Analytical methods

Abstract

To provide an analysis method capable of detecting each of multiple kinds of target substances contained in a sample even without dividing the sample.SOLUTION: Fusions 1A, 1B having coupling substances 3A, 3B having activity of being combined with target substances 7A, 7B, and labeled substances 5A, 5B are mixed with a sample containing the target substances. The fusions not combined with the target substances are eliminated. By detecting a phenomenon generated by the labeled substances contained in the fusions combined with the target substances, the target substances are analyzed. Multiple types exist in the fusions. The target substances combined with the fusions and the phenomenon differ for each type of the fusions.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] This disclosure relates to an analysis method.

Background Art

[0002] Patent Document 1 discloses an analysis method of a target substance using a fusion body. The fusion body includes a binding substance and a labeling substance. The binding substance has an activity of binding to the target substance. The labeling substance causes an observable phenomenon.

[0003] In the analysis method of the target substance, a sample containing the target substance is mixed with the fusion body. Next, the fusion body not bound to the target substance is removed. Next, the phenomenon caused by the fusion body bound to the target substance is detected.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] A sample may contain a plurality of types of target substances. In this case, in order to detect each of the plurality of types of target substances, first, the sample needs to be divided into a plurality of parts, and then, one target substance needs to be detected in each of the divided samples.

[0006] However, when dividing a sample, various problems may occur. For example, when the sample is微量 (I'm not sure what "微量" means precisely in this context, it might be a typo or a very specific term. If it's "trace amount", then: when the sample is in trace amount, it is difficult to divide. Also, the operation of dividing the sample is cumbersome and requires time for dividing the sample. Further, if the sample cannot be accurately divided, the accuracy of the analysis decreases.

[0007] In one aspect of this disclosure, it is preferable to provide an analysis method capable of detecting each of a plurality of types of target substances contained in a sample without dividing the sample. [Means for solving the problem]

[0008] One aspect of this disclosure is a method for analyzing a target substance, comprising mixing a fusion (1A, 1B) having binding substances (3A, 3B) that are active in binding to the target substance, and labeling substances (5A, 5B), with a sample containing the target substance, removing the fusion that is not bound to the target substance, and detecting a phenomenon caused by the labeling substances contained in the fusion that is bound to the target substance.

[0009] There are multiple types of the aforementioned fusion body. The target substance that binds to the fusion body and the phenomenon described above differ depending on the type of fusion body.

[0010] According to one aspect of this disclosure, the analytical method, multiple types of target substances contained in a sample can be detected without dividing the sample. [Brief explanation of the drawing]

[0011] [Figure 1] This is an explanatory diagram showing the composition of the fusion entity. [Figure 2] This is an explanatory diagram illustrating the method of analysis when a sample contains only one type of target substance. [Figure 3] This is an explanatory diagram illustrating the method of analysis when a sample contains two different target substances. [Figure 4] This is a flowchart illustrating the method for synthesizing fusions. [Figure 5] This is a flowchart illustrating the analysis method for the target substance. [Figure 6] This is an explanatory diagram illustrating the state in which two types of composites are formed in a mixture containing a fusion material and a target substance. [Figure 7] This is an explanatory diagram illustrating a state in which one type of composite is formed in a mixture containing a fusion material and a target substance. [Figure 8] This is an explanatory diagram illustrating a state in which one type of composite is formed in a mixture containing a fusion material and a target substance. [Figure 9] It is an explanatory diagram showing a state in which no conjugate is formed in a mixed solution containing a conjugate and a target substance. [Figure 10] It is an explanatory diagram showing the measurement results of absorbance in Example 1. [Figure 11] It is a flowchart showing a method for synthesizing a conjugate. [Figure 12] It is a flowchart showing a method for synthesizing a conjugate. [Figure 13] It is a flowchart showing a method for analyzing a target substance. [Figure 14] It is an explanatory diagram showing the measurement results of absorbance in Example 2.

Mode for Carrying Out the Invention

[0012] Exemplary embodiments of the present disclosure will be described with reference to the drawings. 1. Target Substance The target substance is a substance to be analyzed. Examples of the target substance include proteins, sugars, nucleic acids, low-molecular compounds, lipids, antigens, viruses, etc. Examples of the virus include a novel coronavirus SARS-CoV2 isolate, etc.

[0013] 2. Conjugate In the analysis method of the present disclosure, a conjugate is used. The conjugate includes a binding substance and a labeling substance. The binding substance has an activity of binding to the target substance. The binding substance has, for example, an activity of specifically binding to a specific target substance and is difficult to bind to other substances. Examples of the binding substance include a substance composed of nucleic acid, a substance composed of amino acid, etc. Examples of the binding substance include nucleic acid aptamer, low-molecular protein preparation, peptide aptamer, antibody, nucleic acid, etc. For example, at least a part of the binding substance is a nucleic acid aptamer or a low-molecular protein preparation. The conjugate can bind to the target substance by including the binding substance.

[0014] Labeled substances produce observable phenomena. These observable phenomena include, for example, at least one of the following: oxidation-reduction, color development, fluorescence, light emission, phosphorescence, endothermic reaction, exothermic reaction, precipitation, and changes in ion content. The ions involved in changes in ion content include, for example, at least one of the following: hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, or chloride ions. Ion content changes, for example, through ion production, consumption, or absorption.

