Enzyme-linked aptamer, polyamine detection reagent, polyamine detection device, and polyamine detection method

Abstract

To suppress inhibition of peroxidase activity and to enable selective, highly sensitive, and simple detection of polyamines without additionally adding hydrogen peroxide.SOLUTION: A polyamine detection reagent according to the present invention contains an enzyme-linked aptamer. The enzyme-linked aptamer comprises an enzyme that generates hydrogen peroxide and an aptamer having a sequence that forms a G-quadruplex.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an enzyme-linked aptamer, a polyamine detection reagent, a polyamine detection device, and a polyamine detection method. [Background technology]

[0002] The importance of early diagnosis for various diseases, including infectious diseases and cancer, has been recognized. However, disease diagnosis generally relies on highly invasive testing of subjects' cells and blood in hospitals. In cases far from hospitals, simple tests based on non-invasive specimens such as saliva, urine, and tears are difficult to perform except in certain cases. Detection targets of non-invasive simple testing methods that can help address this issue include antibodies, antigens, viruses, bacteria, and metabolic molecules. For example, the types and amounts of polyamines present in a sample vary depending on the subject's physical condition and the presence of disease. In particular, polyamine concentrations can be elevated in actively proliferating cells, such as cancer cells. Therefore, polyamines can be used as markers in cancer diagnosis. For example, a method for examining the risk of specific cancers based on polyamines contained in saliva has been proposed (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2019-035768 [Patent Document 2] Japanese Patent Publication No. 2022-061489 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the method of Patent Document 1 requires the use of an expensive and highly sensitive mass spectrometer (LC / MS) for analysis, which results in high testing costs. Furthermore, high levels of expertise and experience are required for measurement and analysis of results. Therefore, there is a need for the development of a testing method that can quickly and easily examine trace amounts of marker components such as polyamines.

[0005] In addition, it is desirable to be able to analyze not only the total amount of polyamines but also the presence or absence of specific polyamines, and to be able to selectively analyze the presence or absence and amount of specific polyamines. For example, Patent Document 2 describes the detection of polyamines contained in a liquid by utilizing peroxidase activity in a state where the polyamines are in contact with a specific aptamer.

[0006] However, according to the inventors' investigations, in the method described in Patent Document 2, depending on the color reagent of the test drug used, the test drug may be decolorized by impurities, such as proteins or reducing molecules, contained in the actual specimen, such as saliva or serum, and the detection using peroxidase activity as an indicator may be inhibited. Furthermore, since detection using peroxidase activity requires the addition of hydrogen peroxide to the detection system, there is room for improvement in terms of simplicity.

[0007] The present invention provides a method that suppresses inhibition and enables detection using peroxidase activity as an indicator without adding hydrogen peroxide, thereby providing a method that allows selective, highly sensitive, and simple detection of polyamines. [Means for solving the problem]

[0008] According to the inventors' investigations, the main causes of inhibition by impurities are the consumption of the substrate hydrogen peroxide due to the reducing properties of the impurities and the loss of color due to the reduction of oxidized color dyes. Therefore, the inventors came up with the idea of linking an enzyme involved in the reaction that generates hydrogen peroxide to the aptamer, thereby spontaneously generating the hydrogen peroxide necessary for polyamine detection near the aptamer (approximately 5 nm) without inhibiting contact between the polyamine and the aptamer. By suppressing inhibition by impurities, it is expected that the detection sensitivity of polyamines can be improved.

[0009] The present invention has the following aspects. [1] A polyamine detection reagent, the polyamine detection reagent contains an enzyme-linked aptamer; The enzyme-linked aptamer is a polyamine detection reagent having an enzyme that generates hydrogen peroxide and an aptamer having a sequence that forms a G-quadruplex. [2] The polyamine detection reagent according to [1], wherein the enzyme comprises glucose oxidase. [3] The polyamine detection reagent according to [1] or [2], further containing potassium ions. [4] The polyamine detection reagent according to any one of [1] to [3], further comprising a porphyrin compound. [5] A polyamine detection reagent according to [4], which exhibits peroxidase activity expressed by a G-quartet structure formed by complexing the enzyme-linked aptamer with the porphyrin compound. [6] A polyamine detection reagent according to [4] or [5], which detects the difference in peroxidase activity expressed by a G-quartet structure formed by the complexation of the enzyme-linked aptamer and the porphyrin compound, depending on the presence or absence of polyamines. [7] A polyamine detection device comprising the polyamine detection reagent according to [5] or [6]. [8] A method for detecting polyamines, comprising contacting the polyamine detection reagent according to [5] or [6] with polyamines. [9] Contacting the polyamine detection reagent according to [5] or [6] with a polyamine; measuring peroxidase activity expressed by a G-quartet structure formed by complexing the enzyme-linked aptamer with the porphyrin compound; A method for detecting polyamines, comprising:

[10] An enzyme-linked aptamer in which an enzyme and an aptamer are linked, wherein the enzyme includes an enzyme involved in a reaction that produces hydrogen peroxide, and the aptamer has a sequence that forms a G-quadruplex.

[11] The enzyme-linked aptamer described in

[10] , wherein the enzyme that generates hydrogen peroxide includes glucose oxidase. [Effects of the Invention]

[0010] According to the present invention, inhibition of peroxidase activity is suppressed, and polyamines can be detected selectively, sensitively, and simply without the need to add additional hydrogen peroxide. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram for explaining the stabilization of the G-quadruplex. [Figure 2] Figure 2 shows an example of various structures that a G-quadruplex can take. [Figure 3] FIG. 3 shows an example of various structures that a G-quadruplex can take. [Figure 4] FIG. 4 shows an example of various structures that a G-quadruplex can take. [Figure 5] FIG. 5 shows an example of various structures that a G-quadruplex can take. [Figure 6] FIG. 6 shows an example of various structures that the G-quadruplex can take. [Figure 7] FIG. 7 shows an example of various structures that a G-quadruplex can take. [Figure 8] FIG. 8 shows a schematic diagram of the Gox-linked aptamer obtained in Example 1. [Figure 9]FIG. 9 shows the results of SDS-PAGE analysis of Example 1. [Figure 10] FIG. 10 shows the calibration curve obtained in Example 2. [Figure 11] FIG. 11 shows the calibration curve obtained in Example 3. [Figure 12] FIG. 12 shows the change over time in absorbance (wavelength 414 nm) after addition of ABTS measured in Example 4. [Figure 13] FIG. 13 shows the calibration curve obtained in Example 6. [Figure 14] FIG. 14 shows the calibration curve obtained in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the following terms have the following meanings: The symbol "to" indicating a range of values means that the values before and after it are included as the lower and upper limits.

[0013] Several embodiments of the present invention will be described in detail below. The following description is an example (typical example) of an embodiment, and the present invention is not limited to these, and can be implemented with any modifications within the scope of the gist of the present invention.

