Method for analyzing detection target substance and system for analyzing detection target substance

Abstract

To provide an analysis method of a substance to be detected and an analysis system of the substance to be detected in which analysis sensitivity is hardly reduced even when the substance to be detected is close to another substance.SOLUTION: The fusant 7 includes a binding substance 9 having an activity of binding to the substance to be detected 1, and a label body 11 that is fused with the binding substance and causes an observable phenomenon. The binding substance is a low-molecular protein preparation or a nucleic acid aptamer. The label includes, for example, an enzyme. The enzyme induces, for example, a hydrogen-producing metabolism, a hydrogen-consuming metabolism, or a hydrogen-absorbing metabolism.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to fusions. [Background technology]

[0002] In technical fields such as biotechnology and healthcare, analytical methods are used to analyze target substances in samples. One known analytical method is the enzyme-linked immunosorbent assay (ELISA). The target substances are proteins, etc.

[0003] In the enzyme-linked immunosorbent assay, a fusion product of an immunoglobulin and a label is bound to a substance to be detected. The fusion product is described in Non-Patent Document 1. Immunoglobulins include monoclonal antibodies, polyclonal antibodies, and the like. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Biosensors and Bioelectronics 117 (2018) 175-182 Summary of the Invention [Problem to be solved by the invention]

[0005] In a sample, a substance to be detected may be close to another substance. For example, when a plurality of substances to be detected form a complex, or when substances to be detected are present at high density on an outer shell such as a cell membrane, the substance to be detected is close to the neighboring substance to be detected. Also, when a substance to be detected is present on an outer shell such as a cell membrane and another large substance is present near the substance to be detected, the substance to be detected is close to the other large substance.

[0006] Immunoglobulins are large in size. Therefore, when a substance to be detected is in close proximity to other substances, immunoglobulin-containing fusion products may be subject to interference from the other substances and may not be able to bind to the substance to be detected. As a result, analytical sensitivity of the substance to be detected decreases.

[0007] In one aspect of the present disclosure, it is preferable to provide a fusion product that is less likely to experience a decrease in analytical sensitivity even when the substance to be detected is in proximity to another substance. [Means for solving the problem]

[0008] One aspect of the present disclosure comprises a binding substance (9) having the activity of binding to a substance to be detected (1), and a label (11) fused to the binding substance to produce an observable phenomenon, wherein the binding substance is a fusion substance (7) that is a low molecular weight protein preparation or a nucleic acid aptamer.

[0009] The binding substance contained in the fusion structure according to one aspect of the present disclosure is a small molecule protein preparation or a nucleic acid aptamer. Therefore, the size of the fusion structure according to one aspect of the present disclosure is smaller than that of a fusion structure containing an immunoglobulin. Therefore, even when the analyte is in close proximity to other substances, the fusion structure according to one aspect of the present disclosure is less susceptible to interference from the other substances and more likely to bind to the analyte. As a result, the analytical sensitivity of the analyte can be improved by using the fusion structure according to one aspect of the present disclosure. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is an explanatory diagram showing the structure of a fusion product and a method for analyzing a substance to be detected. [Figure 2] FIG. 1 is an explanatory diagram showing the flow of the process for synthesizing a fusion product. [Figure 3] FIG. 1 is an explanatory diagram showing metabolism caused by the enzymatic activity of alkaline phosphatase. [Figure 4] FIG. 1 is an explanatory diagram showing the flow of an analysis method. [Figure 5] FIG. 10 is an explanatory diagram showing the results of absorbance measurement. [Figure 6]FIG. 1 is an explanatory diagram showing the flow of an analysis method. [Figure 7] FIG. 1 is an explanatory diagram showing the recording results of pH. [Figure 8] FIG. 1 is an explanatory diagram showing the flow of a test for confirming the expression of the function of a labeled substance. [Figure 9] FIG. 1 is an explanatory diagram showing the recording results of pH. DETAILED DESCRIPTION OF THE INVENTION

[0011] Exemplary embodiments of the present disclosure will now be described with reference to the drawings. 1.Fusion (1-1) Composition of the fusion The fusion product comprises a binding substance. The binding substance has the activity of binding to an analyte (hereinafter referred to as binding activity). The binding substance retains its binding activity even when fused with a label.

[0012] The binding substance is a low molecular weight protein preparation or a nucleic acid aptamer. The number of amino acids constituting the low molecular weight protein preparation is, for example, 5 to 200. When the number of amino acids is 200 or less, even if the substance to be detected is close to other substances, the fusion product is less susceptible to interference from other substances and is more likely to bind to the substance to be detected. The low molecular weight protein preparation may be, for example, a combination of multiple units consisting of amino acids. The number of amino acids in each unit is preferably 5 to 200. When the number of amino acids in each unit is 200 or less, even if the substance to be detected is close to other substances, the fusion product is less susceptible to interference from other substances and is more likely to bind to the substance to be detected.

[0013] Examples of small molecule protein preparations include fragment antibodies, single-chain antibodies, diabodies, nanobodies, VHHs, peptide aptamers, etc. Small molecule protein preparations are, for example, neither monoclonal antibodies nor polyclonal antibodies.

[0014] The number of bases in the nucleic acid aptamer is, for example, from 10 to 100. When the number of bases is 100 or less, even if the substance to be detected is close to another substance, the fusion product is less susceptible to interference from the other substance and is more likely to bind to the substance to be detected.