[0015] Labels that induce the production, consumption, or absorption of ions, for example. Examples of labels that induce the production, consumption, or absorption of ions include enzymes. Examples of labels that induce the production, consumption, or absorption of ions include 1,3-propanediol dehydrogenase, 15-hydroxyprostaglandin dehydrogenase, 1H-pyrrole-2-carbonyl-[peptidyl carrier protein]chlorinase, 2,4-dichlorobenzoyl-CoA reductase, 2,5-dioxovalerate dehydrogenase, 2-aminobenzenesulfonic acid 2,3-dioxygenase, 2-iminobutanoate / 2-Iminopropanoate deaminase, 2-enoic acid reductase, 2'-dehydrokanamycin reductase, 3,4-dihydroxyphenylalanine reducing deaminase, 3α-hydroxysteroid 3-dehydrogenase, 3-aminobutyryl-CoA ammonia lyase, 3-oxo-5α-steroid 4-dehydrogenase, 3-oxosteroid-1-dehydrogenase, 3-chloro-D-alanine dehydrochlorinase, 4-chlorophenylacetic acid 3,4-dioxygenase, 4-chlorobenzoyl-CoA dehalogenase, 4-chlorobenzoic acid dehalogenase, 4-trimethylammonium butyraldehyde dehydrogenase, 4-methylaminobutanoate oxidase, 4-methylene glutamate ammonia ligase, 5-phosphooxy-L-lysine phosphorylase, 7,8-didemethyl -8-hydroxy-5-deazariboflavin synthase, 7-carboxy-7-deazaguanine synthase, 7-chloro-L-tryptophan 6-halogenase, 7-cyano-7-deazaguanine synthase, AMP deaminase, CTP synthase, DDT-dehydrochlorinase, dTDP-4-amino-4,6-dideoxy-D-glucose ammonia lyase, D-arabinose-1-dehydrogenase, D-arginine dehydrogenase, D-xylose reductase, D-glucosamine-6-phosphate ammonia lyase, D-serine ammonia lyase, D-lactate dehydrogenase, GDP-4-dehydro-6-deoxy-α-D-mannose 3-dehydratase, GMP synthase, L-2-amino-4-chloropent-4-enoate dehydrochlorinase, L-cystine β-lyase,L-cysteine ​​desulfidase, L-cysteine ​​sulfolylase, L-serine ammonia lyase, L-tryptophan ammonia lyase, L-lysine cyclodeaminase, N1-acetylpolyamine oxidase, N8-acetylspermidine oxidase, NAD+-diphthamide ADP-ribosyltransferase, NAD+ synthase, NAD+-dinitrogen reductase ADP-D-ribosyltransferase, N-succinarylginine dihydrolase, S-(hydroxymethyl)mycotiol dehydrogenase, S-carboxymethylcysteine ​​synthase, UDP-2-acetamido-2,6-β-L-arabinohexyl-4-ose reductase, UDP-N-acetyl-2-amino-2-deoxyglucuronate dehydrogenase, UDP-N-acetyl-D-mannosamine dehydrogenase UDP-N-acetyl-α-D-quinovosamine dehydrogenase, UDP-N-acetylglucosamine 3-dehydrogenase, UDP-N-acetylglucosamine 6-dehydrogenase, UDP-glucuronide dehydrogenase, β-alanyl-CoA ammonia lyase, β-ureidopropionase, β-lactamase, γ-butyrobetaine dioxygenase, asparaginyl-tRNA synthase, asparagine synthase, ammonia aspartate lyase Aze, aspartate-ammonia ligase, aspartate semialdehyde dehydrogenase, aspartate dehydrogenase, adenylyl sulfate-ammonia adenylyltransferase, adenosylchloride synthase, atrazine chlorohydrolase, aminomethyltransferase, alanine dehydrogenase, allantoic acid deiminase, alkaline phosphatase, alcohol dehydrogenase, aldehyde dehydrogenase, ammonia kinase, ammonia monooxygenase, isocitrate dehydrogenase, imidazole glycerol-phosphate synthase, uricase, urease, ureidoglycolate amide hydrolase, uronic acid dehydrogenase, ethanolamine ammonia lyase, erythro-3-hydroxy-L-aspartate ammonia lyase, octopamine dehydratase, ornithine cyclodeaminase, galactose dehydrogenase, carbamate kinase,Carbamoyl-serine ammonia lyase, carbamoyl phosphate synthase, carbonyl reductase, formate dehydrogenase, glycine reductase, glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucose dehydrogenase, glucokinase, glucosamine-6-phosphate ammonia lyase, glucosamine-6-phosphate deaminase, glutaminyl-tRNA synthase, glutamine synthase, glutamate synthase, glutamate dehydrogenase, crotonobetenyl-CoA Hydratase, Chloride peroxidase, Coproporphyrinogen dehydrogenase, Choline oxidase, Choline monooxygenase, Cholesterol oxidase, Diacetyl reductase, Diaminopimelate dehydrogenase, Diaminopropionate ammonia lyase, Shikimate dehydrogenase, Cyclohexane-1,2-diol dehydrogenase, Dichlorochromopyrrolate synthase, Dichloromethane dehalogenase, Cystathionine γ-lyase, Cysteine-S-conjugate β-lyase, Dihydrolipoyl dehydrogenase -ase, diphthin-ammonia ligase, cinnamoyl-coenzyme A-reductase, succinyl glutamate-semialdehyde dehydrogenase, stachydrin N-demethylase, threo-3-hydroxy-D-aspartate ammonia lyase, threo-3-hydroxy-L-aspartate ammonia lyase, threonine ammonia lyase, serine sulfate ammonia lyase, thioredoxin disulfide reductase, tyrosine ammonia lyase, tyrosine phenol lyase, tetrachloroethene reduction dehalogenase, tetracycline 7- Halogenase, tryptophanase, tryptophan 5-halogenase, tryptophan 6-halogenase, tryptophan 7-halogenase, nicotinamide adenine dinucleotide phosphate-dehydrogenase, nicotinamide adenine dinucleotide phosphate-hemeprotein reductase, nitrogenase, vanadium-dependent nitrogenase, haparindol-type alkaloid chlorinase, histidine ammonia lyase, hydrazine synthase, hydrazine dehydrogenase, hydroxymethylvilan synthase, hydroxylamine reductase,At least one of pyridoxal 5'-phosphate synthase, arsenate reductase, phenylalanine / tyrosine ammonia lyase, phenylalanine 2-monooxygenase, phenylalanine ammonia lyase, ferredoxin-nicotinamide adenine dinucleotide phosphate reductase, ferredoxin-nitrite reductase, fumarate reductase, protoporphyrinogen oxidase, betaine aldehyde dehydrogenase, betaine reductase, peroxidase, homocysteine desulfhydrase, homospermidine synthase, formimidoyl tetrahydrofolate cyclodeaminase, myeloperoxidase, methanol dehydrogenase, methane monooxygenase, methionine γ-lyase, methylaspartic acid ammonia lyase, methylamine dehydrogenase, methylenediurea deaminase, lactaldehyde dehydrogenase, ribose-5-phosphate-ammonia ligase, malate dehydrogenase, rubredoxin-nicotinamide adenine dinucleotide phosphate reductase, chlorite O2-lyase, nitrite reductase, sulfite dehydrogenase, chloride peroxidase, chlorate reductase, lipid II isoglutaminyl synthase, carbonic anhydrase, and non-specific polyamine oxidase is included.

[0016] The labeling substance contains, for example, at least one of an enzyme, a DNAzyme, an RNAzyme, a magnetic label, a fluorescent label, a chemiluminescent probe, and nanoparticles.

[0017] The binding substance and the label are linked, for example, via a chemical substituent or a nucleic acid-binding protein. Examples of chemical substituents include at least one of biotin, primary amines, azides, alkynes, dibenzocyclooctin, bicyclononine, 2'-O-propargyl, 2'-O-propargyl, thiols, avidin, streptavidin, neutraavidin, N-hydroxysuccinimide, maleimide, and 5-halouracil. Examples of nucleic acid-binding proteins include zinc fingers and CRISPR. Examples of 5-halouracil include 5-iodouracil and 5-bromouracil. 5-halouracil is a chemical substituent that can be UV crosslinked.

[0018] The analytical method described herein uses multiple types of fusion bodies. The target substance that binds to the fusion body and the observable phenomena differ for each type of fusion body.

[0019] For example, two fusion bodies 1A and 1B are shown in Figure 1. Fusion body 1A comprises a binding substance 3A and a label 5A. Fusion body 1B comprises a binding substance 3B and a label 5B.

[0020] If fusion bodies 1A and 1B are of the same type, then binding substances 3A and 3B are of the same type. If binding substances 3A and 3B are of the same type, then the target substance that binds to binding substance 3A is the same as the target substance that binds to binding substance 3B. Therefore, if fusion bodies 1A and 1B are of the same type, then the target substance that binds to binding substance 3A is the same as the target substance that binds to binding substance 3B.

[0021] Furthermore, if the types of fusion bodies 1A and 1B are the same, then the types of labeling objects 5A and 5B are also the same. If the types of labeling objects 5A and 5B are the same, then the observable phenomena produced by labeling object 5A and the observable phenomena produced by labeling object 5B are the same. Therefore, if the types of fusion bodies 1A and 1B are the same, then the observable phenomena produced by labeling object 5A and the observable phenomena produced by labeling object 5B are the same.

[0022] If the types of fusion bodies 1A and 1B are different, then the types of binding substances 3A and 3B will be different. If the types of binding substances 3A and 3B are different, then the target substance that binds to binding substance 3A will be different from the target substance that binds to binding substance 3B. Therefore, if the types of fusion bodies 1A and 1B are different, then the target substance that binds to binding substance 3A will be different from the target substance that binds to binding substance 3B.

[0023] Furthermore, if the types of fusion bodies 1A and 1B are different, the types of labeling objects 5A and 5B will also be different. If the types of labeling objects 5A and 5B are different, the observable phenomena produced by labeling object 5A will be different from the observable phenomena produced by labeling object 5B. Therefore, if the types of fusion bodies 1A and 1B are different, the observable phenomena produced by labeling object 5A will be different from the observable phenomena produced by labeling object 5B.

[0024] 3.Analysis method In the analytical method described herein, a sample containing the target substance is mixed with several types of fusions. At this time, at least a portion of the fusions binds to the target substance. More specifically, the binding material present in the fusions binds to the target substance.

