WO2023171738A1 - 解析システム、プレート、および解析方法 - Google Patents

解析システム、プレート、および解析方法 Download PDF

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Publication number
WO2023171738A1
WO2023171738A1 PCT/JP2023/008996 JP2023008996W WO2023171738A1 WO 2023171738 A1 WO2023171738 A1 WO 2023171738A1 JP 2023008996 W JP2023008996 W JP 2023008996W WO 2023171738 A1 WO2023171738 A1 WO 2023171738A1
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Prior art keywords
component
signal information
analysis
nucleic acid
plate
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English (en)
French (fr)
Japanese (ja)
Inventor
麻由香 加羽澤
弘志 北
俊平 一杉
孝敏 末松
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Konica Minolta Inc
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Konica Minolta Inc
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Priority to US18/843,058 priority Critical patent/US20250163508A1/en
Priority to CN202380026351.7A priority patent/CN118871772A/zh
Priority to JP2024506391A priority patent/JPWO2023171738A1/ja
Publication of WO2023171738A1 publication Critical patent/WO2023171738A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to an analysis system, a plate, and an analysis method.
  • the data used in the inductive interpretation method is data that is difficult to handle deductively using human thinking, such as scientific data whose basis is not sufficiently established, or data that has not been given sufficient meaning. Scientific data, etc. Furthermore, even if scientific data has a well-established basis or has sufficient meaning, it is difficult for human thinking to process it deductively due to its complexity or large amount of information. Scientific data is included in inductive data. Such inductive data is useful as data for machine learning, but the current rate limiting factor is that it generates a large amount of data.
  • Desirable requirements for such data for machine learning include being multidimensional and large amounts of data, being able to output it easily, and being able to link it to substances and states.
  • changes occur, such as the formation of ionic bonds and coordinate bonds, state changes such as association, aggregation, and crystallization, and quantum state changes such as excitation, transition, and relaxation.
  • the data obtained along with this is suitable as data for machine learning.
  • light and color information can be linked to physical or quantum state changes that occur due to reactions, interactions, complex formation, ionization, etc. of substances.
  • there are a wide variety of light and color measuring devices and they are capable of acquiring multidimensional and large amounts of data, as well as providing simple output.
  • High-throughput methods often use analytical instruments such as DNA microarrays in which single-stranded DNA is placed on various substrates, reacted with a sample having a complementary base sequence, and detected by fluorescence or electric current. This is a method of detecting a target substance using specific interactions, and is used to obtain information about a specific substance.
  • analytical instruments such as DNA microarrays in which single-stranded DNA is placed on various substrates, reacted with a sample having a complementary base sequence, and detected by fluorescence or electric current.
  • This is a method of detecting a target substance using specific interactions, and is used to obtain information about a specific substance.
  • reagents are mixed on a substrate, more complex chemical reactions, ionic reactions, complex formation reactions, interactions, energy transfer, etc. occur, and data with enhanced features related to substances and phenomena can be obtained.
  • the data generated by such reactions is often multidimensional data that integrates multiple reactions, and may not be understandable to humans. Multidimensional data such as the one described above cannot be solved using
  • reaction field that can be applied to such machine learning, where complex interactions and chemical reactions occur with multiple reaction systems, and for analysis methods using this field.
  • reaction field makes it possible to generate multidimensional, large amounts of data.
  • Non-Patent Document 1 describes that a two-dimensional colorimetric sensor is formed by printing a plurality of chemical reagents in a pattern at high density using an inkjet printer. It is described that after a sensor is fabricated, an analysis target is applied by a spray method, and the color of the sensor is subjected to machine learning. That is, in this technique, each region of the colorimetric sensor functions as a kind of reaction field.
  • Patent Document 1 describes that the results containing a variety of information obtained using particles to which a plurality of different light-emitting probes are bound are analyzed using a columnar graph.
  • Non-Patent Document 1 when a reaction field is formed by arranging chemical reagents at a high density, as in the technique of Non-Patent Document 1, when the target substance to be analyzed is applied using a spray method, the chemical Reagents may elute and bleed. As a result, noise and variation increase, making it difficult to perform accurate analysis. Furthermore, in the detection of a target substance, it is common to use a probe that has a specific interaction that is compatible with the target substance. It was necessary to create a probe tailored to the target substance, and it was also necessary to select the target substance to be detected when analyzing mixtures.
  • Patent Document 1 describes an example of identifying a genotype by polymorphism analysis using particles to which a plurality of different probes are bound, but the analysis is performed from a relative luminescence intensity columnar graph. It determines the presence or absence of complementarity, and the use of data that includes diverse information generated from complex interactions and chemical reactions is insufficiently utilized, and includes data generated from the use of specific binding agents. Information is limited, and it is necessary to generate multidimensional data that integrates multiple reaction systems.
  • the present invention detects as a signal the fact that it easily interacts with a target substance and that the emission color and emission spectrum shape change slightly due to the interaction, and converts a large amount of data from the signal in a short and simple manner. Furthermore, an analysis system using a composition for generating a signal, an ink, a measurement chip containing a luminescent probe or a carrier thereof, which can be a molecular probe suitable for acquiring a large amount of data, and an analysis system using the same. The purpose of this study is to provide plates used for this purpose as well as analysis methods.
  • an analysis system reflecting one aspect of the present invention includes a plurality of reaction fields for accommodating a first component and a second component, and a plurality of reaction fields for accommodating a first component and a second component.
  • a plate in which fields are respectively divided at intervals, first signal information from the plate when the first component is accommodated in a plurality of the reaction fields, and a plurality of the reaction fields from which the first signal information has been acquired.
  • a signal information acquisition unit for acquiring second signal information from the plate when the second component is further accommodated in the reaction field and the first component and the second component are accommodated; and the first signal information and an analysis unit for performing machine learning and analyzing the difference in the second signal information, and at least two of the plurality of reaction fields accommodate the first components having mutually different compositions.
  • analysis in which at least one of the plurality of reaction fields is a region for causing a plurality of types of reactions including interactions of the first component and/or the second component. It is a system.
  • a plate reflecting one aspect of the present invention is a plate that has a plurality of reaction fields for accommodating a first component and a second component, and the plurality of reaction fields are separated from each other at intervals, and a plurality of At least two of the reaction fields are regions for respectively accommodating first components having different compositions, and at least one of the plurality of reaction fields is a region for accommodating the first component and/or the first component.
  • This is a plate for use in machine learning, which is an area for producing multiple types of reactions, including interactions of the second component.
  • An analysis method reflecting one aspect of the present invention includes a plate having a plurality of reaction fields for accommodating a first component and a second component, and in which the plurality of reaction fields are separated from each other at intervals. , placing the first component in each of the plurality of reaction fields; acquiring first signal information from the plate on which the first component is placed; further arranging the second component in each of the plurality of reaction fields; obtaining second signal information from the plate on which the first component and the second component are arranged; and the first signal information and machine learning and analyzing the second signal information, and in the step of arranging the first component, arranging the first component having different compositions in at least two of the reaction fields,
  • the method is an analysis method in which, in the step of arranging two components, a plurality of types of reactions including interactions between the first component and/or the second component occur in at least one of the reaction fields.
  • FIG. 1 is a flowchart of an analysis method according to an embodiment of the present invention.
  • FIG. 2 is a diagram for explaining a luminescent probe that can be used in the analysis method of the present invention.
  • FIGS. 3A to 3D are diagrams for explaining the mechanism by which the luminescent probe used in one embodiment of the present invention exerts its effects.
  • 4A and 4B are the results of principal component analysis in Example 1.
  • 5A and 5B are the results of principal component analysis in Example 1.
  • FIG. 6 shows the results of principal component analysis in Example 2.
  • FIG. 7 is a cross-sectional view showing an example of the microarray device used in Example 3.
  • FIG. 8 is a partially enlarged view of the bitmap pattern of the microarray device used in Example 3.
  • FIG. 9 is an overall diagram of the bitmap pattern of the microarray device used in Example 3.