[0015] A nucleic acid aptamer may be, for example, a compound in which multiple nucleic acid units are linked together. The number of bases in each unit is preferably 10 to 100. When the number of bases in each unit is 100 or less, even if the target substance is close to another substance, the fusion product is less susceptible to interference from the other substance and more likely to bind to the target substance.

[0016] Nucleic acid aptamers include, for example, DNA aptamers and RNA aptamers. Binding substances can be chemically synthesized, for example, by in vitro processes. The binding substance binds to the analyte through, for example, an intermolecular interaction of the type that occurs between an antibody and an antigen, such as hydrogen bonding, electrostatic complementarity bonding, hydrophobic contact bonding, and steric bonding.

[0017] The fusion product comprises a label. The label is fused to a binding substance. The label exhibits some function. When the label exhibits its function, an observable phenomenon occurs. Examples of the observable phenomenon include the production, consumption, or absorption of ions. Examples of ions include hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, and chloride ions. Examples of the observable phenomenon include oxidation-reduction, color development, fluorescence, luminescence, phosphorescence, endothermic reaction, exothermic reaction, and precipitation. The label can be detected by observing the phenomenon that occurs, for example, in situ. Furthermore, when the fusion product is bound to a target substance, the target substance can be indirectly detected by detecting the label.

[0018] The label may be, for example, one or more selected from the group consisting of a magnetic label, a fluorescent label, an enzyme, a DNAzyme, an RNAzyme, a chemiluminescent probe, a nanoparticle, etc. The nanoparticle may be, for example, one or more selected from the group consisting of a metal particle, a non-metal particle, a dye particle, a pigment particle, an electrochemically active species, a semiconductor nanocrystal, a quantum dot fluorophore, etc.

[0019] Enzymes have, for example, the activity of inducing metabolism using a substrate as a starting material (hereinafter referred to as enzymatic activity). Examples of metabolism include metabolism that produces ions, metabolism that consumes ions, metabolism that absorbs ions, and metabolism that produces substances that absorb light of a specific wavelength. Examples of ions include hydrogen ions, potassium ions, sodium ions, calcium ions, lithium ions, ammonium ions, and chloride ions.

[0020] When metabolism that produces, consumes, or absorbs ions occurs, the ion concentration in the solution changes. When a substance that absorbs light of a specific wavelength is produced, the solution develops color or emits light. Enzyme activity corresponds to the function exhibited by the enzyme. The change in ion concentration in the solution and the development of color or light in the solution correspond to observable phenomena caused by enzyme activity.

[0021] Enzymes such as 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-enoate reductase, 2'-dehydrokanamycin reductase, 3,4-dihydroxyphenylalanine reductive 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- ...'-dehydrokanamycin reductase, 3,4-dihydroxyphenylalanine reductive deaminase, 3α-hydroxysteroid 3-dehydrogenase, 3-aminobutyryl-CoA Phenylacetate 3,4-dioxygenase, 4-chlorobenzoyl-CoA dehalogenase, 4-chlorobenzoate dehalogenase, 4-trimethylammoniobutyraldehyde dehydrogenase, 4-methylaminobutanoate oxidase, 4-methyleneglutamate-ammonia ligase, 5-phosphooxy-L-lysine phospholyase, 7,8-didemethyl-8-hydroxy-5-deazariboflavin synthase, 7-carboxy-7-deazaguaia N-glucan 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-glucosaminate-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 ​​sulfolyase, 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-succiniarginine dihydrolase, S-(hydroxymethyl)mycothiol dehydrogenase, S-carboxymethylcysteine ​​synthase, UDP-2-acetamido-2,6-β-L-arabino-hexyl-4-ose reductase, UDP-N-acetyl-2-amino-2-deoxyglucuronate dehydrogenase, UDP-N-acetyl-D-mannosamine dehydrogenase, UDP-N-acetyl-α-D-quinoline Bosamine dehydrogenase, UDP-N-acetylglucosamine 3-dehydrogenase, UDP-N-acetylglucosamine 6-dehydrogenase, UDP-glucuronate dehydrogenase, β-alanyl-CoA ammonia-lyase, β-ureidopropionase, β-lactamase, γ-butyrobetaine dioxygenase, asparaginyl-tRNA synthase, asparagine synthase, aspartate ammonia-lyase, aspartate-ammonia ligase, aspartate semialdehyde Dodehydrogenase, aspartate dehydrogenase, adenylyl sulfate-ammonia adenylyltransferase, adenosyl chloride synthase, atrazine chlorohydrolase, aminomethyltransferase, alanine dehydrogenase, allantoate deiminase, alkaline phosphatase, alcohol dehydrogenase, aldehyde dehydrogenase, ammonia kinase, ammonia monooxygenase, isocitrate dehydrogenase, imidazoleglycerol-phosphate synthase, uricase, urease, ureidoglycolate amidohydrolase, uronate dehydrogenase, ethanolamine ammonia-lyase, erythro-3-hydroxy-L-aspartate ammonia-lyase, octopamine dehydratase, ornithine cyclodeaminase, galactose dehydrogenase, carbamate kinase, carbamoyl-serine ammonia-lyase, carbamoylphosphate synthase, carbonyl reductase, formate dehydrogenase, glycine reductase,Glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucose dehydrogenase, glucokinase, glucosaminate ammonia-lyase, glucosamine-6-phosphate deaminase, glutaminyl-tRNA synthase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, crotonobetenyl-CoA hydratase, chloride peroxidase, coproporphyrinogen dehydrogenase, choline oxidase, choline monooxygenase, cholesterol diol oxidase, diacetyl reductase, diaminopimelate dehydrogenase, diaminopropionic acid ammonia-lyase, shikimate dehydrogenase, cyclohexane-1,2-diol dehydrogenase, dichlorochromopyrrolate synthase, dichloromethane dehalogenase, cystathionine γ-lyase, cysteine-S-conjugate β-lyase, dihydrolipoyl dehydrogenase, diphthionine-ammonia ligase, cinnamoyl-coenzyme A-reductase, succinylglutamate-semialdehyde dehydrogenase, stachydori threonine 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 reductive dehalogenase, tetracycline 7-halogenase, tryptophanase, tryptophan 5-halogenase, tryptophan 6-halogenase, tryptophan 7-halogenase, nicotinamide ammonia Adenine dinucleotide phosphate-dehydrogenase, nicotinamide adenine dinucleotide phosphate-heme protein reductase, nitrogenase, vanadium-dependent nitrogenase, hapalindole-type alkaloid chlorinase, histidine ammonia-lyase, hydrazine synthase, hydrazine dehydrogenase, hydroxymethylbilane synthase, hydroxylamine reductase, 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, formimidoyltetrahydrofolate cyclodeaminase, myeloperoxidase, methanol dehydrogenase, methane monooxygenase, methionine γ-lyase, methylasparagine The enzymes include one or more selected from the group consisting of phosphate 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 nonspecific polyamine oxidase.

