1-thioglycerol (Sigma-Aldrich) aqueous solution was prepared, and an appropriate amount of the three types of ferrite fine particles was immersed in 100 μl to introduce hydroxyl groups on the surface of the fine particles.
(3) Using the nucleic acid automatic synthesizer, in addition to the above-mentioned SEQ ID NO: 1 and SEQ ID NO: 2, the following nucleic acids of SEQ ID NO: 5 are sequentially used as probe nucleic acids for the three types of ferrite fine particles introduced with hydroxyl groups. Synthesized.
SEQ ID NO: 5
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 3'
The synthesis was performed by requesting BEX Co., Ltd. All the synthesis protocols were carried out by standard methods.

The fine particles to which these three kinds of nucleic acids were bound were observed with a TOF-SIMS apparatus. As a result, for three types of ferrite fine particles, the atoms as the labeling elements including the three levels of Ni atoms could be detected, respectively, and in addition, PO ⁻ and PO ₂ ⁻ which are fragment ions at the phosphate site of the nucleic acid. , PO ₃ ⁻ ions, and fragment ions of the base were strongly observed. From the microparticles to which the probe of SEQ ID NO: 5 was bound, only thymine-derived fragment ions were observed as base-derived fragment ions. Among the ions, PO ₂ ⁻ and PO ₃ ⁻ were particularly strongly observed.

  From the above, according to the present invention, probe-bound microparticles provided with a labeling atom containing atoms existing over three levels, discrimination of the probe bound to the microparticles by solid-phase sequential synthesis, and the probe on the microparticles It was shown that detection and identification are possible.

Next, hybridization was performed in the same manner as in Example 1 on the three types of ferrite particles to which the nucleic acid probe was bound. The prepared complementary strand is the following SEQ ID NO: 6 which is complementary to SEQ ID NO: 5 in addition to SEQ ID NO: 3 and SEQ ID NO: 4.
SEQ ID NO: 6
5 'A (Br) A (Br) A (Br) A (Br) A (Br) AAAAAAAAAAAAAAAAAAAAAAAAAA 3'
As for 5 bases on the 5 ′ end side of SEQ ID NO: 6, a bromine atom is bonded as in SEQ ID NO: 3 and SEQ ID NO: 4.

Next, the microparticles subjected to hybridization were observed with a TOF-SIMS apparatus based on the above-described method. As a result, it was possible to detect the atomic atom as a label attached to the fine particles including the three levels of Ni atoms from the ferrite fine particles hybridized with the complementary strand. In addition, the fragment ions derived from the nucleic acid probe and the target nucleic acid, and ⁷⁹ Br ⁻ and ⁸¹ Br ⁻ ions derived from the target nucleic acid were observed. Further, from the ferrite particles hybridized with the non-complementary nucleic acid, atoms as a label and fragment ions derived from the nucleic acid probe were observed, but ⁷⁹ Br ⁻ and ⁸¹ Br ⁻ ions were not observed. .

Further, when the reaction was carried out by changing the target nucleic acid concentration at the time of hybridization and observed with a TOF-SIMS apparatus, ⁷⁹ Br ⁻ , ⁸¹ Br ⁻ that were linearly proportional to the target nucleic acid concentration within a certain range of the target nucleic acid concentration. Ionic strength was observed.

  Thus, the target nucleic acid contains a target nucleic acid that specifically binds (hybridizes) to the nucleic acid probe bound by solid phase sequential synthesis to the probe-bound microparticles to which the label of the present invention is attached. It was shown that detection, identification and relative quantification were possible using atoms not contained in the microparticles or probes bound to the microparticles.

  An arbitrary probe can be bound to the 575 types of ferrite fine particles referred to in this example, and used for detection of a target substance that specifically binds to the probe.

Example 4
Nature Biotechnology Vol. No. 18, 438, 2000, two cell lines of human oral squamous cell carcinoma, production of oligonucleotide chip for detecting mutation of exon 7 of genomic DNA of HSC4 and HSC5, and the above exon using this chip For the detection of fluorescently labeled nucleic acids derived from. In this example, in accordance with the above method, ferrite fine particles to which oligonucleotide probes were bound were prepared, the target nucleic acid was synthesized by changing the label from fluorescence to bromine, and further hybridization was performed using this target nucleic acid.