[0014] [Enzyme-linked aptamer] The enzyme-linked aptamer contains an enzyme that generates hydrogen peroxide and an aptamer having a sequence that forms a G-quadruplex. The enzyme contains an enzyme involved in the reaction that generates hydrogen peroxide. This eliminates the need to add additional hydrogen peroxide to the detection system. The aptamer also has a sequence that forms a G-quadruplex (hereinafter also referred to as a "G-quartet structure"). Therefore, in the presence of polyamines and potassium ions, a G-quadruplex-hemin complex, which is important for the expression of peroxidase activity, is formed. The aptamer and enzyme of the embodiment will be described below in turn.

[0015] (Aptama) The aptamer is not particularly limited as long as it has a sequence that forms a G-quadruplex. In the present invention, the aptamer forms a G-quadruplex, which is necessary for the expression of peroxidase activity that depends on the presence or absence and concentration of polyamine. A structure that forms a G-quadruplex-hemin complex is more preferred.

[0016] Aptamers are nucleic acid molecules that specifically bind to target molecules. Aptamers are composed of nucleotide residues, such as ribonucleotide residues and deoxyribonucleotide residues.

[0017] The nucleic acid molecule may be, for example, RNA composed of ribonucleotide residues, DNA composed of deoxyribonucleotide residues, or a nucleic acid molecule containing both deoxyribonucleotides and ribonucleotides. The nucleic acid molecule may be a single-stranded nucleic acid or a double-stranded nucleic acid. Examples of single-stranded nucleic acids include single-stranded RNA and single-stranded DNA. Examples of double-stranded nucleic acids include double-stranded RNA, double-stranded DNA, and double-stranded nucleic acids of RNA and DNA. Among them, the nucleic acid molecule is preferably a single-stranded nucleic acid. In the nucleic acid molecule, each base may be a natural base (non-artificial nucleic acid) such as adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U), or may be an artificial base (non-natural base).

[0018] Examples of artificial bases include modified bases and altered bases. The artificial base is preferably a base having the same function as a natural base (A, C, G, T, or U). Examples of artificial bases having the same function as a natural base include an artificial base that can bind to cytosine (C) instead of guanine (G), an artificial base that can bind to guanine (G) instead of cytosine (C), an artificial base that can bind to thymine (T) or uracil (U) instead of adenine (A), an artificial base that can bind to adenine (A) instead of thymine (T), and an artificial base that can bind to adenine (A) instead of uracil (U). In the detection method and detection device of the present invention, the bases represented by A, G, C, T, and / or U include not only natural bases but also artificial bases having the same function as the respective natural bases.

[0019] Examples of modified bases include methylated bases, fluorinated bases, aminated bases, thiolated bases, etc. Specific examples of modified bases include 2'-fluorouracil, 2'-aminouracil, 2'-O-methyluracil, 2-thiouracil, etc. In the detection method and detection device of the present invention, from the viewpoints of synthesis and stability, DNA aptamers (A, T, C, G) are preferred, and G-quadruplexes having a G-quartet interface are more preferred. Furthermore, systems that do not have various modifications that may cause steric modulation are preferred.

[0020] In particular, to obtain high peroxidase activity, it is preferable to have an aptamer that can have a more stable interaction (coordination) with the prosthetic group described below, specifically, an aptamer having a G-quartet surface that forms a G-quadruplex, and a metal complex that has the potential to stably generate a peroxide adduct using that aptamer.

[0021] Regarding G-quadruplexes, G-rich sequences in telomeric regions have been intensively studied. This is because it has become clear that these sequences can form a quadruplex DNA structure called G4 and further form a complex called a G-quadruplex. Several patterns of G-quadruplexes are known, including a parallel type in which all four DNA strands point in the same direction from the 5' to the 3' end, and an antiparallel type in which two strands point in the same direction but the remaining two point in opposite directions.

[0022] The G-quadruplex has the following characteristics: four G bases form a structure called a G-quartet through Hoogsteen hydrogen bonds, and the structure is maintained by π-π stacking interactions between the G-quartet faces. Figure 1 illustrates how the aptamer interacts with polyamines to stabilize the G-quadruplex structure, forming a complex with hemin and exhibiting peroxidase activity. This complex catalyzes the oxidation of an absorbance-changing substance by hydrogen peroxide, resulting in a color change. The amount of polyamines in saliva can be optically detected by assessing this color change as absorbance. Furthermore, the amount of polyamines in saliva can be assessed by evaluating the charge change resulting from this redox reaction and its accompanying reactions.

[0023] The formation of a G-quadruplex requires the coordination of metal ions between the G-quartet planes, as explained below. K ions, Na ions, etc. are known to coordinate between the G-quartet planes.

[0024] It is known that various polyaromatic ring compounds coordinate to G-quadruplexes. In particular, from the viewpoint of utilizing the mechanism of peroxidase activity of heme proteins and heme enzymes, the planarity is favorable for the peroxidase activity, and the overall size of the polycyclic molecules allows efficient π-π stacking on the G-quartet surface of the G-quadruplex, and O2 is attached to the central metal. 2- A prosthetic group that generates a high-valent metal oxo species from the addition of peroxide is preferred. For example, the central metal is Fe. 2+ , Ni 2+ , Cu 2+ , Co 2+ , Mn 2+ , Zn 2+ are preferred, and those that have a D4h structure and coordinate at the axial position under multiple valences are preferred. Examples include salen-manganese complexes and porphyrin-iron complexes. Metalloporphyrins are preferred because of the planarity of their aromatic rings, the overall size of the polycyclic molecule, and cell permeability. It is also preferable to select a side chain that is as minimally bulky as possible. This is because, in order for the metal complex, which is the prosthetic group, to achieve sufficient peroxidase activity as a result of its complexation with the G-quadruplex, sufficient space above and below to coordinate the components of the G-quartet surface and also to coordinate the peroxide is required.

[0025] The base sequence of the aptamer is not particularly limited as long as it exhibits peroxidase activity when a complex is formed. By arranging A, C, or T between consecutive Gs, such as GGG or GG, in the aptamer sequence, an appropriate distance to the consecutive Gs can be achieved, improving the peroxidase activity described below. Furthermore, partial double-stranded portions (loops) may be generated, and depending on their spatial position, this may reduce the target peroxidase activity. Therefore, the number and order of consecutive Gs in the aptamer sequence, as well as the selection of sequences other than G, are important points for detection sensitivity.

[0026] Therefore, the molecular weight (number of bases) of the aptamer is preferably within an appropriate range. Specifically, 6 to 30 bases are preferred, and 11 to 25 bases are even more preferred, especially when optical detection is performed. The sensitivity of the peroxidase activity can be estimated to some extent from the sequence before conjugation. For example, sequences with four or more (GGG) units tend to be difficult to balance sensitivity, such as poor polyamine selectivity. Furthermore, sequences with one or two (GGG) units tend to be less sensitive. Even in cases with three (GGG) units, sequences such as 5'-GTGGG(NNN)GGG(NNN)GGG(NN)GGN-3' and 5'-GTGGG(NN)GGG(NNN)GGG(NN)GGN-3' tend to be less sensitive. In cases with three (GGG) units, 5'-GTGGG(NN)GGG(NN)GGG(NNN)GGN-3' tends to balance sensitivity.

[0027] Since it can be applied to both optical and electrical detection, examples include the following sequences: In this sequence, one of the (GGG) units may be (GGGG), and N between the (GGG) units is independently A, C, or T.

[0028] SEQ ID NO: 1: 5'-GTGGGTAGGGCGGGTTGGAAGTGUAGGCA-3' Sequence A1: 5'-GTGGGTAGGGCTGGGTTGGAAGTGUAGGCA-3' Sequence A2: 5'-GTGGGTGGGCGGGTTGGAAGTGUAGGCA-3' Sequence A3: 5'-GTGGGTAGGGCGGGTGGAAAGTGUAGGCA-3'

[0029] (enzyme) The enzyme is not particularly limited as long as it is an enzyme that is involved in a reaction that produces hydrogen peroxide. Glucose oxidase is preferred because glucose as a substrate is a stable and easy-to-use substance and is relatively stable both in a dissolved solution state and in a dried solid state. The method for linking the enzyme and the aptamer is not particularly limited, and an example of the linking method will be described below using glucose oxidase (hereinafter also referred to as "GOx") as the enzyme.

[0030] First, GOx and UdgX (uracil DNA glycosylase) are covalently linked. The SpyCatcher / Tag system can be used to link GOx and UdgX. Next, a GOx-UdgX complex can be prepared by mixing SpyCatcher-fused GOx and SpyTag-fused UdgX and allowing the mixture to stand. In the SpyCatcher / Tag system, any enzyme can be covalently linked to UdgX by replacing the enzyme fused to SpyCatcher. It is preferable to prepare an aptamer by synthesis, with uracil partially introduced into its sequence. A Gox-linked aptamer can be prepared by mixing an aptamer with uracil partially introduced into it with the GOx-UdgX complex and allowing the mixture to stand.

[0031] [Polyamine detection method] The polyamine detection method of the present invention (hereinafter also referred to as "the detection method of the present invention") comprises contacting the above-described enzyme-linked aptamer with polyamines. The detection method of the present invention detects polyamines in a state where the polyamines are in contact with the enzyme-linked aptamer. Polyamines can be detected by measuring the peroxidase activity expressed by the G-quartet structure formed by complexing the above-described enzyme-linked aptamer with a porphyrin compound.

[0032] More specifically, polyamines can be detected by utilizing the fact that the peroxidase activity expressed by the porphyrin compound, which is amplified by the appropriate G-quartet interface formed by the complexation of the above-mentioned enzyme-linked aptamer and the porphyrin compound, differs between when the peroxidase activity is suppressed in the absence of polyamines and when it is amplified in the presence of polyamines. More simply, the difference in peroxidase activity due to the presence or absence of polyamines is detected. This peroxidase activity can be measured in the presence of potassium ions. According to the detection method of the present invention, the peroxidase activity expressed by the G-quartet structure formed by complexing the above-mentioned enzyme-linked aptamer with a porphyrin compound is measured using a G-quadruplex folded in the presence of potassium ions within a specific concentration range, thereby measuring the difference in peroxidase activity due to the presence or absence of polyamines in the presence of potassium ions. Here, peroxidase activity is increased in the presence of polyamines compared to its absence.

[0033] The detection of polyamines is carried out by at least one of optical and electrical methods. In one example, the detection method includes a step of preparing a liquid containing polyamines (hereinafter also referred to as a "sample preparation step"), and a step of detecting polyamines in the liquid in contact with an enzyme-linked aptamer by at least one of optical and electrical methods (hereinafter also referred to as a "detection step").

[0034] (Polyamine) The detection method of the present invention detects polyamines contained in a liquid. Polyamines are compounds present in living organisms, and it is known that the type and amount present in a living organism change depending on the subject's physical condition, disease, and other factors. It is known that polyamine concentrations can be particularly high in actively proliferating cells such as cancer cells, and polyamines are also used as markers for cancer diagnosis. Examples of polyamines include spermine, spermidine, and putrescine. The detection method of the present invention is suitable for detecting these polyamines, and is particularly suitable for detecting spermine and spermidine.

[0035] The polyamine to be detected preferably contains at least one of spermine and spermidine. Furthermore, since the sensitivity of the detection method is increased, the polyamine to be detected is preferably a polyamine contained in body fluids such as saliva and blood or excrement, with polyamines contained in saliva being particularly preferred. Therefore, in the sample preparation step, it is preferable to prepare a liquid containing at least one of spermine and spermidine. Furthermore, in the sample preparation step, it is preferable to prepare a liquid containing body fluids such as saliva and blood or excrement, with preparing a liquid containing saliva being particularly preferred.

[0036] (Contact between polyamines and enzyme-linked aptamers) The method for contacting a polyamine with an enzyme-linked aptamer is not particularly limited, as long as the enzyme-linked aptamer and the polyamine come into contact when detecting the polyamine. Preferred examples include a method in which both the polyamine and the enzyme-linked aptamer are contained in the liquid to be tested, and a method in which the enzyme-linked aptamer is present on the surface of the detection unit of a device for detecting polyamine that comes into contact with the liquid to be tested. The amount of the enzyme-linked aptamer relative to polyamine is not particularly limited as long as the presence of the enzyme-linked aptamer improves the detection sensitivity of polyamine.

[0037] The method for adding both polyamine and enzyme-linked aptamer to the liquid to be tested is not particularly limited to the liquid containing polyamine and enzyme-linked aptamer, and the enzyme-linked aptamer may be added to the liquid containing polyamine, or the liquid containing polyamine and the liquid containing enzyme-linked aptamer may be mixed. When an enzyme-linked aptamer is present on the surface of the detection unit of the device for detecting polyamines that comes into contact with the liquid to be tested, the polyamine is brought into contact with the surface. In this case, there are no particular limitations as long as the polyamine comes into contact with the enzyme-linked aptamer when detecting the polyamine.

[0038] (Method for expressing peroxidase activity for highly sensitive polyamine detection by combining a specific range of potassium ion concentrations with an enzyme-linked aptamer of a specific base sequence) As mentioned above, the formation of a G-quadruplex structure requires the coordination of metal ions between the G-quartet planes. The spatial size of the G-quartet planes and between the G-quartets, as well as the ionic radius and charge of the metal ion, correlates with the binding constant between the G-quadruplex structure and the metal ion coordination.

[0039] It is believed that potassium ions have a greater influence on the system than sodium ions. This is because sodium ions have a slightly smaller ionic radius, while rubidium and cesium ions have larger ionic radii, making stable coordination difficult. Potassium ions, in particular, may have the ability to allow a certain degree of freedom in the G-quadruplex structure, as explained below.

[0040] The function of allowing a certain degree of freedom in the G-quadruplex structure is necessary for the following reason: As a result of extensive investigations by the present inventors, it has been found that G4-hemin complexes with high peroxidase activity tend to maintain this activity even in the absence of polyamines, and therefore there is a risk that the difference in the amount of coloration between the presence and absence of polyamines (hereinafter sometimes referred to as the difference in absorbance) may not be sufficient. If a complex and expensive detection system is not used, order estimation of polyamine concentration is possible even with conventional methods. However, according to the findings of the present inventors, for example, 3 Less than a few μmol / dm 3 In the concentration range below 1 μmol / dm 3 It can be difficult to detect the difference in concentration using conventional methods.

[0041] Therefore, the present inventors conceived the idea of constructing a reaction system that intentionally makes it difficult to form the G-quadruplex structure ideal for expressing peroxidase activity in the absence of polyamines, thereby forming a G-quadruplex-hemin complex that facilitates the expression of peroxidase activity with the assistance of the polyamine to be detected. The presence of potassium ions coordinated to the carboxyl group of guanine forms a G-quadruplex-hemin complex that is prone to peroxidase activity.

[0042] By combining a specific sequence with potassium ion concentration, the aforementioned "wide detection sensitivity margin" can be achieved, making it possible to realize a simple test. The interaction between the enzyme-linked aptamer and potassium ions results in the formation of a G-quadruplex-hemin complex, which promotes peroxidase activity.

[0043] There are several timings for forming the G-quadruplex-hemin complex, and they are not particularly limited. It is preferable to form the G-quadruplex-hemin complex in the step of folding the enzyme-linked aptamer.

[0044] Further investigation by the present inventors revealed that by combining a specific sequence of such an enzyme-linked aptamer with a specific concentration range of potassium ions, it is possible to achieve polyamine detection sensitivity that can withstand detection in a simple detection system. Enzyme-linked aptamers that can ensure differences in coloration due to differences in peroxidase activity in the presence or absence of polyamines may have characteristics that can be parameterized in circular dichroism spectra.

[0045] In circular dichroism spectra, G-quadruplexes can take on a variety of structures. These include at least the types of structures shown in Figures 2 to 7. It has previously been thought that hemin with a planar aromatic structure would have a parallel structure. However, G-quadruplexes are originally bound to the ends of telomeres, and their over 700,000 G-quartet structures are involved in controlling various functions in the body. The circular dichroism spectra provide insight into the various structures of G-quadruplexes, where G-quartets are complexed.

[0046] The present inventors have found that in addition to the aforementioned effect of potassium concentration on the promotion of polyamine-induced peroxidase activity, polyamines also inhibit peroxidase activity, and it is believed that this inhibitory effect can result in selectivity for spermine and spermidine in particular. The effect of potassium ion concentration is also significant when peroxidase activity is detected by luminol luminescence, a luminescence-changing substance, instead of optical detection of the coloration level of peroxidase activity as described above. In the present invention, it is possible to suppress peroxidase activity when polyamines are not present in the system. This reduces background. As a result, polyamines in the nM concentration range can be detected using the spontaneous luminescence detection method with the addition of luminol. This provides a simple detection method that can replace mass spectrometers, which require multiple modification steps and large-scale equipment.

[0047] In the present invention, the potassium ion concentration is 0.1 to 50 mmol / dm when detecting a change in the amount of coloration. 3 In combination with an enzyme-linked aptamer, particularly when detection is aimed at using simple equipment, a concentration of 0.5 to 10 mmol / dm 3 is preferred. When detecting spontaneous luminescence, the potassium ion concentration is preferably in a lower concentration range than when detecting changes in the amount of coloration, and the range is 0.1 to 10 mmol / dm 3 is preferred, and 1 to 10 mmol / dm 3 is more preferred. When aiming for simple and rapid detection, such as within 3 minutes in combination with an enzyme-linked aptamer, the potassium ion concentration should be 5 to 10 mmol / dm 3 may be preferred.

[0048] (Coloration of oxidation reaction) As a conjugate, the G4 DNAzyme binds to a target substance, thereby forming a G4 DNAzyme / hemin complex. Thus, a conjugate in which a nucleic acid, a protein, a peptide, or a ligand compound is bound to a G4 DNAzyme can be used. The porphyrin compound and the conjugate are preferably contacted in a buffer solution at 20°C to 30°C under static or agitated conditions for several minutes to several days.

[0049] In the present invention, the peroxidase activity of the polyamine-assisted aptamer is intentionally suppressed in the absence of polyamine, and then the polyamine-assisted aptamer's peroxidase activity is facilitated with the assistance of polyamine. To measure the polyamine concentration, the G-quadruplex is preferably formed by heating the aptamer in a buffer solution (pH 7.5-8.5) with the specific potassium concentration at 90-98°C for 1-10 minutes (followed by the folding step) and then cooling to 20-30°C over 15-40 minutes.

[0050] The contact with hemin can be carried out by adding a hemin solution to the obtained sample-contacted conjugate and leaving it to stand for several minutes to 60 minutes at 20 to 30°C, thereby forming a DNA (G4 DNAzyme) / hemin complex. The formed G4 DNAzyme / hemin complex can be measured or detected by utilizing the redox action of hemin.

[0051] The G4DNAzyme / hemin complex can be detected by, for example, measuring the potential of a conductive electrode to which the G4DNAzyme / hemin complex is bound. Hemin absorbs electrons from the electrode to become reduced, and then reduces hydrogen peroxide to return to its oxidized state. During this process, hydrogen peroxide (HO)W is reduced to water (HO)X, as shown in Figure 1. For example, in differential pulse voltammetry, the detection of a peak cathodic current relative to the reference electrode indicates the presence of a G4 DNAzyme / hemin complex. Furthermore, in differential pulse voltammetry, the intensity of the peak cathodic current relative to the reference electrode can be used as an indicator of the amount of G4 DNAzyme / hemin complex present. The level of the G4 DNAzyme / hemin complex can be measured from the intensity of the peak cathodic current.

[0052] The peroxidase activity of the G4 DNAzyme / hemin complex may be utilized to detect the G4 DNAzyme / hemin complex. In this method, the post-treatment solution is reacted with a peroxidase substrate, and the color development of a redox coloring dye is detected. Detection of dye color indicates the presence of the G4 DNAzyme / hemin complex. Furthermore, the intensity of the dye color development can be used as an indicator of the amount of G4 DNAzyme / hemin complex present. The level of the G4 DNAzyme / hemin complex can be measured from the intensity of the dye color. Color development of the substrate indicates the presence of G4 DNAzyme / hemin, and the degree of color development is an indicator of the amount of G4 DNAzyme / hemin present. In the present invention, the peroxidase activity is deliberately adjusted to allow amplification in the presence of the detection target, thereby obtaining a calibration curve for the detection target.

[0053] When optical detection is performed, the absorbance of the oxidized color dye, which develops color upon reduction of hydrogen peroxide by the peroxidase activity, increases around 390 nm, 410 nm, 550 nm, and 650 nm, for example, in the Examples described below. This change in absorbance can be quantified by irradiating a light source with an emission band of 405 nm, 450 nm, or 650 nm, which is used as an LED backlight source for smartphones, etc., and using software that converts the brightness and color tone into color temperature or RGB coding, or by receiving transmitted light using a CMOS, photodiode, etc. and monitoring the light intensity.

[0054] When detection is performed using an electrical method, for example, an enzyme-linked aptamer can be immobilized on a substrate such as a specific semiconductor or a specific electrode via a linker, a sample is dropped, and the charge generated by the spermine or spermidine contained in the sample is detected by detecting a change in current at an equal voltage due to a shift in the current-voltage curve caused by the field effect of the semiconductor.

[0055] The polyamine detection step is carried out by at least one of an optical method and an electrical method. When detecting by an electrical method, the enzyme-linked aptamer may be immobilized on the surface of an electrode (e.g., a gate electrode) or the like provided in the detection unit. Here, the enzyme-linked aptamer may be immobilized directly on the electrode or via a linker described below. Furthermore, the enzyme-linked aptamer does not necessarily have to be immobilized on the detection unit. The enzyme-linked aptamer may be present in the detection unit so that it can come into contact with the liquid to be detected when it comes into contact with the liquid. Furthermore, when contacting polyamine with the enzyme-linked aptamer, the entire amount of the liquid containing the polyamine may be contacted with the enzyme-linked aptamer, but from the viewpoint of improving accuracy, it is preferable to contact the polyamine in small amounts by dropping, etc.

[0056] (optical method) Polyamine detection is preferably performed in a state where the polyamine is in contact with a porphyrin compound. Furthermore, polyamine detection is preferably performed in the presence of a substance whose absorbance changes due to peroxidase activity. This is presumed as follows: When an enzyme-linked aptamer and a porphyrin compound are present, a complex between the enzyme-linked aptamer and the porphyrin compound is formed, and this complex can oxidize and reduce hydrogen peroxide (peroxidase activity). Here, polyamines interact with the enzyme-linked aptamer to stabilize the G-quadruplex structure of the enzyme-linked aptamer, thereby forming a complex with the porphyrin compound and promoting the expression of peroxidase activity. They function as a catalyst for this redox reaction. Therefore, polyamines can be detected by performing the redox reaction of hydrogen peroxide in the presence of a redox coloring dye (hereinafter sometimes referred to as an "absorbance-changing substance") whose absorbance changes due to this redox reaction. In addition, polyamines can be similarly detected by using a substance that emits fluorescence through this oxidation-reduction reaction (hereinafter sometimes referred to as a "fluorescence-generating substance") and a substance whose luminescence intensity changes through this oxidation-reduction reaction (hereinafter sometimes referred to as a "luminescence-changing substance") (hereinafter, absorbance-changing substances, fluorescence-generating substances, and luminescence-changing substances may be collectively referred to as "absorbance-changing substances, etc.").

[0057] The absorbance-changing substance is not particularly limited as long as it is a substance whose absorbance changes due to the oxidation-reduction reaction of hydrogen peroxide. Specific examples include 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt (ABTS), ortho-phenylenediamine dihydrochloride (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), OxiRed, [Fe(CN)6] 4- The fluorescence-changing substance may be, for example, Amplite™ ADHP, Amplex Red, or the like.

[0058] Examples of porphyrin compounds include protoporphyrin IX, heme, hemin, zinc protoporphyrin, magnesium protoporphyrin, hematoporphyrin, benzoporphyrin, metalloporphyrin, 5-aminolevulinic acid, texaphyrin, chlorin, purpurin, bacteriochlorin, phthalocyanine, naphthalocyanine, and derivatives thereof. Metalloporphyrins are preferred due to the size of the porphyrin compound and the planarity of the aromatic rings of the porphyrin compound, and metalloporphyrins having a non-bulky side chain are more preferred. This is thought to be because, when the metalloporphyrin prosthetic group is coordinated to an enzyme-linked aptamer having a G-quartet surface, space for further coordination of peroxide is more easily secured, facilitating the expression of peroxidase activity. Hemin is particularly preferred as a porphyrin compound.

[0059] The method for contacting the polyamine with the absorbance-changing substance or the like is not particularly limited, as long as the polyamine is contacted when optically detecting the polyamine in the detection step. That is, the polyamine may be contacted with the enzyme-linked aptamer at the same time as the polyamine is contacted with the enzyme-linked aptamer, or may be contacted with the absorbance-changing substance or the like first and then with the enzyme-linked aptamer, or may be contacted with the enzyme-linked aptamer first and then with the absorbance-changing substance or the like. The amount of the absorbance changing substance relative to the polyamine is not limited as long as it can change the absorbance in the oxidation-reduction reaction of hydrogen peroxide.

[0060] (electrical method) When the polyamine detection step is carried out electrically, there are no particular limitations as long as the polyamine in the liquid can be electrically detected in contact with the enzyme-linked aptamer in the detection step. Electrical detection is usually carried out on a liquid in which the voltage, current, etc. change due to the presence of polyamine. Here, the changes in voltage, current, etc. can also be measured by known methods. The electrical detection process for polyamines can be carried out, for example, using a system in which charge transfer occurs through a reaction catalyzed by a complex formed between a porphyrin compound and an enzyme-linked aptamer whose G-quadruplex structure has been stabilized by interaction with the polyamine. Here, the enzyme-linked aptamer is presumed to be able to reduce the distance between the electrode and the porphyrin compound. The electrical detection process can be carried out using a system in which charge transfer occurs through a reaction between the polyamine and a substance that undergoes a redox reaction with the polyamine, such as a porphyrin compound. Here, the enzyme-linked aptamer is presumed to be able to reduce the distance between the electrode and the substance that undergoes a redox reaction with the polyamine.

[0061] (detection sensitivity) The detection method of the present invention enables highly sensitive detection by detecting polyamines in contact with an enzyme-linked aptamer. Specifically, the lower limit of detection sensitivity is usually 1 × 10 -6 mol / dm 3 and 1×10 -7 mol / dm 3 may be 1 x 10 -9 mol / dm 3 may be 1 x 10 -10 mol / dm 3 The upper limit of the detection sensitivity is not particularly limited, but is usually 5 × 10 -3 mol / dm 3 and 2 × 10 -3 mol / dm 3 may be 1 x 10 -3 mol / dm 3 In the case of a simple non-invasive test, the upper limit is 10 × 10, taking into consideration dilution to eliminate the influence of impurities contained in the sample. -4 ~5×10 -5 mol / dm 3 is preferred.

[0062] The detection method of the present invention can detect each type of polyamine by selecting an appropriate combination of enzyme-linked aptamers etc. depending on the type of polyamine. Furthermore, the detection method of the present invention can also be applied to a method for quantifying each polyamine depending on the degree of optical change and electrical change. The amount of polyamines such as spermine and spermidine contained in saliva is usually 1 × 10 -6 ~1×10 -5 mol / dm 3 The amount of polyamines such as spermine and spermidine contained in urine is usually 1 × 10 -8 ~1×10 -7 mol / dm 3 When analyzing polyamines in saliva or urine, the saliva or urine is usually diluted about 10 to 100 times before analysis to eliminate the influence of impurities other than polyamines contained in the saliva or urine. The detection method of the present invention is considered to be applicable to cancer diagnosis using saliva, urine, etc.

[0063] (Other processes) The detection method of the present invention may include steps other than the sample preparation step and detection step described above. Such other steps include a sample holding step of holding a liquid sample introduced into the device in the sample introduction step described above, an irradiation step of irradiating light onto the liquid sample held in the sample holding step, and a measurement step of measuring light that has passed through the liquid sample. In this case, the detection step of detecting polyamines in the liquid described above can also be said to include an irradiation step and a measurement step.

[0064] [Polyamine detection reagent] The polyamine detection reagent of the present invention contains the enzyme-linked aptamer described above. Preferably, the polyamine detection reagent of the present invention further contains potassium ions. Details and preferred embodiments of the potassium ions are as described in the above sections.

[0065] According to the polyamine detection reagent of the present invention, the difference between the peroxidase activity expressed by the porphyrin compound, which is amplified by the appropriate G-quartet surface formed by the complexation of the enzyme-linked aptamer and the porphyrin compound, when it is suppressed in the absence of polyamines and when it is amplified in the presence of polyamines, can be detected in the presence of potassium ions. This peroxidase activity can be measured in the presence of potassium ions. The polyamine detection reagent of the present invention exhibits peroxidase activity expressed by the G-quartet structure formed by complexing the above-mentioned enzyme-linked aptamer with a porphyrin compound. By measuring the peroxidase activity thus expressed using a G-quadruplex folded in the presence of potassium ions within a specific concentration range, the difference in peroxidase activity due to the presence or absence of polyamines can be measured.

[0066] The polyamine detection reagent of the present invention may further contain at least one selected from the group consisting of dye precursors and chemiluminescent reagents. Examples include metalloporphyrin compounds such as hemin; color dyes and dye precursors such as hydrogen peroxide and ABTS; and chemiluminescent reagents such as luminol. These may coexist, or at least a portion of each may be separately impregnated, for example, into test paper or the like.

[0067] The polyamine detection reagent may be prepared from, for example, an enzyme-linked aptamer, a metalloporphyrin compound such as hemin, hydrogen peroxide, a color dye such as ABTS, or a chemiluminescent reagent such as luminol, and these components may coexist or at least some of them may be separately pre-loaded onto a test paper or the like.

[0068] [Polyamine detection device] The polyamine detection device of the present invention (hereinafter also referred to as the "detection device of the present invention") is equipped with the polyamine detection reagent described above. The detection device of the present invention can detect polyamines contained in a liquid in a state where the polyamines are in contact with the enzyme-linked aptamer.

[0069] In one example, the detection device includes a sample introduction section for introducing a liquid containing polyamine into the detection device, and a detection section for detecting polyamine in the liquid, and the polyamine in contact with the enzyme-linked aptamer in the detection section can be detected by at least one of optical and electrical methods. The detection device of the present invention is preferably an optical detection device (hereinafter also referred to as "optical detection device"). Optical detection devices will be described below.

[0070] (Optical detection device) The optical detection device is not particularly limited as long as it can optically detect polyamines in contact with the enzyme-linked aptamer. Specifically, for example, when the above-mentioned absorbance-changing substance is used, an example of the optical detection device includes a sample holding unit that holds a liquid sample introduced into the device from the above-mentioned sample introduction unit, an irradiation unit that irradiates light onto the liquid sample held in the sample holding unit, and a measurement unit that measures the light that has passed through the liquid sample. In this case, the detection unit that detects polyamines in the above-mentioned liquid has an irradiation unit and a measurement unit.

[0071] The sample holder is not particularly limited as long as it can hold a liquid sample and measure its optical properties. Specifically, for example, it may be in the shape of a container for holding a liquid sample, or may be in the shape of a plate or depression on which a liquid sample can be held. When light is irradiated onto the sample introduction section, the sample introduction section may also serve as the sample holder. When the liquid sample is saliva, for example, the subject may hold the sample holder in his or her mouth and apply saliva to it.

[0072] The optical detection device measures the optical properties of the polyamine in contact with the enzyme-linked aptamer in the detection unit. The optical detection device preferably includes at least one of a sample introduction unit configured to allow introduction of a liquid containing the polyamine and the enzyme-linked aptamer, or a sample holding unit configured to allow introduction of the enzyme-linked aptamer.

[0073] The irradiating unit is not particularly limited as long as it can irradiate the liquid sample with light. The irradiating light is light of a wavelength whose absorbance changes in the presence of polyamine in contact with the enzyme-linked aptamer. It is sufficient that light of a wavelength that changes absorbance be irradiated according to the types and combinations of the absorbance-changing substance, polyamine, and enzyme-linked aptamer. For example, when ABTS is used as the absorbance-changing substance, it is preferable that light having a wavelength of 414 nm be irradiated. Furthermore, the light source is not particularly limited as long as it can irradiate light of the desired wavelength, and examples that can be used include a halogen lamp, tungsten lamp, xenon lamp, LED, and laser.

[0074] The measurement unit is not particularly limited as long as it can measure light that has passed through a liquid sample. The absorbance can be calculated from the ratio of the intensity of the irradiated light to the intensity of the transmitted light. The amount of polyamine in the liquid sample can be quantified from the intensity of this absorbance.

[0075] Although several embodiments have been described above, the present invention is not limited to the embodiments disclosed in this specification and can be appropriately modified and implemented without departing from the spirit of the invention. The embodiments disclosed in this specification can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. [Example]

[0076] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following description. In the following description, "fc" means the final concentration of a solution.

[0077] [Polyamine] Spermine: A product from Nacalai Tesque was prepared. Spermidine: A product from Nacalai Tesque was prepared.

[0078] [Absorbance measurement] The absorbance at 414 nm and 405 nm was measured using a plate reader, either Multiskan Sky T (ThermoFisher Scientific) or MPRA-100 (AS ONE).

[0079] [Example 1] In Example 1, the enzyme-linked aptamer was obtained and its formation efficiency was evaluated. First, to form the GOx-UdgX complex by the SpyTag / SpyCatcher reaction, UdgX-ST (fc 10 μM) and GOx-SC (fc 5 μM) were incubated at 4°C for 24 hours. The resulting GOx-UdgX complex was then heated at 95°C for 10 minutes in buffer solution A (10 mM NaH2PO4 / Na2HPO4, 1 mM KCl, 0.003% (v / v), Triton-X, pH 7.0) and slowly cooled to 25°C over 2 hours to allow folding of the aptamer of SEQ ID NO: 1. The aptamer of SEQ ID NO: 1, which had been introduced with the UdgX recognition sequence, was mixed in equal amounts in buffer buffer A. The aptamer of SEQ ID NO: 1 was 5'-GTGGGTAGGGCGGGTTGG AAGTGUAGGCA -3', and the underlined AAGTGUAGGCA at the 3' end is the UdgX recognition sequence of SEQ ID NO:2. The Gox-linked aptamer was obtained by overnight incubation at 4°C, as shown in Figure 8. As shown in Figure 8, in the Gox-linked aptamer 10, an aptamer having a sequence that forms a G-quadruplex 11 is linked to GOx13 via UdgX12.

[0080] The resulting complex was loaded onto an SDS-PAGE gel so that the total protein amount per lane was 500 ng. SDS-PAGE analysis (20 mA, 90 min) was performed, and the gel was stained with Coomassie Brilliant Blue (CBB). The results are shown in Figure 9. In the lane containing UdgX-ST and GOx-SC, a band was observed near the theoretical molecular weight of the UdgX-GOx complex (102.2 kDa). Furthermore, UdgX-GOx complex formation via the SpyTag / SpyCatcher linking module system was confirmed. Furthermore, in the lane containing the aptamer of SEQ ID NO: 1 and GOx-UdgX, a band was observed near the theoretical molecular weight of the Gox-linked aptamer (122.6 kDa), confirming the formation of the Gox-linked aptamer via glycosidic bond formation between UdgX and the recognition sequence (SSU9(5)).

[0081] When the aptamer of SEQ ID NO: 1 was replaced with the aptamer of SEQ ID NO: 3, the efficiency of complex formation between UdgX and the aptamer was slightly reduced. In the detection of spermine described below, although the spermine concentration could be calibrated, the luminescence intensity tended to be about 10 times lower. The aptamer of SEQ ID NO: 3 is 5'-GTGGGTAGGGCGGGTTGG TTTAAGTGUAGGCA -3', and the 3' end of the RNA aptamer is underlined. AAGTGUAGGCA is the UdgX recognition sequence.

[0082] [Example 2] A GOx-linked G4 aptamer was obtained using the same method as in Example 1. SEQ ID NO: 1 was used as the recognition sequence-modified aptamer. The GOx-linked G4 aptamer (fc 100 nM) was added to a 96-well PP white plate. Hemin (fc 400 nM) was then mixed with spermine (fc 0, 0.05, 0.1, 0.5, 0.8, 1 μM) diluted in Tris-HCl buffer (pH 7.0). The mixture was then shaken at room temperature for 1 hour. 50 μL of reaction solution (BM chemiluminescence ELISA substrate (POD) A Solution + fc 200 μM Glucose) was then added using a 12-channel pipette. 50 μL of reaction solution (BM chemiluminescence ELISA substrate (POD) A Solution + fc 200 μM Glucose) was then added. The time course of chemiluminescence was then immediately measured using a plate reader. The change in the chemiluminescence intensity obtained in this manner per minute was plotted to prepare a spermine calibration curve.

[0083] The results are shown in Figure 10. R 2 A highly linear calibration curve was obtained, with a ΔΨ = 0.92. Furthermore, the detection limit was calculated using the 3σ method, resulting in an LOD of 0.26 μM. The saliva of pancreatic cancer patients contains approximately 10 mM spermine, suggesting that this spermine can be quantified.

[0084] [Example 3] The same conditions as in Example 2 were used to obtain Gox-modified G4 aptamers with different folding states, except that buffer solution B was used, which was a 10 mM potassium solution in buffer solution A. Next, 100 nM of Gox-modified G4 aptamer was added to a 96-well PP white plate. Hemin (400 nM) was then mixed with spermine (0, 0.025, 0.005, 0.075, 0.1, 0.5, 0.8, and 1 μM) diluted in Tris-HCl buffer (pH 7.0). Immediately afterwards, 80 μL of reaction solution (BM chemiluminescence ELISA substrate (POD) A Solution + 200 μM glucose) was added, and the time course of chemiluminescence was immediately measured using a plate reader. A spermine calibration curve was created by plotting the change in chemiluminescence intensity per minute.

[0085] The results are shown in Figure 11. Compared to the potassium 1 mM condition, the detection range was shifted to the lower concentration side. 2 A highly linear calibration curve was obtained, with a ΔΨ = 0.99. Furthermore, the detection limit was calculated using the 3σ method, resulting in a LOD of 0.012 μM. This suggests the possibility of quantifying spermine with high sensitivity.

[0086] [Example 4] 2.5 μmol / dm 3 20 μL of the linked aptamer was dissolved in 10 mmol / dm 3 1 μL of Tris-HCl aqueous solution (pH 7.0), 100 μmol / dm 3 2 μL of DMSO solution containing hemin, buffer solution A (10 mmol / dm 3 NaH 2 PO4 / Na2HPO4 (pH 7.0), 1mmol / dm 323 μL of human serum (Sigma Aldrich, H3667) diluted 10-fold with 0.003 vol% Triton X-100 (KCl, 0.003 vol% Triton X-100) was mixed and added to a 384-multiwell plate. After standing at 25°C for 60 minutes, the plate was diluted with 40 mmol / dm 3 4 μL of Buffer A containing ABTS was added, stirred for 30 seconds, and then allowed to react for an additional 60 minutes, during which the absorbance of ABTS at 414 nm was measured using a plate reader (Multiskan Sky T, ThermoFisher Scientific).

[0087] The measurement results are shown in Figure 12. In the figure, the horizontal axis represents the time after the addition of ABTS. The vertical axis represents the change in absorbance (wavelength 414 nm) derived from ABTS oxidized by hydrogen peroxide. Compared to the absence of spermine, the addition of 10 µM spermine significantly increased the absorbance. This demonstrates that spermine can be detected in 10-fold diluted human serum by the colorimetric method using the aptamer of the present invention.

[0088] [Example 5] The GOx-linked aptamer (2.5 μmol / dm 3 ) diluted 4-fold with the buffer solution B, 10 μL, 50 μmol / dm 3 of spermine dissolved in 10mmol / dm 3 2 μL of Tris-HCl buffer (pH 7.0), 10 μmol / dm 3 2 μL of DMSO solution containing hemin was mixed with 37.8 μL of buffer solution B and added to a 96-well plate. After standing at 25°C for 60 minutes, the plate was diluted to 40 mmol / dm 3 4 μL of 625 mmol / dm ABTS-dissolved buffer solution B 32 μL of buffer solution B containing dissolved D-glucose was added, and the mixture was stirred for 30 seconds to allow the reaction to proceed. The change in absorbance at 405 nm was then measured. The absorbance was 0.411 immediately after the reaction, but increased to 0.69, 1.02, 1.342, and 1.642 after 3, 8, 13, and 30 minutes, respectively. After 60 minutes, the absorbance decreased to 1.313. This confirmed that a reaction time of just a few minutes was sufficient to obtain sufficient color for detection.

[0089] On the other hand, the GOx-linked aptamer (2.5 μmol / dm 3 ) 2 μL, 50 μmol / dm 3 of spermine dissolved in 10mmol / dm 3 2 μL of Tris-HCl buffer (pH 7.0), 10 μmol / dm 3 2 μL of DMSO solution containing hemin was mixed with 37.8 μL of buffer solution A and added to a 96-well plate. After standing at 25°C for 60 minutes, the plate was diluted to 40 mmol / dm 3 4 μL of 2 mol / dm ABTS-dissolved buffer solution A was added. 3 2 μL of buffer solution A containing dissolved D-glucose was added, and the mixture was stirred for 30 seconds to allow the reaction to proceed. The change in absorbance at 405 nm was then measured. The absorbance was 0.163 immediately after the reaction, but after 3, 8, 13, and 30 minutes, the changes were small, reaching 0.183, 0.215, and 0.265, respectively. After 30 minutes, the absorbance tended to increase, reaching 0.594 and 0.82.

[0090] [Example 6] The same conditions as in Example 2 were used to obtain Gox-modified G4 aptamers with different folding states, except that buffer solution B was used, which was a 5 mM potassium solution in buffer solution A. Next, 10 μL of GOx-modified G4 aptamer (fc 100 nM) was added to a 96-well PP white plate. Hemin (fc 400 nM) was then mixed with spermine (fc 0, 0.025, 0.005, 0.075, 0.1, 0.5, 0.8, and 1 μM) diluted in Tris-HCl buffer (pH 7.0). Immediately afterwards, 80 μL of reaction solution (BM chemiluminescence ELISA substrate (POD) A Solution + fc 200 μM Glucose) was added, and the time course of chemiluminescence was immediately measured using a plate reader. A spermine calibration curve was created by plotting the change in chemiluminescence intensity per minute.

[0091] The measurement results are shown in Figure 13. As a result of detecting spermine under the 5 mM potassium condition, it was confirmed that the quantification range shifted to the lower concentration side compared to the 1 mM potassium condition, and a calibration curve with a steep slope was obtained that was quantitative up to a final spermine concentration of 0.25 μM. 2 The LOD was calculated to be 0.025 μM, which is approximately 1 / 10 of that at 1 mM potassium. This suggests that the 5 mM potassium condition is the optimal condition for spermine detection.

[0092] [Example 7] Spermine was diluted in artificial saliva (5 mM NaHPO, 5.4 mM CaCl, 5.4 mM KCL, 6.8 mM NaCl, 0.4 g / L mucin for porcine stomach, 66 mM urea, 100 μM uric acid, 10 μM ascorbic acid, pH 7.2) containing ascorbic acid and uric acid, two well-known contaminants with HO decomposition ability. Detection sensitivity in artificial saliva was evaluated using the same method as in Example 6.

[0093] The measurement results are shown in Figure 14. Spermine detection in artificial saliva under 5 mM potassium conditions yielded a linear calibration curve over the spermine concentration range up to 0.25 μM, similar to the detection results in buffer. No significant changes were observed in the slope or error range of each plot. Furthermore, the LOD was calculated to be 0.040 μM, showing no significant decrease from the detection results in buffer. Because this detection sensitivity is significantly lower than the spermine concentration of 10 μM in patient saliva, actual measurements can be performed with saliva diluted approximately 50–100 times, which suggests that the effect of contaminants can be further reduced compared to the results obtained in this study. These findings suggest the feasibility of this detection system for actual measurements in saliva. [Industrial Applicability]

[0094] According to the present invention, inhibition of peroxidase activity is suppressed, and polyamines can be detected selectively, sensitively, and simply without the need to add additional hydrogen peroxide. [Explanation of symbols]

[0095] 10 Gox-linked aptamer 11 G-quadruplex 12 UdgX 13 GOx 21 Aptama 22 Porphyrin compounds 23 Polyamines W Hydrogen peroxide (H2O2) X Water (H2O)

Claims

1. A polyamine detection reagent, comprising: the polyamine detection reagent contains an enzyme-linked aptamer in which an enzyme and an aptamer are linked; The enzyme-linked aptamer is a polyamine detection reagent having an enzyme that generates hydrogen peroxide and an aptamer having a sequence that forms a G-quadruplex.

2. The polyamine detection reagent according to claim 1 , wherein the enzyme that generates hydrogen peroxide comprises glucose oxidase.

3. The polyamine detection reagent according to claim 1 , further comprising potassium ions.

4. The polyamine detection reagent according to claim 1 , further comprising a porphyrin compound.

5. The polyamine detection reagent according to claim 4 , which exhibits peroxidase activity due to a G-quartet structure formed by complexing the enzyme-linked aptamer with the porphyrin compound.

6. The polyamine detection reagent according to claim 4, which detects the difference in peroxidase activity expressed by a G-quartet structure formed by complexing the enzyme-linked aptamer with the porphyrin compound, depending on the presence or absence of polyamines.

7. A polyamine detection device comprising the polyamine detection reagent according to claim 5 or 6.

8. A method for detecting polyamines, comprising contacting the polyamine detection reagent according to claim 5 or 6 with polyamines.

9. contacting the polyamine detection reagent according to claim 5 or 6 with a polyamine; measuring peroxidase activity expressed by a G-quartet structure formed by complexing the enzyme-linked aptamer with the porphyrin compound; A method for detecting polyamines, comprising:

10. An enzyme-linked aptamer in which an enzyme and an aptamer are linked, The enzyme includes an enzyme involved in a reaction that produces hydrogen peroxide, The enzyme-linked aptamer has a sequence that forms a G-quadruplex.

11. The enzyme-linked aptamer of claim 10 , wherein the hydrogen peroxide-generating enzyme comprises glucose oxidase.

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