[0022] For example, the label and the binding substance each comprise a chemical substituent or a nucleic acid-binding protein, and the label is fused to the binding substance by, for example, binding between the chemical substituent or nucleic acid-binding protein of the label and the chemical substituent or nucleic acid-binding protein of the binding substance.

[0023] Examples of chemical substituents that the label may have include one or more selected from the group consisting of biotin, primary amine, azide, alkyne, dibenzocyclooctyne, bicyclononyne, 2'-O-propargyl, 2'-O-propargyl, thiol, avidin, streptavidin, neutravidin, N-hydroxysuccinimide, maleimide, and 5-halouracil. Examples of 5-halouracil include 5-iodouracil and 5-bromouracil. 5-halouracil is a chemical substituent that can be UV-crosslinked. The nucleic acid binding protein contained in the label may be, for example, one or more selected from the group consisting of zinc finger, CRISPR, and the like.

[0024] Examples of chemical substituents that the binding substance may have include one or more selected from the group consisting of biotin, primary amine, azide, alkyne, dibenzocyclooctyne, bicyclononyne, 2'-O-propargyl, 2'-O-propargyl, thiol, avidin, streptavidin, neutravidin, N-hydroxysuccinimide, maleimide, and 5-halouracil. Examples of 5-halouracil include 5-iodouracil and 5-bromouracil. 5-halouracil is a chemical substituent that can be UV-crosslinked. The nucleic acid binding protein contained in the binding substance may be, for example, one or more selected from the group consisting of zinc finger, CRISPR, and the like. The chemical substituent of the label is different from the chemical substituent of the binding substance, for example, if the chemical substituent of the label is biotin, the chemical substituent of the binding substance is avidin, streptavidin, or neutravidin.

[0025] Examples of substances to be detected include proteins, sugars, lipids, nucleic acids, low molecular weight compounds, antigens, cells, and viruses. Examples of antigens include antigens presented by cells or viruses, or antigens contained in cells or viruses. Examples of antigens presented by cells or viruses or antigens contained in cells or viruses include the RBD region (hereinafter referred to as RBD) of the novel coronavirus SARS-CoV-2. Examples of viruses include the novel coronavirus SARS-CoV-2.

[0026] (1-2) Effects of the fusion The fusion product of the present disclosure has the following effects. (1A) Multiple analyte substances may form a complex. Examples of the complex include a dimer, a trimer, and a tetramer. In one complex, each analyte substance is in close proximity to another analyte substance.

[0027] Because the size of the fusion complex is small, when the fusion complex approaches one analyte, it is less susceptible to interference from other analytes. Therefore, the fusion complex can bind to each of the analytes that form a complex. As a result, the analytical sensitivity of the analyte can be improved by using the fusion complex of the present disclosure.

[0028] Proteins in living organisms often form complexes. Examples of proteins in living organisms include membrane proteins and coat proteins. Examples of membrane proteins include receptor proteins. Examples of coat proteins include viral spike proteins. By using the fusion protein of the present disclosure, the analytical sensitivity of proteins in living organisms can be improved even when the proteins in living organisms form complexes.

[0029] (1B) There are cases where multiple target substances are present at high density on the outer shell of a cell membrane, etc. In this case, each target substance is very close to the neighboring target substance. Because the size of the fusion complex is small, when the fusion complex approaches one analyte, it is less susceptible to interference from other analytes. Therefore, the fusion complex can bind to each of the analytes present at high density. As a result, the analytical sensitivity of the analyte can be improved by using the fusion complex of the present disclosure.

[0030] Proteins in living organisms are often present at high density on the outer membrane of cell membranes. Examples of proteins in living organisms include the proteins listed in (1A) above. By using the fusion protein of the present disclosure, the analytical sensitivity of proteins in living organisms can be improved even when the proteins in living organisms are present at high density on the outer membrane of cell membranes.

[0031] (1C) There are cases where the substance to be detected exists on an outer shell such as a cell membrane, and other large substances exist in the vicinity of the substance to be detected. Because the size of the fusion complex is small, it is less susceptible to interference from other large substances when approaching the analyte. Therefore, the fusion complex can bind to the analyte present in the vicinity of other large substances. As a result, the analytical sensitivity of the analyte can be improved by using the fusion complex of the present disclosure.