The specific procedure is described below.
SEQ ID NO: 7
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-GATGGGCCTCCGGTTCAT 3'
SEQ ID NO: 8
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-GATGGGCC A CCGGTTCAT 3'
SEQ ID NO: 9
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-GAT C GGCC A CCGG A TCAT 3'
A base sequence complementary to a part of the base sequence of exon 7 of HSC4 (part including codon No. 248) and having a thiol group for binding to the substrate at the 5 ′ end, SEQ ID NO: Seven single-stranded nucleic acids were synthesized in the same manner as in Example 1 using the probe nucleic acid. In addition, while SEQ ID NO: 7 is completely complementary, a single-stranded nucleic acid having single base and three base mismatch sites (underlined nucleotides) with respect to the target base sequence was also synthesized ( SEQ ID NO: 8, SEQ ID NO: 9), respectively.

From 256 kinds of ferrite fine particles labeled with the atomic group of Co, Ni, Mn, Zn, Cu, Mg, Al, Ti of Example 1, “Ni, Mn, Zn, Cu, Mg, Al, Ti”, Fine particles labeled with “Co, Ni, Mn, Zn, Cu, Mg, Al” and “Co, Ni, Mn, Cu, Mg, Al, Ti” are selected in the same manner as in Example 1, The single-stranded nucleic acids of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 were bound to each other. Discrimination of the produced probe-bound ferrite fine particles as particles and confirmation of the bound probe were performed by the method of Example 1.
SEQ ID NO: 10
E7S: 5 '-ACTGGCCTCATCTTGGGCCT- 3' (exon 7, sense)
SEQ ID NO: 11
E7A: 5 '-TGTGCAGGGTGGCAAGTGGC-3' (exon 7, anti-sense)

5-Bromo-2′-deoxyuridine triphosphate (Br-dUTP)
Next, the exon 7 portion was amplified from the genome of HSC4 by PCR reaction using the PCR primers of SEQ ID NOS: 10 and 11 (requested for synthesis from Bex Corporation). That is, PCR amplification was performed by repeating a cycle of 94 ° C. (30 sec) and 60 ° C. (45 sec) 40 times with 50 μl of a PCR mixture containing 20 ng of genomic DNA and 0.4 μM of each sense and antisense primer. The resulting chain length is designed to be 171 nucleotides.

  Next, using a part of the amplification product as a template, 0.2 μM sense primer (SEQ ID NO: 10) and 5-bromo-2′-deoxy, which is a kind of bromine-labeled nucleotide whose structure is shown in the above figure. Ursine triphosphate (Sigma Aldrich) was added to normal 2′-deoxyuridine triphosphate 10 μM at a concentration of 1/10, and ssPCR (single-stranded PCR) was performed. PCR cycles are 96 ° C. (30 sec), 50 ° C. (30 sec), 60 ° C. (4 min), and 25 cycles. The other three deoxynucleotide triphosphates were used at a concentration of 10 μM. The obtained bromine-labeled single-stranded DNA was purified by gel filtration.

Hybridization was performed by the same method as in Example 1 using the above-described three nucleic acid probe-bound ferrite fine particle chips and the above ssPCR product. However, the concentration of the ssPCR product was 10 nM, and the buffer was heated at 80 ° C. for 10 min using 6 × SSPE (0.9 M NaCl, 60 mM NaH ₂ PO ₄ , 6 mM EDTA) containing 20% formamide, Hybridization was performed at 45 ° C. for 15 hours, and then a washing operation was performed at 55 ° C. using 2 × SSPE. Next, it was appropriately washed with pure water (room temperature), and the ferrite particles were collected and dried under reduced pressure.

When the obtained ferrite fine particles were observed with a TOF-SIMS apparatus, ⁷⁹ Br ⁻ and ⁸¹ Br ⁻ ions were observed with strong ionic strength from fine particles in which a nucleic acid probe completely complementary to the target base sequence portion of the target nucleic acid was bound. Next, from the fine particles to which a nucleic acid probe having a single base mismatch is bound, ⁷⁹ Br ⁻ and ⁸¹ Br ⁻ ionic strengths of about 1/10 of the full match are observed, and a nucleic acid probe having a three base mismatch is bound. ⁷⁹ Br ⁻ and ⁸¹ Br ⁻ ions were not observed from the fine particles.