[0025] Each of the multiple types of fusion compounds will bind to its corresponding target substance, if such a substance exists. The corresponding target substance is a substance that the fusion compound has the activity to bind to.

[0026] For example, the fusions 1A and 1B shown in Figure 1 can be mixed with a sample. Fusions 1A and 1B are different types of fusions. The target substance that binds with fusion 1A is designated as target substance 7A, and the target substance that binds with fusion 1B is designated as target substance 7B. Target substances 7A and 7B are different types of substances.

[0027] If the sample contains the target substance 7A but not the target substance 7B, then, as shown in STEP 1 of Figure 2, when the fusion 1A and 1B are mixed with the sample, the target substance 7A will bind to fusion 1A but not to fusion 1B. Fusion 1B will remain unbound to the target substance.

[0028] Next, when a process is performed to remove fusions that are not bound to the target substance, as shown in STEP 2 of Figure 2, fusion 1A that is bound to the target substance 7A remains, while fusion 1B is removed. In addition, fusion 1A that is not bound to the target substance 7A is also removed. Methods for removing fusions that are not bound to the target substance include, for example, ultrafiltration or the use of a support.

[0029] Next, as shown in STEP 3 of Figure 2, an observable phenomenon (hereinafter referred to as phenomenon A) produced by the fusion product 1A bound to the target substance 7A is detected. Phenomenon A is, for example, the phenomenon in which metabolite 11A is produced from substrate 9A under conditions in which the enzyme contained in label 5A is functional.

[0030] The conditions under which an enzyme functions vary depending on the type of enzyme. Examples of these conditions include a suitable temperature range, a suitable pH range, and the presence of the necessary coenzymes in the solution.

[0031] By detecting phenomenon A, it can be determined that the sample contained the target substance 7A. Furthermore, the amount and concentration of the target substance 7A can be estimated based on the degree of phenomenon A.

[0032] If the sample contains target substances 7A and 7B, then, as shown in STEP 11 of Figure 3, when the fusion 1A and 1B are mixed with the sample, target substance 7A will bind to fusion 1A, and target substance 7B will bind to fusion 1B.

[0033] Next, when the fusion bodies that are not bonded to the target substance are removed, as shown in STEP 12 of Figure 3, fusion body 1A bonded to the target substance 7A and fusion body 1B bonded to the target substance 7B remain. Fusion bodies 1A and 1B that are not bonded to the target substance are removed.

[0034] Next, as shown in STEP 13 of Figure 3, the phenomenon A produced by fusion 1A bonded with the target substance 7A, and the observable phenomenon (hereinafter referred to as phenomenon B) produced by fusion 1B bonded with the target substance 7B are detected.

[0035] By detecting phenomenon A, it can be determined that the sample contained the target substance 7A. Furthermore, the amount and concentration of the target substance 7A can be estimated based on the degree of phenomenon A. Phenomenon B is, for example, the phenomenon in which metabolite 11B is produced from substrate 9B under conditions in which the enzyme contained in label 5B is functional.

[0036] The conditions under which an enzyme functions vary depending on the type of enzyme. Examples of these conditions include a suitable temperature range, a suitable pH range, and the presence of the necessary coenzymes in the solution.

[0037] By detecting phenomenon B, it can be determined that the sample contained the target substance 7B. Furthermore, the amount and concentration of the target substance 7B can be estimated based on the degree of phenomenon B. Since phenomena A and B are different phenomena, the user can distinguish between phenomena A and B and detect them separately.

[0038] For example, if the phenomenon involves the production, consumption, or absorption of hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, or chloride ions, the phenomenon can be detected using, for example, a pH meter or an ion-sensitive field-effect transistor.

[0039] For example, if the phenomenon is coloration, luminescence, fluorescence, or phosphorescence, it can be detected using, for example, a light-receiving device. For example, if the phenomenon is endothermic or exothermic, it can be detected using, for example, a thermal analyzer. For example, if the phenomenon is oxidation-reduction, it can be detected using, for example, a potential measuring instrument or an ion-sensitive field-effect transistor. For example, if the phenomenon is precipitation, it can be detected using, for example, a mass spectrophotometer, absorbance spectrophotometer, or spectrophotometer.

[0040] 4. Effects of the analysis method (1A) The analytical method of this disclosure allows for the detection of multiple target substances without dividing the sample by using multiple types of fusions. The reason for this is as follows: The analytical method of this disclosure allows for the mixing of a sample with multiple types of fusions. If the sample contains multiple types of target substances, each type of target substance will bind to the corresponding type of fusion. For example, if the sample contains multiple types of target substances 7A and 7B, target substance 7A will bind to fusion 1A, and target substance 7B will bind to fusion 1B. Fusions 1A and 1B are different types of fusions. Fusion 1A has the activity to specifically bind to target substance 7A. Fusion 1B has the activity to specifically bind to target substance 7B.

[0041] A fusion 1A formed by bonding with the target substance 7A produces phenomenon A, which is specific to the type of fusion 1A. A fusion 1B formed by bonding with the target substance 7B produces phenomenon B, which is specific to the type of fusion 1B. Since phenomena A and B are different, phenomena A and B can be distinguished and detected.

[0042] By detecting phenomenon A, it can be determined that the sample contained the target substance 7A. Furthermore, the amount and concentration of the target substance 7A can be estimated based on the degree of phenomenon A. By detecting phenomenon B, it can be determined that the sample contained the target substance 7B. Furthermore, the amount and concentration of the target substance 7B can be estimated based on the degree of phenomenon B.

[0043] Therefore, according to the analytical method of this disclosure, multiple types of target substances contained in a sample can be detected without dividing the sample.

[0044] (1B) At least a portion of the binding substance is, for example, a nucleic acid or a nucleic acid aptamer. In this case, since the size of the binding substance is small, it is less susceptible to steric interference during binding between the target substance and the binding substance, and it binds easily to the target substance. As a result, analytical sensitivity can be improved. In addition, the binding substance can be easily synthesized artificially.

[0045] (1C) At least a portion of the target substance is, for example, a protein, sugar, nucleic acid, small molecule compound, or lipid. A variety of target substances can be detected according to the analytical method of this disclosure.

[0046] (1D) In ​​the analytical method of this disclosure, for example, the number of types of fusions mixed with the sample is greater than or equal to the number of types of target substances contained in the sample. In this case, all types of target substances contained in the sample can be detected.

[0047] (1E) The observable phenomena produced by the fusion formed with the target substance are, for example, at least one of the following: oxidation-reduction, color development, fluorescence, luminescence, phosphorescence, endothermic, exothermic, precipitation, and changes in ion content. The ions in the changes in ion content include, for example, at least one of the following: hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, and chloride ions. The ion content changes, for example, due to ion production, consumption, or absorption. If the observable phenomena are these, the target substance contained in the sample can be detected.

[0048] (1F) The label is, for example, a label that induces the production, consumption, or absorption of ions. In this case, the target substance contained in the sample can be detected with high sensitivity.

[0049] (1G) The label includes, for example, at least one of enzymes, DNAzymes, RNAzymes, magnetic labels, fluorescent labels, chemiluminescent probes, and nanoparticles. In this case, the target substance contained in the sample can be detected.

[0050] The (1H) binding substance and the label are bound, for example, via a chemical substituent or a nucleic acid-binding protein. Examples of nucleic acid-binding proteins include zinc fingers and CRISPR. Chemical substituents include, for example, at least one of biotin, primary amines, azides, alkynes, dibenzocyclooctin, bicyclononine, 2'-O-propargyl, 2'-O-propargyl, thiols, avidin, streptavidin, neutraavidin, N-hydroxysuccinimide, maleimide, and 5-halouracil. In this case, the binding substance and the label readily bind.