[0032] Proteins in living organisms are often present on outer shells such as cell membranes, and other large substances are often present in the vicinity of the proteins. Examples of proteins in living organisms include the proteins listed in (1A) above. By using the fusion protein of the present disclosure, the analytical sensitivity of proteins in living organisms can be improved even when other large substances are present in the vicinity of the proteins in living organisms.

[0033] (1D) The epitope of a substance to be detected may change due to factors such as mutation. If the epitope of a substance to be detected changes, the current binding substance may lose its binding activity. In particular, when the substance to be detected is a protein expressed by an RNA virus such as influenza virus or coronavirus, mutations occur frequently, and the epitope changes frequently.

[0034] The binding substance of the fusion construct of the present disclosure can be chemically synthesized, for example, by an in vitro process, which makes it easier to develop a binding substance that has binding activity for an analyte with an altered epitope than by synthesizing a monoclonal or polyclonal antibody from scratch. 2. Analysis method for detected substances (2-1) Procedure for analyzing the detected substance The method for analyzing the substance to be detected can be carried out, for example, by the following procedure.

[0035] (i) As shown in (1) of FIG. 1, a substance to be detected 1 is prepared. The substance to be detected 1 exists, for example, in a solution 3. The substance to be detected 1 and the solution 3 constitute a sample 5. (ii) Next, as shown in (2) of Figure 1, a process is performed to bind at least a portion of the fusion product 7 to the analyte 1. For example, by causing the fusion product 7 and the analyte 1 to coexist in a solution 3, at least a portion of the fusion product 7 and the analyte 1 are bound to each other.

[0036] The fusion compound 7 comprises a binding substance 9 and a label 11. The fusion compound 7 is as described above in the section "1. Fusion Compound." The binding substance 9 contained in at least a portion of the fusion compound 7 binds to the analyte 1 to form a conjugate.

[0037] (iii) Next, from among the fusion products 7, fusion products 7 in which the binding substance 9 does not bind to the analyte 1 are removed. (iv) Next, the function of the label 11 provided in the fusion product 7 is expressed. When the function of the label 11 is expressed, an observable phenomenon occurs. Examples of the function of the label 11 include enzyme activity, chemiluminescence, and fluorescence generation.

[0038] The enzyme activity induces metabolism using substrate 13 as a starting material, for example, as shown in (3) of Figure 1. The metabolism using substrate 13 as a starting material produces product 15. Product 15 contains, for example, hydrogen ions. Alternatively, product 15 consumes hydrogen ions. Therefore, the metabolism using substrate 13 as a starting material either produces hydrogen ions or consumes hydrogen ions. When metabolism that produces or consumes hydrogen ions occurs, the hydrogen ion concentration in solution 3 changes. The change in hydrogen ion concentration corresponds to an observable phenomenon that occurs when the function of labeled compound 11 is expressed.

[0039] The product 15 absorbs, for example, light of a specific wavelength. As a result, the solution 3 containing the fusion product 7 develops a color. The color development of the solution 3 corresponds to an observable phenomenon that occurs when the function of the label 11 is expressed.

[0040] (v) Next, the labeled substance 11 is detected. Since the fusion product 7 is bound to the analyte 1, detecting the labeled substance 11 is equivalent to detecting the analyte 1. In order to detect the labeled entity 11, a phenomenon that occurs when the function of the labeled entity 11 is expressed is observed. If the phenomenon can be observed, it means that the labeled entity 11 has been detected.

[0041] For example, if the phenomenon occurring in the labeled substance is 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, an ion sensor, or an ion-sensitive field-effect transistor. When the phenomenon caused by the label is color development, luminescence, fluorescence, or phosphorescence, the phenomenon can be detected using, for example, a light-receiving device.When the phenomenon caused by the label is endothermic or exothermic, the phenomenon can be detected using, for example, a thermal analyzer. When the phenomenon in which the label is produced is oxidation-reduction, the phenomenon can be detected using, for example, a potential measuring device, an ion sensor, or an ion-sensitive field-effect transistor.When the phenomenon in which the label is produced is precipitation, the phenomenon can be detected using, for example, a mass spectrometer, an absorptiometer, or a spectrophotometer. For example, if the function of the labeled compound 11 is an enzyme activity that produces hydrogen ions or induces metabolism that consumes hydrogen ions, the change in hydrogen ion concentration is measured using a measuring device 17 shown in (3) of Figure 1. If there is a change in hydrogen ion concentration, it means that the labeled compound 11 has been detected.

[0042] Furthermore, if the function of the label 11 is an enzyme activity that causes the solution to develop color, the degree of color development is measured using a measuring device 17. If color development occurs, it means that the label 11 has been detected. Furthermore, if the function of the label 11 is to generate chemiluminescence or fluorescence, the amount of light is measured using a measuring device 17. If the generation of chemiluminescence or fluorescence can be observed, the label 11 can be detected.

[0043] (2-2) Effects of the analytical method for the detected substance The method for analyzing an analyte substance according to the present disclosure has the above-mentioned advantages (1A) to (1D) and further has the following advantages.

[0044] (2A) According to the method for analyzing an analyte substance of the present disclosure, the concentration of the analyte substance in a sample can be measured easily and with high sensitivity. (2B) When the function of the label is an enzyme activity that causes the solution to develop color, the substance to be detected can be detected by the color development of the solution.

