Method for concentrating organic substance, method for detecting organic substance, and kit for use in concentration of organic substance

Abstract

This method for concentrating an organic substance comprises: a step for mixing a liquid sample containing an organic substance, a polysaccharide, and a polyether to obtain a first mixed liquid that contains a first phase containing the largest amount of the polysaccharide and a second phase containing the largest amount of the polyether; and a step for mixing the first phase with a concentration polymer to obtain a second mixed liquid. The concentration polymer is a compound in which a group represented by formula R7 is introduced into dextran, and the average molecular weight of the concentration polymer excluding the group represented by formula R7 is 5,000-100,000.

Description

Method for concentrating organic matter, method for detecting organic matter, and kit for use in concentrating organic matter

[0001] The present invention relates to a method for concentrating organic substances, a method for detecting organic substances, and a kit for use in concentrating organic substances. This application claims priority to Japanese Patent Application No. 2024-100713, filed on June 21, 2024, the contents of which are incorporated herein by reference.

[0002] Conventionally, a technique for concentrating organic substances such as nucleic acids and proteins by liquid-liquid phase separation of an aqueous two-phase system has been known.

[0003] For example, Non-Patent Document 1 describes that in an environment separated into a dextran-rich phase and a polyethylene glycol-rich phase, organic substances such as nucleic acids and proteins can be concentrated in the dextran-rich phase.

[0004] This document discloses that ALP molecules and Cas13 / RNA complexes can be concentrated 30 times or more in a dextran-rich phase compared to a polyethylene glycol-rich phase, and that this concentration can improve the detection sensitivity of digital bioassays.

[0005] Y. Minagawa et al., On-Chip Enrichment System for Digital Bioassay Based on Aqueous Two-Phase System. ACS Nano, 17(1):212-220, 2022.

[0006] However, when the amount of the target organic substance contained in the sample is very small, the concentration technique using liquid-liquid phase separation described in Non-Patent Document 1 may not be sufficient to concentrate the substance, and as a result, the target organic substance may not be detected.

[0007] It is also possible to concentrate organic substances using antibodies that specifically bind to the organic substances to be concentrated, but antibodies have the problem of being expensive.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a technique for concentrating organic substances that can concentrate target organic substances contained in a sample at low cost and with higher efficiency than conventional concentration techniques using liquid-liquid phase separation.

[0009] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0010] [1] A method for concentrating an organic substance, comprising the steps of: mixing a liquid sample containing the organic substance with a polysaccharide and a polyether to obtain a first mixture containing a first phase containing the polysaccharide in the largest amount and a second phase containing the polyether in the largest amount; and mixing the first phase with a concentrating polymer to obtain a second mixture, wherein the concentrating polymer is a polymer having a structure represented by the following formula R ７ is a compound having a group represented by the following formula R ７ a method for concentrating an organic substance, wherein the average molecular weight of the concentrating polymer excluding the group represented by the formula (I) is 5,000 to 100,000; (R ８ n is a divalent hydrocarbon group having 1 to 20 carbon atoms which may contain one or more bonds selected from the group consisting of an ester bond, an ether bond, a carbonyl bond, and an amide bond. ２ is on average 2 to 250. ３ is the reaction of the above formula R ７ R represents the number of groups represented by the formula (I) introduced, and is more than 1 and 10 or less on average per the average molecular weight of 20,000, and when the average molecular weight is less than 20,000, it is 1 or more and 10 or less on average. ９ is a hydrocarbon group having 1 to 5 carbon atoms or a hydrogen atom.

[0011] [2] The R ８ is a group selected from the group consisting of the following general formulas (1-1) to (1-9): (R in formulas (1-1) to (1-9) １０ is a divalent group selected from the group consisting of the following formulas (2-1) to (2-9):

[0012] [3] The method for concentrating organic matter described in [1] or [2], wherein the polysaccharide is at least one selected from the group consisting of dextran, chitin, inulin, heparin, cellulose, methylcellulose, alginic acid, and salts thereof.

[0013] [4] The method for concentrating organic matter according to any one of [1] to [3], wherein the polysaccharide is dextran.

[0014] [5] The polyether is at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and mixtures thereof. [1] The method for concentrating organic matter according to any one of [4] to [5].

[0015] [6] The method for concentrating organic matter according to any one of [1] to [5], wherein the polyether is polyethylene glycol.

[0016] [7] The method for concentrating an organic substance according to any one of [1] to [6], wherein the concentration polymer is a compound represented by the following formula (3): (n in formula (3) １ The average is 16 to 320. ２ The average is 2 to 250. ３ is the formula R ７ The number of groups represented by the formula R is more than 1 and 10 or less on average per the average molecular weight of 20,000, and when the average molecular weight is less than 20,000, the number is 1 or more and 10 or less on average. ７ The carbon atom to which the group represented by the formula R ７ When a group represented by the formula R ７ An OH group is bonded in place of the group represented by R ９ is a hydrogen atom or a methyl group.

[0017] [8] In the formula (3), n １ is on average 80 to 180, and n ２ is on average 60 to 110, and n ３ The method for concentrating organic matter according to [7], wherein the average is 5 to 9.

[0018] [9] The method for concentrating organic matter described in any one of [1] to [8], wherein the organic matter is an organic matter selected from the group consisting of nucleic acids, proteins, and viruses.

[0019]

[10] The method for concentrating organic matter according to any one of [1] to [9], further comprising a step of centrifuging the second mixture.

[0020]

[11] The method for concentrating organic matter according to any one of [1] to

[10] , wherein in the step of obtaining the first mixture, the liquid sample, the polysaccharide, and the polyether are mixed and centrifuged.

[0021]

[12] The method for concentrating organic matter according to any one of [1] to

[11] , wherein the concentration of the concentration polymer in the second mixture is 1 mass% or more.

[0022]

[13] A method for detecting organic matter, comprising a step of detecting the organic matter contained in the phase containing the most of the concentrating polymer among multiple phases obtained after centrifugal separation of the second mixture obtained by the method for concentrating organic matter described in

[10] .

[0023]

[14] A kit comprising a polysaccharide, a polyether, and the concentration polymer according to [1], for use in concentrating organic matter by the method for concentrating organic matter according to any one of [1] to

[12] .

[0024]

[15] A method for concentrating organic matter, comprising: a first concentration step of mixing a liquid sample containing the organic matter with a polysaccharide and a polyether to produce a first mixture containing a first phase containing the polysaccharide (dextran) in the largest amount and a second phase containing the polyether in the largest amount, and transferring the organic matter from the second phase to the first phase to concentrate it; and a second concentration step of mixing the first phase with a concentration polymer to produce a second mixture, and transferring the organic matter from the first phase to the concentration polymer to concentrate it.

[0025]

[16] The enrichment polymer is a compound in which a group represented by the above formula R7 is introduced into dextran, and ７The method for concentrating organic matter according to

[15] , wherein the average molecular weight of the concentrating polymer excluding the group represented by the formula: is 5,000 to 100,000.

[0026] According to the present invention, the organic matter to be concentrated is concentrated into a first phase containing the most polysaccharide, and then the first phase is mixed with a concentrating polymer to obtain a second mixture, which is then separated into a liquid-liquid phase containing the most concentrating polymer. Thus, a technique for concentrating organic matter can be provided that can concentrate target organic matter contained in a sample inexpensively and more efficiently than conventional techniques.