[0051] 5. Example 1 (5-1) Synthesis of Fusions Fusions 1A and 1B were synthesized using the method shown in Figure 4. First, in step S21 of Figure 4, the conjugates 3A and 3B were chemically synthesized by an in vitro process. Conjugate 3A was a DNA aptamer having the nucleotide sequence of Sequence ID No. 1 and with biotinylation modification at its 5' end. The nucleotide sequence of conjugate 3A was "5'-CAGCACCGAC CTTGTGCTTT GGGAGTGCTG GTCCAAGGGC GTTAATGGAC A-3'". The nucleotide sequence of Sequence ID No. 1 is described in Anal.Chem.2020,92,9895-9900 (hereinafter referred to as Reference 1).

[0052] According to Reference 1, the DNA aptamer having the base sequence of Sequence ID No. 1 is a DNA aptamer whose target substance is the RBD in the spike glycoprotein of the novel coronavirus SARS-CoV-2.

[0053] The binding agent 3B was a DNA aptamer having the nucleotide sequence of Sequence ID No. 2 and with biotinylation modification at its 5' end. The nucleotide sequence of binding agent 3B was "5'-TCGATTCGGGTGGGTGGGAGGGGGTGGAGGTTGGGGGTTGGACGCAGAGTGC-3'". The nucleotide sequence of Sequence ID No. 2 is described in PLoS ONE 10(4):e0125060.Doi:10.1371 / journal.Pone.0125060 (hereinafter referred to as Reference 2). According to Reference 2, the DNA aptamer having the nucleotide sequence of Sequence ID No. 2 is a DNA aptamer that targets hemagglutinin from influenza A.

[0054] Binding agent 3A was dissolved in phosphate buffer (hereinafter referred to as 1xPBS / T) to prepare a solution of binding agent 3A. Similarly, binding agent 3B was dissolved in 1xPBS / T to prepare a solution of binding agent 3B.

[0055] The concentration of binding substance 3A in the binding substance 3A solution and the concentration of binding substance 3B in the binding substance 3B solution were both 10 μmol / l.

[0056] 1xPBS / T was a phosphate buffer solution prepared by diluting 10xPBS / T (Fujifilm Wako Pure Chemical Industries, Ltd., product number MB-075-1000) 10 times with ultrapure water. The binding agent 3A solution and the binding agent 3B solution were heated at 95°C and then slowly cooled.

[0057] Next, in step S22 of Figure 4, labeled product 5A was prepared. Labeled product 5A was a streptavidin-alkaline phosphatase conjugate (Thermofisher Scientific, product number S921). Labeled product 5A was an enzyme. The streptavidin-alkaline phosphatase conjugate is obtained by modifying alkaline phosphatase with streptavidin. Streptavidin is a chemical substituent that modifies alkaline phosphatase. Labeled product 5A was dissolved in 1x PBS / T to prepare a solution of labeled product 5A. The concentration of labeled product 5A in the solution was 20 μmol / l.

[0058] A 100 μL solution of conjugate 3A and a 5 μL solution of label 5A were mixed and allowed to stand at room temperature for 1 hour. During this time, due to the interaction between biotin and streptavidin, conjugate 3A and label 5A fused, and fusion product 1A was synthesized.

[0059] Furthermore, in step S22 of Figure 4, labeled substance 5B was prepared. Labeled substance 5B was a horseradish peroxidase-streptavidin conjugate (Thermofisher Scientific, product number 43-4323). Labeled substance 5B was an enzyme. The horseradish peroxidase-streptavidin conjugate is obtained by modifying horseradish peroxidase with streptavidin. Streptavidin is a chemical substituent that modifies horseradish peroxidase. Labeled substance 5B was dissolved in 1x PBS / T to prepare a labeled substance 5B solution. The concentration of labeled substance 5B in the labeled substance 5B solution was 20 μmol / l.

[0060] 100 μL of a solution of conjugate substance 3B and 5 μL of a solution of label 5B were mixed and allowed to stand at room temperature for 1 hour. During this time, due to the interaction between biotin and streptavidin, conjugate substance 3B and label 5B fused, and fusion product 1B was synthesized.

[0061] Next, in step S23 of Figure 4, fusions 1A and 1B were purified and recovered. Specifically, the binding substance 3A that did not fuse with the labeled substance 5A was separated from fusion 1A using an ultrafiltration filter. Then, fusion 1A was recovered by dissolving it in 1xPBS / T.

[0062] Furthermore, the binding substance 3B that did not fuse with the labeled substance 5B was separated from the fusion 1B using an ultrafiltration filter. Next, the fusion 1B was recovered by dissolving it in 1xPBS / T.

[0063] Note that binding substances 3A and 3B may be binding substances other than DNA aptamers. Binding substances 3A and 3B may be, for example, small molecular weight protein preparations or RNA aptamers. Examples of small molecular weight protein preparations include fragment antibodies, single-chain antibodies, diabodies, nanobodies, VHHs, and peptide aptamers.

[0064] Furthermore, the base sequences of the DNA aptamers constituting binding substances 3A and 3B may be base sequences other than those of sequence numbers 1 and 2. The base sequences of the DNA aptamers can be selected according to the target substance. In addition, the fusion of binding substance 3A and label 5A, and the fusion of binding substance 3B and label 5B may be fusions other than those based on biotin-streptavidin interactions. Also, when nucleic acid aptamers are used as binding substances 3A and 3B, the ends of the nucleic acid aptamers other than the 5' end may fuse with the labels 5A and 5B.

[0065] Furthermore, when using small molecular weight protein preparations as binding substances 3A and 3B, the N-terminus of the small molecular weight protein preparation may be fused with the labeled substances 5A and 5B, or the C-terminus may be fused with the labeled substances 5A and 5B.

[0066] Fusion 1A and Fusion 1B are of different types. That is, the target substance 7A that binds to Fusion 1A and the target substance 7B that binds to Fusion 1B are different substances. Furthermore, the phenomenon A that produces Fusion 1A and the phenomenon B that produces Fusion 1B are different phenomena.

[0067] (5-2) Implementation of the analytical method for the target substance As shown in Figure 5, the analysis method for the target substance was carried out. In S31 of Figure 5, target substances 7A and 7B were prepared. In this embodiment 1, the number of types of fusions 1A and 1B is 2. The number of types of target substances 7A and 7B is 2. Therefore, the number of types of fusions 1A and 1B is greater than or equal to the number of types of target substances 7A and 7B.

[0068] The target substance 7A was SARS-CoV-2 (2019-nCoV) Spike S1-His Recombinant Protein (Sinobiological, product number 40591-V08H). The amino acid sequence of target substance 7A includes RBD. A solution of target substance 7A was prepared by dissolving target substance 7A in 1x PBS / T. The concentration of target substance 7A in the solution was 4 μg / mL.

[0069] The target substance 7B was Influenza A H1N1 (A / California / 04 / 2009) Hemagglutinin / HA0 Protein (SinoBiological, product number 11055-VNAB). A solution of target substance 7B was prepared by dissolving it in 1x PBS / T. The concentration of target substance 7B in the solution was 4 μg / mL.

[0070] Next, 200 μL of solution of target substance 7A and 200 μL of solution of target substance 7B were mixed to prepare solution of target substance 7AB. The concentration of target substance 7A in solution of target substance 7AB was 2 μg / mL, and the concentration of target substance 7B was 2 μg / mL.

[0071] Next, 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, and 100 μL of target substance 7AB solution were mixed and allowed to stand at room temperature for 1 hour to prepare a fusion-target substance 7AB mixture. In the fusion-target substance 7AB mixture, the concentration of fusion 1A was 5 μmol / L, the concentration of fusion 1B was 5 μmol / L, the concentration of target substance 7A was 1 μg / mL, and the concentration of target substance 7B was 1 μg / mL.

