Method for immobilizing nucleic acid compounds, reagent kit and sensor

Abstract

To provide a method and reagent kit as well as sensor for immobilizing nucleic acid compound mores simply and at higher density, and to provide a sensor for performing the method or using the reagent kit.SOLUTION: The method for immobilizing a nucleic acid compound according to the embodiment is a method for immobilizing a nucleic acid compound on the surface of a sensor element composed of graphene, graphene oxide, carbon nanotube, or graphite, the method comprising: preparing an aqueous solution containing a nucleic acid compound and sodium chloride, the nucleic acid compound comprising a polycyclic aromatic moiety composed of a polycyclic aromatic skeleton and a linker structure bonded to the polycyclic aromatic skeleton, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety; and dropping the prepared aqueous solution onto the surface of the sensor element.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for immobilizing a nucleic acid compound, a reagent kit, and a sensor. [Background technology]

[0002] To improve the sensitivity of sensors, it is necessary to use a simpler method to efficiently immobilize aptamers on sensors and form high-density probes. Summary of the Invention [Problem to be solved by the invention]

[0003] An object of the present invention is to provide a method and a reagent kit for immobilizing nucleic acid compounds more easily and at higher density, as well as a sensor for carrying out the method or using the reagent kit. [Means for solving the problem]

[0004] A method for immobilizing a nucleic acid compound according to an embodiment is a method for immobilizing a nucleic acid compound on the surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, and includes preparing an aqueous solution containing a nucleic acid compound and sodium chloride, where the nucleic acid compound comprises a polycyclic aromatic moiety composed of a polycyclic aromatic backbone having affinity for the sensor surface and a linker structure bonded to the polycyclic aromatic backbone, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety, and dropping the aqueous solution onto the sensor surface.

[0005] A reagent kit according to an embodiment is a reagent kit used to immobilize a nucleic acid compound on the surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, and includes a first container containing a nucleic acid compound comprising a polycyclic aromatic moiety composed of a polycyclic aromatic backbone having affinity for the sensor element surface and a linker structure bound to the polycyclic aromatic backbone, and a nucleic acid moiety bound to the linker structure of the polycyclic aromatic moiety, and a second container containing an aqueous sodium chloride solution.

[0006] A sensing device according to an embodiment comprises a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, a first container for containing a first solution, a second container for containing a second solution, a first flow path for supplying the first solution from the first container to the surface of the sensor element, a second flow path for supplying the second solution from the second container to the surface of the sensor element, and a third flow path for discharging liquid from the surface of the sensor element, wherein the first solution is an aqueous solution containing a nucleic acid compound and sodium chloride, wherein the nucleic acid compound comprises a polycyclic aromatic moiety composed of a polycyclic aromatic skeleton having affinity for the surface of the sensor element and a linker structure bonded to the polycyclic aromatic skeleton, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety, and the second solution is an aqueous solution containing at least one of a buffer solution, an ionic liquid, a surfactant, and a chelating agent. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a flow chart showing an example of the method for immobilizing nucleic acid compounds according to the first embodiment. [Figure 2] FIG. 2 is a flowchart showing an example of the method for immobilizing a nucleic acid compound according to the first embodiment. [Figure 3] FIG. 3 is a schematic diagram showing an example of a sensor according to the third embodiment. [Figure 4]FIG. 4 is a schematic cross-sectional view showing an example of a sensor element portion of a sensor according to the third embodiment, where (A) shows the state of the sensor according to the third embodiment before it is put into use, and (B) shows the state of the sensor according to the third embodiment after it has been put into use. [Figure 5] FIG. 5 is a schematic diagram showing an example of a sensor according to the third embodiment. [Figure 6] FIG. 6 is a graph showing the experimental results of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0008] Various embodiments will be described below with reference to the drawings. Each figure is a schematic diagram for facilitating understanding of the embodiment, and the shapes, dimensions, and comparisons may differ from the actual ones. However, these may be appropriately modified in design, taking into consideration the following explanations and known technologies.