[0027] 1 is a flow chart showing a method for concentrating organic matter according to a preferred embodiment of the present invention. FIG. 1 is a schematic diagram of multiple types of concentration polymers with different dextran molecular weights and PEG group molecular weights. FIG. 2 is a confocal microscope image of an aqueous dispersion containing a concentration polymer, dextran, and polyethylene glycol immediately after shaking and stirring. FIG. 3 is a graph showing the relative DNA amount in each phase after macrophase separation with the addition of 500 bp DNA. FIG. 4 is a graph showing the relative DNA amount in each phase after macrophase separation with the addition of 48,500 bp DNA. FIG. 5 is a graph showing the partition coefficient for each DNA length. FIG. 6 is a schematic longitudinal cross-sectional view showing the process of placing concentration polymer A in a microwell of a microarray chamber. FIG. 7 is a fluorescent image of a microarray chamber viewed from diagonally above. FIG. 8 is a schematic longitudinal cross-sectional view showing the process of concentrating RNA in a microwell of a microarray chamber. FIG. 9 is a fluorescent image of a microarray chamber containing RNA in the microwell. FIG. 10 is a fluorescent image of a control microarray chamber in which no RNA was placed in the microwell. FIG. 11 is a fluorescent image showing the results of RNA detection using a microarray chamber in Experimental Example 6. Fluorescence images showing the results of RNA detection after a first concentration step using liquid-liquid phase separation of dextran and PEG. Fluorescence images showing the results of RNA detection after a second concentration step using liquid-liquid phase separation of enrichment polymer A and dextran. Graphs showing the percentage of microwells in which fluorescence was detected for each concentration method. Graphs showing fluorescence plots of digital bioassays for 6 fM and 6 aM RNA.

[0028] Preferred embodiments of the present invention will be described in detail below, with reference to the drawings as needed, although the present invention is not limited to the following embodiments.

[0029] [Method 1 for Concentrating Organic Substances] FIG. 1 is a flow diagram illustrating a method for concentrating organic substances according to a preferred embodiment of the present invention. As shown in FIG. 1 , the method for concentrating organic substances according to this embodiment includes a step S1 (hereinafter also referred to as the "first mixing step") of mixing a liquid sample containing organic substances to be concentrated (the organic substance to be concentrated) with polysaccharides and polyethers to obtain a first mixed solution containing a first phase containing the most polysaccharides and a second phase containing the most polyethers, and a step S2 (hereinafter also referred to as the "second mixing step") of mixing the first phase with a concentrating polymer to obtain a second mixed solution. Note that the method for concentrating organic substances according to this embodiment is not limited to concentration for the purpose of detecting organic substances, and may also be used for concentration for other purposes.

[0030] <<First Mixing Step S1>> <Organic Matter> The type of organic matter to be concentrated is not particularly limited, and examples thereof include nucleic acids such as RNA and DNA, proteins, phages, viruses, cells, polymers, vitamins, nanoparticles such as extracellular vesicles, etc. The organic matter to be concentrated may be any one selected from the group consisting of nucleic acids, proteins, sugars, and viruses.

[0031] When the organic matter to be concentrated is a nucleic acid, the longer the nucleotide length, the greater the concentration effect in the phase containing the most concentrating polymer in the second mixing step S2 described in detail below. For this reason, the nucleic acid is preferably 300 bp or more, more preferably 500 bp or more, even more preferably 10,000 bp or more, and particularly preferably 45,000 bp or more.

[0032] (Lower limit of molecular weight of organic substance) The molecular weight of the organic substance to be concentrated is not particularly limited, but may be, for example, 500 or more, 1,000 or more, 3,000 or more, 5,000 or more, 8,000 or more, or 10,000 or more.

[0033] (Upper limit of molecular weight of organic substance) The molecular weight of the organic substance to be concentrated is not particularly limited, but may be, for example, 10,000 or less, 30,000 or less, 100,000 or less, 200,000 or less, 300,000 or less, 500,000 or less, or 800,000 or less. The above-mentioned lower limit and upper limit of molecular weight can be combined in any manner.

[0034] In this specification, unless otherwise specified, the terms "molecular weight" and "average molecular weight" refer to the number average molecular weight (Mn). The number average molecular weight is the polystyrene-equivalent number average molecular weight determined by gel permeation chromatography (GPC).

[0035] The liquid sample to be subjected to the concentration method of this embodiment is not particularly limited as long as it contains (or may contain) the organic matter to be concentrated, but examples thereof include aqueous solutions and aqueous dispersions containing organic matter.

[0036] Examples of such samples include, but are not limited to, serum, plasma, lymph, interstitial fluid, urine, ascites, pleural effusion, and cerebrospinal fluid, as well as extracts extracted from various animals, plants, bacteria, viruses, etc. Furthermore, liquid samples may be, for example, water, saline, or a buffer solution containing the organic substance to be concentrated. Examples of buffer solutions include, but are not limited to, phosphate buffer, phosphate-buffered saline, acetate buffer, citrate buffer, Tris buffer, and HEPES buffer.

[0037] <Polysaccharides> The polysaccharides may be homopolysaccharides consisting of a single monosaccharide, heteropolysaccharides consisting of multiple types of monosaccharides, or salts thereof, such as sodium salts and potassium salts.

[0038] When the polysaccharide is a homopolysaccharide, the polysaccharide may be a homopolysaccharide consisting of one member selected from the group consisting of glucose, fructose, galactose, and glucosamine.

[0039] When the polysaccharide is a heteropolysaccharide, the polysaccharide may be composed of two or more types selected from the group consisting of glucose, fructose, galactose, mannose, glucosamine, and idose.

[0040] The polysaccharide may be at least one selected from the group consisting of dextran, chitin, inulin, heparin, cellulose, methylcellulose, alginic acid, and salts thereof. Examples of the salts of these polysaccharides include sodium salts and potassium salts. The polysaccharide is preferably dextran.

[0041] Dextran is a polysaccharide in which glucose is linked. Dextran may have a structure in which glucose is linked by an α-1,6-glycosidic bond, a structure in which glucose is linked by an α-1,6-glycosidic bond and an α-1,4-glycosidic bond, or a structure in which glucose is linked by an α-1,6-glycosidic bond and an α-1,3-glycosidic bond.

[0042] (Lower Limit of Average Molecular Weight of Polysaccharide) The average molecular weight of the polysaccharide is not particularly limited, and may be, for example, 3,000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 200,000 or more, 300,000 or more, 400,000 or more, or 500,000 or more.

[0043] (Upper limit of average molecular weight of polysaccharide) The upper limit of the average molecular weight of the polysaccharide is not particularly limited, and may be, for example, 10 million or less, 5 million or less, 4 million or less, 3 million or less, 2 million or less, 1 million or less, 800,000 or less, or 500,000 or less. The above upper limit and lower limit can be combined in any manner.

[0044] The average molecular weight of a polysaccharide can be determined by comparing it with a molecular weight standard substance of the same type as the polysaccharide to be measured, which has a known molecular weight distribution range, through gel filtration chromatography analysis.

[0045] The state of the polysaccharide when mixed with the liquid sample and the polyether is not particularly limited, and may be, for example, a solid (e.g., a dried polysaccharide) or may be dissolved in water, physiological saline, a buffer solution, etc. Examples of the buffer solution include those mentioned above.

[0046] <Polyether> Polyether is a polymer containing multiple ether bonds. Preferably, the polyether is a polymer in which alkylene glycol units are linked by ether bonds. The alkylene is a linear alkylene having 1 to 6 carbon atoms or a branched alkylene having 3 to 6 carbon atoms.

[0047] The polyether is preferably at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, polytetramethylene glycol and mixtures thereof, and is most preferably polyethylene glycol.

[0048] (Upper limit of average molecular weight of polyether) The average molecular weight of the polyether is not particularly limited, but is preferably 20,000 or less, and may be 10,000 or less, or may be 5,000 or less.

[0049] (Upper limit of average molecular weight of polyether) The average molecular weight of the polyether may be, for example, 1500 or more, 2,000 or more, 2500 or more, 5,000 or more, or 8,000 or more. The upper limit and lower limit of the polyether can be combined in any manner.

[0050] The state of the polyether when mixed with the liquid sample and the polysaccharide is not particularly limited, and may be, for example, a solid, a liquid, or a dissolved state in water, physiological saline, a buffer solution, or the like.

[0051] In the first mixing step S1, the order in which the liquid sample, the polysaccharide, and the polyether are mixed is not particularly limited. Thus, for example, the liquid sample and the polysaccharide may be mixed together and then the polyether may be mixed together, the liquid sample and the polyether may be mixed together and then the polysaccharide may be mixed together, the polysaccharide and the polyether may be mixed together and then the liquid sample may be mixed together, or the liquid sample, the polysaccharide, and the polyether may all be mixed together at the same time.

[0052] As described above, the polysaccharide and polyether to be subjected to the first mixing step S1 may each be in the form of a solution. Here, the first mixture, which is a mixture of a liquid sample, polysaccharide, and polyether, must be in a liquid state. Therefore, if the mixture has a high concentration and is in a gel-like state, for example, it is preferable to add water, a buffer solution, or the like to reduce the viscosity. This can promote the concentration of organic matter in the first phase of the first mixture.

[0053] Examples of buffer solutions include, but are not limited to, phosphate buffer, phosphate buffered saline, acetate buffer, citrate buffer, Tris buffer, and HEPES buffer.

[0054] <Concentration of Polysaccharide in First Mixed Liquid> The concentration of the polysaccharide in the first mixed liquid is preferably 0.1 to 40% by mass as a final concentration (concentration immediately before subjecting to the second mixing step) relative to the total mass of the first mixed liquid, and examples of the lower limit include 0.2% by mass, 0.5% by mass, 0.7% by mass, 1% by mass, 1.5% by mass, 2% by mass, 2.5% by mass, 3% by mass, 3.5% by mass, 4% by mass, 4.5% by mass, and 5% by mass.

[0055] The upper limit of the final concentration of the polysaccharide in the first mixture may be, for example, 35 mass%, 30 mass%, 25 mass%, 20 mass%, 15 mass%, 10 mass%, or 5 mass%. The above-mentioned lower and upper limits of the polysaccharide may be combined in any desired manner.

[0056] <Concentration of Polyether in First Mixed Liquid> The concentration of the polyether in the first mixed liquid, as a final concentration (concentration immediately before subjecting the first phase to the second mixing step), is preferably 0.1 to 40 mass% relative to the total mass of the first mixed liquid, and examples of the lower limit include 0.2 mass%, 0.5 mass%, 0.7 mass%, 1 mass%, 1.5 mass%, 2 mass%, 2.5 mass%, 3 mass%, 3.5 mass%, 4 mass%, 4.5 mass%, and 5 mass%.

[0057] The upper limit of the final concentration of the polyether in the first mixed solution may be, for example, 35 mass%, 30 mass%, 25 mass%, 20 mass%, 15 mass%, 10 mass%, or 5 mass%. The above-mentioned lower and upper limits of the polyether may be combined in any desired manner.

[0058] <Mixing Ratio of Polysaccharide to Polyether> The mass ratio of polysaccharide to polyether (polysaccharide:polyether) in the first mixed solution is not particularly limited, but may be, for example, 1:20 to 20:1. The mass ratio of polysaccharide to polyether (polysaccharide:polyether) may also be 1:9 to 9:1, 2:8 to 8:2, 3:7 to 7:3, 4:6 to 6:4, or 1:1.

[0059] In the first mixing step S1, a first phase rich in polysaccharides and a second phase rich in polyethers are formed by liquid-liquid phase separation. As a result, the organic matter to be concentrated in the second phase is passively transferred to the first phase, allowing the organic matter to be concentrated in the first phase. The number of phases contained in the first mixture may be two in total, including the first and second phases, or may be three or more.

[0060] <Centrifugation Treatment> In the first mixing step S1, the liquid sample, polysaccharide, and polyether may be mixed, and then the resulting first mixture may be centrifuged (first centrifugation treatment step). By centrifuging the first mixture, the boundary between two or more fractions including the first phase and the second phase, which differ in organic concentration, can be clearly defined, making it easier to identify the first phase to be subjected to the second mixing step S2 (macrophase separation). Typically, the centrifugation treatment results in the first phase being formed in the lower part of the first mixture, and the second phase being formed in the upper part.

[0061] (Centrifugal Acceleration) The centrifugal acceleration (centrifugal force) in the centrifugation process of the first mixed liquid is not particularly limited, but may be, for example, from 1,000 x g to 10,000 x g, from 1,000 x g to 3,000 x g, or from 5,000 x g to 10,000 x g.

[0062] (Centrifugation Processing Time) The centrifugation processing time in the centrifugation processing of the first mixed liquid is not particularly limited, but may be, for example, 1 to 10 minutes, 1 to 5 minutes, or 2 to 4 minutes.

[0063] The temperature during the centrifugation of the first mixture is not particularly limited, but may be, for example, 2 to 40°C, 2 to 15°C, 15 to 30°C, or 15 to 40°C.

[0064] <Stirring Treatment> In the first mixing step S1, the first mixture may be stirred after mixing the liquid sample, polysaccharide, and polyether and before centrifugation. Stirring the first mixture can promote the formation of the first and second phases by liquid-liquid phase separation and the concentration of organic matter. Note that the stirring treatment may be performed even when centrifugation is not performed.

[0065] The stirring method is not particularly limited, but may be, for example, shaking and stirring using a vortex mixer, or so-called pipetting.

[0066] <Temperature of First Mixed Liquid> The temperature of each liquid (liquid sample, polysaccharide-containing liquid, polyether-containing liquid, first mixed liquid) in the first mixing step S1 may be 100°C or less, preferably 10 to 40°C, and may be room temperature or around 37°C.

[0067] <pH of First Mixed Liquid> The pH of each liquid in the first mixing step S1 is not particularly limited, and may be 1 to 14, may be in the range of pH 3 to 10, may be in the range of pH 5 to 9, or may be around pH 7.

[0068] <<Second Mixing Step S2>> In the second mixing step S2, the first phase containing the most polysaccharides obtained in the first mixing step S1 described above is mixed with a concentration polymer, which will be described in detail later, to obtain a second mixture.

[0069] As described above, the organic matter passively transferred from the second phase is concentrated in the first phase. When the first phase is mixed with the concentration polymer in the second mixing step S2, a phase containing the most polysaccharide (hereinafter also referred to as the "third phase") and a phase containing the most concentration polymer (hereinafter also referred to as the "fourth phase") are formed by liquid-liquid phase separation.

[0070] As a result, the organic matter to be concentrated in the third phase is passively transferred to the fourth phase, thereby enabling the organic matter to be highly efficiently concentrated in the fourth phase. The number of phases contained in the second mixture may be two in total, including the third phase and the fourth phase, or may be three or more.

[0071] In the second mixing step S2, the first phase, which is the fraction containing the most polysaccharide, may be recovered from the first mixed liquid and mixed with the concentration polymer or a liquid containing the concentration polymer, or other phases other than the first phase may be removed from the first mixed liquid, and then the first phase may be mixed with the concentration polymer or a liquid containing the concentration polymer.

[0072] Examples of the liquid containing the concentrating polymer include, but are not limited to, a liquid in which the concentrating polymer is dissolved or dispersed in water, physiological saline, or the above-mentioned buffer solution. The concentrating polymer of this embodiment will be described in detail below.

[0073] <Concentration polymer> The concentration polymer is a polymer obtained by adding a compound of the following formula R to dextran. ７ In the enrichment polymer, a compound having a group represented by the formula R ７ The average molecular weight of dextran excluding the group represented by is 5,000 to 100,000. In this specification, the wavy lines in each formula represent bonds. When multiple PEG groups are present in the enrichment polymer, the PEG groups may be the same or different. [R ８n is a divalent hydrocarbon group having 1 to 20 carbon atoms which may contain one or more bonds selected from the group consisting of an ester bond, an ether bond, a carbonyl bond, and an amide bond. ２ is on average 2 to 250. ３ is the above formula R ７ R represents the number of groups introduced, and is, on average, more than 1 and 10 or less per 20,000 of the average molecular weight of dextran excluding the PEG group (when the average molecular weight is 20,000 or more), and is 1 or more and 10 or less when the average molecular weight is less than 20,000. ９ is a hydrocarbon group having 1 to 5 carbon atoms or a hydrogen atom. ７ In the above, the wavy line represents a bond to the dextran skeleton (the carbon skeleton of the tetrahydropyran ring).

[0074] (R ８ In the PEG group, R ８ The left bond of the formula binds to the tetrahydropyran ring of dextran.

[0075] The above R ８ may be, for example, a group selected from the group consisting of the following general formulas (1-1) to (1-9): ８ may be the same group or different groups.

[0076]

[0077] [R in formulas (1-1) to (1-9)] １０ is a divalent group selected from the group consisting of the following (2-1) to (2-9):

[0078] R represented by formulas (1-1) to (1-9) ８ The wavy line to the right of the group indicates the n in the PEG group. ２ The bond to the oxygen atom of each ethylene glycol unit is referred to as the bond.

[0079] (R １０ The wavy lines on the left and right of the divalent group selected from the above (2-1) to (2-9) represent R １０ and the bond to the adjacent atom.

[0080] (R ９ ) R ９ is a hydrocarbon group having 1 to 5 carbon atoms or a hydrogen atom. As the hydrocarbon group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms is preferred, a methyl group, an ethyl group or an n-propyl group is more preferred, and a methyl group or an ethyl group is even more preferred. In the concentration polymer, each R ９ may be the same group or different groups.

[0081] The dextran into which the PEG group is introduced is a polysaccharide containing glucose as a constituent element (constituent monosaccharide), and may have a structure in which glucose is bonded via an α-1,6-glycosidic bond, a structure in which glucose is bonded via an α-1,6-glycosidic bond and an α-1,4-glycosidic bond, or a structure in which glucose is bonded via an α-1,6-glycosidic bond and an α-1,3-glycosidic bond.

[0082] (Average molecular weight of dextran in the PEG group and n ３ The average molecular weight of dextran excluding the PEG group is preferably 20,000 to 80,000. ３ is preferably 2 to 30 on average. The average molecular weight of dextran excluding the PEG group is particularly preferably 30,000 to 50,000, and in this case, n ３ is preferably 3 to 15, more preferably 5 to 9, on average. The average molecular weight of dextran excluding the PEG group is most preferably 35,000 to 45,000, in which case n ３ is preferably 5 to 9, more preferably 6 to 8 on average. ３ The average is the average for all PEG groups introduced into the dextran.

[0083] (n ２ ) n indicates the degree of polymerization of ethylene glycol in the PEG group ２ is preferably 60 to 110, more preferably 70 to 100, and particularly preferably 80 to 95, on average. ２ The average is the average for all PEG groups introduced into the dextran.

[0084] (Dextran to which a PEG group is introduced) Dextran generally has a structure represented by the following general formula (2), １ ~R ６ are each an OH group, and the enrichment polymer is a polymer in which one or more PEG groups are introduced into dextran as substituents for the hydroxyl groups of the dextran (the number of PEG groups introduced depends on the molecular weight of the dextran). Therefore, in the enrichment polymer represented by general formula (2), R １ ~R ６ are each independently a hydroxy group or the above R ７ R is a group represented by the formula: １ ~R ６ When all of the above are hydroxy groups, the structure is one in which glucose is linked via an α-1,6-glycosidic bond.

[0085]

[0086] The average molecular weight of the dextran in the enrichment polymer, i.e., ７ The average molecular weight of the enrichment polymer excluding the group represented by n is 5,000 to 100,000. １ The average value of n is in the range corresponding to the average molecular weight of 5,000 to 100,000, about 16 to 320, preferably 50 to 200, more preferably 80 to 180, more preferably 100 to 160, even more preferably 110 to 150, particularly preferably 120 to 140, and most preferably 125 to 135. １ The average is the average for all dextrans into which PEG groups have been introduced. The condensing polymer, excluding the group represented by the formula R7, does not include the oxygen atom of the PEG group that is directly bonded to the tetrahydropyran ring of the dextran.

[0087] The enrichment polymer may be a compound represented by the following formula (3): [In formula (3), n １ , n ２ , n ３ and R ９ are the same as the above formula R ７ or n in formula (2) １ , n ２and n ３ and n １ The average is 16 to 320. ２ is 2 to 250 on average. ７ n indicates the number of groups (groups represented by ３ is, on average, more than 1 and 10 or less per 20,000 average molecular weight of the condensing polymer excluding the PEG group, and when the average molecular weight is less than 20,000, it is 1 or more and 10 or less. ７ When a PEG group is not introduced, an OH group is bonded to the carbon atom (carbon atom constituting the tetrahydropyran ring) to which the group represented by the formula R7 is bonded, and the group represented by the formula R8 is bonded to the carbon atom (carbon atom constituting the tetrahydropyran ring) to which the group represented by the formula R7 is bonded. ９ is a hydrocarbon group having 1 to 5 carbon atoms or a hydrogen atom.

[0088] (n １ , n ３ In formula (3), n １ is 80 to 180 on average, and n ３ is preferably 3 to 25 on average. １ is on average 100 to 160, and n ３ is preferably 3 to 15 on average. １ is on average 110 to 150, and n ３ More preferably, n is 4 to 12 on average. １ is on average 120 to 140, and n ３ is particularly preferably 5 to 9 on average. １ is on average 125-135, and n ３ is most preferably 6 to 8 on average.

[0089] (n １ In formula (3), n １ is preferably 50 to 200, more preferably 80 to 180, more preferably 100 to 160, even more preferably 110 to 150, particularly preferably 120 to 140, and most preferably 125 to 135, on average.

[0090] (n ２ In formula (3), n ２ is preferably 60 to 110, more preferably 70 to 100, and particularly preferably 80 to 95 on average.

[0091] (n ３ In formula (3), n represents the number of PEG groups introduced. ３ is preferably 2 to 7, more preferably 2 to 5, on average, per 20,000 average molecular weight of the enrichment polymer excluding the PEG group. ３ is preferably 5 to 9, particularly preferably 6 to 8, in the entire enrichment polymer. ７ When a PEG group is not introduced, an OH group is bonded to the carbon atom (a carbon atom constituting the tetrahydropyran ring) to which the group represented by the formula R7 is bonded. In other words, one or more PEG groups are introduced into dextran as substituents for the hydroxyl groups of the dextran.

[0092] (R ９ In formula (3), R ９ is the above formula R ７ R in ９ and preferably a hydrogen atom or a methyl group.

[0093] (Concentration of Concentrating Polymer) The concentration of the concentrating polymer in the second mixed liquid is not particularly limited, but may be, for example, 0.1 to 30% by mass relative to the total mass of the second mixed liquid.

[0094] The concentration of the concentration polymer may be 0.1% by mass or more, 0.3% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, or 4% by mass or more, relative to the total mass of the second mixed solution. The upper limit of the concentration of compound (P) in the mixed solution is not particularly limited, and may be, for example, 30% by mass or less, 20% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 2% by mass or less, or 1% by mass or less, relative to the total mass of the mixed solution. The above upper and lower limits can be arbitrarily combined. The concentration of the concentration polymer relative to the total mass of the second mixed solution is preferably 3 to 10% by mass.

[0095] (Production of Concentrating Polymer) The concentrating polymer can be produced by reacting dextran with polyethylene glycol. For details, see, for example, JP-A-2023-156995.

[0096] (Mixing ratio of polysaccharide to concentrating polymer) The mass ratio of polysaccharide to concentrating polymer (polysaccharide:concentrating polymer) in the second mixed solution is not particularly limited, but may be, for example, 1:20 to 20:1. The mass ratio of polysaccharide to concentrating polymer (polysaccharide:concentrating polymer) may also be 1:9 to 9:1, 2:8 to 8:2, 3:7 to 7:3, 4:6 to 6:4, or 1:1.

[0097] <Centrifugation Step> The organic matter concentrating method of this embodiment may further include a step of centrifuging the second mixture (second centrifugation step) after the second mixing step S2. By centrifuging the second mixture, the boundary between two or more fractions including the third and fourth phases, which differ in organic matter concentration, can be clearly defined, making it easier to identify the fourth phase in which the organic matter is concentrated (macrophase separation). Typically, the centrifugation step forms the third phase in the lower part of the second mixture, and the fourth phase in the upper part.

[0098] (Centrifugal Acceleration) The centrifugal acceleration (centrifugal force) in the centrifugation process of the second mixture is not particularly limited, but may be, for example, 1,000 x g or more and 10,000 x g or less, 1,000 x g or more and 3,000 x g or less, or 5,000 x g or more and 10,000 x g or less.

[0099] (Centrifugation Processing Time) The centrifugation processing time in the centrifugation processing of the second mixture is not particularly limited, but may be, for example, 1 to 10 minutes, 1 to 5 minutes, or 2 to 4 minutes.

[0100] The temperature at which the second mixture is centrifuged is not particularly limited, but may be, for example, 5 to 40°C, or 15 to 30°C.

[0101] <Stirring Treatment> In the second mixing step S2, the second mixture may be stirred after mixing the first phase and the concentration polymer and before centrifugal separation. Stirring the second mixture can promote the formation of a third phase and a fourth phase by liquid-liquid phase separation and the concentration of organic matter. Note that the stirring treatment may be performed even when centrifugal separation is not performed.

[0102] The stirring method is not particularly limited, but may be, for example, shaking and stirring using a vortex mixer, or so-called pipetting.

[0103] <Temperature of Second Mixed Liquid> The temperature of the liquid (first phase liquid, liquid to which the concentration polymer has been added) in the second mixing step S2 may be 100°C or lower, preferably 10 to 40°C, and may be room temperature or around 37°C.

[0104] <pH of second mixed liquid> The pH of each liquid in the second mixing step S2 is not particularly limited, and may be 1 to 14, may be in the range of pH 3 to 10, may be in the range of pH 5 to 9, or may be around pH 7.

[0105] According to the method for concentrating organic matter of this embodiment, in addition to being able to concentrate organic matter into the first phase in the first mixing step S1 by about 30 times, it is also possible to further concentrate organic matter into the fourth phase in the second mixing step S2 by several times. Therefore, organic matter can be concentrated inexpensively and efficiently without using antibodies.

[0106] [Method 2 for Concentrating Organic Substances] In another embodiment, the present invention provides a method for concentrating organic substances, comprising a first concentration step and a second concentration step.

[0107] The first concentration step is a step of mixing a liquid sample containing organic matter with polysaccharides and polyether to produce a first mixed liquid containing a first phase containing the most polysaccharides and a second phase containing the most polyethers, and then transferring the organic matter from the second phase to the first phase to concentrate it.

[0108] The first concentration step is similar to the first mixing step S1 described above, in which a first phase and a second phase are formed by liquid-liquid phase separation, and organic matter contained in the liquid sample is passively transferred to the first phase and concentrated therein.

[0109] In this embodiment, the organic-containing liquid sample, polysaccharide, polyether, first phase, second phase, and first mixed solution are the same as the organic-containing liquid sample, polysaccharide, polyether, first phase, second phase, and first mixed solution in the above-described embodiment of [Organic Substance Concentration Method 1]. Therefore, in this embodiment, the polysaccharide may be, for example, dextran, and the polyether may be, for example, polyethylene glycol.

[0110] The second concentration step is a step in which the first phase is mixed with a concentration polymer to produce a second mixture, and the organic matter is transferred from the first phase to the concentration polymer (in other words, the phase containing the most concentration polymer) to concentrate it.

[0111] The operation in the second concentration step is the same as the operation in the second mixing step S2 described above, in which two phases are formed by liquid-liquid phase separation, and the organic matter is passively transferred to the phase containing the most enriched enrichment polymer and concentrated therein. However, the enrichment polymer in this embodiment may be different from the enrichment polymer in the above-described [Method 1 for Enriching Organic Matter].

[0112] The concentration polymer in this embodiment is a polymer that can transport the organic matter contained in the first phase of the second mixture to the phase containing the largest amount of the concentration polymer, and is, for example, a polymer that contains ethylene glycol units (-CH ２ CH ２ The enrichment polymer in this embodiment may be a compound having a repeating unit represented by the formula R ７It is a compound having a PEG group introduced therein, represented by the following formula:

[0113] When the concentrating polymer is a compound in which PEG groups have been introduced into dextran, the average molecular weight of the concentrating polymer excluding the PEG groups is preferably 5,000 to 100,000. In this case, the concentrating polymer and second mixed solution of this embodiment are the same as those in the embodiment of the above-mentioned [Method for concentrating organic substances 1], and the second concentrating step corresponds to the second mixing step S2.

[0114] [Method for detecting organic matter] Another preferred embodiment of the method for detecting organic matter of the present invention includes a step (hereinafter also referred to as an "organic matter detection step") of detecting organic matter contained in a phase containing the above-mentioned concentrating polymer most abundantly (phase 4 in the case of the concentration method according to [Method for concentrating organic matter 1]) among multiple phases obtained after centrifugal treatment of the second mixture (after the second centrifugation treatment step) by the concentration method according to the embodiment of the above-mentioned [Method for concentrating organic matter 2]. The organic matter to be detected is the organic matter to be concentrated.

[0115] The method for detecting organic matter in the organic matter detection process is not particularly limited. For example, when the organic matter is a nucleic acid, a virus, a bacterium, or the like, examples of the method include the PCR method, the RT-PCR method, the invader method, the LAMP method, and the microarray method.

[0116] Furthermore, for example, when the organic substance is a protein, ELISA or Western blotting may be used.

[0117] [Kit for concentrating organic matter] A kit for concentrating organic matter according to another preferred embodiment of the present embodiment includes a polysaccharide, a polyether, and a concentrating polymer, and is a kit for use in concentrating organic matter by the concentrating method according to the embodiment of [Method 1 for concentrating organic matter] described above or the concentrating method according to the embodiment of [Method 2 for concentrating organic matter] described above.

[0118] The polysaccharide of this embodiment is the same as the polysaccharide described in detail in the embodiment of [Method for concentrating organic matter 1] above. The polyether of this embodiment is the same as the polyether described in detail in the embodiment of [Method for concentrating organic matter 1] above. The concentrating polymer of this embodiment is the same as the concentrating polymer described in detail in the embodiment of [Method for concentrating organic matter 1] above or the concentrating polymer described in detail in the embodiment of [Method for concentrating organic matter 2] above. The organic matter concentrated using the kit of this embodiment is the same as the organic matter concentrated by the concentration method according to the embodiment of [Method for concentrating organic matter 1] or [Method for concentrating organic matter 2] above.

[0119] According to the kit for concentrating organic matter of this embodiment, organic matter can be concentrated inexpensively and efficiently.

[0120] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0121] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. Each experiment was carried out at room temperature of 25° C. unless otherwise specified.

[0122] [Experimental Example 1] Figure 2 is a schematic diagram of several types of concentrating polymers with different dextran molecular weights and PEG group molecular weights. In this experiment, the localization of dextran and polyethylene glycol during liquid-liquid phase separation was examined for each of the following polymers: concentrating polymer A, in which an average of seven PEG groups 2 each having an average molecular weight of 4,000 have been introduced into dextran 1 having an average molecular weight of 40,000; concentrating polymer B, in which an average of 2.2 PEG groups 2 each having an average molecular weight of 20,000 have been introduced into dextran 1 having an average molecular weight of 40,000; and concentrating polymer C, in which an average of eight PEG groups 2 each having an average molecular weight of 4,000 have been introduced into dextran 1 having an average molecular weight of 550,000. Concentrating polymers A to C are each compounds represented by the above formula (3), and R ９ is a methyl group.

[0123] Specifically, an aqueous dispersion containing 2% by mass of the concentrating polymer, 0.004% by mass of the concentrating polymer labeled with green fluorescent FITC, 4% by mass of dextran (average molecular weight 550,000), 0.0004% by mass of dextran labeled with red fluorescent rhodamine (average molecular weight 550,000), and 4% by mass of PEG (polyethylene glycol, average molecular weight 35,000) was shaken and stirred using a vortex mixer. Three systems using the concentrating polymers A, B, and C were prepared.

[0124] Figure 3 shows confocal microscopic images of an aqueous dispersion containing a concentrating polymer, dextran, and polyethylene glycol immediately after shaking and stirring. In Figure 3, the scale bars indicate 50 μm.

[0125] The upper row of Fig. 3 shows images of a system using the above-mentioned A as the concentration polymer, the middle row of Fig. 3 shows images of a system using the above-mentioned B as the concentration polymer, and the lower row of Fig. 3 shows images of a system using the above-mentioned C as the concentration polymer. In addition, the left column of Fig. 3 shows observation images of green fluorescence derived from FITC, the central column in the horizontal direction of Fig. 3 shows observation images of red fluorescence derived from rhodamine, and the right column of Fig. 3 shows a three-dimensional image combining the observation images of green fluorescence and red fluorescence.

[0126] As shown in the top left image of Figure 3, the bright areas of the concentrating polymer A, which exhibit green fluorescence, are located outside (in PEG) the circular droplets of dextran, which are shown in dark color.

[0127] In contrast, as shown in the image on the middle left, the bright green fluorescent areas of enrichment polymer B are present both inside and outside the dextran droplets, and as shown in the image on the bottom left, the bright green fluorescent areas of enrichment polymer C are present inside the dextran droplets, and it can be seen that the inside of the circular droplets glows brightly.

[0128] These results revealed that enrichment polymer A, in which an average of seven PEG groups with an average molecular weight of 4,000 have been introduced into dextran with an average molecular weight of 40,000, undergoes phase separation from dextran.

[0129] Therefore, the inventors came up with the idea that the organic matter concentrated from the PEG-rich phase to the dextran-rich phase could be further concentrated by passively transferring it to the concentrating polymer A. In each of the subsequent experiments, A was used as the concentrating polymer.

[0130] [Experimental Example 2] In this experiment, an attempt was made to concentrate DNA, which is an example of an organic substance, using the concentrating polymer A and dextran.

[0131] Specifically, a first mixture was prepared, which was a mixture (aqueous mixture) of the concentrating polymer A and dextran, and a second mixture was prepared, which was a mixture (aqueous mixture) of the concentrating polymer A, dextran, and PEG. The compositions of each mixture were as follows: First mixture: 5% by mass of the concentrating polymer A, 5% by mass of dextran with an average molecular weight of 550,000; Second mixture: 2% by mass of the concentrating polymer A, 2% by mass of dextran with an average molecular weight of 550,000, 6% by mass of PEG with an average molecular weight of 35,000.

[0132] Next, 500 bp or 48,500 bp DNA (0.05 mg / mL) was mixed with the first mixture and the second mixture, respectively, and centrifuged at 10,000 rpm for 3 minutes, causing macrophase separation into a phase of enrichment polymer A (upper layer; in the second mixture, a phase of enrichment polymer A and PEG) and a phase of dextran (lower layer).

[0133] Thereafter, SYBR®-Gold, which binds to DNA, was diluted 1 / 15 and mixed, and the DNA in each of the upper and lower liquid layers was quantified using the absorption spectrum at 495 nm derived from SYBR®-Gold.

[0134] Figure 4 is a graph showing the relative DNA amount in each phase after macrophase separation with 500 bp DNA added, and Figure 5 is a graph showing the relative DNA amount in each phase after macrophase separation with 48,500 bp DNA added.

[0135] In a two-phase system consisting of a phase of concentrating polymer A and a phase of dextran (see the left side of each of Figures 4 and 5), DNA was highly localized in the concentrating polymer A ("DEX / PEG" in the figures). Furthermore, as shown in Figure 5, the difference in distribution amount between the two phases was more pronounced for 48,500 bp, demonstrating that DNA was more efficiently concentrated in the phase of concentrating polymer A.

[0136] In contrast, in the case of a two-phase system consisting of a phase of PEG and concentrating polymer A and a phase of dextran (see the right side of each of Figures 4 and 5), DNA was highly localized in the dextran phase.

[0137] [Experimental Example 3] In this experiment, the concentration efficiency of a plurality of DNAs having different lengths was confirmed in a two-phase system of concentrating polymer A and dextran.

[0138] Specifically, DNA of 10 bp, 500 bp, or 48,500 bp was added to a mixture of 5.0% by mass of concentrating polymer A and 5.0% by mass of dextran (average molecular weight 550,000), followed by centrifugation to determine the partition coefficient of DNA into the upper phase of concentrating polymer A, which was the macrophase-separated layer. The partition coefficient is the ratio of DNA present in the phase of concentrating polymer A to that of the dextran phase (concentration efficiency into the phase of concentrating polymer A).

[0139] Figure 6 is a graph showing the partition coefficient for each DNA length. As shown in Figure 6, it was confirmed that the partition coefficient, i.e., the concentration efficiency into the concentration polymer A phase, increases with increasing DNA length. For example, with 48,500 bp DNA, a concentration of more than 14 times was achieved.

[0140] [Experimental Example 4] In this experiment, it was confirmed whether or not the concentrating polymer A could be accommodated in each microwell in a microarray chamber having a large number of microwells formed on the inner upper surface.

[0141] FIG. 7 is a schematic longitudinal cross-sectional view showing the process of placing the enrichment polymer A into the microwells of the microarray chamber.

[0142] As shown in FIG. 7 , first, a mixture 5 containing 8% by mass of enrichment polymer A labeled with FITC and 2% by mass of PEG (average molecular weight 35,000) was poured into a microarray chamber 10, and each microwell 10 a formed on the inner upper surface was filled with the mixture 5.

[0143] Next, a 30% by mass aqueous solution 6 of dextran (average molecular weight 550,000) was poured into the microarray chamber 10, and the bottom end of each microwell 10a was blocked with the aqueous dextran solution 6. Thereafter, a fluorescent image of the microarray chamber 10 was obtained using a confocal microscope.

[0144] Figure 8 is a fluorescent image of the microarray chamber viewed from diagonally above. As shown in Figure 8, bright green fluorescence was observed in each of the regularly arranged microwells.

[0145] This result revealed that the aqueous solution of the concentrating polymer A could be contained in each of the microwells 10 a formed on the inner upper surface of the microarray chamber 10 .

[0146] [Experimental Example 5] In this experiment, RNA was concentrated in the microarray chamber 10 used in Experimental Example 4.

[0147] 9 is a schematic longitudinal cross-sectional view showing the process of concentrating RNA in the microwells of a microarray chamber. As shown in Fig. 9, first, a mixture 7 containing 8% by mass of concentrating polymer A and 2% by mass of PEG (average molecular weight 35,000) was poured into the microarray chamber 10, filling each microwell 10a with the mixture 7.

[0148] Next, an aqueous solution 8 containing RNA 9 (1.55 μM (mol / L)) fluorescently labeled with 100×SYBR®-Gold and dextran (27.5% by mass) having an average molecular weight of 550,000 was flowed into the microarray chamber 10. As a control, a separate system was also prepared in which an aqueous solution not containing fluorescently labeled RNA 9 was flowed into the microarray chamber 10.

[0149] After 1 minute, an aqueous solution 11 containing dextran (27.5% by mass) with an average molecular weight of 550,000 was supplied to the microarray chamber 10 to wash away the aqueous solution 8 located below the microwells 10a. Thereafter, a fluorescent image of the microarray chamber 10 was obtained using a confocal microscope.

[0150] Fig. 10 is a fluorescence image of the microarray chamber 10 in which RNA 9 was accommodated in the microwell 10a. Fig. 11 is a fluorescence image of the control microarray chamber 10 in which RNA 9 was not accommodated in the microwell 10a. As shown in Fig. 11, in the control system in which fluorescently labeled RNA 9 was not flowed into the microarray chamber 10, no fluorescence was observed inside the microwell 10a.

[0151] In contrast, as shown in Figure 10, in the system in which fluorescently labeled RNA 9 was flowed into the microarray chamber 10, fluorescence was confirmed inside each microwell 10a. The above results revealed that the RNA contained in the aqueous solution 8 was passively transferred to the microwell 10a containing the concentration polymer A. Although the mixed solution 7 also contained PEG, the amount was small compared to the amount of concentration polymer A, which is thought to be why the RNA 9 was able to be concentrated in the microwell 10a.

[0152] [Experimental Example 6] In this experiment, FAM-labeled RNA at a low concentration of 6 fM was concentrated by liquid-liquid phase separation of dextran and PEG, and then concentrated in each microwell 10a using concentration polymer A and the microarray chamber 10, followed by a digital bioassay.

[0153] After concentrating the RNA in the reactor, the mixture from the CRISPR / Cas9 assay kit was poured in, and the bound FAM was activated by degradation of the target RNA, and fluorescence was detected. In the digital bioassay, the solution was supplied to the microarray chamber 10 and evaluated according to the following steps 1 to 5. Step 1: BSA blocking solution: 3 μL of 5 mg / mL BSA, 0.6 μL of 25 μM FAM probe, 0.6 μL of 25 μM Cy5 probe, buffer (HEPES-NaOH = 20 mM, NaCl = 60 mM, MgCl ２ Step 2 BSA blocking solution: 20 μL of 30% polymer A for concentration, 10 μL of ultrapure water. Step 3 Dextran aqueous solution containing polymer A for concentration: 20 μL of 30% dextran with an average molecular weight of 550,000, 1.8 μL of 2% polymer A for concentration, 8.2 μL of ultrapure water. Step 4 Target RNA solution: 967 μL of 30% PEG, 967 μL of buffer (HEPES-NaOH = 20 mM, NaCl = 60 mM, MgCl ２ = 6 mM) 75 μL, target RNA 10 pM 0.87 μL, ultrapure water 407 μL Step 5 Cas13 solution: Cas13a 0.5 μM 1.35 μL, crRNA 450 nM 0.75 μL, FAM probe 100 μM 0.45 μL, Cy5 probe 100 μM 1.5 μL, buffer (HEPES-NaOH = 20 mM, NaCl = 60 mM, MgCl ２ = 6 mM) 0.75 μL, dextran having an average molecular weight of 550,000 30% by mass 20 μL, polymer A for concentration 2% by mass 0.2 μL

[0154] Fig. 12 is a fluorescence image showing the results of RNA detection using the microarray chamber 10 in Experimental Example 6. As shown in Fig. 12, even with RNA at a low concentration of 6 fM, a large amount of green fluorescence (light-colored areas in Fig. 10) indicating the presence of RNA was confirmed through the first-stage concentration by liquid-liquid phase separation between dextran and PEG, and the second-stage concentration by liquid-liquid phase separation between the dextran phase obtained in the first-stage concentration and the phase of concentrating polymer A.

[0155] [Experimental Example 7] In this experiment, FAM-labeled RNA at an extremely low concentration of 600 a (atto)M was concentrated by liquid-liquid phase separation between dextran and PEG, and then concentrated in each microwell 10a using enrichment polymer A and the microarray chamber 10, followed by a digital bioassay. Concentration and detection were performed in the same manner as in Experimental Example 6, except that the target RNA concentration in the target RNA solution in Step 4 was set to 1 pM. Two systems were prepared: one in which RNA detection was performed after a first stage of enrichment by liquid-liquid phase separation between dextran and PEG, and the other in which RNA detection was performed after a second stage of enrichment by liquid-liquid phase separation between enrichment polymer A and dextran in addition to the first stage.

[0156] Figure 13 is a fluorescent image showing the results of RNA detection after the first concentration step by liquid-liquid phase separation of dextran and PEG, and Figure 14 is a fluorescent image showing the results of RNA detection after the second concentration step by liquid-liquid phase separation of concentrating polymer A and dextran.

[0157] As shown in FIG. 13, in the system in which RNA was detected after the first stage of concentration by liquid-liquid phase separation of dextran and PEG, only a few microwells 10a exhibited bright fluorescence indicating the presence of RNA.

[0158] In contrast, as shown in Figure 14, in a system in which a second concentration step involving liquid-liquid phase separation of concentrating polymer A and dextran was performed in addition to the first concentration step, the number of areas showing bright fluorescence increased significantly.

[0159] These results demonstrate that by performing the second concentration step using liquid-liquid phase separation between the concentrating polymer A and dextran, RNA can be concentrated more efficiently than by performing only the first concentration step.

[0160] FIG. 15 is a graph showing the percentage of microwells 10a in which fluorescence was detected for each concentration method. In FIG. 15, P positive on the vertical axis represents the number of microwells 10a in which fluorescence was confirmed divided by the total number of microwells 10a (the percentage of microwells 10a in which fluorescence was detected). The horizontal axis represents the concentration of target RNA. Also, a solid line represents an approximation line of the actual measured value, and a dashed line represents a line based on the theoretical value. Furthermore, in FIG. 15, of the two dashed lines with a constant P positive value on the vertical axis, the background in "Previous Research +DEX / PEG droplet" is represented by a dense dashed line, and the background in "Previous Research -DEX / PEG droplet" is represented by a coarse dashed line.

