Nucleic acid probe, chemical sensor, and detection method

Abstract

To provide a nucleic acid probe, a chemical sensor, and a detection method, each enabling a target substance to be detected with high sensitivity.SOLUTION: According to the embodiment, a nucleic acid probe comprises a first and a second nucleic acid. The first nucleic acid has a first to a seventh base sequence. The second base sequence binds to the first base sequence. The third base sequence binds to the second base sequence. The fourth base sequence is complementary to the third base sequence. The fifth base sequence is complementary to the second base sequence. The sixth base sequence binds to the fifth base sequence. The seventh base sequence is complementary to the sixth base sequence. A binding site for capturing a target substance is located between the fourth base sequence and the seventh base sequence. The second nucleic acid has an eighth base sequence complementary to at least a part of the first base sequence and at least a part of the second base sequence. The number of bases in the eighth base sequence is greater than the number of bases in the second base sequence.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] FIELD OF THE DISCLOSURE Embodiments of the present invention relate to nucleic acid probes, chemical sensors, and detection methods. [Background technology]

[0002] For example, Patent Document 1 proposes a cocaine aptamer capable of detecting cocaine. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-74995 A Summary of the Invention [Problem to be solved by the invention]

[0004] An object of embodiments of the present invention is to provide a nucleic acid probe, a chemical sensor, and a detection method capable of detecting a target substance with high sensitivity. [Means for solving the problem]

[0005] According to an embodiment of the present invention, a nucleic acid probe includes a first nucleic acid and a second nucleic acid. The first nucleic acid has a binding site for capturing a target substance. The second nucleic acid pairs with a portion of the first nucleic acid. The first nucleic acid has a first base sequence, a second base sequence, a third base sequence, a fourth base sequence, a fifth base sequence, a sixth base sequence, and a seventh base sequence. The second base sequence binds to the first base sequence. The third base sequence binds to the second base sequence. The fourth base sequence is complementary to the third base sequence. The fifth base sequence is complementary to the second base sequence. The sixth base sequence binds to the fifth base sequence. The seventh base sequence is complementary to the sixth base sequence. The binding site is located at least between the fourth base sequence and the seventh base sequence. The second nucleic acid has an eighth base sequence. The eighth base sequence is complementary to at least a portion of the first base sequence and at least a portion of the second base sequence. The number of bases in the eighth base sequence is greater than the number of bases in the second base sequence. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is a schematic diagram illustrating a chemical sensor according to an embodiment. [Diagram 2] FIG. 1 is a schematic diagram showing a nucleic acid probe according to an embodiment. [Diagram 3] FIG. 2 is a schematic diagram showing the structure of a nucleic acid probe according to an embodiment in a first state. [Figure 4] FIG. 2 is a schematic diagram showing a structure of a nucleic acid probe according to an embodiment in a state between a first state and a second state. [Diagram 5] FIG. 4 is a schematic diagram showing the structure of the nucleic acid probe according to the embodiment in a second state. [Figure 6] 6(a) to 6(d) are schematic diagrams illustrating a detection method according to an embodiment. [Figure 7] 1 is a table showing the simulation results of Reference Examples 1 to 8. [Figure 8] 13 is a table showing the simulation results of Reference Examples 9 to 13. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings are schematic or conceptual, and the relationship between the thickness and width of each part, the size ratio between parts, etc. are not necessarily the same as those in reality. Even when the same part is shown, the dimensions and ratios of each part may be different depending on the drawing. In this specification and each drawing, elements similar to those described above with reference to the previous drawings are given the same reference numerals and detailed descriptions thereof will be omitted as appropriate.

[0008] (Chemical Sensor) FIG. 1 is a schematic diagram illustrating a chemical sensor according to an embodiment. As shown in FIG. 1, a chemical sensor 100 according to the embodiment includes a substrate 10, a sensor element 20, a nucleic acid probe 30, a first electrode 41, and a second electrode .