[0009] (First embodiment) The method for immobilizing a nucleic acid compound according to the embodiment is a method for immobilizing a nucleic acid compound on the surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, and as shown in FIG. 1, (S1) preparing an aqueous solution containing a nucleic acid compound and sodium chloride; (S2) Drop the prepared aqueous solution onto the surface of the sensor element. wherein the nucleic acid compound comprises a polycyclic aromatic moiety composed of a polycyclic aromatic backbone having affinity for the surface of the sensor element and a linker structure bonded to the polycyclic aromatic backbone, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety.

[0010] The term "polycyclic aromatic skeleton" refers to molecules of aromatic compounds and their derivatives having two or more ring structures within the molecule. The polycyclic aromatic skeleton may be a structure having fused rings such as acenes (e.g., naphthalene and anthracene), phenanthrene, and pyrene, or a structure having two or more separate rings (e.g., biphenyl, terphenyl, and triphenylmethane). The polycyclic aromatic skeleton may be a molecule of a heterocyclic compound (e.g., quinoline, coumarin, etc.). The polycyclic aromatic skeleton may also be a molecule of a non-benzene-based aromatic compound (e.g., azulene, etc.).

[0011] The polycyclic aromatic skeletons exemplified above have cyclically delocalized π electrons to form thermodynamically stable ring systems, and therefore undergo π-π interactions with graphene, graphene oxide, carbon nanotubes, and graphite, which also have cyclically delocalized π electrons. Therefore, the polycyclic aromatic skeletons are easily adsorbed to graphene, graphene oxide, carbon nanotubes, and graphite, and have affinity for graphene, graphene oxide, carbon nanotubes, and graphite.

[0012] The polycyclic aromatic moiety in the nucleic acid compound immobilization method of the embodiment refers to a compound composed of the polycyclic aromatic skeleton and a linker structure bonded to the polycyclic aromatic skeleton. The linker structure suppresses steric interference between the nucleic acid and the sensor element surface that occurs when the nucleic acid is directly bonded to the aromatic ring of the polycyclic aromatic skeleton. As long as the polycyclic aromatic skeleton maintains its adsorption to the sensor element surface, the nucleic acid compound does not need to include a linker structure. The linker structure contains at least two carbon atoms between the bonding position to the polycyclic aromatic skeleton and the bonding position to the nucleic acid moiety. The linker structure contains at least one carbon-carbon single bond between the bonding position to the polycyclic aromatic skeleton and the bonding position to the nucleic acid moiety. At least one carbon atom constituting the linker structure, such as the carbon atom closest to the bonding position to the nucleic acid moiety or the carbon atom bonded to the nucleic acid moiety, is not included in the plane formed by the carbon atoms constituting the polycyclic aromatic. The end of the linker structure is preferably a hydrophilic group that easily bonds to the nucleic acid moiety. If the linker structure contains a phosphate group as the hydrophilic group, the nucleic acid moiety can be synthesized by adding nucleotides starting from the phosphate group. The end of the linker structure can be bound to, for example, the 5' end or 3' end of the nucleic acid portion via a phosphate bond, a phosphoester bond, or the like.

[0013] The linker structure bonded to the polycyclic aromatic skeleton is, for example, a linker structure in which the carbon at the 5-position of the pyrimidine ring of deoxyuridine is alkynylated (the following formula (1)), or a linker structure in which a phosphate group is bonded to the carbon at the 2-position of the pyrrolidine ring (the following formula (2)).

[0014] [ka]

[0015] [ka]

[0016] The nucleic acid constituting the nucleic acid portion may be a single-stranded nucleic acid, and is not limited to DNA or RNA molecules, but may be various artificial nucleic acids such as GNA, LNA, PNA, and TNA. The nucleic acid portion may also be an aptamer that binds to a specific substance. The nucleic acid portion may also be optionally modified, and an optional protecting group may be introduced. The base length of the nucleic acid portion is not particularly limited, and may be from a few bases to several hundred bases.