  • FIG. 10 is a graph showing the origin identification rate for each number of microdot light emitting parts used in the microarray device used in Example 3.
  • FIG. 11A shows a linear discriminant analysis model plot when 95 brands of beverages of 7 types were analyzed using luminescent probes 1 to 15, and FIG. 11B is a confusion matrix at this time.
  • FIG. 12A shows a linear discriminant analysis model plot when 95 brands of 7 types of drinks were analyzed using luminescent probes 1 and 17 to 31, and FIG. 12B is a confusion matrix at this time.
  • FIG. 13A shows a linear discriminant analysis model plot when 95 brands of 7 types of drinks were analyzed using luminescent probes 1 to 15 and 17 to 31, and FIG.
  • FIG. 13B is a confusion matrix at this time.
  • FIG. 14A shows a linear discriminant analysis model plot when 95 brands of 7 types of drinks were analyzed using luminescent probes 1 and 32 to 45, and FIG. 14B is a confusion matrix at this time.
  • FIG. 15A shows a linear discriminant analysis model plot when 95 brands of 7 types of drinks were analyzed using the luminescent probes 46 to 60, and FIG. 15B is a confusion matrix at this time.
  • FIG. 16A shows a linear discriminant analysis model plot when seven types and 95 brands of beverages were analyzed by replacing the explanatory variables when creating the discriminant model, and FIG. 16B is the confusion matrix at this time.
  • acting non-specifically means that an analysis substance such as a luminescent probe described below can act not only on one type of substance but also on multiple substances, or on a specific substance. It refers to being able to act on multiple positions rather than just one specific one, such as being able to act on multiple positions in a substance instead of just one.
  • the plurality of types of reactions are interactions that occur nonspecifically between a first component described below and a second component described below.
  • the analysis method of this embodiment uses a plate that has a plurality of reaction fields for causing the first component and the second component to interact, and in which the plurality of reaction fields are separated from each other at intervals. I will do it.
  • a flowchart showing each step of the analysis method of this embodiment is shown in FIG.
  • a first component is placed in a plurality of reaction fields on the plate (S101, hereinafter also referred to as “first component placement step”).
  • first signal information is acquired from the plate on which the first component is placed (S102, hereinafter also referred to as "first signal acquisition step”).
  • the second component is placed in each of the plurality of reaction fields of the plate (S103, hereinafter also referred to as “second component placement step”).
  • second signal information is acquired from the plate on which the first component and the second component are placed (S104, hereinafter also referred to as “second signal acquisition step”).
  • the analysis unit performs machine learning and analyzes the difference between the first signal information and the second signal information (S105, hereinafter also referred to as "analysis step”).
  • the first component and the second component used in this embodiment may be components that can interact with each other.
  • one of the first component and the second component may contain the target substance to be analyzed, and the other may contain a substance that can interact with the target substance (hereinafter also referred to as "analysis substance").
  • analysis substance a substance that can interact with the target substance
  • the first component contains the substance for analysis and the second component contains the target substance, but these may be reversed.
  • first component and the second component may each contain the analysis substance or the target substance alone, but the first component and the second component may further contain various substances such as a solvent and impurities. You can stay there.
  • first component does not refer to a specific compound or a specific composition, but is a general term for various compounds and various compositions that are placed in the reaction field in the first component placement step. It is.
  • second component does not refer to a specific compound or a specific composition, but is a general term for various compounds and various compositions that are placed in the reaction field in the second component placement step.
  • the type of target substance to be analyzed by the analysis method of this embodiment is not particularly limited, and for example, it may be a substance whose structure is known or a substance whose structure is unknown. Further, it may be a mixture of various compounds, or it may be a substance, compound, or composition belonging to any field such as the medical field, industrial field, food field, etc.
  • target substances belonging to the medical field include proteins, antibodies, antibody-attached beads, tumor markers, and the like.
  • examples of target substances belonging to the industrial field include metal nanoparticles, carbon nanotubes, magnetic fluids, nanosilica, crystalline zirconia, and the like.
  • target substances that belong to the food sector include agricultural products and processed products thereof.
  • the type of substance for analysis is appropriately selected depending on the signal acquisition method in the first signal information acquisition step and the second signal information acquisition step, and the type of substance to be analyzed is appropriately selected according to the signal acquisition method in the first signal information acquisition step and the second signal information acquisition step, and is obtained in the second signal information acquisition step by interaction with the target substance. It is sufficient if the second signal information obtained in the first signal acquisition step is different from the first signal information obtained in the first signal acquisition step.
  • the substance for analysis may be an organic compound or an inorganic compound. However, organic compounds are more preferable from the viewpoint that signal information is likely to change when interacting with the target substance.
  • the analysis substance includes a "luminescence probe" that emits light when irradiated with a predetermined light, but the analysis substance is not limited to the luminescence probe.
  • Examples of luminescent probes include compounds that have a molecular weight of 10,000 or less and can interact with the target substance.
  • the molecular weight of the luminescent probe is more preferably 100 or more and 10,000 or less.
  • the luminescent probe easily enters the inside of the target substance and interacts with the target substance.
  • the luminescent probe is a compound with a relatively low molecular weight, the specificity between the luminescent probe and the target substance is low, and the luminescent probe tends to interact with multiple positions and multiple structures of the target substance. As a result, the second signal information tends to change significantly from the first signal information.
  • luminescent probes include common fluorophores.
  • Examples of common fluorophores include fluorescein isothiocyanate (FITC), derivatives of rhodamine (TRITC), coumarin, cyanine, CF dyes, FluoProbes, DyLight Fluors, Oyester (dyes), Atto dyes, HiLyte Fluors, and Alexa Fluor.
  • fluorophores may also be quantum dots, proteins (e.g. green fluorescent protein (GFP)), or small molecule dyes.
  • small molecule dyes include xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin, Texas red, etc.), cyanine derivatives (cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (cascade blue, etc.), BODIPY (Invitrogen), oxazine derivatives (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc.), acridine derivatives (Proflavin, Acridine Orange
  • a luminescent probe includes a compound having a nucleic acid structure and one or more chromophores or luminophores attached to the backbone of the nucleic acid structure.
  • the type of nucleic acid structure is not particularly limited. For example, it may have an artificial nucleic acid structure.
  • An artificial nucleic acid structure refers to a nucleic acid structure in which a non-natural portion is introduced into a natural nucleic acid, or a nucleic acid structure is synthesized using only a natural portion.
  • Artificial nucleic acid structures are molecules that usually do not exist in nature and are completely artificially synthesized. Therefore, it has the feature that it is difficult to be recognized by nucleolytic enzymes, etc. that exist in the air, and is difficult to be degraded.
  • nucleic acid structure When the nucleic acid structure has such an artificial nucleic acid structure, it becomes possible to stabilize the luminescent probe, increase the luminescence caused by the chromophore or luminophore, and so on.
  • nucleic acid structures include DNA, RNA, phosphorothioate oligodeoxynucleotides, 2'-O-(2-methoxy)ethyl-modified nucleic acids, siRNA, cross-linked nucleic acids, peptide nucleic acids, and morpholino antisense nucleic acids, acyclic Threoninol Nucleic Acid (aTNA), Serinol Nucleic Acid (SNA), Peptide Nucleic Acid (PNA), Glycol Nucleic Acid (GNA), Locked Nucleic Ac id (LNA), etc., but is not limited to these.
  • aTNA Threoninol Nucleic Acid
  • SNA Serinol Nucleic Acid
  • PNA
  • the luminophore or chromophore may be attached to at least a portion of the main chain (for example, on one end side) of the nucleic acid structure, and there may be a region not containing the luminophore or chromophore on the other side.
  • luminescent probes having the above nucleic acid structure include a main chain having one or more structural units including a pentose- or hexose-derived sugar structure and a phosphate ester bond bonded to the sugar structure, and one or more color-producing molecules. Some have a group or a luminophore.
  • the structure or position of one or more chromophores or luminophores is not particularly limited, but it is preferred that they are bound to a sugar structure. Furthermore, it is preferable that the labeling substance has a site capable of interacting with the target object.
  • the interaction part of the luminescent probe that interacts with the target substance has a nucleic acid structure that can non-specifically interact with the target substance.
  • interactions between interacting parts and objects include hydrogen bonds (hybridization) between nucleic acids, hydrogen bonds with protein amide groups and amino acids in biological materials such as enzymes and tumor markers, hydrogen bonds with acids, This includes interactions due to the shape of the formed three-dimensional structure.
  • the structure or position of the chromophore or luminophore contained in the luminescent probe may be located at a position separate from the interaction portion via another nucleic acid structure.
  • the chromophore or luminophore be located close to the interaction part, and preferably at the end of the interaction part or between the bases of the nucleic acid structure of the interaction part.
  • chromophore refers to a structure that absorbs light with a wavelength of 300 nm or more
  • a “luminophore” refers to a structure that absorbs light with a wavelength of 300 nm or more and emits light.
  • the number of chromophores or luminophores included in the luminescent probe is not particularly limited, and may be one or more.
  • the chromophores or luminophores may interact with each other to cause self-quenching when the luminescent probe is not interacting with the target substance, or when the luminescent probe interacts with the target substance. When doing so, it becomes possible to increase the luminous intensity.
  • the chromophore contained in the luminescent probe and the type of dye that can be a luminophore are not particularly limited, and examples thereof include common fluorescent dyes (e.g., cyanine dyes, merocyanine dyes, acridine dyes, coumarin dyes, and ethidium dyes). dyes, flavin dyes, fused aromatic ring dyes, xanthene dyes, etc.).
  • common fluorescent dyes e.g., cyanine dyes, merocyanine dyes, acridine dyes, coumarin dyes, and ethidium dyes.
  • examples of chromophores or luminophores that emit fluorescence include structures derived from fluorescein, rhodamine, boron dipyrromethene, and the like.
  • examples of chromophores or luminophores that emit phosphorescence include structures derived from iridium complexes, platinum complexes, and the like.
  • Examples of excimer-emitting chromophores or luminophores include structures derived from pyrene, anthracene, perylene, and the like.
  • Examples of exciplex-emitting chromophores or luminophores include structures derived from pyrene-dimethylaniline and the like.
  • Examples of chromophores or luminophores that emit heat-activated delayed fluorescence include structures derived from 4CzIPN, DABNA, and the like.
  • Examples of chromophores or luminophores that emit excited-state intramolecular proton emission include structures derived from hydroxyphenylbenzoxazole and the like.
  • Examples of chromophores or luminophores that emit triplet triplet annihilation luminescence include structures derived from 9,10-diphenylanthracene, rubrene, and the like.
  • Examples of chromophores or luminophores that emit twisted intramolecular charge transfer luminescence include structures derived from diaminoanthracene, diaminonaphthalene, and the like.
  • Examples of chromophores or luminophores that emit aggregated organic luminescence include structures derived from tetraphenylethene, hexaphenylsilole, and the like.
  • the above-mentioned luminescent probes respond to a single excitation light with fluorescence, phosphorescence, excimer emission, exciplex emission, thermally activated delayed fluorescence, excited state intramolecular proton emission, triplet triplet annihilation emission, and twisted intramolecular charge emission. It may be a compound that emits two or more types of luminescence selected from the group consisting of mobile luminescence and aggregated organic luminescence.
  • the sugar structure is preferably ribose or deoxyribose, although it is not particularly limited as long as it is a structure that can interact with the target substance.
  • the target substance is natural DNA or the like
  • the DNA has a ⁇ -form of deoxyribose linked to nucleobases. Therefore, when the target substance is natural DNA, it is preferable that 50% or more of the sugar structure to which the chromophore or luminophore is bound is the ⁇ -form.
  • the number of chromophores or luminophores that the luminescent probe has may be only one, or two or more, as long as the plurality of types of luminescence described above can be obtained. Furthermore, when there are two or more chromophores or luminophores, there may be only one type of chromophore or luminophore, or there may be two or more types of chromophores or luminophores. In addition, when there are multiple chromophores or luminophores, one or more chromophores or luminophores may be bonded to all structural units of the main chain, but some structural units may have chromophores or luminophores bonded to them. Groups do not need to be joined.
  • a luminescent probe is a compound that emits multiple types of light in response to a single excitation light
  • complex luminescence will occur due to the interaction between the target substance and the luminescent probe.
  • a luminescent probe is a compound that exhibits three different types of luminescence: fluorescence, phosphorescence, and excimer luminescence
  • the interaction between the target substance and the luminescent probe causes fluorescence, phosphorescence, and excimer luminescence.
  • the process by which light is emitted changes, and the wavelength and lifetime of each light change.
  • complex data is obtained by combining these lights depending on the structure, state, etc. of the target material, and by analyzing this, it becomes possible to understand the structure, state, etc. of the target material.
  • the luminescent probe of this embodiment is assumed to be a molecule having four sugar structures and four luminophores as shown in FIG. 3A. All four R's may be pyrene (Py in the figure), dimethylaminobiphenyl (N), or a mixture thereof. Furthermore, one or two of the four R's may be hydrogen atoms. Such a molecular structure allows the construction of such a complex fluorescent dye molecule.
  • the sample (target substance) does not contain any component that interacts with pyrene, excimer emission of pyrene shown in the center diagram of FIG. 3B is obtained.
  • a target substance (fluorescent substance) in the specimen (target substance) that has an energy level close to the band gap of pyrene that interacts with pyrene, pyrene Exciplex light emission by the fluorescent substance can be obtained.
  • the luminescent probe has the above-mentioned phosphate group
  • the phosphoric acid group present in the main chain portion of the luminescent probe Forms a chelate between As shown in FIG. 3D, the intermolecular distance between pyrenes changes depending on the size of the metal ion. Therefore, the emission color (emission spectrum) of the excimer emission itself also changes.
  • R is dimethylaminobiphenyl (N)
  • the emission color (spectrum) of the fluorescent dye (N) changes due to the proximity of the acid and base as shown in FIG. 3C. This is different from acid-base ion pairing, such as on/off switching between mineral acids (such as sulfuric acid and nitric acid) and alkali metals.
  • mineral acids such as sulfuric acid and nitric acid
  • alkali metals such as on/off switching between mineral acids (such as sulfuric acid and nitric acid) and alkali metals.
  • using this luminescence phenomenon as a signal leads to an expansion of the dynamic range.
  • dimethylaminobiphenyl (N) shown in Figure 3A is a Lewis base, it has similar interactions with Lewis acidic substances (for example, triarylborane, trialkylaluminium, tetraalkoxytitanium, etc.) in addition to protic acidic substances. wake up Therefore, a specific luminescence color change occurs even for such a target substance.
  • Lewis acidic substances for example, triarylborane, trialkylaluminium, tetraalkoxytitanium, etc.
  • Such a new concept is the fundamental concept principle of this embodiment, and will be used in various future research and development and production processes, as well as complex and mysterious specimens (target substances) such as cell culture, waste liquid, wastewater, and sludge treatment. ) is extremely useful as a new method for state description.
  • the luminescent probe described above can be synthesized by the following method.
  • a monomer in which the above chromophore or luminophore and a phosphate ester are bonded to a pentose or hexose is prepared.
  • the monomers are polymerized in a desired sequence using a phosphoramidide method using a DNA/RNA synthesizer or the like. According to such a method, a wide variety of luminescent probes can be synthesized depending on the type of target substance. The analysis method of this embodiment will be explained in detail below.
  • First component arrangement step In the first component placement step, the first component is placed in each reaction field of a plate having a plurality of reaction fields for causing the first component and the second component to interact.
  • the plate used in this step may have a plurality of reaction fields for causing the first component and the second component to interact, and the plurality of reaction fields are separated from each other at intervals.
  • the plate may be flat or may have unevenness, but is particularly preferably flat. Further, the material, size, shape, etc. of the plate are appropriately selected depending on the purpose of analysis, the types of the first component, the second component, etc.
  • the positions of each reaction field may be determined at intervals so that adjacent reaction fields do not come into contact with each other.
  • the interval is appropriately selected depending on the size of the reaction field, the types of the first component and the second component, the arrangement method, and the like.
  • the size of each reaction field is preferably 100 ⁇ m or less in diameter. Further, at this time, the interval between adjacent reaction fields can be 200 ⁇ m or less.
  • first component placement step and the second component placement step are performed by a machine (for example, an inkjet device, etc.), marks (forming an uneven structure or markings) indicating the position of each reaction field are formed on the plate. It doesn't have to be done. On the other hand, if marks (formation of uneven structures or markings) indicating the positions of each reaction field are formed on the plate, when performing the first component placement step or the second component placement step, it is easier to locate the desired position (reaction field). It becomes easier to accurately place the first component and the second component in the field).
  • reaction fields when each reaction field is formed in a concave shape or when a partition wall is arranged around each reaction field, there is an advantage that the first component and the second component of adjacent reaction fields are difficult to mix. . Further, for example, when a water repellent treatment section is arranged around a reaction field, it becomes difficult for the first component and the second component of adjacent reaction fields to mix.
  • a plate in which a plurality of wells are regularly arranged is used. In a plate having such wells, the wells (reaction fields) are physically separated from each other by partition walls.
  • the number of reaction fields that one plate has is appropriately selected depending on the type of target substance to be analyzed, the type of luminescent probe that labels it, etc.
  • the number of reaction fields may be two or more, but the larger the number, the more multi-dimensional data can be acquired and the more precise analysis can be performed.
  • the first component preferably includes a luminescent probe
  • the second component preferably includes a target substance to be analyzed.
  • the method of arranging the first component in each reaction field is not particularly limited, and is appropriately selected depending on the type, physical properties, etc. of the first component.
  • methods for disposing the first component include coating with an inkjet device, coating with a dispenser, disposing a carrier supporting the first component, directly fixing the first component to a reaction field, and the like.
  • coating with an inkjet device, arrangement of a carrier supporting the first component, or direct fixation of the first component to a reaction field are particularly preferred. According to the inkjet method, it is possible to efficiently arrange the liquid first component in a large number of areas to form a reaction field. This makes it possible to acquire a large amount of data.
  • a compound or composition having the same composition may be placed as the first component in a plurality of reaction fields.
  • first components having mutually different compositions are placed in two or more of the plurality of reaction fields. That is, first components having different compositions are placed in different reaction fields.
  • the more first components having different compositions are arranged in the reaction field, the more data can be acquired, so it is preferable.
  • the first component is a luminescent probe, it is preferable to arrange a plurality of luminescent probes in one reaction field in the first component placement step, since this allows a large number of multidimensional data to be obtained.
  • first signal information acquisition step first signal information is acquired from a plate in which a plurality of first components are arranged in a plurality of reaction fields.
  • the first signal information acquired in this step is not particularly limited as long as it is useful information for the analysis described below.
  • the first signal information may be acquired from all the reaction fields of the plate at once, or the first signal information may be acquired from each reaction field.
  • Examples of the first signal information include absorption spectra of ultraviolet and visible light obtained with an ultraviolet-visible absorption meter, fluorescence spectra and fluorescent fingerprints obtained with a fluorescent fingerprint measuring device, and absorbance obtained with a circular dichroism dispersion meter. , a chromatogram obtained by high-performance liquid chromatography (HLPC), and changes in spectral distribution and chromaticity over time when irradiated with specific excitation light. A combination of two or more of these may be used as the first signal information.
  • these acquisition methods will be specifically explained, but the method of acquiring signal information is not limited to these methods.
  • the plate on which the first component is placed is irradiated with excitation light in a specific wavelength range, and the wavelength of the light (fluorescence or phosphorescence) emitted by the plate (first component) and its Measure the intensity with a spectrophotometer. Then, the wavelength range of the excitation light emitted by the excitation light source is shifted by a desired width (for example, 10 nm), and the wavelength of the light and its intensity are similarly measured. Repeat these steps to obtain a large amount of data. The wavelength of the excitation light and the wavelength and intensity of the light emitted by the plate (first component) are then converted into three-dimensional data to form a fluorescent fingerprint.
  • a desired width for example, 10 nm
  • a data obtained by converting the wavelength and intensity of phosphorescence emitted by the plate (first component) is also referred to as a "fluorescent fingerprint.”
  • Acquisition of a fluorescent fingerprint is very useful when the above-mentioned compound having a molecular weight of 10,000 or less and capable of interacting with a target substance is used as a luminescent probe. When such a luminescent probe is used, the fluorescent fingerprint is likely to change due to the interaction between the luminescent probe and the target substance, making it easier to analyze the target substance.
  • the plate on which the first component is arranged is irradiated with excitation light of a specific wavelength for a short time. Thereafter, the spectral distribution is measured with a spectrophotometer, either continuously or intermittently.
  • the plate on which the first component is arranged is irradiated with excitation light of a specific wavelength for a short time. After that, an image is acquired using a known CCD camera, CMOS camera, etc., and chromaticity is acquired from the obtained image.
  • luminescent probes with a main chain having one or more structural units containing the above-mentioned pentose- or hexose-derived sugar structure and a phosphate ester bond bonded to the sugar structure. , one or more chromophores or luminophores attached to the sugar structure.
  • the luminescent probe exhibits multiple types of luminescence, and the lifetime and intensity of each luminescence are likely to change depending on the interaction between the luminescent probe and the target substance. Therefore, analysis of the target substance becomes easier by acquiring the spectral distribution and chromaticity change.
  • the second components are placed in each of the reaction fields of the plate described above.
  • a second component having a different composition may be placed in some or all of the reaction fields.
  • the second component having the same composition may be placed in all reaction fields.
  • the combination of the first component and the second component is adjusted so that a plurality of reactions including interactions between the first component and/or the second component occur within at least one reaction field.
  • Multiple reactions may be multiple types of chemical bonding reactions caused by non-specific reactions of the first component and the second component. Specifically, it may be a reaction in which one luminescent probe is chemically bonded non-specifically to different positions within the molecule of the target substance.
  • the reaction may be such that the first component or the second component (in this embodiment, the first component) contains a plurality of light-emitting probes, and these light-emitting probes are chemically bonded to different positions of the target substance.
  • multiple reactions may be multiple types of light-emitting reactions caused by the interaction of the first component and the second component.
  • weak non-covalent interactions such as hydrogen bonds, ⁇ - ⁇ stacking interactions, and metal coordination bonds, to change the absorption and emission characteristics of the multiple types of luminescent reactions. It is possible to generate multi-dimensional, large amounts of data and obtain more information.
  • the reaction may be such that multiple types of light emission are generated due to the interaction between the luminescent probe and the target substance.
  • luminescent reactions are preferably luminescent reactions with different luminescent probes, wavelengths, and types of luminescence in order to generate multidimensional and large amounts of data.
  • complex data is obtained by combining these lights, and by analyzing this data, it becomes possible to understand the structure, state, etc. of the target substance.
  • the method of arranging the second component is not particularly limited, and is appropriately selected depending on the type and properties of the second component.
  • the method can be similar to the method for arranging the first component described above. Particularly preferred is application using an inkjet device.
  • the liquid second component can be placed in a large number of reaction fields by precise spotting for each reaction field, making it difficult for the reaction fields to mix with each other.
  • the second component placement step the second component may be placed not only in the reaction field on the plate but also in a region other than the reaction field, that is, a region on the plate where the first component is not arranged. On the other hand, the second component may not be placed in a part of the reaction field where the first component is placed.
  • second signal information acquisition step second signal information is obtained from the plate on which the first component is placed.
  • the second signal information acquired in this step is not particularly limited as long as it is information useful for analysis in the analysis step described below. Usually, it is preferable that the information be acquired by the same method as the information acquired in the first signal information acquisition step. Also, in the second signal information acquisition step, the second signal information may be acquired from all the reaction fields of the plate at once, or the second signal information may be acquired from each reaction field.
  • Machine learning refers to learning regularities and judgment criteria from data, and predicting and making judgments about unknown things based on that learning.
  • the machine learning performed in this step may be supervised learning or unsupervised learning.
  • supervised learning refers to a learning method that learns the "relationship between input and output" from learning data with correct answer labels.
  • Unsupervised learning refers to a learning method that learns the "structure of a data group" from training data without correct labels, and refers to methods such as clustering and dimension reduction using principal component analysis.
  • analysis data data obtained by subtracting the first signal information from the second signal information
  • this is subjected to machine learning to analyze the state of the target substance, etc.
  • the method for analyzing the analysis data in this step is appropriately selected depending on the purpose, the type of the analysis data, and the like.
  • first component arrangement step processes similar to the above-mentioned first component arrangement step, first signal information acquisition step, second component arrangement step, second signal information acquisition step, etc. are performed, and standard data are obtained.
  • the standard data may be prepared and compared with the analysis data to identify the state, structure, etc. of the target substance.
  • the target substance may be in a good state or in a bad state. You may create standard data for each case and compare it with these.
  • the comparison result between the standard data and the data for analysis is converted into a distance matrix, and a heat map (weighted
  • the distance matrix can be analyzed by principal component analysis (also known as PCA, weighting with emphasis on anisotropy), analysis by DL (weighting with emphasis on isotropy), etc. It's okay.
  • the standard data may be a trained model created in advance.
  • the trained model can be created, for example, by a trained model generation process described below. By using the trained model, it is possible to perform more appropriate analysis of the target substance.
  • the prediction result may be obtained as, for example, classification, regression, clustering, abnormality detection (outlier detection), or the like.
  • a plurality of predictive models are constructed based on the difference (data for analysis) between the above-mentioned second signal information and first signal information. Then, by combining the results of a plurality of prediction models, information regarding the target substance (for example, structure, amount, etc.) may be predicted.
  • the above prediction model should perform machine learning using the characteristics of the analysis data as explanatory variables and the structure and amount of the target substance as objective variables. It can be constructed with As explanatory variables, numerical values representing the characteristics of the above-mentioned analysis data and numerical values calculated from them can be used.
  • the first signal information or the second signal is a fluorescent fingerprint
  • analysis data obtained from the fluorescence intensity of the fluorescent fingerprint for each excitation wavelength can be employed as an explanatory variable.
  • the target variable can be selected as appropriate depending on the purpose of the analysis, and is not limited to the structure or amount of the target substance, but may also be any other variable related to the target substance.
  • Machine learning includes, for example, linear regression (multiple regression analysis, partial least squares (PLS) regression, LASSO regression, Ridge regression, principal component regression (PCR), etc.), random forests, decision trees, support vector machines (SVM), A prediction model constructed by an analysis method selected from support vector regression (SVR), neural network, discriminant analysis, etc. can be applied.
  • linear regression multiple regression analysis, partial least squares (PLS) regression, LASSO regression, Ridge regression, principal component regression (PCR), etc.
  • PLS partial least squares
  • PCR principal component regression
  • SVM support vector machines
  • a prediction model constructed by an analysis method selected from support vector regression (SVR), neural network, discriminant analysis, etc. can be applied.
  • the method of performing the first component placement step, the first signal acquisition step, the second component placement step, the second signal information acquisition step, and the analysis step has been described.
  • the signal obtained from the region where only the first component is arranged or the region where only the second component is arranged is treated as the above-mentioned first signal
  • the signal obtained from the reaction field containing the first component and the second component is treated as the above-mentioned first signal.
  • RGB data and hyperspectral data may be extracted from the digital image data acquired as the first signal information and the second signal information.
  • the above analysis method uses a plate that has a plurality of reaction fields for interacting the first component and the second component, and in which the plurality of reaction fields are separated from each other at intervals; Signal information acquisition for acquiring first signal information from the plate when the first component is accommodated in the plate, and second signal information from the plate when the first component and the second component are accommodated in the plurality of reaction fields.
  • a machine learning section that performs machine learning on the first signal information and the second signal information, and an analysis section that performs the analysis.
  • the analysis system of this embodiment may further include components other than the plate, the signal information acquisition section, and the analysis section, and may further include an inkjet printing section for applying the first component and the second component. may have.
  • the analysis system of this embodiment will be described below. Note that the plate is the same as that explained in the above-mentioned analysis method, so a detailed explanation will be omitted here.
  • the signal information acquisition unit is means for acquiring the above-mentioned first signal information and second signal information.
  • the configuration of the signal acquisition unit and the type of signal information acquired by the signal acquisition unit are selected as appropriate depending on the type of target substance, the purpose of analysis, etc.
  • the signal information acquisition section may be an ultraviolet-visible light absorption meter or a fluorescent fingerprint measuring device. Further, it may be a circular dichroism dispersion meter, a high performance liquid chromatograph (HPLC), or a device that combines a predetermined excitation light source with a spectrophotometer or an imaging section. Further, the signal information acquisition section may be a combination of two or more of these.
  • a fluorescent fingerprint measurement device includes, for example, an excitation light source for irradiating excitation light, and a spectrophotometer for measuring the wavelength and intensity of fluorescence emitted by the first component or a mixture of the first component and the second component. , the wavelength of the excitation light, the wavelength of the light emitted by the first component or a mixture of the first component and the second component, and a calculation unit for converting the intensity into three-dimensional data, etc.
  • Examples of the above light sources include supercontinuum light sources (a broadband pulsed light source that uses the nonlinear effect of optical fibers to emit intense light with a uniform phase over a very wide wavelength range, and is also called an "SC light source”). This includes LEDs, etc. According to these light sources, since the amount of light can be increased, fluorescent fingerprints tend to become clearer. Note that the fluorescent fingerprint measuring device may include multiple light sources and multiple spectrophotometers.
  • the arithmetic unit for converting the obtained data into three-dimensional data can be a general information processing device, such as a personal computer.
  • the excitation light source in a device that combines a predetermined excitation light source with a spectrophotometer or an imaging section is not particularly limited as long as it is a means that can irradiate the plate with light of a desired wavelength for a desired period of time.
  • preferred light sources include picosecond diode lasers, wavelength tunable lasers, supercontinuum light sources, LED light sources, and the like. According to these light sources, the plate can be irradiated with light of a predetermined wavelength for a short period of time.
  • the imaging unit is not particularly limited as long as it is a means capable of acquiring changes over time in the luminescence state of the luminescence control material, and may be a known CCD that captures images of the plate intermittently or continuously over a plurality of times. It may be a camera, a CMOS camera, or the like.
  • the analysis section analyzes the target substance by performing machine learning on the difference between the first signal information and the second signal information acquired by the signal information acquisition section. Specifically, any configuration may be used as long as it is capable of calculating the difference between the first signal information and the second signal information and performing various arithmetic operations.
  • the analysis unit may create a learned model and analyze the target substance based on the learned model.
  • Such an analysis unit includes storage means such as a hard disk drive (HDD), solid state drive (SSD), and read-only memory (ROM) that store programs and data, and a central processing unit that executes programs and performs calculation processing.
  • storage means such as a hard disk drive (HDD), solid state drive (SSD), and read-only memory (ROM) that store programs and data
  • ROM read-only memory
  • a general computer general-purpose computer equipped with a device (CPU) can be used. Further, the computer may further include input means such as a keyboard and mouse, and output means such as a monitor and a printer.
  • the inkjet printing unit may be used as long as it is capable of ejecting the first component and the second component described above to a predetermined position (reaction field) on the plate.
  • the inkjet printing section can have a similar configuration to a general inkjet device.
  • the plate of the present embodiment is not particularly limited as long as it has a plurality of reaction fields for causing the first component and the second component to interact, and the plurality of reaction fields are separated from each other at intervals.
  • the structure of the plate is similar to the plate described in the analysis method above.
  • at least two of the plurality of reaction fields are regions for accommodating first components having mutually different compositions.
  • at least one of the plurality of reaction fields is a region for causing a plurality of types of reactions including interactions between the first component and/or the second component.
  • the first component and the second component are respectively arranged in a plurality of reaction fields arranged at intervals to obtain signal information. Therefore, the first component and the second component do not mix between adjacent reaction fields, and noise and variation are less likely to occur during signal acquisition. Therefore, various information such as the detailed structure of the target substance can be obtained from signal information generated by the interaction between the first component and the second component.
  • Example 1 (1) Preparation of target substance-containing liquid (second component)
  • D(-)-fructose also referred to as “target substance a” or “Fru”
  • N-acetylneuraminic acid also referred to as “Substance b” or “Neu5Ac”
  • target substance-containing solution D(-)-fructose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with 0.1 mol/L phosphate buffer pH 7.4 (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and dimethyl sulfoxide (Kanto
  • a target substance-containing solution a (20mM fructose-containing solution) was prepared by dissolving the target substance in a mixed solution (volume ratio 80:20) with Kagaku Co., Ltd.).
  • target substance-containing solution b N-acetylneuraminic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with 0.1 mol/L phosphate buffer pH 7.4 (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and dimethyl sulfoxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.).
  • a target substance-containing solution b (20mM N-acetylneuraminic acid-containing solution) was prepared by dissolving the target substance in a mixed solution (volume ratio: 80:20) with Kanto Kagaku Co., Ltd.).
  • luminescent probe-containing solution B Alizarin Red S (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., molecular weight 342.26, a substance that emits fluorescence) was mixed with 0.1 mol/L phosphate buffer pH 7.4 and dimethyl sulfoxide (Kanto Kagaku Co., Ltd.)
  • a luminescent probe-containing solution B (0.00266mM alizarin red S-containing solution) was prepared by dissolving the probe in a mixed solution (volume ratio 80:20) with the following products (manufactured by Akihabara Corporation).
  • Luminescent probe-containing liquids A and B were mixed at a volume ratio of 1:1 to prepare luminescent probe-containing liquid A+B.
  • Luminescent probe-containing liquids A and C were mixed at a volume ratio of 1:1 to prepare luminescent probe-containing liquid A+C.
  • First component placement step A 96-well microplate was prepared in which wells with an opening diameter of 7 mm were arranged in 12 columns x 8 rows with an interval of 9 mm. 100 ⁇ l of each of the first components shown in Table 1 below were placed in the 96-well microplate using an automatic dispensing device (NichiMart CUBE) to form a plurality of reaction fields.
  • an automatic dispensing device NeichiMart CUBE
  • Second component placement step 100 ⁇ l of the second component was placed in each 96-well microplate after the first signal information acquisition step using an automatic dispensing device (NichiMart CUBE) as shown in Table 1 below.
  • Second signal information acquisition step Set the 96-well microplate with the above-mentioned first and second components in a SPARK multi-detection mode microplate reader (manufactured by TECAN), The intensity of fluorescence (wavelengths from 280 to 740 nm) from the plate was measured when irradiating each excitation wavelength by changing the wavelength in steps. Detected values near the excitation wavelength ⁇ 30 nm during fluorescence measurement were excluded because they were significantly affected by leakage of excitation light. Then, the excitation wavelength, fluorescence wavelength, and fluorescence intensity were converted into three-dimensional data to create a fluorescence fingerprint. Fluorescent fingerprints were made three times for each sample.
  • Principal component analysis was performed on the data frame calibrated above. Then, a dot plot was drawn with principal component 1 (PC1) and principal component 2 (PC2) as axes. The results of the principal component analysis are shown in FIGS. 4A, 4B, 5A, and 5B. Principal component analysis was performed on wells 16, 17, 1, and 2 of Table 1 above ( Figures 4A and 5A), wells 16, 17, 10, and 11 of Table 1 above ( Figure 4B), and wells of Table 1 above. 16, 17, 13, and 14 (Fig. 5B). The graph represents data dispersion within the data space, with data points plotted close together exhibiting similar fluorescence fingerprint changes. On the other hand, data points plotted far apart represent relatively large changes in the fluorescent fingerprint.
  • Example 2 (1) Synthesis of the first component (luminescent probe) All reactions were performed in oven-dried glassware under a nitrogen atmosphere unless otherwise specified. All chemicals were purchased from Aldrich or TCI or Kanto Chemical and used as received without further purification.
  • Each oligonucleotide solid phase support obtained by automatic synthesis was reacted with ammonium water at room temperature for 2 hours, cut out from the solid phase, the solvent was dried in a centrifugal dryer, and ultrapure water was added to each luminescent probe 1 to 16.
  • First components 1 to 16 containing the following were obtained. It was confirmed that the luminescent probes 1 to 16 emit fluorescence and excimer when exposed to specific excitation light (for example, light with a wavelength of 350 nm).
  • Second component arrangement step Automatically dispense three types of soft drinks (second components 1 to 3) into the 96-well microplate after the first signal information acquisition step (NichiMart CUBE, manufactured by NICHIRYO) 100 ⁇ l each was placed.
  • Second signal information acquisition step Fluorescence spectra obtained when excitation light (wavelength 350 nm) was irradiated to the 96-well microplate in which the above-described first component and second component were placed were acquired as second signal information.
  • Example 3 Preparation of microarray device (plate) A microwell is prepared on an OLED substrate having a microdot light emitting part by the following method, and a microarray device (100) having a plateau unit (10) having the structure shown in FIG. ) was created.
  • a gas barrier layer was formed on the entire surface of one side of a polyethylene naphthalate film (manufactured by Teijin DuPont, hereinafter abbreviated as "PEN film").
  • PEN film a polyethylene naphthalate film
  • an atmospheric pressure plasma discharge treatment apparatus having the configuration described in JP-A No. 2004-68143 was used.
  • the material of the gas barrier layer was silicon oxide (SiO x ; 1 ⁇ X ⁇ 4).
  • the thickness of the gas barrier layer was 500 nm.
  • an ITO (indium tin oxide) film having an area of 30 mm x 30 mm and a thickness of 120 nm was formed by sputtering.
  • patterning was performed by photolithography to form a 20 mm x 10 mm anode (11) and extraction electrode (not shown).
  • the support (1) on which the anode (11) was formed was washed and subjected to lyophilic treatment using an atmospheric pressure plasma discharge treatment apparatus.
  • Argon gas was used as a discharge gas
  • oxygen gas was used as a reactive gas, and they were supplied at 25° C. and at a rate of 1 L/(min ⁇ cm).
  • the power source used for plasma generation was PHF2-K manufactured by Heiden Laboratories, and a voltage of approximately 2 kV was applied to generate plasma.
  • ink 1 for forming an insulating part having the following composition was injected by an inkjet method under conditions such that the layer thickness after drying was 100 nm so as to cover the entire anode surface.
  • ink 2 for forming an insulating portion was injected under conditions such that the layer thickness after drying was 20 nm.
  • a piezo type inkjet printer head "KM1024i" manufactured by Konica Minolta was used for ejecting inks 1 and 2. This formed the insulating portion (12) of the receiving layer (19).
  • Propylene glycol monomethyl ether acetate 1,000 parts by mass
  • microdot light emitting parts (13) 1 to 9 On the insulating part (12) of the above-mentioned receptor layer (19), inks 1 to 9 for forming a light emitting part having the following compositions were injected using an inkjet printer head "KM1024i" in the same manner as described above.
  • the bitmap pattern shown in FIG. 8 was used as input data.
  • microdot light emitting parts (13) 1 to 9 were formed in circles corresponding to the production positions of microwell structures (22) with a diameter of 500 ⁇ m at 60 dpi and every 6 pixels, which will be produced later.
  • microdot light emitting parts 1 to 9 were formed at 360 dpi, every 2 pixels, 100 ⁇ m in diameter, and in a 3 ⁇ 3 arrangement.
  • the broken line circle corresponds to the manufacturing position of the microwell structure (22).
  • the solid circle is the microdot light emitting section (13).
  • the light-emitting part forming inks 1 to 9 each contain one or more of the following light-emitting dopants (light-emitting compounds) Dp-1 to 9.
  • each microdot light emitting part (13) the above-mentioned insulating part (12) is once dissolved by the ejected ink, and the luminescent dopant and host compound in the light emitting part forming ink are mixed and dried again to form microdots.
  • a light emitting section (13) is formed. Ink was injected onto the insulating part (12) under conditions such that the layer thickness of the microdot light emitting part (13) thus formed was 30 nm. Next, microdot light-emitting portions (13) were formed by drying at 120° C. for 30 minutes under nitrogen.
  • the light emitting part forming inks 1 to 9 are applied to positions corresponding to the microwell structure parts (22) in the patterns shown in FIG. 9 and Table 3, respectively, and the microdot light emitting parts (13) are A dot light emitting section (13) was produced.
  • the maximum emission wavelengths of the luminescent dopants (luminescent compounds) Dp-1 to Dp-9 are as follows.
  • green to red light region Dp-7 362 nm
  • blue light region Dp-9 765 nm
  • a take-out anode (not shown) and a take-out cathode (not shown) were connected to an anode pad and a cathode pad of an on-board NCF tag IC: NTAG213F (manufactured by NXP Corporation) (17), respectively, to obtain an anode member.
  • On-board NCF tag IC: NTAG213F (17) is a power receiving antenna serving as a power receiving section (18).
  • ⁇ Preparation of cathode member> (Preparation of cathode film) A separately prepared PEN film was attached to a vacuum evaporation device. Further, a tungsten resistance heating boat filled with silver was attached to a vacuum evaporation apparatus, and the pressure of the vacuum chamber was reduced to 4 ⁇ 10 ⁇ 5 Pa. Thereafter, the boat was heated by electricity, and silver was deposited to form a cathode (14) with a thickness of 100 nm. The following electron injection adhesive layer forming ink was spin coated at 500 rpm on the silver surface of the cathode film taken out from the vacuum evaporation apparatus. Then, it was dried on a hot plate at 120°C for 10 minutes. Next, the anode (11) was covered and cut into a size that could be connected to an extraction electrode to obtain a cathode film.
  • PFN-Br (Ink for forming electron injection adhesive layer): 20 parts by mass Branched polyethyleneimine (manufactured by Aldrich, molecular weight 10,000): 20 parts by mass 2-propanol: 1,000 parts by mass
  • a flexible base material (16) having gas barrier properties was separately prepared.
  • a thermosetting adhesive shown below as a sealing adhesive was uniformly applied to a thickness of 20 ⁇ m along the barrier surface of the substrate using a dispenser. This was dried under a vacuum of 100 Pa or less for 12 hours.
  • the sealing member (15) was moved to a nitrogen atmosphere with a dew point temperature of ⁇ 80° C. or lower and an oxygen concentration of 0.8 ppm, and was dried for 12 hours or more. The moisture content of the sealing adhesive was adjusted to 100 ppm or less.
  • thermosetting adhesive an epoxy adhesive mixed with the following (A) to (C) was used.
  • the above-mentioned cathode film was placed on the adhesive layer (sealing member (15)) so that the cathode (14) was exposed to form a sealing bonding member.
  • the cathode (14) of the sealing bonding member, the receiving layer surface including the light emitting portion (13) of the anode member, and the extraction electrode on which the NFC tag (17) was arranged were arranged and brought into close contact.
  • the product was tightly sealed under pressure conditions of 90° C. and 0.1 MPa to obtain an OLED base material.
  • ink for forming a microwell structure having the following composition was injected at a head heating temperature of 50°C. .
  • Bitmap patterns as shown in FIGS. 8 and 9 were used as input data. According to these bitmap patterns, the outside of the circle was painted solid at 60 dpi, every 6 pixels, so that the inside of the circle with a diameter of 500 ⁇ m was blank. The injection was performed in two steps with the inkjet printing running direction reversed by 90 degrees.
  • the hole portion surrounded by the hardened composition was defined as a microwell structure portion (22).
  • the layer of the cured composition is the cured layer (21).
  • ⁇ Ink for forming microwell structure Diphenyl (2,4,6-trimethylbenzoyl)phosphine oxide (manufactured by Sigma-Aldrich): 2 parts by mass 2-phenoxyethyl acrylate (manufactured by TCI): 45 parts by mass Phenoxydiethylene glycol acrylate (manufactured by Shin Nakamura Chemical): 45 Parts by mass Polyacrylic acid (manufactured by Sigma-Aldrich, molecular weight 450,000): 8 parts by mass
  • the number of microdot light emitting parts used increased from one to two, microdot light emitting parts with a maximum emission wavelength of less than 380 nm, microdot light emitting parts with a maximum emission wavelength of 380 nm or more and less than 500 nm, and maximum emission wavelengths. It has been found that analysis accuracy is greatly improved by having two or more types of microdot light-emitting portions with a diameter of 500 nm or more.
  • microdot light emitting units used has increased from two to three, including microdot light emitting units with a maximum emission wavelength of less than 380 nm, microdot light emission units with a maximum emission wavelength of 380 nm or more and less than 500 nm, and maximum emission wavelengths. It has been found that the accuracy of analysis can be greatly improved by aligning all the microdot light-emitting parts with a diameter of 500 nm or more.
  • microarray device it is possible to analyze the state of interaction between a specimen (target substance) and a luminescent probe in a high-density and multidimensional manner using a simple process and energy-saving detection method using a CMOS sensor of a general-purpose camera. be.
  • Example 4 4-1 Synthesis of luminescent probes 1 to 60 (1) Synthesis of monomer 3 Based on the reaction formula below, monomer 3 having a main chain containing a phosphate ester and a luminophore bonded to the main chain is synthesized through intermediates 7 to 11. Synthesized.
  • Monomer 4 was a reagent having the structure shown below and was purchased from Glen Research (Sterling, Virginia). Thymidine contained in the monomer 4 is a type of natural base.
  • Monomer 5 was synthesized according to a non-patent document (J. Am. Chem. Soc. 1996, 118, 7671-7678.).
  • monomer 5 is a structural isomer of the above-mentioned monomer 1, and is a monomer whose sugar structure is ⁇ -form.
  • Luminescent probes 1 to 16 were prepared in the same manner as in Example 1 above.
  • ⁇ Target substance arrangement step After the first signal information acquisition step, 95 brands of 7 types of beverages (type I: 12 brands, type II: 23 brands, type III: 6 brands, type IV: 10 brands) are placed in the 96-well microplate after the first signal information acquisition step. , type V: 20 brands, type VI: 13 brands, type VII: 11 brands) were placed in 20 ⁇ l portions each by the same method as above.
  • Second signal information acquisition step Fluorescence spectra when excitation light (wavelength 350 nm) was irradiated to the 96-well microplate in which the above-mentioned first component and second component were placed were acquired as second signal information.
  • the first signal information acquired in the first signal information acquisition step was subtracted from the second signal information acquired in the second signal information acquisition step to calculate data for analysis. Then, the analysis data was used as an explanatory variable, the type data of each beverage was learned as an objective variable, and a discriminant model was created by linear discriminant analysis (LDA). The resulting linear discriminant analysis model plot is shown in FIG. 11A. Afterwards, we calculated the accuracy rate and created a confusion matrix using 6-fold cross-validation to quantify the generalization performance of the discriminant model. The confusion matrix is shown in FIG. 11B. -Results As shown in FIG. 11B, the above discrimination model was able to classify the types of 95 brands of beverages with an accuracy of about 65%.
  • LDA linear discriminant analysis
  • the above discrimination model was able to classify the types of 95 brands of beverages with an accuracy of approximately 45%.
  • the reason why the accuracy was lower than that of other analyzes is that the proportion of the ⁇ -form sugar structure in the luminescent probe was small.