[0009] The sensor element 20, the first electrode 41, and the second electrode 42 are each provided on the substrate 10. The first electrode 41 and the second electrode 42 are each electrically connected to the sensor element 20.

[0010] The chemical sensor 100 has, for example, a field effect transistor (FET) structure. One of the first electrode 41 and the second electrode 42 functions as a drain electrode. The other of the first electrode 41 and the second electrode 42 functions as a source electrode. A current flows between the first electrode 41 and the second electrode 42 through the sensor element 20.

[0011] The sensor element 20 is, for example, graphene. The substrate 10 is, for example, a silicon substrate. The graphene can be provided, for example, on the substrate 10 via an undercoat film (not shown). For example, a silicon oxide film can be used as the undercoat film. The undercoat film can also have a chemical catalytic function for forming graphene. The sensor element 20 can be, for example, an ion-sensitive field effect transistor (ISFET).

[0012] The nucleic acid probe 30 specifically recognizes the target substance 1. More specifically, the nucleic acid probe 30 has a binding site (binding site 31s described below) that captures the target substance 1. When the binding site captures the target substance 1, the structure of the nucleic acid probe 30 changes, allowing the target substance 1 to be detected. Note that the nucleic acid probe 30 may be capable of recognizing not only the target substance 1, but also substances having a similar structure, such as derivatives of the target substance 1. The structure of the nucleic acid probe 30 will be described below.

[0013] The nucleic acid probe 30 is located on the surface of the sensor element 20. The nucleic acid probe 30 is fixed to the surface of the sensor element 20, for example, via a linker (or a scaffold molecule) 25. The nucleic acid probe 30 is bonded to the linker 25. For example, pyrene can be used as the linker 25. The nucleic acid probe 30 being located on the surface of the sensor element 20 means that the nucleic acid probe 30 is bound, adsorbed, or brought into close proximity to the surface of the sensor element 20 by chemical or charge-induced attraction, hydrophobic interaction, or the like, and the nucleic acid probe 30 is fixed to the surface of the sensor element 20.

[0014] The surface of the sensor element 20 and the nucleic acid probe 30 are immersed in a solution 50. The solution 50 is, for example, an aqueous solution. The solution 50 is, for example, a buffer solution. Alternatively, the solution may contain, as its main component, a water-soluble organic solvent such as DMSO (dimethyl sulfoxide), DMF (N,N-dimethylformamide), or a lower alcohol.

[0015] The first electrode 41 and the second electrode 42 are covered with a protective film (not shown). The protective film prevents the first electrode 41 and the second electrode 42 from directly contacting the nucleic acid probe 30, the target substance 1, or the solution 50. The surface of the graphene may be covered with an insulator such as a phospholipid film, as necessary.

[0016] When the nucleic acid probe 30 captures the target substance 1, the electronic state of the sensor element 20 changes due to a structural change in the nucleic acid probe 30 caused by the capture of the target substance 1. By detecting this as a change in the current flowing between the first electrode 41 and the second electrode 42, the presence or absence of the target substance 1 in the sample and the concentration of the target substance 1 can be detected.

[0017] (Nucleic acid probe) FIG. 2 is a schematic diagram showing a nucleic acid probe according to an embodiment. 2, the nucleic acid probe 30 has a first nucleic acid 31 and a second nucleic acid 32. The first nucleic acid 31 has a binding site 31s that captures the target substance 1. The second nucleic acid 32 is capable of pairing with a portion of the first nucleic acid 31.

[0018] The first nucleic acid 31 and the second nucleic acid 32 are, for example, DNA (deoxyribonucleic acid). In the figure, "A" means adenine, "T" means thymine, "G" means guanine, and "C" means cytosine. The first nucleic acid 31 and the second nucleic acid 32 may be RNA (ribonucleic acid). In this case, uracil is included instead of thymine.