[0017] The nucleic acid compound according to the embodiment of the method for immobilizing a nucleic acid compound is a compound formed by binding the polycyclic aromatic moiety and the nucleic acid moiety to the end of a linker structure bonded to the polycyclic aromatic backbone constituting the polycyclic aromatic moiety. For example, when the polycyclic aromatic backbone is pyrene, the linker structure is the structure shown in formula (1), and the nucleic acid moiety is DNA, the nucleic acid compound is the compound shown in formula (3) below.

[0018] [ka]

[0019] Furthermore, for example, when the polycyclic aromatic skeleton is pyrene, the linker structure is the structure shown in formula (2) above, and the nucleic acid portion is DNA, the nucleic acid compound is a compound shown in formula (4) below.

[0020] [ka]

[0021] Since the nucleic acid compound contains the polycyclic aromatic skeleton, it is easily adsorbed to graphene, graphene oxide, carbon nanotubes, and graphite, and has affinity for graphene, graphene oxide, carbon nanotubes, and graphite. Therefore, in the method for immobilizing a nucleic acid compound according to the embodiment, a solution containing the nucleic acid compound is dropped onto the surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, whereby the nucleic acid compound, and in turn the nucleic acid moiety constituting the nucleic acid compound, can be immobilized on the surface of the sensor element.

[0022] The nucleic acid compound-containing solution used in the nucleic acid compound immobilization method according to the embodiment contains water as the solvent and sodium chloride as a solute other than the nucleic acid compound. That is, the nucleic acid compound-containing solution is an aqueous sodium chloride solution. As will be described later, sodium chloride has the effect of promoting the immobilization of nucleic acid compounds on the surface of a sensor element, so the more sodium chloride is added, the more the immobilization of the nucleic acid moiety can be promoted. Therefore, the concentration of sodium chloride in the nucleic acid compound-containing solution is preferably higher, for example, 150 mM or higher.

[0023] The solution containing the nucleic acid compound may contain any solute, but the concentrations of phosphate ions and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (hereinafter referred to as "HEPES") are preferably lower, and it is even more preferable that the solution does not contain phosphate ions or HEPES. This is because, as will be described later, phosphate ions and HEPES have the effect of inhibiting the immobilization of the nucleic acid compound on the surface of the sensor element.

[0024] Conventionally, a method for immobilizing a nucleic acid compound on the surface of a sensor element made of graphene has been carried out by the following steps (a) to (d): (a) Dropping an organic solvent containing 1-pyrenebutanoic acid and a succinimide ester onto the surface of the sensor element; (b) after step (a), cleaning the surface of the sensor element; (c) after step (b), dripping a buffer solution containing NH2-DNA onto the surface of the sensor element; and (d) After step (c), an ethanolamine solution is added to the surface of the sensor element.

[0025] Here, in the above step (a), 1-pyrenebutanoic acid is bound to the surface of the sensor element, and a succinimide ester is bound to the 1-pyrenebutanoic acid bound to the surface of the sensor element, thereby immobilizing the scaffold molecule (pyrene derivative) of the nucleic acid molecule on the surface of the sensor element.

[0026] In the above step (b), 1-pyrenebutanoic acid and succinimide ester that are not immobilized as scaffold molecules for nucleic acid molecules are removed from the surface of the sensor element.

[0027] In the above step (c), the scaffold molecule immobilized on the surface of the sensor element through the above step (a) is bound to the nucleic acid molecule NH2-DNA, thereby immobilizing the nucleic acid molecule on the surface of the sensor element.

[0028] In the above step (d), scaffold molecules that are not bound to nucleic acid molecules are inactivated, and the surface of the sensor element is washed.

[0029] As described above, conventional nucleic acid probe immobilization methods have the drawback of requiring a large number of steps and being cumbersome. Furthermore, there is also the risk that the organic solvent used in step (a) may cause the graphene to peel off from the surface of the sensor element.

[0030] On the other hand, the method for immobilizing a nucleic acid compound according to the embodiment can immobilize a nucleic acid compound on the surface of a sensor element simply by dropping an aqueous solution containing a nucleic acid compound and sodium chloride onto the surface of the sensor element.

[0031] Because a nucleic acid compound is a compound in which a polycyclic aromatic skeleton, a linker structure, and a nucleic acid moiety are bonded, the method for immobilizing a nucleic acid compound according to the embodiment does not require the step of bonding the polycyclic aromatic skeleton and the linker structure on the surface of a sensor element (i.e., step (a) in the conventional method for immobilizing a nucleic acid compound). Furthermore, because steps (b) and (d) in the conventional method for immobilizing a nucleic acid compound are steps that are necessary in addition to step (a), the method for immobilizing a nucleic acid compound according to the embodiment does not require steps corresponding to steps (b) and (d) in the conventional method for immobilizing a nucleic acid compound. Therefore, the method for immobilizing a nucleic acid compound according to the embodiment has fewer steps than the conventional method for immobilizing a nucleic acid compound, and can immobilize a nucleic acid compound more easily.

[0032] Furthermore, since the method for immobilizing a nucleic acid compound according to the embodiment does not require an organic solvent, there is no risk of the graphene being peeled off from the surface of the sensor element due to the organic solvent, which is preferable.

[0033] In a further embodiment, step (S3) of washing the sensor element surface may be carried out after step (S2), as shown in Fig. 2. The purpose of step (S3) is to remove nucleic acid compounds and the like that are not immobilized on the sensor element surface and remain in the solution. The sensor element surface may be washed, for example, by replacing the solution with an aqueous solution that does not contain nucleic acid compounds.

[0034] (Second embodiment) The reagent kit according to the second embodiment is a reagent kit used for immobilizing a nucleic acid compound on the surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite.

[0035] The reagent kit according to the second embodiment includes a first container containing a nucleic acid compound comprising a polycyclic aromatic moiety composed of a polycyclic aromatic backbone having affinity for the surface of a sensor element and a linker structure bonded to the polycyclic aromatic backbone, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety, and a second container containing an aqueous sodium chloride solution.

[0036] The nucleic acid compound in the reagent kit according to the second embodiment is the same as the nucleic acid compound in the nucleic acid compound immobilization method according to the first embodiment. The nucleic acid compound contained in the first container is more stable when it is in a solid and dry state. Therefore, the first container is preferably configured so that the nucleic acid compound contained therein is stored in a moisture-proof state.

[0037] When using the reagent kit according to the second embodiment, the composition contained in the first container is weighed, and the aqueous sodium chloride solution stored in the second container is added in accordance with the weighed amount, or a predetermined amount of the aqueous sodium chloride solution stored in the second container is added to the first container, thereby preparing an aqueous solution containing the composition and sodium chloride.

[0038] By dropping an aqueous solution containing the composition prepared as described above and sodium chloride onto the surface of the sensor element, the polycyclic aromatic skeleton constituting the nucleic acid compound is adsorbed onto the surface of the sensor element, and thus the nucleic acid portion can be immobilized on the surface of the sensor element.

[0039] In a further embodiment, the nucleic acid compound may be stored in the first container in a state of being dissolved in a liquid, and the liquid may further contain a stabilizer for the nucleic acid compound.

[0040] (Third embodiment) The sensor according to the third embodiment is a sensor for use in carrying out the method of the first embodiment or in using the reagent kit according to the second embodiment. The structure of the sensor according to the third embodiment will be described in detail below with reference to Figs. 3 and 4.

[0041] 3, the sensor 1 includes a first container 2 for containing an aqueous solution (first solution) containing a nucleic acid compound and sodium chloride, a second container 3 for containing a measurement solution (second solution), a sensor element 4 made of graphene, graphene oxide, carbon nanotubes, or graphite, and a third container 5 for containing a liquid discharged from the surface of the sensor element 4. The nucleic acid compound in the sensor according to the third embodiment is the same as the nucleic acid compound described in the first and second embodiments.

[0042] The second solution may contain, for example, at least one of an ionic liquid that enhances the measurement sensitivity of the sensor 1, a buffer solution that enhances the stability of the sensor 1, and a surfactant / chelating agent that enhances the stability of the nucleic acid moiety. The second solution may contain, as the ionic liquid, any ionic liquid, such as choline dihydrogen phosphate, imidazolium salt-based ionic liquid, pyrrolidinium salt-based ionic liquid, pyridinium salt-based ionic liquid, piperidinium salt-based ionic liquid, ammonium salt-based ionic liquid, phosphonium salt-based ionic liquid, or phosphonate-based ionic liquid. The second solution may contain, as the buffer, any buffer, such as phosphate buffer or HEPES buffer. The second solution may contain, as the chelating agent, an aminocarboxylate such as EDTA.

[0043] 4 shows an example of the sensor element portion of the sensing device of the third embodiment. As shown in (A) of FIG. 4, before the sensor according to the third embodiment is put into use, i.e., before the first solution and the second solution are supplied to the surface of the sensor element 4, the nucleic acid compound, and hence the nucleic acid portion, is not immobilized on the surface of the sensor element 4.

[0044] When the sensor according to the third embodiment is put into use, a first solution is supplied to the surface of the sensor element 4 via the first flow path 21 extending from the first container 2. By supplying the first solution, nucleic acid compounds 11 are immobilized on the surface of the sensor element 4, as shown in Fig. 4(B). Nucleic acid compounds that are not bound to the surface of the sensor element 4 are discharged together with the supply of the second solution.

[0045] The sensor 1 according to the third embodiment, in which the nucleic acid compound 11 and thus the nucleic acid portion 10 are immobilized on the surface of the sensor element 4 as shown in Figure 4 (B), can be used, for example, as a sensor for capturing and measuring a specific target substance, and the nucleic acid portion 10 can be used as a probe in such a sensor that specifically binds to and captures the target substance.

[0046] When the sensor 1 according to the third embodiment is used as a sensor for capturing and measuring a specific target substance, it is preferable to replace the first solution on the sensor element 4 with the second solution after immobilizing the nucleic acid moiety 10 on the surface of the sensor element 4. This replacement is performed by discharging the first solution on the sensor element 4 through the third flow path 23 and supplying the second solution onto the sensor element 4 through the second flow path 22. By replacing the first solution on the sensor element 4 with the second solution, it is possible to prevent a decrease in measurement sensitivity due to binding of the nucleic acid moiety constituting the compound with the target substance when a nucleic acid compound that has not adsorbed to the sensor element 4 remains in the first solution.

[0047] In a further embodiment, the sensor further includes a fourth container 6 containing an aqueous sodium chloride solution, and the first container 2 contains a nucleic acid compound in a solid state rather than a first solution. In this case, as shown in Fig. 5 , the first container 2 and the fourth container 6 are connected by a fourth flow path 24, and when the sensor is put into use, the aqueous sodium chloride solution is supplied from the fourth container 6 to the first container 2 through the fourth flow path 24. In the first container 2 supplied with the aqueous sodium chloride solution, the nucleic acid compound dissolves in the aqueous sodium chloride solution to prepare a first solution. The first solution prepared in the first container is supplied onto the surface of the sensor element 4 through the first flow path 21. [example] The following describes experiments carried out using the nucleic acid compound immobilization method of the embodiment. Example 1: Comparison of the amount of nucleic acid compounds immobilized under different solutes Nine types of aqueous solutions (referred to as aqueous solutions A to I, respectively) with different solutes and concentrations were prepared. The compositions of each are shown in Table 1 below.

[0048] [Table 1]

[0049] Here, aqueous solutions A to H all contain a nucleic acid compound represented by the following formula (5) at a concentration of 1 μM as a solute, as shown in Table 1. Aqueous solution I is pure water and does not contain any solute. In the table, "D-PBS(-)" refers to a buffer solution containing KCl at concentrations of 200 mg / L, NaCl at 8000 mg / L, KH2PO4 at 200 mg / L, and Na2HPO4 at 1150 mg / L, and "PB" refers to a phosphate buffer solution.

[0050] [ka]

[0051] Here, the DNA in the above formula (5) is a single-stranded DNA of 40 bases.

[0052] Each of the aqueous solutions A to I was dropped onto a different graphene surface. 60 minutes after the dropping, each graphene surface was analyzed by X-ray photoelectron spectroscopy (XPS). The XPS analysis conditions were: excitation X-ray: monochromatic Al Kα 1,2 The X-ray diameter was 100 μm, and the photoelectron detection angle was 45° (the inclination of the detector relative to the sample surface).

[0053] The atomic ratio (N / Si or P / Si) was calculated from the elemental composition (atomic %), and the P / Si value was used as an index of the amount of immobilization. The relative ratio of the amount of immobilization was calculated by setting the P / Si value in NaCl (150 mM) as 1. The relative ratio of the amount of immobilization of nucleic acid compounds when each solute was included was calculated by setting the P / Si value in NaCl (150 mM) as 1.

[0054] The calculation results of the relative ratio of the amount of nucleic acid compound immobilized are shown in Figure 6. Referring to Figure 6, it can be seen that the immobilization methods using aqueous solutions A and B immobilized a higher amount of nucleic acid compound than the immobilization methods using aqueous solutions C to I. This shows that sodium chloride immobilized a higher amount of nucleic acid compound than other solutes.

[0055] 6, it can be seen that the amount of immobilization when aqueous solution A, which has a composition close to that of a saturated aqueous solution of NaCl, is used is greater than the amount of immobilization when aqueous solution B is used. This indicates that the higher the concentration of sodium chloride, the greater the amount of immobilization of nucleic acid compounds, and that sodium chloride has the effect of promoting the immobilization of nucleic acid compounds.

[0056] Furthermore, referring to Figure 6, it can be seen that the immobilization amount was lower in the immobilization methods using aqueous solution F or aqueous solution G than in the immobilization method using aqueous solution D. Furthermore, it can be seen that almost no immobilization of nucleic acid compounds was observed when aqueous solution H was used. Therefore, since the amount of immobilized nucleic acid compounds tends to decrease when PB or HEPES is used, it was inferred that phosphate ions and HEPES may have an inhibitory effect on the immobilization of nucleic acid compounds.

[0057] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims. The inventions described in the claims of the original application are as follows: [1] A method for immobilizing a nucleic acid compound on a surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, comprising: providing an aqueous solution containing a nucleic acid compound and sodium chloride, wherein the nucleic acid compound comprises a polyaromatic moiety composed of a polyaromatic backbone and a linker structure attached to the polyaromatic backbone, and a nucleic acid moiety attached to the linker structure; and dropping the aqueous solution onto the surface of the sensor element; The method comprises: [2] 2. The method of claim 1, wherein the polycyclic aromatic skeleton is pyrene. [3] 3. The method according to claim 1, wherein the linker structure of the polycyclic aromatic moiety has a phosphate group at a terminal thereof, and the nucleic acid compound is bound to the nucleic acid moiety and the linker structure via the phosphate group. [4] The method according to claim 3, wherein the linker structure of the polycyclic aromatic moiety is a linker structure represented by the following formula (1) or the following formula (2): [ka]

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[10] A reagent kit used to immobilize a nucleic acid compound on a surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, comprising: a first container containing a nucleic acid compound comprising a polycyclic aromatic moiety composed of a polycyclic aromatic skeleton and a linker structure bonded to the polycyclic aromatic skeleton, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety; a second container containing an aqueous sodium chloride solution; The reagent kit comprises:

[11] The reagent kit according to claim 10, wherein the nucleic acid moiety is DNA or RNA.

[12] 12. The reagent kit according to claim 10, wherein the polycyclic aromatic skeleton is pyrene or a derivative thereof.

[13] 13. The reagent kit according to claim 10, wherein the linker structure of the polycyclic aromatic moiety is a linker structure represented by the following formula (5) or (6):

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[14] 14. The reagent kit according to claim 10, wherein the nucleic acid compound is a compound represented by the following formula (7) or (8):

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[15] 15. The reagent kit according to claim 10, wherein the concentration of the aqueous sodium chloride solution is 150 mM or more.

[16] 16. The reagent kit according to claim 10, wherein the aqueous sodium chloride solution does not contain phosphate ions and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid.

[17] 17. The reagent kit according to claim 10, wherein the aqueous sodium chloride solution does not contain an organic solvent.

[18] A sensor for use in carrying out the method according to any one of claims 1 to 9 or in using the reagent kit according to any one of claims 10 to 16, A sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite; a first container containing a first solution; a second container containing a second solution; a first flow path for supplying the first solution from the first container to a surface of the sensor element; a second flow path for supplying the second solution from the second container to the surface of the sensor element; a third flow path for draining liquid from the surface of the sensor element; Equipped with the first solution is an aqueous solution containing a nucleic acid compound and sodium chloride, wherein the nucleic acid compound comprises a polycyclic aromatic moiety composed of a polycyclic aromatic backbone and a linker structure bonded to the polycyclic aromatic backbone, and a nucleic acid moiety bonded to the linker structure of the polycyclic aromatic moiety; The sensor, wherein the second solution is an aqueous solution containing at least one of a buffer solution, an ionic liquid, a surfactant, and a chelating agent.

[19] A sensor for use in carrying out the method according to any one of claims 1 to 9 or in using the reagent kit according to any one of claims 10 to 17, A sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite; a first container containing a composition of a nucleic acid compound and sodium chloride, wherein the nucleic acid compound comprises a polyaromatic moiety composed of a polyaromatic backbone and a linker structure attached to the polyaromatic backbone, and a nucleic acid moiety attached to the linker structure of the polyaromatic moiety; a second container containing a second solution containing at least one of a buffer solution, an ionic liquid, a surfactant, and a chelating agent; a third container for receiving liquid discharged from the sensor element; a fourth container containing an aqueous sodium chloride solution; Equipped with the fourth container and the first container are connected by a fourth flow path for supplying the sodium chloride aqueous solution to the first container; the first container and the sensor element are connected by a first flow path for supplying a first solution, which is generated in the first container by dissolving the nucleic acid compound in the sodium chloride aqueous solution supplied by the fourth flow path, to a surface of the sensor element; the second container and the sensor element are connected by a second flow path for supplying the second solution from the second container to the surface of the sensor element; The sensor, wherein the sensor element and the third container are connected by a third flow path for discharging liquid from the surface of the sensor element. [Explanation of symbols]

[0058] 1. Sensor 2. First container 3. Second container 4. Sensor element 5. Third container 6. Fourth Container 7. Sensor element surface 8. Polycyclic aromatic skeleton 9. Linker structure (linker molecule) 10...Nucleic acid part 11... Nucleic acid compounds 21. First flow path 22 Second flow path 23. Third Flow Path 24. Fourth Flow

Claims

1. A method for immobilizing a nucleic acid compound on a surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, comprising: providing an aqueous solution containing a nucleic acid compound and sodium chloride, but free of phosphate ions and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid, wherein the nucleic acid compound comprises a polyaromatic moiety composed of a polyaromatic backbone and a linker structure bonded to the polyaromatic backbone, and a DNA moiety bonded to the linker structure; and dropping the aqueous solution onto the surface of the sensor element; Including, The sodium chloride concentration of the aqueous solution is 150 mM or more. The method.

2. The method of claim 1 , wherein the polycyclic aromatic skeleton is pyrene.

3. The method according to claim 1 or 2, wherein the linker structure of the polycyclic aromatic moiety has a phosphate group at its terminal, and the DNA moiety and the linker structure of the nucleic acid compound are bonded via the phosphate group.

4. The method according to claim 3 , wherein the linker structure of the polycyclic aromatic moiety is a linker structure represented by the following formula (1) or the following formula (2): 【Chemistry 1】 【Chemistry 2】

5. The method according to claim 4, wherein the nucleic acid compound is a compound represented by the following formula (3) or (4): 【Transformation 3】 【Chemistry 4】

6. The method according to any one of claims 1 to 5, wherein the aqueous solution does not contain an organic solvent.

7. The method according to any one of claims 1 to 6, further comprising: washing the surface of the sensor element by dropping a sodium chloride aqueous solution onto the surface of the sensor element onto which the aqueous solution has been dropped, thereby replacing the aqueous solution.

8. A reagent kit used to immobilize a nucleic acid compound on a surface of a sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite, comprising: a first container containing a nucleic acid compound comprising a polycyclic aromatic moiety composed of a polycyclic aromatic skeleton and a linker structure bonded to the polycyclic aromatic skeleton, and a DNA portion bonded to the linker structure of the polycyclic aromatic moiety; a second container containing an aqueous sodium chloride solution having a concentration of 150 mM or more; Equipped with The reagent kit does not contain phosphate ions and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid.

9. The reagent kit according to claim 8 , wherein the polycyclic aromatic skeleton is pyrene.

10. The reagent kit according to claim 8 or 9, wherein the linker structure of the polycyclic aromatic moiety is a linker structure represented by the following formula (5) or (6): 【Transformation 5】 【Transformation 6】

11. The reagent kit according to any one of claims 8 to 10, wherein the nucleic acid compound is a compound represented by the following formula (7) or the following formula (8): 【Transformation 7】 【Transformation 8】

12. The reagent kit according to any one of claims 8 to 11, wherein the aqueous sodium chloride solution does not contain an organic solvent.

13. A sensor for use in carrying out the method according to any one of claims 1 to 7 or in using the reagent kit according to any one of claims 8 to 12, A sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite; a first container containing a first solution; a second container containing a second solution; a first flow path for supplying the first solution from the first container to a surface of the sensor element; a second flow path for supplying the second solution from the second container to the surface of the sensor element; a third flow path for draining liquid from the surface of the sensor element; Equipped with the first solution is an aqueous solution containing a nucleic acid compound and sodium chloride at a concentration of 150 mM or more, but not containing phosphate ions or 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid, wherein the nucleic acid compound comprises a polycyclic aromatic moiety composed of a polycyclic aromatic skeleton and a linker structure bonded to the polycyclic aromatic skeleton, and a DNA moiety bonded to the linker structure of the polycyclic aromatic moiety; The sensor, wherein the second solution is an aqueous solution containing at least one of a buffer solution, an ionic liquid, a surfactant, and a chelating agent.

14. A sensor for use in carrying out the method according to any one of claims 1 to 7 or in using the reagent kit according to any one of claims 8 to 12, A sensor element made of graphene, graphene oxide, carbon nanotubes, or graphite; a first container containing a nucleic acid compound comprising a polycyclic aromatic moiety composed of a polycyclic aromatic skeleton and a linker structure bonded to the polycyclic aromatic skeleton, and a DNA portion bonded to the linker structure of the polycyclic aromatic moiety; a second container containing a second solution containing at least one of a buffer solution, an ionic liquid, a surfactant, and a chelating agent; a third container for receiving liquid discharged from the sensor element; a fourth container containing an aqueous sodium chloride solution having a concentration of 150 mM or more; Equipped with the nucleic acid compound and the sodium chloride aqueous solution do not contain phosphate ions and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid; the fourth container and the first container are connected by a fourth flow path for supplying the sodium chloride aqueous solution to the first container; the first container and the sensor element are connected by a first flow path for supplying a first solution, which is generated in the first container by dissolving the nucleic acid compound in the sodium chloride aqueous solution supplied by the fourth flow path, to a surface of the sensor element; the second container and the sensor element are connected by a second flow path for supplying the second solution from the second container to the surface of the sensor element; The sensor, wherein the sensor element and the third container are connected by a third flow path for discharging liquid from the surface of the sensor element.

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