  • the discriminant model in which the explanatory variables were randomly replaced was able to classify the types of 95 brands of beverages at approximately 25% rate. This result indicates that the above-mentioned accuracy was not obtained by chance in the discriminant model in which the luminescent probe was used and the explanatory variables and objective variables were set correctly.
  • the results show that various compounds can be analyzed with high accuracy using a discriminant model in which the luminescent probe is used and explanatory variables and target variables are set correctly.
  • Example 5 Adjustment of luminescent probe-containing solutions 1 to 16
  • the above luminescent probes 1 to 16 were prepared in a solution containing Na 2 HPO 4 /NaH 2 PO 4 (150 mM) and NaCl (50 mM), each having a pH of 8.5, and a Tween 20 concentration of 0.01%. to prepare luminescent probe-containing solutions 1 to 16.
  • Luminescent probe-containing solutions 1-16 were spotted onto PolyAn 3D NHS slides at 20-24° C. and 70% relative humidity. A humidity chamber was used for immobilization and rehydration of the spotted luminescent probe-containing solutions 1 to 16. A humidity chamber was filled with 50-100 ml of 1xSSC, the spotted glass slide was placed in the chamber, and the spots were allowed to rehydrate for 24 hours.
  • the above slide glass was blocked for 2 hours at pH 9 using ethanolamine (50mM) and Tris (100mM).
  • the above slide glass was placed in a solution containing NaCl (137mM), KCl (2.7mM), Na2HPO4 (4.3mM), and KH2PO4 ( 14mM ), the pH was 7.5, and the concentration of Tween20 was Washed with a solution that was 0.05%.
  • the slide glass was washed with a solution containing NaCl (137 mM), KCl (2.7 mM), Na 2 HPO 4 (4.3 mM), and KH 2 PO 4 (14 mM) and having a pH of 7.5. .
  • the above slide glass was spin-dried for 3 minutes using a centrifuge (1000 rpm) to prepare a microarray in which each of the luminescent probes 1 to 16 was immobilized.
  • a centrifuge 1000 rpm
  • wine was dropped as a specimen (target substance) in the same manner as in Example 3, and similar analysis was performed from the RGB values of the obtained luminescence image. It was possible to distinguish the origin of each sample wine as a category.
  • the analysis method and analysis system described above it is possible to easily analyze the target substance by using data obtained from multiple types of reactions including interactions between the first component and/or the second component. . It is also possible to easily acquire multidimensional and large amounts of data all at once. Therefore, it is very useful in analysis in various fields such as the medical field, industrial field, food field, etc.
  • luminescent probes that emit multiple types of luminescence or carriers containing them can be used as indicators for testing by making them into a solution or dispersion state, and furthermore, they can be used as indicators for inspections using ink jets, automatic dispensing machines, etc. This makes it possible to acquire a large amount of data in a short period of time, making it possible to greatly contribute to the revitalization and speeding up of industries, such as data-driven research and development using inverse problem solving, and data-driven testing and diagnosis. .
  • the fluorescent dye molecules of the present invention can generate a large amount of real data that is highly compatible with machine learning and deep learning simply by measuring light and color.
  • Another feature is that it does not require expensive and large equipment analyzers, and due to the various features mentioned above, it can be used in medical settings where liquid substances such as blood and saliva are donated, and in food processing such as alcoholic beverages and fruit juice.
  • the Japanese government will be able to collect data by bringing it to various work sites, including manufacturing sites in the chemical industry that require sewage treatment, water treatment plants, and even farms that collect milk and raw milk. It is expected that this technology will develop into a new technology that is consistent with the digital garden city-state concept advocated by Japan.
  • Support 10 Light source unit 11 Anode 12 Insulating section 13 Microdot light emitting section 14 Cathode 15 Sealing member 16 Barrier material 17 NFC tag 18 Power receiving section 19 Receptive layer 20 Multiwell unit 21 Hardening layer 22 Microwell structure section 100 Microarray device

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024116670A1 (ja) * 2022-11-30 2024-06-06 コニカミノルタ株式会社 マイクロアレイデバイス、マイクロアレイデバイスの製造方法、検査キット、検査システム、及び検査方法
WO2026009615A1 (ja) * 2024-07-05 2026-01-08 コニカミノルタ株式会社 解析システムおよび解析方法

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002189026A (ja) * 2000-08-25 2002-07-05 Stmicroelectronics Srl Ｄｎａマイクロアレイのイメージなどを自動解析するシステム
JP2002191372A (ja) * 2000-09-26 2002-07-09 National Institute Of Advanced Industrial & Technology 新規核酸プローブ並びにそれを用いる核酸測定方法、及びその方法によって得られるデータを解析する方法
US20020160411A1 (en) * 1999-12-14 2002-10-31 Research Corporation Technologies, Inc. Fluorescent nucleoside analogs and combinatorial fluorophore arrays comprising same
JP2003319799A (ja) * 2002-05-02 2003-11-11 Japan Science & Technology Corp 核酸塩基配列の解析方法
WO2007047408A2 (en) * 2005-10-12 2007-04-26 Pathologica, Llc. Promac signature application
JP2008003004A (ja) * 2006-06-23 2008-01-10 Ricoh Co Ltd 反応性物質の反応方法、その反応装置、及び基板
US20120309651A1 (en) * 2010-06-07 2012-12-06 Firefly Bioworks, Inc. Nucleic acid detection and quantification by post-hybridization labeling and universal encoding
US20120309107A1 (en) * 2009-12-14 2012-12-06 Eric Todd Kool Direct Sensing of Molecular Species by Polyfluors on a DNA Backbone
JP2016534107A (ja) * 2013-08-22 2016-11-04 ソニー株式会社 水溶性蛍光染料又は有色染料及びその使用方法
JP2016534723A (ja) * 2013-10-22 2016-11-10 キム・ソンチョン 生体分子と核酸の結合情報を生成するためのマーカー、その製造方法、並びにそれを用いた生体分子分析方法及び装置

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020160411A1 (en) * 1999-12-14 2002-10-31 Research Corporation Technologies, Inc. Fluorescent nucleoside analogs and combinatorial fluorophore arrays comprising same
JP2002189026A (ja) * 2000-08-25 2002-07-05 Stmicroelectronics Srl Ｄｎａマイクロアレイのイメージなどを自動解析するシステム
JP2002191372A (ja) * 2000-09-26 2002-07-09 National Institute Of Advanced Industrial & Technology 新規核酸プローブ並びにそれを用いる核酸測定方法、及びその方法によって得られるデータを解析する方法
JP2003319799A (ja) * 2002-05-02 2003-11-11 Japan Science & Technology Corp 核酸塩基配列の解析方法
WO2007047408A2 (en) * 2005-10-12 2007-04-26 Pathologica, Llc. Promac signature application
JP2008003004A (ja) * 2006-06-23 2008-01-10 Ricoh Co Ltd 反応性物質の反応方法、その反応装置、及び基板
US20120309107A1 (en) * 2009-12-14 2012-12-06 Eric Todd Kool Direct Sensing of Molecular Species by Polyfluors on a DNA Backbone
US20120309651A1 (en) * 2010-06-07 2012-12-06 Firefly Bioworks, Inc. Nucleic acid detection and quantification by post-hybridization labeling and universal encoding
JP2016534107A (ja) * 2013-08-22 2016-11-04 ソニー株式会社 水溶性蛍光染料又は有色染料及びその使用方法
JP2016534723A (ja) * 2013-10-22 2016-11-10 キム・ソンチョン 生体分子と核酸の結合情報を生成するためのマーカー、その製造方法、並びにそれを用いた生体分子分析方法及び装置

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024116670A1 (ja) * 2022-11-30 2024-06-06 コニカミノルタ株式会社 マイクロアレイデバイス、マイクロアレイデバイスの製造方法、検査キット、検査システム、及び検査方法
WO2026009615A1 (ja) * 2024-07-05 2026-01-08 コニカミノルタ株式会社 解析システムおよび解析方法

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