  Based on these results, it is shown that the probe-coupled microparticles of the present invention can detect and detect single-base mismatches by using the target cell nucleic acid labeling method with the incorporation label during PCR, in combination with the actual cell genome-derived gene. It was done.

  In addition, arbitrary probes can be couple | bonded with 256 types of ferrite fine particles referred to in the present Example, and it can use for the detection of the target substance couple | bonded specifically with this probe.

(Example 5)
SEQ ID NO: 12
_{E7S: 5 '- A (Br} ) CTGGCCTCA (Br) TCTTGGGCCT- 3' (exon 7, sense)
Instead of the incorporation label of Example 4, as a primer for ssPCR, bromine-labeled by the same method as the bromine-labeled model target nucleic acid of Example 1, the above SEQ ID NO: 12 (base sequence is the same as SEQ ID NO: 10) The target nucleic acid was labeled with single stranded nucleic acid.

  Other than that, hybridization and TOF-SIMS observation were performed in the same manner as in Example 4. As a result, although the ionic strength was generally weaker than that of Example 4, the same result as that of Example 4 was obtained as a tendency.

  These results indicate that the probe-coupled microparticles of the present invention can detect and detect single-base mismatches by using the target cell nucleic acid labeling method with primer labeling in the actual cell genome. It was done.

  By using the object labeling method according to the present invention, for example, a large number of minute objects such as a fine particulate substrate used for immobilization of probe molecules used in bioassays can be used as necessary. It is possible to attach a seed mark. In other words, by applying the object labeling method according to the present invention, a predetermined particle is immobilized on the surface of the labeled fine particle by using a fine particulate substrate that has been previously labeled. Thus, a label that can be used for discriminating the types of individual probe molecules can be easily applied to a large number of types of probe molecules fixed to a fine particle. The bioassay can be performed by using, for example, microparticles labeled by the labeling method.

Claims (36)