[0045] According to the method for analyzing a substance to be detected disclosed herein, only the fusion complex bound to the substance to be detected causes the solution to develop a color. Therefore, the higher the concentration of the substance to be detected, the stronger the color development of the solution. Therefore, according to the method for analyzing a substance to be detected disclosed herein, the concentration of the substance to be detected can be quantified based on the intensity of the color development of the solution.

[0046] (2C) When the function of the label is an enzyme activity that induces metabolism that produces, consumes, or absorbs hydrogen ions, the target substance can be detected by detecting a change in hydrogen ion concentration in the solution.

[0047] According to the method for analyzing a substance to be detected disclosed herein, only the fusion complex bound to the substance to be detected induces a metabolic process that produces, consumes, or absorbs hydrogen ions. Therefore, the higher the concentration of the substance to be detected, the greater the change in hydrogen ion concentration in the solution. Therefore, according to the method for analyzing a substance to be detected disclosed herein, the concentration of the substance to be detected can be quantified based on the change in hydrogen ion concentration in the solution.

[0048] 3. Working Example (3-1) Synthesis of fusion compounds A DNA aptamer having the base sequence of SEQ ID NO: 1 (hereinafter referred to as the unmodified DNA aptamer) was chemically synthesized by an in vitro process. The base sequence of the unmodified DNA aptamer was "5'-CAGCACCGAC CTTGTGCTTT GGGAGTGCTG GTCCAAGGGC GTTAATGGAC A-3'". The unmodified DNA aptamer is described in Anal. Chem. 2020, 92, 9895-9900 (hereinafter referred to as Reference 1).

[0049] According to the description in Reference 1, the unmodified DNA aptamer is a DNA aptamer that detects the RBD in the spike glycoprotein of the novel coronavirus SARS-CoV-2. Next, the 5' end of the unmodified DNA aptamer was chemically modified with biotin via a C6 spacer.

[0050] The DNA aptamer was obtained through the above process. The DNA aptamer corresponds to the binding substance. The DNA aptamer was dissolved in phosphate buffer (hereinafter referred to as 1xPBS / T) to prepare a DNA aptamer solution. The concentration of the DNA aptamer in the DNA aptamer solution was 10 μmol / L.

[0051] 1x PBS / T contained 0.05% (v / v) of the surfactant Tween 20. 1x PBS / T also contained 137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.7 mmol / L KCl, and 1.47 mmol / L KHPO. A streptavidin-alkaline phosphatase conjugate (Thermofisher Scientific, product number S921) was prepared as the label. This label is an enzyme. The streptavidin-alkaline phosphatase conjugate is obtained by modifying alkaline phosphatase with streptavidin. Streptavidin is a chemical substituent that modifies the label. The label was dissolved in 1x PBS / T to prepare a label solution. The concentration of the label in the label solution was 20 μmol / L.

[0052] 100 μL of the DNA aptamer solution was mixed with 5 μL of the label solution and left to stand at room temperature for 1 hour, at which time the DNA aptamer and label were fused together through biotin-streptavidin interaction, resulting in the synthesis of a fusion product.

[0053] The fusion product was then purified and recovered. Specifically, the unfused DNA aptamer was separated from the fusion product using an ultrafiltration filter, and the fusion product was recovered. The flow chart for synthesizing the fusion product is shown in Figure 2.

[0054] The binding substance may be a binding substance other than the DNA aptamer. The binding substance may be, for example, a low molecular weight protein preparation or an RNA aptamer. Examples of low molecular weight protein preparations include fragment antibodies, single-chain antibodies, diabodies, nanobodies, VHHs, and peptide aptamers.

[0055] Furthermore, the base sequence of the DNA aptamer may be a base sequence other than the base sequence of SEQ ID NO: 1. The base sequence of the DNA aptamer can be selected depending on the analyte to be analyzed. The fusion between the binding substance and the label may be a fusion other than that based on the biotin-streptavidin interaction. When a nucleic acid aptamer is used as the binding substance, an end of the nucleic acid aptamer other than the 5' end may be fused to the label.

[0056] When a low molecular weight protein preparation is used as a binding substance, the N-terminus or C-terminus of the low molecular weight protein preparation may be fused with a label. (3-2) Implementation of the analytical method for the detected substance Spike S1-His Recombinant Protein (Sinobiological Co., Ltd., product number 40591-V08H) was prepared as the substance to be detected. This substance to be detected will be referred to as S1 below. The amino acid sequence of S1 includes RBD.

[0057] S1 was dissolved in 0.1 M carbonate buffer to prepare S1-A solution and S1-B solution. The concentration of S1 in S1-A solution was 5 μg / mL. The concentration of S1 in S1-B solution was 1 μg / mL. The pH of the 0.1 M carbonate buffer was adjusted to 9.6.

[0058] Additionally, α-amylase (Lee Biosolutions, product number 120-17) was prepared as a negative control substance to be detected. α-amylase was dissolved in 0.1 M carbonate buffer to prepare an α-amylase solution. The concentration of α-amylase in the α-amylase solution was 5 μg / mL.

[0059] The buffers used to prepare the S1-A solution, S1-B solution, and α-amylase solution may be buffers other than the 0.1 M carbonate buffer, such as commonly used buffers such as 1x PBS / T, 1x PBS, 1x TBS / T, and 1x TBS.

[0060] Next, 100 μL of S1-A solution was added dropwise to some of the ELISA wells of a 96-well ELISA plate, H type (Sumitomo Bakelite Co., Ltd., product number MS-8896F), and the plate was left standing overnight at 4°C. The ELISA wells to which the S1-A solution was added are hereinafter referred to as S1-A wells. At this time, S1 adhered to the S1-A wells.

[0061] In addition, 100 μL of S1-B solution was dropped into some of the ELISA wells other than the S1-A wells and left to stand overnight at 4°C. The ELISA wells to which the S1-B solution was dropped are hereinafter referred to as S1-B wells. At this time, S1 was fixed to the S1-B wells.

[0062] In addition, 100 μL of α-amylase solution was added dropwise to an ELISA well that was neither an S1-A nor an S1-B well, and the well was left standing overnight at 4°C. The ELISA well to which the α-amylase solution was added will be referred to as the α-amylase well below. At this time, α-amylase was fixed to the α-amylase well.

[0063] Next, the S1-A well, the S1-B well, and the α-amylase well were each washed with 200 μL of 1×PBS / T. Washing was performed three times. Next, bovine serum albumin (Fujifilm Wako Pure Chemical Industries, Ltd., product number 013-15104) was dissolved in 1x PBS / T to prepare a BSA solution. Hereinafter, bovine serum albumin will be referred to as BSA. The concentration of BSA in the BSA solution was 3% (w / v).

[0064] Next, 200 μL of BSA solution was dropped into each of the S1-A well, S1-B well, and α-amylase well, and the plates were left to stand at room temperature for 2 hours, at which time blocking was performed.

[0065] Next, the S1-A well, the S1-B well, and the α-amylase well were each washed with 200 μL of 1×PBS / T. Washing was performed three times. Next, 50 μL of the fusion solution was dropped into each of the S1-A well, S1-B well, and α-amylase well, and allowed to stand at room temperature for 1 hour. The fusion solution contained the fusion synthesized in the above "(3-1) Synthesis of fusion protein." At this time, a portion of the fusion protein bound to S1 in the S1-A well and S1-B well.

[0066] Next, the S1-A well, S1-B well, and α-amylase well were each washed with 200 μL of 1x PBS / T three times. At this time, the fusion protein that was not bound to the analyte was removed.

[0067] 4-Nitrophenyl phosphate disodium hexahydrate (Tokyo Chemical Industry Co., Ltd., product number N0241) was prepared. 4-Nitrophenyl phosphate disodium hexahydrate, hereafter referred to as pNPP, is a substrate for alkaline phosphatase-induced metabolism.

[0068] Next, pNPP was dissolved in a solution containing carbonate buffer and magnesium sulfate to prepare a pNPP solution. The concentration of pNPP in the pNPP solution was 10 mmol / L. The solution containing carbonate buffer and magnesium sulfate contained 1 mmol / L carbonate buffer and 1 mmol / L magnesium sulfate. The pH of the solution containing carbonate buffer and magnesium sulfate was adjusted to 9.6.

[0069] The solution for dissolving pNPP may be a solution other than a solution containing a carbonate buffer 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, Tricine, Bicine, TAPS, CHES, CAPSO, and CAPS.

[0070] The pH of the solution in which pNPP is dissolved may be a value other than 9.6. The solution in which pNPP is dissolved is preferably a weakly alkaline solution. The pH of the solution in which pNPP is dissolved is preferably 8 or more and 11 or less. When the solution in which pNPP is dissolved is a weakly alkaline solution, the pH change resulting from metabolism using pNPP as a substrate becomes large. When the pH of the solution in which pNPP is dissolved is 8 or more and 11 or less, the pH change resulting from metabolism using pNPP as a substrate becomes large.

[0071] Next, 100 μL of pNPP solution was dropped into each of the S1-A well, S1-B well, and α-amylase well, and the wells were left to stand at 37° C. for 10 minutes. As shown in FIG. 3, in the S1-A well and S1-B well, the enzymatic activity of alkaline phosphatase contained in the fusion protein induced metabolism using pNPP as a substrate, resulting in the separation of a phosphate group from pNPP.

[0072] As a result, inorganic phosphoric acid and p-nitrophenol were produced in wells S1-A and S1-B. The absorption maximum wavelength of p-nitrophenol is 405 nm. Therefore, the solution containing p-nitrophenol turned yellow. Furthermore, the produced inorganic phosphoric acid ionized in the solution, releasing hydrogen ions. As a result, the pH of the solution decreased.

[0073] Next, 5 μL of ethylenediaminetetraacetic acid at a final concentration of 0.5 mol / L was added dropwise to each of the S1-A well, S1-B well, and α-amylase well, at which time the metabolism by alkaline phosphatase was stopped.

[0074] Next, the absorbance of light at a wavelength of 405 nm was measured using a microplate reader in each of the S1-A well, S1-B well, and α-amylase well. The absorbance was also measured in the blank well in the same manner.

[0075] The above process flow is shown in Figure 4. The absorbance measurement results are shown in Figure 5. "S1 5 μg / mL" in Figure 5 corresponds to the S1-A well. "S1 1 μg / mL" in Figure 5 corresponds to the S1-B well. "α-amylase 5 μg / mL" in Figure 5 corresponds to the α-amylase well. As shown in Figure 5, color development was observed only in the S1-A and S1-B wells.

[0076] The absorbance measurement results showed the following: The alkaline phosphatase contained in the fusion product, even when fused with the DNA aptamer, had the enzymatic activity of inducing metabolism using pNPP as a substrate. Furthermore, the DNA aptamer contained in the fusion product, even when fused with the label, had binding activity to S1. Furthermore, the DNA aptamer contained in the fusion product did not have binding activity to α-amylase.