[0161] As shown in Figure 15, in the detection after the first stage of concentration by liquid-liquid phase separation of dextran (DEX) and PEG ("Previous Research +DEX / PEG droplet" in the figure), the proportion of microwells 10a in which fluorescence could be detected was approximately 30 times higher than when the sample was subjected to detection without the concentration ("Previous Research -DEX / PEG droplet" in the figure).

[0162] Furthermore, when RNA was detected after a first stage of liquid-liquid phase separation between dextran and PEG and a second stage of concentration by liquid-liquid phase separation between the concentration polymer A and dextran ("first and second stage concentration" in the figure), the proportion of microwells 10a in which fluorescence could be detected was approximately three times higher than when RNA was detected after the first stage of concentration.

[0163] Figure 16 is a graph showing the fluorescence plots of the digital bioassay described above for 6 fM and 6 aM RNA. In Figure 16, in addition to the fluorescence of FAM derived from RNA, the fluorescence intensity when Cy5 was bound to RNA that was not degraded by CRISPR / Cas9 was plotted to examine the influence of fluorescence derived from spurious signals.

[0164] The digital bioassay in the absence of target RNA was carried out in the same manner as in Experimental Examples 5 and 6 above, except that the solution supplied to the microarray chamber 10 in Step 4 did not contain target RNA.

[0165] The number of plots where the Cy5 fluorescence intensity on the horizontal axis is lower than 1500 and the FAM fluorescence intensity on the vertical axis is 1000 or higher indicates the number of RNAs that were normally detected without contamination. As shown in Figure 16, a large number of RNAs were normally detected, demonstrating that the digital bioassay was performed accurately for both the 6 fM and 6 aM RNA samples.

[0166] According to the present invention, organic matter to be concentrated is concentrated into a first phase containing the most polysaccharide, and then the first phase is mixed with a concentrating polymer to obtain a second mixture, which is separated into liquid-liquid phases and the organic matter can be concentrated into the phase containing the most concentrating polymer. Thus, the present invention is industrially applicable.

[0167] 1... dextran, 2... polyethylene glycol, 5... mixture containing FITC-labeled enrichment polymer A and polyethylene glycol, 6... aqueous dextran solution, 7... mixture of enrichment polymer A and polyethylene glycol, 8... aqueous solution containing fluorescently labeled RNA and dextran, fluorescently labeled RNA, 10... microarray chamber, 10a... microwell, 11... aqueous solution containing dextran

Claims

1. A method for concentrating organic matter, comprising: a step of mixing a liquid sample containing the organic matter with a polysaccharide and a polyether to obtain a first mixture containing a first phase containing the polysaccharide in the largest amount and a second phase containing the polyether in the largest amount; and a step of mixing the first phase with a concentrating polymer to obtain a second mixture, wherein the concentrating polymer is a polymer having a structure represented by the following formula R ７ and a compound having a group represented by the following formula R ７ the average molecular weight of the concentrating polymer excluding the group represented by the formula (I) is 5,000 to 100,000; (R ８ is a divalent hydrocarbon group having 1 to 20 carbon atoms which may contain one or more bonds selected from the group consisting of an ester bond, an ether bond, a carbonyl bond, and an amide bond. ２ is on average 2 to 250. ３ is the reaction of the above formula R ７ R represents the number of groups represented by the formula (I) introduced, and is more than 1 and 10 or less on average per the average molecular weight of 20,000, and when the average molecular weight is less than 20,000, it is 1 or more and 10 or less on average. ９ is a hydrocarbon group having 1 to 5 carbon atoms or a hydrogen atom.

2. The above R ８ is a group selected from the group consisting of the following general formulas (1-1) to (1-9): (R in formulas (1-1) to (1-9) １０ is a divalent group selected from the group consisting of the following formulas (2-1) to (2-9):

3. The method for concentrating organic matter according to claim 1, wherein the polysaccharide is at least one selected from the group consisting of dextran, chitin, inulin, heparin, cellulose, methylcellulose, alginic acid, and salts thereof.

4. The method for concentrating organic matter according to claim 3, wherein the polysaccharide is dextran.

5. The method for concentrating organic matter according to claim 1, wherein the polyether is at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and mixtures thereof.

6. The method for concentrating organic matter according to claim 5, wherein the polyether is polyethylene glycol.

7. The method for concentrating organic matter according to claim 1, wherein the concentrating polymer is a compound represented by the following formula (3): (n in formula (3) １ The average is 16 to 320. ２ The average is 2 to 250. ３ is the formula R ７ The number of groups represented by the formula R is more than 1 and 10 or less on average per the average molecular weight of 20,000, and when the average molecular weight is less than 20,000, the number is 1 or more and 10 or less on average. ７ The carbon atom to which the group represented by the formula R ７ When a group represented by the formula R ７ An OH group is bonded in place of the group represented by R ９ is a hydrogen atom or a methyl group.

8. In the formula (3), n １ is on average 80 to 180, and n ２ is on average 60 to 110, and n ３ The method for concentrating organic matter according to claim 7, wherein the average is 5 to 9.

9. A method for concentrating organic matter according to any one of claims 1 to 8, wherein the organic matter is selected from the group consisting of nucleic acids, proteins, and viruses.

10. The method for concentrating organic matter according to any one of claims 1 to 8, further comprising a step of centrifuging the second mixture.

11. A method for concentrating organic matter according to any one of claims 1 to 8, wherein in the step of obtaining the first mixture, the liquid sample, the polysaccharide, and the polyether are mixed and centrifuged.

12. A method for concentrating organic matter according to any one of claims 1 to 8, wherein the concentration of the concentration polymer in the second mixed liquid is 1 mass % or more.

13. A method for detecting organic matter, comprising a step of detecting the organic matter contained in the phase containing the most of the concentrating polymer among multiple phases obtained after centrifugal separation of the second mixture obtained by the method for concentrating organic matter described in claim 10.

14. A kit for use in concentrating organic matter by the method for concentrating organic matter according to claim 1, comprising a polysaccharide, a polyether, and the concentrating polymer according to claim 1.

15. A method for concentrating organic matter, comprising: a first concentration step of mixing a liquid sample containing the organic matter with a polysaccharide and a polyether to produce a first mixed liquid containing a first phase containing the polysaccharide (dextran) in the largest amount and a second phase containing the polyether in the largest amount, and transferring the organic matter from the second phase to the first phase to concentrate it; and a second concentration step of mixing the first phase with a concentrating polymer to produce a second mixed liquid, and transferring the organic matter from the first phase to the concentrating polymer to concentrate it.

16. The enrichment polymer is a compound of the following formula R ７ and a compound having a group represented by the following formula R ７ The method for concentrating organic matter according to claim 15, wherein the average molecular weight of the concentrating polymer excluding the group represented by the formula (I) is 5,000 to 100,000; (R ８ is a divalent hydrocarbon group having 1 to 20 carbon atoms which may contain one or more bonds selected from the group consisting of an ester bond, an ether bond, a carbonyl bond, and an amide bond. ２ is on average 2 to 250. ３ is the reaction of the above formula R ７ R represents the number of groups represented by the formula (I) introduced, and is more than 1 and 10 or less on average per the average molecular weight of 20,000, and when the average molecular weight is less than 20,000, it is 1 or more and 10 or less on average. ９ is a hydrocarbon group having 1 to 5 carbon atoms or a hydrogen atom.

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