[0072] Furthermore, 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, 50 μL of target substance 7A solution, and 50 μL of 1xPBS / T were mixed and allowed to stand at room temperature for 1 hour to prepare a fusion-target substance 7A mixture. In the fusion-target substance 7A mixture, the concentration of fusion 1A was 5 μmol / L, the concentration of fusion 1B was 5 μmol / L, and the concentration of target substance 7A was 1 μg / mL.

[0073] Furthermore, 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, 50 μL of target substance 7B solution, and 50 μL of 1xPBS / T were mixed and allowed to stand at room temperature for 1 hour to prepare a fusion-target substance 7B mixture. In the fusion-target substance 7B mixture, the concentration of fusion 1A was 5 μmol / L, the concentration of fusion 1B was 5 μmol / L, and the concentration of target substance 7B was 1 μg / mL.

[0074] Furthermore, a control mixture was prepared by mixing 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, and 100 μL of 1xPBS / T, and allowing it to stand at room temperature for 1 hour. The concentration of fusion 1A in the control mixture was 5 μmol / l, and the concentration of fusion 1B was also 5 μmol / l.

[0075] At this time, as shown in S32 of Figure 5, a composite 33A of the target substance 7A and the fusion 1A, or a composite 33B of the target substance 7B and the fusion 1B was formed. That is, as shown in Figure 6, in the fusion-target substance 7AB mixture, a composite 33A of the target substance 7A and the fusion 1A was formed, as well as a composite 33B of the target substance 7B and the fusion 1B.

[0076] Furthermore, as shown in Figure 7, in the fusion-target substance 7A mixture, a compound 33A was formed between the target substance 7A and the fusion 1A. Also, as shown in Figure 8, in the fusion-target substance 7B mixture, a compound 33B was formed between the target substance 7B and the fusion 1B. Furthermore, as shown in Figure 9, no compounds 33A or 33B were formed in the control mixture.

[0077] Next, in step S33 of Figure 5, fusions 1A and 1B that were not bound to target substances 7A and 7B were separated from the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture using an ultrafiltration filter. As a result, bound substances 33A and 33B were recovered.

[0078] Furthermore, in step S33 of Figure 5, the conjugates recovered from the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture were dispensed into two microcentrifuge tubes. These two dispensed solutions were designated as Solution 1 and Solution 2.

[0079] In Figure 5, at S34, 4-nitrophenyl disodium hexahydrate phosphate (Tokyo Chemical Industries, Ltd., product number N0241) was prepared. Hereafter, 4-nitrophenyl disodium hexahydrate phosphate will be referred to as pNPP. pNPP is a substrate for alkaline phosphatase-induced metabolism.

[0080] Next, pNPP was dissolved in a solution containing carbonate pH standard solution (Fujifilm Wako Pure Chemical Industries, Ltd., product number 037-16145) and magnesium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., product number 137-12335) to prepare a pNPP solution. The concentration of pNPP in the pNPP solution was 10 mmol / l. The solution containing carbonate pH standard solution and magnesium sulfate contained 1 mmol / l of carbonate pH standard solution and 1 mmol / l of magnesium sulfate. The pH of the solution containing carbonate pH standard solution and magnesium sulfate was adjusted to 9.6.

[0081] Note that the solution used to dissolve pNPP may be a solution other than one containing a carbonate pH standard solution and magnesium sulfate. Examples of solutions for dissolving pNPP include Tris buffer, phosphate buffer, and Good's buffer. Examples of Good's buffer include MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricin, Bicine, TAPS, CHES, CAPSO, and CAPS.

[0082] The pH of the solution used to dissolve pNPP may be a value other than 9.6. The solution used to dissolve pNPP is preferably a weakly alkaline solution. The pH of the solution used to dissolve pNPP is preferably between 8 and 11. When the solution used to dissolve pNPP is weakly alkaline, the pH change due to metabolism using pNPP as a substrate becomes larger. When the pH of the solution used to dissolve pNPP is between 8 and 11, the pH change due to metabolism using pNPP as a substrate becomes larger.

[0083] Furthermore, in S34 of Figure 5, a 1-Step Ultra TMB-ELISA (Thermofisher Scientific, product number 34028) was prepared. The 1-Step Ultra TMB-ELISA contains substrates for the metabolism induced by horseradish peroxidase.

[0084] Next, 100 μL of pNPP solution was added to each of the first solutions obtained from the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture, and the mixture was allowed to stand at 37°C for 15 minutes. If the first solution contains fusion substance 1A, the enzymatic activity of the alkaline phosphatase present in fusion substance 1A induces metabolism using pNPP as a substrate, and the phosphate group is separated from pNPP. As a result, inorganic phosphate and p-nitrophenol are produced. The absorption maximum wavelength of p-nitrophenol is 405 nm. Therefore, the solution containing p-nitrophenol turns yellow. In addition, the produced inorganic phosphate dissociates in the solution and releases hydrogen ions. Therefore, the pH of the solution decreases.

[0085] Furthermore, in S34 of Figure 5, 100 μL of 1-Step Ultra TMB-ELISA was added to the second solution obtained from each of the following: the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture, and the mixtures were allowed to stand at room temperature for 15 minutes.

[0086] Next, in step S35 of Figure 5, 5 μL of 0.5 mol / l ethylenediaminetetraacetic acid (Fujifilm Wako Pure Chemical Industries, product number 311-90075) was added dropwise to the first solution obtained from each of the fusion-target substance 7AB mixture, fusion-target substance 7A mixture, fusion-target substance 7B mixture, and control mixture to stop the alkaline phosphatase enzyme reaction.

[0087] Furthermore, in step S35 of Figure 5, 50 μL of 1 mol / l H2SO4 was added to the second solution obtained from each of the fusion-target substance 7AB mixture, fusion-target substance 7A mixture, fusion-target substance 7B mixture, and control mixture to stop the horseradish peroxidase enzyme reaction.

[0088] Next, in S36 of Figure 5, the absorbance at 405 nm was measured for the first solution obtained from each of the following: the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture. In addition, the absorbance at 450 nm was measured for the second solution obtained from each of the following: the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture.

[0089] The absorbance measurement results are shown in Figure 10. In Figure 10, "○" means that the absorbance is greater than that of the first or second solution obtained from the control mixture. "×" means that the absorbance is the same as or less than that of the first or second solution obtained from the control mixture.

[0090] The absorbance measurement results show the following: In Example 1, a solution of target substances 7AB containing target substances 7A and 7B was mixed with fusions 1A and 1B. Next, fusions 1A and 1B that were not bound to either target substance 7A or 7B were removed. Phenomenon A, in which fusion 1A bound to target substance 7A was formed, and phenomenon B, in which fusion 1B bound to target substance 7B was formed, were detected. Phenomenon A is characterized by high absorbance at 405 nm. Phenomenon B is characterized by high absorbance at 450 nm. Therefore, in Example 1, target substances 7A and 7B could be detected.

[0091] In Example 1, a solution of target substance 7A containing the target substance 7A was mixed with fusions 1A and 1B. Next, fusions 1A and 1B that were not bound to the target substance 7A were removed. The phenomenon of A, in which fusion 1A bound to the target substance 7A was formed, was detected. Therefore, in Example 1, the target substance 7A could be detected.

[0092] In Example 1, a solution of target substance 7B containing the target substance 7B was mixed with fusions 1A and 1B. Next, fusions 1A and 1B that were not bound to the target substance 7B were removed. The phenomenon of fusion 1B being bound to the target substance 7B was detected. Therefore, in Example 1, the target substance 7B could be detected.

[0093] Furthermore, the label 5A present in fusion 1A possessed enzymatic activity that induced metabolism using pNPP as a substrate, even when fused with the binding substance 3A. In addition, the binding substance 3A present in fusion 1A possessed binding activity to the target substance 7A, even when fused with the label 5A.

[0094] The label 5B present in fusion 1B retained enzymatic activity that induced substrate metabolism even when fused with binding substance 3B. Furthermore, the binding substance 3B present in fusion 1B retained binding activity to the target substance 7B even when fused with label 5B.

[0095] Furthermore, binding substance 3A did not bind to target substance 7B. In other words, binding substance 3A did not have binding activity to target substance 7B. Also, binding substance 3B did not bind to target substance 7A. In other words, binding substance 3B did not have binding activity to target substance 7A.

[0096] 6. Example 2 (6-1) Synthesis of Fusion 1A Fusion 1A was synthesized using the method shown in Figure 11. First, in step S41 of Figure 11, the binder 3A was chemically synthesized by an in vitro process. The binder 3A was the same as the binder 3A in Example 1.

[0097] A solution of binding agent 3A was prepared by dissolving binding agent 3A in 1x PBS / T. The concentration of binding agent 3A in the solution was 10 μmol / L. The solution of binding agent 3A was heated to 95°C and then slowly cooled.

[0098] Next, in step S42 of Figure 11, labeled substance 5A was prepared. Labeled substance 5A was the same as labeled substance 5A in Example 1. Labeled substance 5A was dissolved in 1x PBS / T to prepare a labeled substance 5A solution. The concentration of labeled substance 5A in the labeled substance 5A solution was 20 μmol / l.

[0099] A 100 μL solution of conjugate 3A and a 5 μL solution of label 5A were mixed and allowed to stand at room temperature for 1 hour. During this time, due to the interaction between biotin and streptavidin, conjugate 3A and label 5A fused, and fusion product 1A was synthesized.

[0100] Next, in step S43 of Figure 11, fusion 1A was purified and recovered. Specifically, the binding substance 3A that did not fuse with the labeled substance 5A was separated from fusion 1A using an ultrafiltration filter. Then, fusion 1A was recovered by dissolving it in 1xPBS / T.

[0101] (6-2) Synthesis of Fusion 1B Fusion 1B was synthesized using the method shown in Figure 12. First, in step S51 of Figure 12, the binding substance 3B was chemically synthesized by an in vitro process. Binding substance 3B was a DNA aptamer having the base sequence of Sequence ID No. 2 and having an azidation modification at its 5' end.

[0102] A solution of binding agent 3B was prepared by dissolving binding agent 3B in 1x PBS / T. The concentration of binding agent 3B in the solution was 10 μmol / L. The solution of binding agent 3B was heated to 95°C and then slowly cooled.

[0103] In step S52 of Figure 12, labeled product 5B was synthesized as follows: Urease (Fujifilm Wako Pure Chemical Industries, product number 210-00781) was prepared. Urease is an enzyme. Urease was dissolved in 1x PBS / T to prepare a urease solution. The concentration of urease in the urease solution was 100 μmol / L.

[0104] Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (Sigma, product number 761524-50MG) (hereinafter referred to as DBCO-NHS) was prepared. DBCO-NHS was diluted in a 0.1 mol / l NaHCO3 solution to prepare a DBCO-NHS solution. The concentration of DBCO-NHS in the DBCO-NHS solution was 100 μmol / l.

[0105] 80 μL of DBCO-NHS solution and 20 μL of urease solution were mixed and allowed to stand at room temperature for 1 hour. During this time, the interaction between NHS and the amino group caused DBCO-NHS to bind to urease, resulting in the synthesis of DBCO-NHS-modified urease. The DBCO-NHS-modified urease was designated as label 5B. In other words, label 5B was a DBCO-NHS-modified enzyme.

[0106] Next, in step S53 of Figure 12, the labeled product 5B was purified and recovered. Specifically, DBCO-NHS that did not fuse with urease was separated from the labeled product 5B using an ultrafiltration filter, and the labeled product 5B was recovered.

[0107] Next, in step S54 of Figure 12, 100 μL of the binding agent 3B solution and 5 μL of the label 5B were mixed and allowed to stand at room temperature for 1 hour. During this time, the binding agent 3B and the label 5B fused together via Azide-DBCO click chemistry, and fusion product 1B was synthesized.

[0108] Next, in step S55 of Figure 12, the fusion product 1B was purified and recovered. Specifically, the binding substance 3B that did not fuse with the labeled substance 5B was separated from the fusion product 1B using an ultrafiltration filter, and the fusion product 1B was recovered.

[0109] Note that binding substances 3A and 3B may be binding substances other than DNA aptamers. Binding substances 3A and 3B may be, for example, small molecular weight protein preparations or RNA aptamers. Examples of small molecular weight protein preparations include fragment antibodies, single-chain antibodies, diabodies, nanobodies, VHHs, and peptide aptamers.

[0110] Furthermore, the base sequences of the DNA aptamers constituting binding substances 3A and 3B may be base sequences other than those of sequence numbers 1 and 2. The base sequences of the DNA aptamers can be selected according to the target substance. Also, the fusion of binding substance 3A and label 5A may be a fusion other than that based on the biotin-streptavidin interaction. Also, the fusion of binding substance 3B and label 5B may be a fusion other than that based on the click chemistry of Azide-DBCO. In addition, when nucleic acid aptamers are used as binding substances 3A and 3B, the ends of the nucleic acid aptamers other than the 5' end may be fused with the labels 5A and 5B.

[0111] Furthermore, when using small molecular weight protein preparations as binding substances 3A and 3B, the N-terminus of the small molecular weight protein preparation may be fused with the labeled substances 5A and 5B, or the C-terminus may be fused with the labeled substances 5A and 5B.

[0112] Fusion 1A and Fusion 1B are of different types. That is, the target substance 7A that binds to Fusion 1A and the target substance 7B that binds to Fusion 1B are different substances. Furthermore, the phenomenon A that produces Fusion 1A and the phenomenon B that produces Fusion 1B are different phenomena.

[0113] (6-2) Implementation of the analytical method for the target substance As shown in Figure 13, the analysis method for the target substance was carried out. In S61 of Figure 13, target substances 7A and 7B were prepared. Target substances 7A and 7B were the same as target substances 7A and 7B in Example 1. In this Example 2, the number of types of fusion products 1A and 1B is 2. The number of types of target substances 7A and 7B is 2. Therefore, the number of types of fusion products 1A and 1B is greater than or equal to the number of types of target substances 7A and 7B.

[0114] A solution of target substance 7A was prepared by dissolving target substance 7A in 1x PBS / T. The concentration of target substance 7A in the solution was 4 μg / mL. Similarly, a solution of target substance 7B was prepared by dissolving target substance 7B in 1x PBS / T. The concentration of target substance 7B in the solution was 4 μg / mL.

[0115] Next, 200 μL of solution of target substance 7A and 200 μL of solution of target substance 7B were mixed to prepare solution of target substance 7AB. The concentration of target substance 7A in solution of target substance 7AB was 2 μg / mL, and the concentration of target substance 7B was 2 μg / mL.

[0116] Next, 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, and 100 μL of target substance 7AB solution were mixed and allowed to stand at room temperature for 1 hour to prepare a fusion-target substance 7AB mixture. In the fusion-target substance 7AB mixture, the concentration of fusion 1A was 5 μmol / L, the concentration of fusion 1B was 5 μmol / L, the concentration of target substance 7A was 1 μg / mL, and the concentration of target substance 7B was 1 μg / mL.

[0117] Furthermore, 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, 50 μL of target substance 7A solution, and 50 μL of 1xPBS / T were mixed and allowed to stand at room temperature for 1 hour to prepare a fusion-target substance 7A mixture. In the fusion-target substance 7A mixture, the concentration of fusion 1A was 5 μmol / L, the concentration of fusion 1B was 5 μmol / L, and the concentration of target substance 7A was 1 μg / mL.

[0118] Furthermore, 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, 50 μL of target substance 7B solution, and 50 μL of 1xPBS / T were mixed and allowed to stand at room temperature for 1 hour to prepare a fusion-target substance 7B mixture. In the fusion-target substance 7B mixture, the concentration of fusion 1A was 5 μmol / L, the concentration of fusion 1B was 5 μmol / L, and the concentration of target substance 7B was 1 μg / mL.