[0077] (3-3) Implementation of the analytical method for the detected substance The novel coronavirus SARS-CoV-2 (hereafter referred to as SARS-CoV-2) was prepared as the substance to be detected. Five types of suspensions were produced by suspending SARS-CoV-2 in 1x PBS / T. The five suspensions differed only in the number of copies of SARS-CoV-2 contained in the suspension. The copy numbers of SARS-CoV-2 in the five suspensions, calculated by quantitative PCR, were 10^1, 10^2, 10^3, 10^4, and 10^5 per μL of suspension, respectively. The following steps were performed for each of the five suspensions.

[0078] Next, 25 μL of the fusion product solution was added dropwise to 25 μL of the suspension and allowed to stand at room temperature for 10 minutes. The fusion product solution contained the fusion product synthesized in the above "(3-1) Synthesis of fusion product." The concentration of the fusion product in the fusion product solution was 10 nmol / L. At this time, some of the fusion product bound to the target substance.

[0079] Next, the entire suspension was dropped onto a Nanosep centrifugal filter device with a molecular weight cutoff of 300K (PALL, product number OD300C34) and centrifuged at high speed to recover fusion proteins that had bound to the target substance and separate those that had not.

[0080] Next, the Nanosep centrifugal filter device with a molecular weight cutoff of 300K was washed three times with 1xPBS / T, which removed fusion proteins that were not bound to SARS-CoV-2. Next, pNPP was dissolved in a solution containing carbonate buffer and magnesium sulfate to prepare a pNPP solution. The concentration of pNPP in the pNPP solution was 10 mmol / L. The solution containing carbonate buffer and magnesium sulfate contained 1 mmol / L carbonate buffer and 1 mmol / L magnesium sulfate. The pH of the solution containing carbonate buffer and magnesium sulfate was adjusted to 9.6.

[0081] Next, the pNPP solution was dropped onto a Nanosep centrifugal filter device with a molecular weight cutoff of 300K and allowed to stand at 37°C for 10 minutes. As shown in Figure 3, the enzymatic activity of alkaline phosphatase induced metabolism using pNPP as a substrate, resulting in the separation of a phosphate group from pNPP. This resulted in the production of inorganic phosphate and p-nitrophenol. The absorption maximum wavelength of p-nitrophenol is 405 nm. Therefore, the solution containing p-nitrophenol turned yellow. Furthermore, the inorganic phosphate produced ionized in the solution, releasing hydrogen ions. This caused the pH of the solution to decrease. Next, the solution in the Nanosep centrifugal filtration device with a molecular weight cutoff of 300K was dropped onto a pH meter. The pH meter was a compact pH meter LAQUAtwin (Horiba, product number pH-33B). The pH of the solution dropped onto the pH meter was recorded. The flow of the above process is shown in Figure 6. The recorded pH results are shown in Figure 7. The "copy number" in Figure 7 refers to the number of copies per μL of SARS-CoV-2 in the suspension.

[0082] As shown in Figure 7, the higher the copy number of SARS-CoV-2 in the suspension, the greater the change in pH. The change in pH refers to the change in pH relative to the blank. The pH recording results showed that the DNA aptamer in the fusion product retained binding activity to SARS-CoV-2 even when fused with the label. Furthermore, the alkaline phosphatase in the fusion product retained the enzyme activity of inducing metabolism using pNPP as a substrate, even when fused with the DNA aptamer. Furthermore, inorganic phosphate produced by metabolism using pNPP as a substrate ionized in the solution, releasing hydrogen ions. As a result, the pH of the solution decreased. Furthermore, when the copy number of SARS-CoV-2 in the suspension was 10^3 or greater, the amount of pH change was even greater.

[0083] To remove fusion products that have not bound to the analyte, a centrifugal filtration device other than the Nanosep centrifugal filtration device with a molecular weight cutoff of 300K may be used. The centrifugal filtration device has, for example, a molecular weight cutoff value equivalent to that of the Nanosep centrifugal filtration device with a molecular weight cutoff of 300K.

[0084] Furthermore, the method for removing fusion products that have not bound to the analyte may be a method other than centrifugal filtration, such as a sandwich-type removal method using an ELISA plate or magnetic beads, a removal method by filtration chromatography using filter paper or gel, or a removal method by affinity chromatography using an antibody supported on a carrier such as gel.

[0085] Furthermore, the solution for dissolving pNPP may be a solution other than a solution containing a carbonate buffer 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, Tricine, Bicine, TAPS, CHES, CAPSO, and CAPS.

[0086] Furthermore, the pH of the solution in which pNPP is dissolved may be a value other than 9.6. The solution in which pNPP is dissolved is preferably a weakly alkaline solution. The pH of the solution in which pNPP is dissolved is preferably 8 or more and 11 or less. When the solution in which pNPP is dissolved is a weakly alkaline solution, the pH change resulting from metabolism using pNPP as a substrate becomes large. When the pH of the solution in which pNPP is dissolved is 8 or more and 11 or less, the pH change resulting from metabolism using pNPP as a substrate becomes large.

[0087] (3-4) Tests to confirm the expression of the labeled substance's function Three types of fusion-pNPP solutions were prepared by adding pNPP solution to the fusion. The fusion was synthesized in the above "(3-1) Synthesis of fusion." The pNPP solution was prepared in the above "(3-2) Implementation of the method for analyzing the target substance."