[0019] The first nucleic acid 31 is, for example, a cocaine aptamer. The cocaine aptamer is, for example, an aptamer capable of specifically recognizing a cocaine molecule. The cocaine aptamer is, for example, an aptamer capable of specifically interacting with a cocaine molecule. When the first nucleic acid 31 is a cocaine aptamer, the target substance 1 is a cocaine molecule or a methyl benzoate molecule having a structure similar to that of a cocaine molecule.

[0020] The first nucleic acid 31 has the first to seventh base sequences 31a to 31g. The number of bases in the first to seventh base sequences 31a to 31g is, for example, 20 or less. The first base sequence 31a is located, for example, at the 3' end or the 5' end of the first nucleic acid 31. In the following, an example in which the first base sequence 31a is located at the 3' end of the first nucleic acid 31 will be described, but the opposite is also possible. In the following, when describing the base sequence, it will be described in order from the 5' side to the 3' side. In this example, the "AAGAGAGA" portion corresponds to the first base sequence 31a. The number of bases in the first base sequence 31a is 2 or more. The number of bases in the first base sequence 31a may be, for example, 6 to 8. When the number of bases in the first base sequence 31a is 4 or more, it is preferable that the first base sequence 31a is not complementary to other base sequences in the first nucleic acid 31. In the following, an example in which the first nucleic acid 31 is composed of one chain will be described. The first nucleic acid 31 may be composed of multiple strands, with the first to third base sequences 31a to 31c being contained in the same strand, the fourth and seventh base sequences 31d and 31g being contained in the same strand, and the fifth and sixth base sequences 31e and 31f being contained in the same strand.

[0021] The second base sequence 31b is bound to the 5' end of the first base sequence 31a. The second base sequence 31b is continuous with the first base sequence 31a. In this example, the "GTCGCC" portion corresponds to the second base sequence 31b. The number of bases in the second base sequence 31b is 2 or more. Preferably, the number of bases in the second base sequence 31b is 4 or more.

[0022] The third base sequence 31c is bound to the 5' end of the second base sequence 31b via a spacer sequence (GG). The second base sequence 31b is located between the first base sequence 31a and the third base sequence 31c. In this example, the "GT" portion corresponds to the third base sequence 31c. The number of bases in the third base sequence 31c is 2 or more.

[0023] The fourth base sequence 31d is complementary to the third base sequence 31c. In this example, the fourth base sequence 31d is bound to the 5' end of the third base sequence 31c via the first loop portion 31p. In this example, the "GAA" portion corresponds to the first loop portion 31p. In this example, the "AC" portion corresponds to the fourth base sequence 31d. The number of bases in the fourth base sequence 31d is 2 or more. Note that the first loop portion 31p may not be included. The third base sequence 31c and the fourth base sequence 31d may be separate chains.

[0024] The fifth base sequence 31e is complementary to the second base sequence 31b. The fifth base sequence 31e is located, for example, at the end of the first nucleic acid 31 opposite to the first base sequence 31a. In this example, the fifth base sequence 31e is located at the 5' end of the first nucleic acid 31. Note that another base sequence may be bound to the 5' end of the fifth base sequence 31e. In this example, the "GGCGAC" portion corresponds to the fifth base sequence 31e. The number of bases in the fifth base sequence 31e is 2 or more.

[0025] The sixth base sequence 31f is linked to the fifth base sequence 31e via a spacer sequence (A). In this example, the "AGGA" portion corresponds to the sixth base sequence 31f. The sixth base sequence 31f has two or more bases.

[0026] The seventh base sequence 31g is complementary to the sixth base sequence 31f. In this example, the seventh base sequence 31g is bound to the 3' end of the sixth base sequence 31f via the second loop portion 31q. In this example, the "AAA" portion corresponds to the second loop portion 31q. In this example, the "TCCT" portion corresponds to the seventh base sequence 31g. The seventh base sequence 31g has two or more bases. The second loop portion 31q may not be included. The sixth base sequence 31f and the seventh base sequence 31g may be separate chains.

[0027] The binding site 31s is present between the stems of the first nucleic acid 31. The binding site 31s is composed of bases between the second sequence 31b and the third sequence 31c, bases between the fourth base sequence 31d and the seventh base sequence 31g, and bases between the fifth base sequence 31e and the sixth base sequence 31f. In this example, the "TCA" portion corresponds to the binding site 31s.

[0028] The second nucleic acid 32 has an eighth base sequence 32a. The number of bases in the eighth base sequence 32a is, for example, 20 or less. The number of bases in the second base sequence 32a may be, for example, 10 to 13. The eighth base sequence 32a is complementary to at least a part of the first base sequence 31a and at least a part of the second base sequence 31b. In this example, the eighth base sequence 32a is complementary to the entire first base sequence 31a and a part of the second base sequence 31b. In this example, the part "TCTCTCTTGGCGA" (the entire second nucleic acid 32) corresponds to the eighth base sequence 32a. Of these, the part "GGCGA" is a part complementary to the second base sequence 31b, and the part "TCTCTCTT" is a part complementary to the first base sequence 31a. The second nucleic acid 32 may have another base sequence bound to the 3' end of the eighth base sequence 32a, or may have another base sequence bound to the 5' end of the eighth base sequence 32a.

[0029] The number of bases in the eighth base sequence 32a is greater than the number of bases in the second base sequence 31b and the number of bases in the fifth base sequence 31e. The number of bases in the eighth base sequence 32a is 4 or more. The number of bases in the eighth base sequence 32a is preferably 10 or more. The number of bases in the portion of the eighth base sequence 32a that is complementary to the first base sequence 31a is 2 or more. The number of bases in the portion of the eighth base sequence 32a that is complementary to the second base sequence 31b is preferably 2 or more than the number of bases in the second base sequence 31b. The number of bases in the portion of the 8th base sequence 32a that is complementary to the first base sequence 31a may be more than the number of bases in the portion of the 8th base sequence 32a that is complementary to the second base sequence 31b, may be less than the number of bases in the portion of the 8th base sequence 32a that is complementary to the second base sequence 31b, or may be the same as the number of bases in the portion of the 8th base sequence 32a that is complementary to the second base sequence 31b.

[0030] A spacer sequence may be provided between each of the base sequences. The number of bases contained in the spacer sequence is, for example, 1 to 3.

[0031] FIG. 3 is a schematic diagram showing the structure of the nucleic acid probe according to the embodiment in a first state. FIG. 4 is a schematic diagram showing the structure of the nucleic acid probe according to the embodiment in a state between a first state and a second state. FIG. 5 is a schematic diagram showing the structure of the nucleic acid probe according to the embodiment in a second state. As shown in FIGS. 3 to 5, the nucleic acid probe 30 has a first state and a second state.

[0032] 3, the first state is a state in which the second nucleic acid 32 is paired with the first nucleic acid 31. As shown in FIG. 5, the second state is a state in which the second nucleic acid 32 is dissociated from the first nucleic acid 31.

[0033] In the second state, the first nucleic acid 31 forms a higher-order structure having a first stem portion 31x, a second stem portion 31y, and a third stem portion 31z. The first stem portion 31x is formed by pairing the second base sequence 31b with the fifth base sequence 31e. The second stem portion 31y is formed by pairing the third base sequence 31c with the fourth base sequence 31d. The second stem portion 31y forms a stem-loop structure together with the first loop portion 31p, for example. The third stem portion 31z is formed by pairing the sixth base sequence 31f with the seventh base sequence 31g. The third stem portion 31z forms a stem-loop structure together with the second loop portion 31q, for example.

[0034] In the first state, the first nucleic acid 31 forms a structure that does not have at least the first stem portion 31x. In this example, in the first state, the first nucleic acid 31 forms a structure that has the second stem portion 31y and the third stem portion 31z, and does not have the first stem portion 31x. In the first state, the first nucleic acid 31 may not have the second stem portion 31y and the third stem portion 31z. In the first state, the second nucleic acid 32 inhibits the formation of the first stem portion 31x by pairing with the second base sequence 31b of the first nucleic acid 31.

[0035] When the binding site 31s captures the target substance 1 in the first state, the nucleic acid probe 30 enters the second state. More specifically, as shown in Fig. 3 and Fig. 4, when the binding site 31s captures the target substance 1 in the first state, the second base sequence 31b of the first nucleic acid 31 pairs with the fifth base sequence 31e, and the pairing between the eighth base sequence 32a of the second nucleic acid 32 and the second base sequence 31b of the first nucleic acid 31 is dissolved. As shown in Fig. 5, when the second base sequence 31b of the first nucleic acid 31 pairs completely with the fifth base sequence 31e, the pairing between the eighth base sequence 32a of the second nucleic acid 32 and the first base sequence 31a of the first nucleic acid 31 is dissolved, and the second nucleic acid 32 is dissociated from the first nucleic acid 31.

[0036] For example, when the nucleic acid probe 30 is in the second state, it maintains the second state even if the binding site 31s does not capture the target substance 1. For example, when the concentration of the second nucleic acid 32 around the first nucleic acid 31 becomes higher than a predetermined value in the second state, the nucleic acid probe 30 is in the first state. As a result, for example, the nucleic acid probe 30 (in the second state) after detecting the target substance 1 can be brought into contact with a solution containing a high concentration of the second nucleic acid 32, and then the concentration of the second nucleic acid 32 can be reduced or the excess second nucleic acid 32 can be removed by washing, thereby returning the nucleic acid probe 30 to the first state. In this way, the state of the nucleic acid probe 30 can be reversibly changed between the first state and the second state, allowing the nucleic acid probe 30 to be used repeatedly.

[0037] In this example, the higher-order structure is a three-way junction. The higher-order structure may be a four-way junction. That is, in the second state, the first nucleic acid 31 may form a higher-order structure having other stem portions in addition to the first to third stem portions 31x to 31z.

[0038] The melting temperature (Tm) of the first nucleic acid 31 in the second state is preferably 25° C. or higher. If the melting temperature (Tm) of the first nucleic acid 31 in the second state is 25° C. or higher, the nucleic acid probe 30 can be in the second state at room temperature (the temperature at which the detection of the target substance 1 is performed). In other words, the nucleic acid probe 30 is easy to use at room temperature (the temperature at which the detection of the target substance 1 is performed). In this example, the melting temperature of the first nucleic acid 31 in the second state is 52.3° C. The melting temperature can be calculated, for example, by OligoAnalyzer Tool, a calculation tool published on the website of Integrated DNA Technologies acquires Archer. Alternatively, it can be experimentally measured by ultraviolet absorption measurement, circular dichroism measurement, differential calorimetry, or the like.

[0039] As described above, the nucleic acid probe 30 is fixed on the surface of the sensor element 20. It may be the first nucleic acid 31 or the second nucleic acid 32 that is fixed on the surface of the sensor element 20. When the first nucleic acid 31 is fixed on the surface of the sensor element 20, the 3' end of the first nucleic acid 31 may be fixed on the surface of the sensor element 20, or the 5' end of the first nucleic acid 31 may be fixed on the surface of the sensor element 20. When the second nucleic acid 31 is fixed on the surface of the sensor element 20, the 3' end of the second nucleic acid 32 may be fixed on the surface of the sensor element 20, or the 5' end of the second nucleic acid 32 may be fixed on the surface of the sensor element 20.

[0040] In order to detect a target substance with high sensitivity, it is preferable that the nucleic acid probe 30 can stably maintain the first state (a state in which the target substance is not captured) until the binding site 31s captures the target substance 1. However, depending on the structures of the first nucleic acid 31 and the second nucleic acid 32, there are cases in which the first state cannot be stably maintained until the binding site 31s captures the target substance 1.