[0119] Furthermore, a control mixture was prepared by mixing 50 μL of fusion 1A solution, 50 μL of fusion 1B solution, and 100 μL of 1xPBS / T, and allowing it to stand at room temperature for 1 hour. The concentration of fusion 1A in the control mixture was 5 μmol / l, and the concentration of fusion 1B was also 5 μmol / l.

[0120] At this time, as shown in S62 of Figure 13, a composite 33A of the target substance 7A and the fusion 1A, or a composite 33B of the target substance 7B and the fusion 1B was formed. That is, as shown in Figure 6, in the fusion-target substance 7AB mixture, a composite 33A of the target substance 7A and the fusion 1A was formed, as well as a composite 33B of the target substance 7B and the fusion 1B.

[0121] Furthermore, as shown in Figure 7, in the fusion-target substance 7A mixture, a compound 33A was formed between the target substance 7A and the fusion 1A. Also, as shown in Figure 8, in the fusion-target substance 7B mixture, a compound 33B was formed between the target substance 7B and the fusion 1B. Furthermore, as shown in Figure 9, no compounds 33A or 33B were formed in the control mixture.

[0122] Next, in step S63 of Figure 13, fusions 1A and 1B that were not bound to target substances 7A and 7B were separated from each of the fusion-target substance 7AB mixture, fusion-target substance 7A mixture, fusion-target substance 7B mixture, and control mixture using an ultrafiltration filter. As a result, bound substances 33A and 33B were recovered.

[0123] Furthermore, in step S63 of Figure 13, the conjugates recovered from the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture were dispensed into two microcentrifuge tubes. These two dispensed solutions were designated as Solution 1 and Solution 2.

[0124] Steps S64, S65, and S66 in Figure 13 are processes performed using the first liquid obtained from the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture, respectively. Steps S67, S68, and S69 in Figure 13 are processes performed using the second liquid obtained from the fusion-target substance 7AB mixture, the fusion-target substance 7A mixture, the fusion-target substance 7B mixture, and the control mixture, respectively.

[0125] In the flowchart in Figure 13, for the sake of explanation, the process is shown to branch after the process in S63, with processes S64, S65, and S66 being followed by processes S67, S68, and S69. For example, processes S64, S65, and S66 could be performed first, followed by processes S67, S68, and S69.

[0126] In step S64 of Figure 13, pNPP was dissolved in a solution containing carbonate pH standard solution (Fujifilm Wako Pure Chemical Industries, Ltd., product number 037-16145) and magnesium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., product number 137-12335) to prepare a pNPP solution. The concentration of pNPP in the pNPP solution was 10 mmol / l. The solution containing carbonate pH standard solution and magnesium sulfate contained 1 mmol / l of carbonate pH standard solution and 1 mmol / l of magnesium sulfate. The pH of the solution containing carbonate pH standard solution and magnesium sulfate was adjusted to 9.6.

[0127] Note that the solution used to dissolve pNPP may be a solution other than one containing a carbonate pH standard solution and magnesium sulfate. Examples of solutions for dissolving pNPP include Tris buffer, phosphate buffer, and Good's buffer. Examples of Good's buffer include MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricin, Bicine, TAPS, CHES, CAPSO, and CAPS.

[0128] The pH of the solution used to dissolve pNPP may be a value other than 9.6. The solution used to dissolve pNPP is preferably a weakly alkaline solution. The pH of the solution used to dissolve pNPP is preferably between 8 and 11. When the solution used to dissolve pNPP is weakly alkaline, the pH change due to metabolism using pNPP as a substrate becomes larger. When the pH of the solution used to dissolve pNPP is between 8 and 11, the pH change due to metabolism using pNPP as a substrate becomes larger.

[0129] Next, 100 μL of pNPP solution was added to the first solution and allowed to stand at 37°C for 15 minutes. If the first solution contains fusion 1A, the enzymatic activity of the alkaline phosphatase present in fusion 1A induces metabolism using pNPP as a substrate, and the phosphate group is separated from pNPP. As a result, inorganic phosphate and p-nitrophenol are produced. The absorption maximum wavelength of p-nitrophenol is 405 nm. Therefore, the solution containing p-nitrophenol turns yellow. In addition, the produced inorganic phosphate dissociates in the solution and releases hydrogen ions. Therefore, the pH of the solution decreases.

[0130] Next, in step S65 of Figure 13, 5 μL of 0.5 mol / l ethylenediaminetetraacetic acid (Fujifilm Wako Pure Chemical Industries, product number 311-90075) was added dropwise to the first solution to stop the alkaline phosphatase enzyme reaction.

[0131] Next, in S66 of Figure 13, the absorbance at 405 nm was measured for the first solution.

[0132] In Figure 13, at step S67, the QuantiChrom Urease Assay Kit (BioAssay BioAssay Systems, product number DURE-100) was prepared. The QuantiChrom Urease Assay Kit contains substrates for urease-induced metabolism.

[0133] Next, 10 μL of urea, included in the QuantiChrom Urease Assay Kit, was added to the second solution and allowed to stand for 10 minutes.

[0134] Next, in step S68 of Figure 13, 100 μL of Reagent A from the QuantiChrom Urease Assay Kit was added to the second solution and mixed thoroughly. Then, 50 μL of Reagent B from the QuantiChrom Urease Assay Kit was added to the second solution and mixed thoroughly, after which the mixture was allowed to stand in the dark for 30 minutes. The urea contained in the QuantiChrom Urease Assay Kit is metabolized by urease, and then, upon addition of Reagent A and subsequently Reagent B from the QuantiChrom Urease Assay Kit, it transforms into a product with an absorption wavelength of 670 nm. Furthermore, the pH of the solution increases due to the ammonia produced.

[0135] Next, in S69 of Figure 13, the absorbance at 670 nm was measured for the second solution. The absorbance measurement results are shown in Figure 14. In Figure 14, "○" means that the absorbance is greater than that of the first or second solution obtained from the control mixture. "×" means that the absorbance is the same as or less than that of the first or second solution obtained from the control mixture.

[0136] The absorbance measurement results show the following. In Example 2, a solution of target substances 7AB containing target substances 7A and 7B was mixed with fusions 1A and 1B. Next, fusions 1A and 1B that were not bound to either target substance 7A or 7B were removed. Phenomenon A, in which fusion 1A bound to target substance 7A was formed, and phenomenon B, in which fusion 1B bound to target substance 7B was formed, were detected. Phenomenon A is characterized by high absorbance at 405 nm. Phenomenon B is characterized by high absorbance at 670 nm. Therefore, in Example 2, target substances 7A and 7B could be detected. Furthermore, since phenomena A and B are different, it was possible to distinguish and detect phenomena A and B.

[0137] In Example 2, a solution of target substance 7A containing the target substance 7A was mixed with fusions 1A and 1B. Next, fusions 1A and 1B that were not bound to the target substance 7A were removed. The phenomenon of A, in which fusion 1A bound to the target substance 7A was formed, was detected. Therefore, in Example 2, the target substance 7A could be detected.

[0138] In Example 2, a solution of target substance 7B containing the target substance 7B was mixed with fusions 1A and 1B. Next, fusions 1A and 1B that were not bound to the target substance 7B were removed. The phenomenon of fusion 1B being bound to the target substance 7B was detected. Therefore, in Example 2, the target substance 7B could be detected.

[0139] Furthermore, the label 5A present in fusion 1A possessed enzymatic activity that induced metabolism using pNPP as a substrate, even when fused with the binding substance 3A. In addition, the binding substance 3A present in fusion 1A possessed binding activity to the target substance 7A, even when fused with the label 5A.