An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The sign is
Including at least two types of atoms selected from a plurality of types of preselected atoms;
The probe binding object, wherein the label is identified by at least one of mass spectrometry, X-ray photoelectron spectroscopy, and Auger electron spectroscopy. An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types (n) of a plurality of types of atoms selected in advance and the presence / absence of the content of each of the selected atoms, the composition conditions of contained atoms corresponding to numerical information of binary n digits are configured. And then
Using a indicator having a composition that satisfies the composition conditions of the contained atoms,
The probe coupled object, characterized by being attached with the discrete identification information expressed as the binary n-digit numerical information. An object combined with a probe,
The probe is coupled to the surface of the object;
The object is labeled,
The label attaches discrete identification information that makes it possible to discriminate each of the objects, that is, each of the probes, with respect to a plurality of objects to be labeled, that is, a plurality of probes coupled to the object.
Using the number of types (n) of a plurality of types of atoms selected in advance and the content level of each of the selected atoms, the composition conditions of the contained atoms corresponding to at least binary n-digit numerical information are configured And then
Using a indicator having a composition that satisfies the composition conditions of the contained atoms,
The probe coupled object, wherein the discrete identification information expressed as numerical information of at least binary n digits is attached. In the sign,
The number of types (n) of plural types of atoms selected in advance is 5 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are The probe coupling object according to claim 2 or 3, wherein a positive integer) is 31 or more. In the sign,
The number of types (n) of plural types of atoms selected in advance is 8 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are The probe coupling object according to claim 4, wherein a positive integer) is 255 or more. In the sign,
The number of types (n) of plural types of atoms selected in advance is 12 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are The probe coupling object according to claim 5, wherein a positive integer) is 4095 or more. In the sign,
The number of types (n) of plural types of atoms selected in advance is 16 or more,
Numerical information type N displayed by the number (n) of atoms and the content of each selected atom; N ≧ ΣnCm = 2 ⁿ −1 (1 ≦ m ≦ n, m, n are The probe coupling object according to claim 6, wherein a positive integer) is 65535 or more.   The probe coupled object according to any one of claims 1 to 7, wherein the shape of the object to be labeled is selected from amorphous fine particles, regular fine particles, fibers, or sheets.   The probe coupled object according to claim 8, wherein the object is a magnetic body. As the size of the object,
When the object is in the form of fine particles, the particle size equivalent size is in the range of 1 nm to 10 μm.
The object according to any one of claims 1 to 9, wherein when the object is in the shape of a fiber or a piece, at least two sizes are selected in the range of 1 nm to 10 µm among the length, width, and height. The probe coupling object according to claim 1.   Probe is nucleic acid, DNA, RNA, cDNA, cRNA, oligodeoxyribonucleotide, polydeoxyribonucleotide, oligoribonucleotide, polyribonucleotide, peptide nucleic acid (PNA), oligopeptide nucleic acid, peptide, oligopeptide, polypeptide, protein, antigen, The antibody, enzyme, ligand, ligand receptor, saccharide, sugar chain, or any other member capable of recognizing and binding to each other. Probe coupling object. A method for producing a probe-coupled object according to any one of claims 1 to 11,
Binding of the probe to the labeled object surface is
According to the probe binding object, characterized by sequentially supplying corresponding monomers to the surface of the object to which the probe is bound according to the base sequence of the probe, and synthesizing a nucleic acid chain by a solid phase synthesis method. Manufacturing method. A method for producing a probe-coupled object according to any one of claims 1 to 11,
Binding of the probe to the labeled object surface is
A method for producing a probe-binding object, comprising applying a nucleic acid chain synthesized and purified in advance according to the base sequence of the probe to the surface of the object. A method for discriminating the type of probe-bound object attached with the label according to any one of claims 2 to 11, that is, the type of probe bound to the object,
Corresponding to at least binary n-digit numerical information by using the number of types of atoms selected in advance (n) and the presence or absence of the content of each selected atom, or the content level, After configuring the composition conditions of the contained atoms,
A label attached to each object using a labeler having a composition that satisfies the composition conditions of the contained atoms,
For each of the plurality of types of preselected atoms, the plurality of types of preselected atoms in the composition of the labeler using means capable of detecting the presence or absence of the content or the level of the content. The presence or absence of the individual content of the atom, or the content level,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
A method for discriminating probe coupled objects, wherein the type of each probe coupled object is identified based on the discrete identification information expressed as numerical information of at least binary n digits. The probe-bound object according to claim 14, wherein mass spectrometry is used as means for detecting the presence or absence of the content of each of the plurality of types of preselected atoms or the content level. How to determine. 16. The method for discriminating a probe-bound object according to claim 15, wherein the mass spectrometry is time-of-flight secondary ion mass spectrometry (TOF-SIMS). Using X-ray photoelectron spectroscopy (XPS) or Auger electron spectroscopy (AES) as a means capable of detecting the presence or absence or the level of the content of each of the plurality of preselected atoms. The method for discriminating a probe coupled object according to claim 14, wherein: If the object is on a plane,
When identifying the presence or absence of the content of each of the atoms, or the level of content for the plurality of preselected atoms in the composition of the indicator,
To locate the object on the plane,
The method for discriminating a probe coupled object according to any one of claims 14 to 17, further comprising a step of performing two-dimensional imaging on a plane on which the object is located. The method according to claim 18, wherein the plurality of objects are located in different regions on the same plane. A method for identifying, detecting or quantifying a probe bound to the probe binding object according to any one of claims 1 to 11, comprising:
The type of object, i.e. the type of probe coupled to the object,
The label of the object, which is attached using the indicator, is used to detect the presence or absence of the content or the level of the content of each of the plurality of preselected atoms. With respect to the plurality of preselected atoms in the composition of the indicator, the presence / absence of the content of each of the atoms, or the content level is identified,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
Discriminating by identifying based on the discrete identification information expressed as numerical information of at least binary n digits,
Detection is performed by using, as an indicator derived from the probe, an atom, a molecule, or a substance that is included in the probe and not included in the object. Alternatively, a method for identifying, detecting or quantifying a probe bound to an object, characterized in that quantification is performed. 21. The probe bound to an object according to claim 20, wherein an atom that is used as an index when detecting or quantifying the probe and that is included in the probe and not included in the object is a phosphorus atom. Identification, detection or quantification method. A mass spectrometric method is used as a means capable of detecting the presence or absence of the content of each of the plurality of types of preselected atoms, or a content level, and a means for detecting an index derived from the probe. The method for specifying, detecting or quantifying a probe bound to an object according to claim 21. The method for identifying, detecting or quantifying a probe bound to an object according to claim 22, wherein the mass spectrometry is time-of-flight secondary ion mass spectrometry (TOF-SIMS). X-ray photoelectron spectroscopy (XPS) is used as a means capable of detecting the presence or absence or the level of the content of each of the plurality of preselected atoms, and a means for detecting an index derived from the probe. Alternatively, Auger electron spectroscopy (AES) is used. The method for identifying, detecting, or quantifying a probe bound to an object according to claim 20. If the object is on a plane,
In the composition of the labeler, regarding the plurality of preselected atoms, when identifying the presence / absence or level of the content of each of the atoms, and as a probe that binds to the surface of the object When detecting the derived index,
At least to locate the object on the plane,
The method for identifying, detecting or quantifying a probe bound to an object according to any one of claims 20 to 24, further comprising a step of performing two-dimensional imaging on a plane on which the object is located. 26. The method for identifying, detecting or quantifying a probe bound to an object according to claim 25, wherein the plurality of objects are located in different regions on the same plane. A method for detecting, identifying, or quantifying a target nucleic acid using a probe-binding object,
At least the following steps (1) and (2):
(1) a step of reacting the probe-binding object according to any one of claims 1 to 11 with a target substance to obtain a hybrid,
(2) Using at least binary n-digit numerical information by using the number of types (n) of a plurality of types of atoms selected in advance and the presence / absence or level of content of each of the selected atoms. After configuring the composition conditions of the corresponding atoms,
The label attached to the binding object on which the hybrid is formed using a labeling composition that satisfies the composition conditions of the contained atoms, the content of each of the plurality of types of preselected atoms. Using the means capable of detecting the presence or absence, or the level of the content, the presence or absence of the content of the individual atoms or the content of the plurality of types of the preselected atoms in the composition of the labeler Identify the level,
Discriminating the composition conditions of the contained atoms that are satisfied in the composition of the indicator, and extracting the corresponding at least binary n-digit numerical information;
If necessary, the method of claims 20 to 26 detects, identifies and quantifies the probe bound to the binding object on which the hybrid is formed,
At the same time or subsequently, the target substance forming the hybrid is detected and specified by using as an index the atoms, molecules or substances contained in the target substance and not included in the probe binding object and the probe. Quantifying;
A method for detecting, identifying and quantifying a target substance characterized by comprising:   28. The method according to claim 27, wherein the atoms included in the target substance and not included in the probe binding object and the probe are a halogen atom and a metal atom.   DATP, dGTP, dCTP, dTTP (or a target substance is a nucleic acid, the nucleic acid is obtained by a primer extension reaction based on a template nucleic acid, and a halogen atom or a metal atom is used for the primer or primer extension reaction. 28. The method of claim 27, wherein the method is bound to (dUTP), ddATP, ddGTP, ddCTP, ddTTP (or ddUTP) ATP, GTP, CTP, UTP.   The probe is a peptide nucleic acid or an oligopeptide nucleic acid, the target substance is a nucleic acid having a sugar phosphate backbone, and the atoms that are contained in the target substance and that are not contained in the probe-binding object and probe are phosphorus 28. The method of claim 27, wherein the method is an atom. For each of the plurality of types of preselected atoms, mass spectrometry is used as means for detecting the presence / absence or level of content, probe detection means, and target substance detection means. The method according to any one of claims 27 to 30.   The method according to claim 31, wherein the mass spectrometry is time-of-flight secondary ion mass spectrometry (TOF-SIMS).   Using X-ray photoelectron spectroscopy (XPS) or Auger electron spectroscopy (AES) as a means capable of detecting the presence or absence or the level of the content of each of the plurality of preselected atoms. 31. A method according to claim 27-30. If the object is on a plane,
When identifying the presence or absence of the content of each of the atoms or the level of content of the plurality of preselected atoms in the composition of the label, and detecting the labeling substance,
At least to locate the object on the plane,
The method according to any one of claims 27 to 33, further comprising a step of performing two-dimensional imaging on a plane on which the object is located.   The method of claim 34, wherein the plurality of objects are located in different regions on the same plane.   After a treatment that binds a substance that specifically binds to a plurality of objects to which a plurality of different probes are respectively bound, only the object to which the target substance that specifically binds is bound by the probe. On the other hand, after discriminating the object and the probe bound to the object, the probe is detected, identified and quantified as necessary, and further, the target substance bound to the probe is detected, identified and quantified. 36. The method according to any one of claims 27 to 35, wherein: