Method for analyzing trace substance

Abstract

This method for analyzing a trace substance involves: a step A for separating a trace substance with a separation member including a porous material; a step B for binding, to the separated trace substance, an enzyme-labeled antibody or an enzyme-labeled nucleic-acid probe, wherein the enzyme-labeled antibody includes an enzyme α and an antibody specific to the trace substance, and the enzyme-labeled nucleic-acid probe includes an enzyme α and a nucleic-acid probe specific to the trace substance; a step C for making the enzyme α of the enzyme-labeled antibody or the enzyme α of the enzyme-labeled nucleic-acid probe act on a compound β which is a substrate of the enzyme α to produce a compound γ which is a substrate of a dehydrogenase; a step D for producing thio-NAD(P)H through an enzyme cycling reaction using the produced compound γ, a dehydrogenase, NAD(P)H, and thio-NAD(P); and a step E for detecting the trace substance by the produced thio-NAD(P)H.

Description

Analytical methods for trace substances

[0001] The present invention relates to a method for analyzing a trace substance.

[0002] Highly sensitive methods for analyzing biopolymers such as proteins and nucleic acids include enzyme immunoassays and nucleic acid probe assays such as Southern blotting, Northern blotting and Western blotting.

[0003] Patent Document 1 discloses an enzyme measurement method using an antibody-enzyme conjugate, in which the reaction products of the antibody-enzyme conjugate are quantified by generating thio-NADH and / or thio-NADPH through an enzymatic cycling reaction using NADH and / or NADPH, thio-NAD and / or thio-NADP, and dehydrogenase (DH), and measuring the amount of the generated thio-NADH and / or thio-NADPH or by measuring the color change caused by the generated thio-NADH and / or thio-NADPH. Patent Document 1 states, "Therefore, an object of the present invention is to provide a measurement method in which sensitivity is geometrically increased using colorimetric detection, which is the simplest method."

[0004] WO 2008 / 117816

[0005] The present invention provides a method for analyzing trace substances, which can easily separate the target trace substances and can analyze the target trace substances with high sensitivity.

[0006] The present invention provides the following method for analyzing trace substances: 1. A method for analyzing trace substances, comprising: Step A: separating trace substances using a separation member containing a porous material; Step B: binding an enzyme-labeled antibody consisting of an antibody specific to the trace substance and enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and enzyme α, to the separated trace substances; Step C: allowing the enzyme α of the enzyme-labeled antibody or the enzyme α of the enzyme-labeled nucleic acid probe to act on compound β, which is a substrate of enzyme α, to generate compound γ, which is a substrate of dehydrogenase; Step D: generating thioNAD(P)H by an enzyme cycling reaction using the generated compound γ, dehydrogenase, NAD(P)H, and thioNAD(P); and Step E: detecting the trace substances using the generated thioNAD(P)H. 2. A method for analyzing trace substances, wherein the porous material comprises a porous membrane. 3. The method for analyzing trace substances according to 2., wherein the porous membrane comprises one or more resins selected from the group consisting of polyester-based resins, polyolefin-based resins, fluorine-based resins, and polystyrene-based resins. 4. The method for analyzing trace substances according to 2. or 3., wherein the average pore size of the porous membrane measured by observation with a scanning electron microscope is 1 nm or more and 5,000 nm or less. 5. The method for analyzing trace substances according to any of 1. to 4., comprising, between step A and step B, step F of recovering the separated trace substances from the separation member onto a plate. 6. The method for analyzing trace substances according to 5., wherein in step A, the separation member has a cover on one surface, and in step F, the cover is removed from the separation member, the surface of the separation member that had the cover is placed on the plate, and the trace substances are then recovered from the separation member onto the plate. 7. The method for analyzing a trace substance according to 5. or 6., wherein at least a portion of the surface of the plate contains a cationic polymer. 8. The method for analyzing a trace substance according to any of 2. to 7., wherein in step A, the separating member is placed in a space where the trace substances are present, thereby causing the trace substances to adhere to the porous material.9. The method for analyzing trace substances according to 1., wherein the porous material comprises a plate-shaped porous member, and the plate-shaped porous member comprises holes penetrating from the top surface to the bottom surface of the plate-shaped porous member. 10. The method for analyzing trace substances according to 9., wherein the plate-shaped porous member comprises glass. 11. The method for analyzing trace substances according to 9. or 10., wherein the average pore diameter of the pores in the plate-shaped porous member measured by observation with a scanning electron microscope is 1 nm or more and 5000 nm or less. 12. The method for analyzing trace substances according to any of 9. to 11., wherein the average depth of the pores in the plate-shaped porous member measured by observation with a scanning electron microscope is 0.5 μm or more and 50 mm or less. 13. The method for analyzing trace substances according to any of 9. to 12., wherein the plate-shaped porous member contains a cationic polymer on at least a portion of the bottom surface of the pores. 14. 9. to 13., wherein steps B to E are performed inside the pores. 15. The method for analyzing a trace substance according to any one of 1. to 14., wherein the trace substance comprises one or more species selected from the group consisting of viruses and exosomes.

[0007] According to the present invention, it is possible to provide a method for analyzing trace substances, which can easily separate the trace substances to be analyzed and can analyze the trace substances to be analyzed with high sensitivity.

[0008] 1A is a schematic diagram showing a separation member of this embodiment having a cover on one side, and FIG. 1B is a schematic diagram showing how the side of the separation member of this embodiment on which the cover of this embodiment was applied is placed on the plate of this embodiment. FIG. 1B is a schematic diagram showing a porous material including a plate-shaped porous member. FIG. 1C is a schematic diagram showing how the method for analyzing a trace substance of this embodiment is performed inside a hole. FIG. 1D is a schematic diagram showing step B when the trace substance to be analyzed is an antigen. FIG. 1F is a schematic diagram showing step B when the trace substance to be analyzed is a nucleic acid. FIG. 1G is a schematic diagram showing step C. FIG. 1H is a schematic diagram showing step D. FIG. 1I is a photograph of a separation member taken in Example 2.

[0009] Hereinafter, embodiments of the present invention will be described.

[0010] In the present embodiment, when a numerical range is described in stages, the upper and lower limits of each numerical range can be combined in any way.

[0011] In this embodiment, NAD(P) means NAD and / or NADP, and NAD(P)H means NADH and / or NADPH.

[0012] In this embodiment, thioNAD(P) means thioNAD and / or thioNADP, and thioNAD(P)H means thioNADH and / or thioNADPH.

[0013] The method for analyzing trace substances of this embodiment includes the following steps: Step A: separating the trace substances using a separation member containing a porous material; Step B: binding an enzyme-labeled antibody composed of an antibody specific to the trace substance of this embodiment and enzyme α, or an enzyme-labeled nucleic acid probe composed of a nucleic acid probe specific to the trace substance of this embodiment and enzyme α, to the separated trace substance of this embodiment; Step C: allowing enzyme α of the enzyme-labeled antibody of this embodiment or enzyme α of the enzyme-labeled nucleic acid probe of this embodiment to act on compound β, which is a substrate of enzyme α, to produce compound γ, which is a substrate for dehydrogenase; Step D: producing thioNAD(P)H by an enzyme cycling reaction using the produced compound γ, dehydrogenase, NAD(P)H, and thioNAD(P); and Step E: detecting the trace substance of this embodiment using the produced thioNAD(P)H.

[0014] <When the porous material includes a porous membrane> Hereinafter, a case where the porous material of this embodiment includes a porous membrane will be described.

[0015] The porous material of this embodiment preferably includes a porous membrane, from the viewpoint of being able to separate trace substances to be analyzed more easily.

[0016] From the viewpoint of enabling even easier separation of trace substances to be analyzed, the porous membrane of this embodiment preferably contains one or more resins selected from the group consisting of polyester-based resins, polyolefin-based resins, fluorine-based resins, and polystyrene-based resins, more preferably contains a polyolefin-based resin, and even more preferably contains a polyethylene resin.

[0017] The average pore size of the porous membrane of the present embodiment, measured by observation with a scanning electron microscope, is preferably 1 nm or more and 5,000 nm or less, more preferably 5 nm or more and 2,000 nm or less, even more preferably 10 nm or more and 1,000 nm or less, even more preferably 50 nm or more and 500 nm or less, even more preferably 80 nm or more and 300 nm or less, and even more preferably 100 nm or more and 200 nm or less, from the viewpoint of enabling even easier separation of the trace substances to be analyzed.

[0018] By making the average pore size of the porous membrane of this embodiment close to the size of the trace substances to be separated, it is possible to separate the trace substances based on their size.

[0019] An example of a commercially available porous membrane of this embodiment is Miraim (registered trademark) manufactured by Teijin Limited.

[0020] From the viewpoint of enabling the trace substance to be analyzed to be separated more easily, the method for analyzing trace substances of this embodiment preferably includes a step F between step A of this embodiment and step B of this embodiment, in which the separated trace substance of this embodiment is recovered from the separation member of this embodiment onto a plate.

[0021] The plate of this embodiment is not particularly limited, and may be, for example, a commercially available microplate or petri dish.

[0022] In step F of this embodiment, from the viewpoint of improving the efficiency of recovering the trace substances to be analyzed that have been recovered on the separation member of this embodiment into the plate, it is preferable to recover the trace substances of this embodiment from the separation member of this embodiment into the plate of this embodiment by applying negative pressure to the separation member of this embodiment or by pouring a liquid onto the separation member of this embodiment.

[0023] FIG. 1A is a schematic diagram showing a separating member 11 of this embodiment having a cover 13 on one surface 11a, and FIG. 1B is a schematic diagram showing the state in which the surface 11a of the separating member 11 of this embodiment, to which the cover 13 of this embodiment has been attached, is superimposed on the plate 15 of this embodiment.

[0024] In step A of this embodiment, from the viewpoint of preventing impurities other than the trace substances to be analyzed from being mixed into the plate, the separation member 11 of this embodiment has a cover 13 on one surface 11a, and in step F of this embodiment, it is preferable to remove the cover 13 of this embodiment from the separation member 11 of this embodiment, place the surface 11a of the separation member 11 of this embodiment to which the cover 13 of this embodiment was attached on the plate 15 of this embodiment, and then recover the trace substances of this embodiment from the separation member 11 of this embodiment to the plate 15 of this embodiment.

[0025] The cover 13 of this embodiment is not particularly limited, and may be a commercially available plastic film or the like.

[0026] The location where steps B to E of this embodiment are performed is not particularly limited. For example, the solution containing the trace substance separated in step A (hereinafter referred to as the sample solution) may be collected on a plate such as a petri dish, and steps B to E of this embodiment may be performed at a location other than the plate. For example, an ELISA method is an example of a method for performing steps B to E of this embodiment at a location other than the plate. In the ELISA method, the sample solution collected on the plate is transferred to a reaction vessel, and then the sample solution and an enzyme-labeled antibody are reacted in the reaction vessel, and steps B to E are performed in the reaction vessel. In addition, examples of methods for performing steps B to E of this embodiment at a location other than the plate include Western blotting, Northern blotting, and Southern blotting. In these methods, the sample solution collected on the plate is separated by electrophoresis and then transferred to a membrane containing an enzyme-labeled antibody, and steps B to E are performed on the membrane.

[0027] Steps B to E of this embodiment may be performed directly on the plate from which the sample solution was collected, thereby making the analysis even easier.

[0028] From the viewpoint of making it easier to retain the trace substance of this embodiment on the plate of this embodiment, the plate of this embodiment preferably contains a cationic polymer, more preferably contains polylysine, and even more preferably contains poly-D polylysine on at least a portion of the surface of the plate of this embodiment.

[0029] In step A of this embodiment, from the viewpoint of enabling even easier separation of the trace substance to be analyzed, it is preferable to attach the trace substance to the porous material of this embodiment by placing the separating member of this embodiment in the space where the trace substance of this embodiment is present. For example, if the trace substance to be analyzed is avian influenza virus, the separating member of this embodiment can be placed in a poultry house, which is the space where the virus is present, to attach the avian influenza virus to the porous material of this embodiment.

[0030] In step A of this embodiment, the period for which the separation member of this embodiment is placed in the space where the trace substance of this embodiment is present is preferably 1 hour or more, more preferably 2 hours or more, even more preferably 4 hours or more, and even more preferably 8 hours or more, from the viewpoint of being able to sufficiently adhere the trace substance of this embodiment to the porous material of this embodiment, and from the viewpoint of being able to more easily separate the trace substance to be analyzed, it is preferably 240 hours or less, even more preferably 180 hours or less, even more preferably 120 hours or less, and even more preferably 72 hours or less.From the viewpoint of being able to sufficiently adhere the trace substance of this embodiment to the porous material of this embodiment and from the viewpoint of being able to more easily separate the trace substance to be analyzed, it is preferably 1 hour or more and 240 hours or less, more preferably 2 hours or more and 180 hours or less, even more preferably 4 hours or more and 120 hours or less, and even more preferably 8 hours or more and 72 hours or less.

[0031] <When the porous material includes a plate-shaped porous member> Hereinafter, a case where the porous material of this embodiment includes a plate-shaped porous member will be described.

[0032] In the method for analyzing trace substances of this embodiment, from the viewpoint of being able to separate the trace substances to be analyzed more easily, it is preferable that the porous material of this embodiment includes a plate-shaped porous member, and that the plate-shaped porous member of this embodiment includes holes that penetrate from the top surface to the bottom surface of the plate-shaped porous member of this embodiment.

[0033] 2 is a schematic diagram showing a porous material including a plate-shaped porous member 21. The plate-shaped porous member 21 includes holes 23 that penetrate from the top surface to the bottom surface of the plate-shaped porous member 21.

[0034] The plate-shaped porous member of this embodiment preferably contains glass, from the viewpoint of being able to separate trace substances to be analyzed more easily.

[0035] The average pore size of the pores in the plate-shaped porous member of this embodiment, measured by observation with a scanning electron microscope, is preferably 1 nm or more and 5,000 nm or less, more preferably 5 nm or more and 2,000 nm or less, even more preferably 10 nm or more and 1,000 nm or less, even more preferably 50 nm or more and 500 nm or less, even more preferably 80 nm or more and 300 nm or less, and even more preferably 100 nm or more and 200 nm or less, from the viewpoint of enabling even easier separation of the trace substances to be analyzed.

[0036] By making the average pore size of the plate-shaped porous member of this embodiment close to the size of the trace substances to be separated, it is possible to separate the trace substances based on their size.

[0037] The average depth of the pores of the plate-shaped porous member of this embodiment, as measured by observation with a scanning electron microscope, is preferably 0.5 μm or more and 50 mm or less, more preferably 1 μm or more and 10 mm or less, even more preferably 5 μm or more and 5 mm or less, even more preferably 10 μm or more and 1 mm or less, and even more preferably 20 μm or more and 500 μm or less, from the viewpoint of enabling even easier separation of the trace substances to be analyzed.

[0038] Although the location where steps B to E of this embodiment are performed is not particularly limited, it is preferable that steps B to E of this embodiment be performed inside the hole of this embodiment, which makes it possible to perform the analysis more easily.

[0039] 3 is a schematic diagram showing how the method for analyzing trace substances according to this embodiment is performed inside a hole 23. In Fig. 3, a plate-shaped porous member 21 includes a hole 23 penetrating from the top surface to the bottom surface, and a plate-shaped member 25 is disposed on the bottom surface of the plate-shaped porous member 21. A trace substance 27 to be analyzed is held inside the hole 23.

[0040] In addition, if the average pore size of the plate-shaped porous member of this embodiment is made close to the size of the trace substance to be separated and steps B to E are performed inside the pores, it is possible to retain one to several trace substances inside each pore. This makes it possible to roughly estimate the number of trace substances from the number of pores in which the trace substances are retained, and to quantitatively analyze the trace substances.

[0041] From the viewpoint of making it easier to retain the trace substance of this embodiment inside the pores 23 of this embodiment, the porous material of this embodiment preferably contains a cationic polymer on at least a portion of the bottom surface of the pores 23 of this embodiment, more preferably contains polylysine, and even more preferably contains poly-D polylysine.

[0042] In step A of this embodiment, from the viewpoint of being able to separate the trace substances to be analyzed more easily, it is preferable to immerse the separation member of this embodiment in a liquid containing the trace substances of this embodiment, or to pour a liquid containing the trace substances of this embodiment onto the separation member of this embodiment.

[0043] <Trace Substance> The trace substance of this embodiment will be described below.

[0044] The trace substance of this embodiment includes, for example, one or more species selected from the group consisting of viruses and exosomes.

[0045] The virus of this embodiment includes, for example, one or more viruses selected from the group consisting of influenza virus, coronavirus, adenovirus, and RS virus.

[0046] The exosomes of this embodiment include, for example, one or more exosomes selected from the group consisting of cancer cell-derived exosomes and neuron-derived exosomes, and are contained in body fluids such as blood, urine, tears, and saliva.

[0047] <Steps B to E> Steps B to E of this embodiment will be described below.

[0048] First, step B will be described separately for the case where the trace substance to be analyzed is an antigen and the case where the trace substance to be analyzed is a nucleic acid. When the trace substance to be analyzed is an antigen, an enzyme-labeled antibody consisting of an antibody that specifically binds to the antigen to be analyzed and enzyme α is prepared, and the antibody of the enzyme-labeled antibody is allowed to bind to the antigen to be analyzed, as shown in Figure 4. When the trace substance to be analyzed is a nucleic acid, an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe that specifically binds to the nucleic acid to be analyzed and enzyme α is prepared, and the nucleic acid probe of the enzyme-labeled nucleic acid probe is allowed to bind to the nucleic acid to be analyzed, as shown in Figure 5.

[0049] In step C, as shown in FIG. 6, enzyme α of the enzyme-labeled antibody or enzyme α of the enzyme-labeled nucleic acid probe is allowed to act on compound β, which is a substrate of enzyme α, to produce compound γ, which is a substrate of dehydrogenase.

[0050] In step D, as shown in FIG. 7 , thioNAD(P)H is produced by an enzymatic cycling reaction using the produced compound γ, dehydrogenase, NAD(P)H, and thioNAD(P). Specifically, when reduced compound γ serves as the starting point of the enzymatic cycling reaction, reduced compound γ is first oxidized to produce oxidized compound γ. At this time, thioNAD(P)H is produced from thioNAD(P). Then, oxidized compound γ is reduced by dehydrogenase to produce reduced compound γ. At this time, NAD(P) is produced from NAD(P)H. This completes one cycle of enzymatic cycling. As long as thioNAD(P) and NAD(P)H are supplied, this cycle is repeated, and thioNAD(P)H is amplified.

[0051] In step D, the thioNAD(P) may include polymerized thioNAD(P).

[0052] Since polymerized thioNAD(P) is bulky, when thioNAD(P) contains polymerized thioNAD(P), step D of the present embodiment is preferably performed in a space of a certain size or larger. Specifically, it is preferably performed in a Petri dish or a microplate.

[0053] In this embodiment, polymerized thioNAD(P) means a polymer containing a unit derived from thioNAD(P) represented by the following general formula (1) (hereinafter referred to as a thioNAD(P) unit), and polymerized thioNAD(P)H means a polymer containing a unit derived from thioNAD(P)H represented by the following general formula (2) (hereinafter referred to as a thioNAD(P)H unit).

[0054]

[0055] In general formulas (1) and (2), R １ is a hydroxy group or a phosphate group.

[0056] The structure of the polymerized thioNAD(P) is not particularly limited, but the polymerized thioNAD(P) is a N-type thioNAD represented by the following general formula (3): ６ It is preferable that the NAD-containing compound contains a structure derived from -carboxymethyl-thioNAD, and the NAD-containing compound is represented by the following general formula (4): ６ It is more preferable that the compound contains a structure derived from -[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propioamide].

[0057]

[0058] In general formulas (3) and (4), R １ is a hydroxy group or a phosphate group.

[0059] The structure of the polymerized thioNAD(P) is not particularly limited, but it is preferable that the polymerized thioNAD(P) contains a structure derived from acrylamide.

[0060] The method for preparing polymerized thioNAD(P) is not particularly limited, and it can be prepared with reference to methods such as those described in: Lindberg M, Larsson PO, Mosbach K. A new immobilized NAD+ analogue, its application in affinity chromatography and as a functioning coenzyme. Eur J Biochem. 1973 Dec 3;40(1):187-93; and Muramatsu M, Urabe I, Yamada Y, Okada H. Synthesis and kinetic properties of a new NAD+ derivative carrying a vinyl group. Eur J Biochem. 1977 Oct 17;80(1):111-7.

[0061] In step E, the trace substance to be analyzed is detected by the thio-NAD(P)H amplified in step D. Because thio-NAD(P)H is amplified in step D, highly sensitive analysis can be performed even if the amount of the analyte is very small.

[0062] When an enzyme-labeled antibody is used, as shown in FIG. 4, the antibody of the enzyme-labeled antibody is bound to an antigen, which is a trace substance to be analyzed.

[0063] The antigen of this embodiment is not particularly limited in type, and may be any antigen capable of binding to an enzyme-labeled antibody, such as a protein, sugar, lipid, peptide, or low-molecular-weight compound that induces an immune response in a living body, preferably a protein. Here, the term "protein and sugar" includes proteins and sugars bound to lipids and / or nucleic acids.

[0064] When an enzyme-labeled nucleic acid probe is used, as shown in FIG. 5, the nucleic acid probe of the enzyme-labeled nucleic acid probe is bound to a nucleic acid, which is a trace substance to be analyzed.

[0065] In step E of this embodiment, the produced thioNAD(P)H is detected. The detection method is not particularly limited, and for example, the produced thioNAD(P)H may be quantitatively analyzed.

[0066] The method for quantitatively analyzing thio-NAD(P)H in this embodiment is not particularly limited, and can be quantitatively analyzed by absorbance, for example. Note that because thio-NAD(P)H exhibits absorption in the visible range (maximum absorption wavelength: 400 nm, molar extinction coefficient = 11,900), it can be measured using, for example, a commonly used absorptiometer or a colorimetric microplate reader.

[0067] The produced thioNAD(P)H is detected in step E. The detection method is not particularly limited, and for example, the produced thioNAD(P)H may be subjected to qualitative analysis.

[0068] The method for qualitative analysis of thioNAD(P)H is not particularly limited, and for example, the produced thioNAD(P)H can be qualitatively analyzed visually. As described above, thioNAD(P)H exhibits absorption in the visible range, and therefore visual qualitative analysis is possible.

[0069] The enzyme α of the enzyme-labeled antibody or enzyme-labeled nucleic acid probe is not particularly limited, but preferably includes one or more enzymes selected from the group consisting of transferases represented by EC numbers 2.-, hydrolases represented by EC numbers 3.-, elimination-addition enzymes represented by EC numbers 4.-, and isomerases represented by EC numbers 5.-, and more preferably includes alkaline phosphatase (ALP).

[0070] The compound β is not particularly limited as long as it can be a substrate for the enzyme α. However, from the viewpoint of combination with a dehydrogenase described later, the compound β preferably contains a compound having an androsterone skeleton, and more preferably contains androsterone 3-phosphate (A3P).

[0071] The dehydrogenase used in the enzymatic cycling reaction of step C is not particularly limited and may include, for example, one or more dehydrogenases selected from group X consisting of malate dehydrogenase, lactate dehydrogenase, glucose dehydrogenase, galactose dehydrogenase, 3α-hydroxysteroid dehydrogenase (abbreviated as 3α-HSD), galactose dehydrogenase, glucose dehydrogenase, testosterone-17β-dehydrogenase, and glycerol-3-phosphate dehydrogenase. From the viewpoint of improving the reaction rate, it is preferable to include a hydroxysteroid dehydrogenase, and it is more preferable to include a 3α-HSD.

[0072] The combination of dehydrogenase and compound β is not particularly limited, but from the perspective of improving the reaction rate, a combination of 3α-HSD and its substrate, a compound having an androsterone skeleton, is preferred. To explain the improvement in reaction rate using a specific example, while the reaction rate of many dehydrogenases when thioNAD is used as a coenzyme is within a few percent of that when NAD is used, in the dehydrogenation reaction of androsterone by 3α-HSD, the reaction rate with thioNAD is approximately 59% of the reaction rate with NAD, making 3α-HSD a preferable enzyme for the cycling reaction according to this embodiment. Specific examples of compounds having an androsterone skeleton include androsterone 3-phosphate (A3P), androsterone, 11β-hydroxyandrosterone, 11-oxoandrosterone, 11α-hydroxyandrosterone, etiocholanolone, tetrahydrocortisol, pregnanediol, lithocholic acid, deoxycholate, chenodeoxycholic acid, and cholic acid. Among the combinations of 3α-HSD and compounds having an androsterone skeleton, the combination of A3P and 3α-HSD is preferred from the viewpoint that it can be combined with ALP, which is a general-purpose enzyme used in enzyme-labeled antibodies or enzyme-labeled nucleic acid probes.

[0073] Compound γ is not particularly limited as long as it can be a substrate for an enzymatic cycling reaction using dehydrogenase, and may be either a reduced substrate or an oxidized substrate, but preferably includes a compound having an androsterone skeleton.

[0074] In the method for analyzing trace substances of this embodiment, the properties of the enzyme α, compound β, and dehydrogenase are not particularly limited, but preferably have the following properties: The turnover number of the labeling enzyme is large. Commercially available and widely used labeling enzymes can be used. The turnover number of the enzyme cycling is large. The dehydrogenase used in the enzyme cycling reaction is free from contamination of the labeling enzyme and has no activity similar to that of the labeling enzyme. The substrate of the labeling enzyme is a commercially available compound or can be easily synthesized and is highly stable.

[0075] In the method for analyzing trace substances of this embodiment, the concentration of the enzyme-labeled antibody or enzyme-labeled nucleic acid probe in the reaction system is not particularly limited, but is, for example, 1 nM or more, preferably 10 nM or more, more preferably 20 nM or more, and is, for example, 100 nM or less, preferably 50 nM or less, more preferably 40 nM or less.

[0076] In the method for analyzing a trace substance of this embodiment, the concentration of compound β in the reaction system is not particularly limited, but is, for example, 0.001 mM or more, preferably 0.01 mM or more, more preferably 0.05 mM or more, and even more preferably 0.1 mM or more, and is, for example, 500 mM or less, preferably 100 mM or less, more preferably 50 mM or less, even more preferably 10 mM or less, even more preferably 5 mM or less, even more preferably 1 mM or less, and even more preferably 0.5 mM or less.

[0077] In the method for analyzing trace substances of this embodiment, the concentration of NAD(P)H in the reaction system is not particularly limited, but is, for example, 0.001 mM or more, preferably 0.01 mM or more, more preferably 0.05 mM or more, even more preferably 0.1 mM or more, even more preferably 0.5 mM or more, even more preferably 1.0 mM or more, and is, for example, 500 mM or less, preferably 100 mM or less, more preferably 50 mM or less, even more preferably 10 mM or less, even more preferably 5 mM or less.

[0078] In the method for analyzing trace substances of this embodiment, the concentration of thioNAD(P)H in the reaction system is not particularly limited, but is, for example, 0.001 mM or more, preferably 0.01 mM or more, more preferably 0.05 mM or more, even more preferably 0.1 mM or more, even more preferably 0.5 mM or more, and even more preferably 1.0 mM or more, and is, for example, 500 mM or less, preferably 100 mM or less, more preferably 50 mM or less, even more preferably 10 mM or less, and even more preferably 5 mM or less.

[0079] In the method for analyzing trace substances of this embodiment, the concentration of dehydrogenase in the reaction system is not particularly limited, but is, for example, 0.01 U / mL or more and 5000 U / mL or less, and from the viewpoint of improving the S / N ratio (signal / noise ratio), it is preferably 0.1 U / mL or more and 500 U / mL or less, more preferably 1 U / mL or more and 100 U / mL or less, and even more preferably 20 U / mL or more and 40 U / mL or less.

[0080] In the method for analyzing trace substances of this embodiment, the reaction temperature is not particularly limited, but is, for example, 0°C or higher, preferably 10°C or higher, more preferably 20°C or higher, and is, for example, 100°C or lower, preferably 80°C or lower, more preferably 60°C or lower, and even more preferably 40°C or lower.

[0081] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention.

[0082] The usefulness of the present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples.

[0083] Example 1 First, one side of a separation element (manufactured by Teijin Limited, product name: Miraim (registered trademark), material: polyethylene, average pore size: 150 nm, length 210 mm x width 297 mm x thickness 50 μm) was covered with a polyvinylidene chloride wrap film, and the element was placed in a space (width 50 cm x depth 40 cm x height 60 cm, temperature: 25°C) in which seasonal influenza virus (type A) was suspended. The element was placed for 24 hours. The seasonal influenza virus (type A) used had been inactivated by ultraviolet irradiation. Next, the wrap film was removed from the separation element, and the surface that had been covered with the wrap film was then placed on a 96-well plate (manufactured by Thermo Scientific, product name: ImmunoModule, the bottom of each well was coated with 5 μg / cm ２ The wells were then placed on a 96-well plate (coated with 100 mM poly-D-lysine), and then 100 mM Tris-HCl buffer was applied to the separation member, and negative pressure was applied from the side of the 96-well plate. The wells were then washed with 100 mM Tris-HCl buffer. A solution of seasonal influenza virus (type A) antibody labeled with alkaline phosphatase (composition: 100 mM Tris-HCl buffer containing 0.1% BSA and 0.02% Tween 20) was then poured into the wells and incubated at 25°C for 60 minutes. The solution in the wells was then removed by suction, and the wells were then washed with 100 mM Tris-HCl buffer containing 0.05% Tween 20. Next, 100 μL of Solution A below was added to the wells, and while heating at 37°C, the absorbance was measured 60 minutes after adding Solution A using a microplate reader (manufactured by Corona Electric Co., Ltd., model name: SH-1000, equipped with a 405 nm filter). The absorbance value was 0.8 Abs.

[0084] Here, solution A is a solution of pH 9.0 containing the following components: Tris-HCl (100 mM), thio-NAD (2 mM), NADH (1.0 mM), androsterone 3-phosphate (A3P) (0.1 mM), and 3α-hydroxysteroid dehydrogenase (3α-HSD) (30 U / mL).

[0085] If separation using a separation member is not performed, many impurities will get in, making it difficult to detect the trace substance to be analyzed. Therefore, according to the method for analyzing a trace substance of this embodiment, the trace substance to be analyzed can be easily separated and the trace substance to be analyzed can be analyzed with high sensitivity.

[0086] Example 2 First, a separation member (manufactured by Akagawa Hard Glass Industry Co., Ltd., material: glass, average pore diameter: 150 nm, average pore depth: 1 μm) including holes penetrating from the top surface to the bottom surface was prepared, and 5 μg / cm ２ A glass plate coated with poly-D-lysine (PDL-Lysine) was placed and fixed. The separation element was then immersed in a seasonal influenza virus (type A) solution (composition: 100 mM Tris-HCl buffer containing 0.1% BSA) and rocked at 25°C for 60 minutes. The seasonal influenza virus (type A) used was inactivated by ultraviolet irradiation. The separation element was then immersed in a Tris-HCl buffer (100 mM) and rocked at 25°C for 60 minutes to wash the inside of the holes. The separation element was then immersed in a solution of seasonal influenza virus (type A) antibody labeled with alkaline phosphatase (composition: 100 mM Tris-HCl buffer containing 0.1% BSA and 0.02% Tween 20) and rocked at 25°C for 60 minutes to incubate the inside of the holes. Next, the separation member was immersed in 100 mM Tris-HCl buffer containing 0.05% Tween 20 and rocked at 25°C for 60 minutes to wash the inside of the holes. Next, the separation member was immersed in the above solution A and rocked at 37°C for 1 minute, and then, while heating at 37°C, the separation member was photographed using a microplate reader (manufactured by Corona Electric Co., Ltd., model name: SH-1000, equipped with a 405 nm filter). The photograph taken is shown in Figure 8.

[0087] Figure 8 shows that there are pores that emit fluorescence (pores that appear white in Figure 8) and pores that do not. It is believed that seasonal influenza virus (type A) is held inside the fluorescent pores, and an enzymatic cycling reaction is occurring. Therefore, it is possible to roughly estimate the amount of virus in the virus solution from the number of fluorescent pores.

[0088] This application claims priority based on Japanese Patent Application No. 2023-218264, filed December 25, 2023, the disclosure of which is incorporated herein in its entirety by reference.

[0089] REFERENCE SIGNS LIST 11 Separation member 11a One surface of separation member 13 Cover 15 Plate 21 Plate-shaped porous member 23 Hole 25 Plate-shaped member 27 Trace substance

Claims

1. Step A of separating trace substances using a separation member containing a porous material; Step B of binding to the separated trace substances an enzyme-labeled antibody composed of an antibody specific to the trace substances and enzyme α, or an enzyme-labeled nucleic acid probe composed of a nucleic acid probe specific to the trace substances and enzyme α; Step C of allowing the enzyme α of the enzyme-labeled antibody or the enzyme α of the enzyme-labeled nucleic acid probe to act on compound β which is a substrate of the enzyme α to generate compound γ which is a substrate of dehydrogenase; Step D of generating thio-NAD(P)H by an enzyme cycling reaction using the generated compound γ, dehydrogenase, NAD(P)H, and thio-NAD(P); and Step E of detecting the trace substances using the generated thio-NAD(P)H. A method for analyzing trace substances comprising these steps.

2. The method for analyzing trace substances according to claim 1, wherein the porous material includes a porous membrane.

3. The method for analyzing trace substances according to claim 2, wherein the porous membrane includes one or more selected from the group consisting of polyester resins, polyolefin resins, fluorine resins, and polystyrene resins.

4. The method for analyzing trace substances according to claim 2 or 3, wherein the average pore diameter of the porous membrane measured by observation with a scanning electron microscope is 1 nm or more and 5000 nm or less.

5. The method for analyzing trace substances according to any one of claims 1 to 4, further including Step F of recovering the separated trace substances from the separation member onto a plate between Step A and Step B.

6. In Step A, the separation member has a cover on one surface. In Step F, the cover is removed from the separation member, the surface of the separation member where the cover was provided is overlaid on the plate, and then the trace substances are recovered from the separation member onto the plate. The method for analyzing trace substances according to claim 5.

7. The method for analyzing trace substances according to claim 5 or 6, wherein at least a part of the surface of the plate contains a cationic polymer.

8. In Step A, the separation member is placed in the space where the trace substances are present to cause the trace substances to adhere to the porous material. The method for analyzing trace substances according to any one of claims 2 to 7.

9. The method for analyzing trace substances according to claim 1, wherein the porous material includes a plate-shaped porous member, and the plate-shaped porous member includes pores penetrating from the top surface to the bottom surface of the plate-shaped porous member.

10. The method for analyzing trace substances according to claim 9, wherein the plate-shaped porous member includes glass.

11. The method for analyzing trace substances according to claim 9 or 10, wherein the average pore diameter of the pores of the plate-shaped porous member measured by observation with a scanning electron microscope is 1 nm or more and 5000 nm or less.

12. The method for analyzing trace substances according to any one of claims 9 to 11, wherein the average depth of the pores of the plate-shaped porous member measured by observation with a scanning electron microscope is 0.5 µm or more and 50 mm or less.

13. The method for analyzing trace substances according to any one of claims 9 to 12, wherein the plate-shaped porous member includes a cationic polymer in at least a part of the bottom surface of the pores.

14. The method for analyzing trace substances according to any one of claims 9 to 13, wherein steps B to E are performed inside the pores.

15. The method for analyzing trace substances according to any one of claims 1 to 14, wherein the trace substances include one or more selected from the group consisting of viruses and exosomes.

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