[0140] The label 5B present in fusion 1B retained enzymatic activity that induced substrate metabolism even when fused with binding substance 3B. Furthermore, the binding substance 3B present in fusion 1B retained binding activity to the target substance 7B even when fused with label 5B.

[0141] Furthermore, binding substance 3A did not bind to target substance 7B. In other words, binding substance 3A did not have binding activity to target substance 7B. Also, binding substance 3B did not bind to target substance 7A. In other words, binding substance 3B did not have binding activity to target substance 7A.

[0142] 7. Other Embodiments Although embodiments of the present disclosure have been described above, the present disclosure is not limited to the embodiments described above and can be implemented in various modified forms.

[0143] (1) The number of types of fusions to be mixed with the sample is not particularly limited and can be, for example, 2, 3, 4, 5, 6, 7, 8... The number of types of target substances contained in the sample is not particularly limited and can be, for example, 2, 3, 4, 5, 6, 7, 8...

[0144] (2) The sample may be divided, and the analytical method of this disclosure may be performed on a portion of the divided sample.

[0145] (3) Multiple functions of one component in the above embodiment may be realized by multiple components, or one function of one component may be realized by multiple components. Also, multiple functions of multiple components may be realized by one component, or one function realized by multiple components may be realized by one component. Furthermore, some of the configurations of the above embodiment may be omitted. Also, at least some of the configurations of the above embodiment may be added to or replaced with the configurations of other above embodiments.

[0146] (4) In addition to the analytical methods described above, this disclosure can also be implemented in various forms, such as fused bodies, groups consisting of multiple types of fused bodies, methods for manufacturing fused bodies, and methods for manufacturing groups of fused bodies.

[0147] [Technical concepts disclosed in this specification] [Item 1] A fusion (1A, 1B) comprising a binding substance (3A, 3B) having activity to bind to the target substance (7A, 7B), and a label (5A, 5B), is mixed with a sample containing the target substance. Remove the fusion that is not bonded to the target substance, A method for analyzing a target substance, comprising detecting a phenomenon caused by a label contained in the fusion body bonded with the target substance, There are multiple types of the aforementioned fusion body. The target substance bonded to the fusion body and the phenomenon differ depending on the type of fusion body. Analysis method. [Item 2] The analysis method described in item 1, The binding substance comprises at least one of nucleic acids and amino acids. Analysis method. [Item 3] The analytical method described in item 1 or 2, At least a portion of the binding substance is a nucleic acid aptamer or a low molecular weight protein preparation. Analysis method. [Item 4] The analysis method described in any one of items 1 to 3, At least a portion of the aforementioned target substance is a protein, sugar, nucleic acid, low molecular weight compound, or lipid. Analysis method. [Item 5] The analysis method described in any one of items 1 to 4, The labeled substance comprises at least one of the following: an enzyme, a DNAzyme, an RNAzyme, a magnetic label, a fluorescent label, a chemiluminescent probe, and nanoparticles. Analysis method. [Item 6] The analysis method described in any one of items 1 to 5, The number of types of the fusion is greater than or equal to the number of types of the target substance contained in the sample. The aforementioned phenomenon is at least one of the following: oxidation-reduction, color development, fluorescence, light emission, phosphorescence, endothermic, exothermic, precipitation, and change in ion content. The ions in the aforementioned change in ion quantity include at least one of hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, and chloride ions. The amount of ions changes due to the production, consumption, or absorption of the ions. Analysis method. [Item 7] The analysis method described in any one of items 1 to 6, The binding substance and the label are linked via a chemical substituent or a nucleic acid-binding protein. The chemical substituent includes at least one of biotin, primary amine, azide, alkyne, dibenzocyclooctin, bicyclononine, 2'-O-propargyl, 2'-O-propargyl, thiol, avidin, streptavidin, neutraavidin, N-hydroxysuccinimide, maleimide, and 5-halouracil. Analysis method. [Item 8] The analysis method described in any one of items 1 to 7, If the phenomenon is the production, consumption, or absorption of hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, or chloride ions, the phenomenon is detected using a pH meter or an ion-sensitive field-effect transistor. If the phenomenon is coloration, light emission, fluorescence, or phosphorescence, the phenomenon is detected using a light-receiving device. If the phenomenon is endothermic or exothermic, the phenomenon is detected using a thermal analyzer. If the aforementioned phenomenon is oxidation-reduction, the phenomenon is detected using a potential measuring instrument or an ion-sensitive field-effect transistor. If the phenomenon is precipitation, the phenomenon is detected using a mass spectrometer, absorbance spectrophotometer, or spectrophotometer. Analysis method. [Explanation of symbols]

[0148] 1A, 1B… Fusion, 3A, 3B… Binding Substance, 5A, 5B… Marker, 7A, 7B… Target Substance, 9A, 9B… Matrix, 11A, 11B… Metabolite, 33A, 33B… Binding

Claims

1. A fusion body (1A, 1B) comprising a binding substance (3A, 3B) having activity to bind to the target substance (7A, 7B), and a label (5A, 5B), is mixed with a sample containing the target substance. Remove the fusion that is not bonded to the target substance, A method for analyzing a target substance, comprising detecting a phenomenon caused by a label contained in the fusion body bonded with the target substance, There are multiple types of the aforementioned fusion body. The target substance bonded to the fusion body and the phenomenon differ depending on the type of fusion body. The labeled substance comprises at least one of the following: an enzyme, a DNAzyme, an RNAzyme, a magnetic label, a chemiluminescent probe, and nanoparticles. Analysis method.

2. A fusion body (1A, 1B) comprising a binding substance (3A, 3B) having activity to bind with the target substance (7A, 7B), and a label (5A, 5B), is mixed with a sample containing the target substance. Remove the fusion that is not bonded to the target substance, A method for analyzing a target substance, comprising detecting a phenomenon caused by a label contained in the fusion body bonded with the target substance, There are multiple types of the aforementioned fusion body. The target substance bonded to the fusion body and the phenomenon differ depending on the type of fusion body. The number of types of the fusion is greater than or equal to the number of types of the target substance contained in the sample. The aforementioned phenomenon is at least one of the following: oxidation-reduction, color development, luminescence, phosphorescence, endothermic, exothermic, precipitation, and change in ion content. The ions in the aforementioned change in ion quantity include at least one of hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, and chloride ions. The amount of ions changes due to the production, consumption, or absorption of the ions. Analysis method.

3. The analytical method according to claim 1 or 2, The binding substance comprises at least one of nucleic acids and amino acids. Analysis method.

4. The analytical method according to claim 1 or 2, At least a portion of the binding substance is a nucleic acid aptamer or a low molecular weight protein preparation. Analysis method.

5. The analytical method according to claim 1 or 2, At least a portion of the aforementioned target substance is a protein, sugar, nucleic acid, low molecular weight compound, or lipid. Analysis method.

6. The analytical method according to claim 1 or 2, The binding substance and the label are linked via a chemical substituent or a nucleic acid-binding protein. The chemical substituent includes at least one of biotin, primary amine, azide, alkyne, dibenzocyclooctin, bicyclononine, 2'-O-propargyl, 2'-O-propargyl, thiol, avidin, streptavidin, neutraavidin, N-hydroxysuccinimide, maleimide, and 5-halouracil. Analysis method.

7. The analytical method according to claim 1 or 2, If the phenomenon is the production, consumption, or absorption of hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, or chloride ions, the phenomenon is detected using a pH meter or an ion-sensitive field-effect transistor. If the phenomenon is coloration, light emission, or phosphorescence, the phenomenon is detected using a light-receiving device. If the phenomenon is endothermic or exothermic, the phenomenon is detected using a thermal analyzer. If the aforementioned phenomenon is oxidation-reduction, the phenomenon is detected using a potential measuring instrument or an ion-sensitive field-effect transistor. If the phenomenon is precipitation, the phenomenon is detected using a mass spectrometer, absorbance spectrophotometer, or spectrophotometer. Analysis method.

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