[0088] The three fusion-pNPP solutions differed only in the concentration of the fusion, which was 1 pmol / L, 10 pmol / L, and 100 pmol / L, respectively.

[0089] In each of the three fusion protein-pNPP solutions, alkaline phosphatase contained in the fusion protein metabolized pNPP as a substrate. The following steps were performed for each of the three fusion protein-pNPP solutions.

[0090] Immediately after preparation of the fusion complex-pNPP solution, the fusion complex-pNPP solution was added dropwise to a pH meter, which was a compact pH meter LAQUAtwin (Horiba, product number pH-33B).

[0091] The pH of the fusion-pNPP solution was recorded 10 minutes, 20 minutes, and 30 minutes after addition. The flow of the above steps is shown in Figure 8. The recorded pH results are shown in Figure 9. In Figure 9, "final fusion concentration" refers to the concentration of the fusion in the fusion-pNPP solution.

[0092] As shown in Figure 9, the higher the concentration of the fusion product in the fusion product-pNPP solution, the greater the change in pH. The change in pH is the amount of change in pH relative to the pH immediately after addition. The pH recording results show the following: The alkaline phosphatase contained in the fusion product, even when fused to the DNA aptamer, has the enzymatic activity to induce metabolism using pNPP as a substrate. Furthermore, inorganic phosphate produced by metabolism using pNPP as a substrate ionizes in the solution, releasing hydrogen ions. As a result, the pH of the solution decreases.

[0093] 4. Other Embodiments Although the embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiments and can be implemented in various modified forms.

[0094] (4-1) Multiple functions possessed by one component in the above embodiments may be realized by multiple components, or one function possessed by one component may be realized by multiple components. Also, multiple functions possessed by multiple components may be realized by one component, or one function realized by multiple components may be realized by one component. Also, part of the configuration of the above embodiments may be omitted. Also, at least part of the configuration of the above embodiments may be added to or substituted for the configuration of another of the above embodiments.

[0095] (4-2) In addition to the fusion products described above, the present disclosure can also be realized in various forms, such as a component having the fusion product as a constituent element, a method for producing the fusion product, and a method for analyzing a substance to be detected. [Explanation of symbols]

[0096] 1...Detected substance, 3...Solution, 5...Sample, 7...Fusion, 9...Binding substance, 11...Label, 13...Substrate, 15...Product, 17...Measuring device

Claims

1. A method for analyzing a substance to be detected (1) using a fusion compound (7) comprising a binding substance (9) having an activity of binding to the substance to be detected (1) and a label (11) fused to the binding substance and causing an observable phenomenon, comprising: the fusion product and the target substance are allowed to coexist in a sample, thereby binding at least a portion of the fusion product to the target substance; removing the fusion bodies that do not bind to the target substance from the fusion bodies; Observing the phenomenon, the substance to be detected is a spike protein of the novel coronavirus SARS-CoV-2, the binding substance is a nucleic acid aptamer having 10 to 100 bases, the binding substance and the label are fused via biotin contained in the binding substance and streptavidin contained in the label, the labeled substance is alkaline phosphatase, the phenomenon is that in the sample, the enzyme activity of alkaline phosphatase induces metabolism using 4-nitrophenyl phosphate disodium hexahydrate as a substrate, resulting in the production of hydrogen ions; Observing the phenomenon using a pH meter; A method for analyzing the substance to be detected.

2. A method for analyzing a substance to be detected according to claim 1, comprising: the greater the number of the target substance per unit volume in the sample, the greater the amount of change in pH caused by the phenomenon; A method for analyzing the substance to be detected.

3. A method for analyzing a substance to be detected according to claim 1 or 2, comprising: This phenomenon occurs when the sample solution is replaced with another solution in which 4-nitrophenyl phosphate disodium hexahydrate is dissolved and the pH is between 8 and 11. A method for analyzing the substance to be detected.

4. A method for analyzing a substance to be detected according to claim 3, comprising: The amount of change in pH in the phenomenon increases over time after the replacement with the other solution. A method for analyzing the substance to be detected.

5. An analytical system for a substance to be detected, comprising: a fusion body (7) comprising a binding substance (9) having an activity of binding to a substance to be detected (1); and a label (11) fused to the binding substance and causing an observable phenomenon; and a pH meter, the substance to be detected is a spike protein of the novel coronavirus SARS-CoV-2, the binding substance is a nucleic acid aptamer having 10 to 100 bases, the binding substance and the label are fused via biotin contained in the binding substance and streptavidin contained in the label, the labeled substance is alkaline phosphatase, the phenomenon is that, in a sample containing the substance to be detected, the enzyme activity of alkaline phosphatase induces metabolism using 4-nitrophenyl phosphate disodium hexahydrate as a substrate, resulting in the production of hydrogen ions; The pH meter observes the phenomenon. An analytical system for the substance to be detected.

6. An analytical system for a substance to be detected according to claim 5, the greater the number of the target substance per unit volume in the sample, the greater the amount of change in pH caused by the phenomenon; An analytical system for the substance to be detected.

7. An analytical system for a substance to be detected according to claim 5 or 6, This phenomenon occurs when the sample solution is replaced with another solution in which 4-nitrophenyl phosphate disodium hexahydrate is dissolved and the pH is between 8 and 11. An analytical system for the substance to be detected.

8. An analytical system for a substance to be detected according to claim 7, The amount of change in pH in the phenomenon increases over time after the replacement with the other solution. An analytical system for the substance to be detected.

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