[0041] Therefore, in the embodiment, the number of bases of the eighth base sequence 32a, which is the portion of the second nucleic acid 32 that pairs with the first nucleic acid 31, is made larger than the number of bases of the second base sequence 31b, which is the portion forming the first stem portion 31x of the first nucleic acid 31. This makes it possible to make the number of base pairs that pair between the first nucleic acid 31 and the second nucleic acid 32 larger than the number of base pairs that pair between the second base sequence 31b and the fifth base sequence 31e. Therefore, the first state in which the second nucleic acid 32 pairs with the first nucleic acid 31 can be stably maintained until the binding site 31s captures the target substance 1. This makes it possible to detect the target substance 1 with high sensitivity.

[0042] (Detection method) 6(a) to 6(d) are schematic diagrams illustrating a detection method according to an embodiment. 6(a) to 6(d), the detection method according to the embodiment is a method for detecting a target substance 1 using the nucleic acid probe 30 according to the embodiment described above. The detection method according to the embodiment includes first to fourth steps.

[0043] 6(a) shows the first step. The first step is a step of immobilizing one of the first nucleic acid 31 and the second nucleic acid 32 on the surface of the sensor element 20. In this example, the first nucleic acid 31 is immobilized on the surface of the sensor element 20 in the first step. For example, by contacting the surface of the sensor element 20 with a reagent (solution) containing one of the first nucleic acid 31 and the second nucleic acid 32, it is possible to immobilize one of the first nucleic acid 31 and the second nucleic acid 32 on the surface of the sensor element 20. Excess reagent can be removed by washing.

[0044] FIG. 6(b) shows the second step. The second step is performed after the first step. The second step is a step of pairing one of the first nucleic acid 31 and the second nucleic acid 32 immobilized on the surface of the sensor element 20 with the other of the first nucleic acid 31 and the second nucleic acid 32 by contacting the surface of the sensor element 20 with a first solution 51 in which the concentration of the other of the first nucleic acid 31 and the second nucleic acid 32 is a first concentration. In this example, in the second step, the first solution 51 in which the concentration of the second nucleic acid 32 is a first concentration is contacted with the surface of the sensor element 20 to pair the first nucleic acid 31 immobilized on the surface of the sensor element 20 with the second nucleic acid 32. The first concentration is, for example, 100 nM or more, more preferably 1 μM or more.

[0045] FIG. 6(c) shows the third step. The third step is performed after the second step. The third step is a step of replacing the surface of the sensor element 20 with a second solution 52 having a second concentration in which the concentration of the other of the first nucleic acid 31 and the second nucleic acid 32 is lower than the first concentration. In this example, in the third step, the surface of the sensor element 20 is replaced with a second solution 52 having a second concentration in which the concentration of the released second nucleic acid 32 is lower than the first concentration. The second concentration is, for example, 100 fM or more and 10 nM or less. Alternatively, the second solution 52 may not contain the released second nucleic acid 32.

[0046] FIG. 6(d) shows the fourth step. The fourth step is performed after the third step. The fourth step is a step of detecting the target substance 1 with the surface of the sensor element 20 covered with the second solution 52. A solution containing a specimen is introduced and brought into contact with the sensor element 20, or a gas containing a specimen is brought into contact with the second solution 52 and dissolved. If the specimen contains the target substance 1, the state of the first nucleic acid 31 and the second nucleic acid 32 changes irreversibly in the vicinity of the sensor element 20. By examining the change in the electrical properties between the first electrode 41 and the second electrode 42, the target substance 1 can be detected with high sensitivity.

[0047] With this method, a nucleic acid probe 30 can be formed on the surface of the sensor element 20 using a reagent (solution) containing the first nucleic acid 31 and a reagent (solution) containing the second nucleic acid 32. Furthermore, by detecting the target substance 1 with the surface of the sensor element 20 covered with the second solution 52, the first nucleic acid 31 is more likely to change from the first state to the second state when the target substance 1 is captured.

[0048] In this example, in the first step, the first nucleic acid 31 is fixed on the surface of the sensor element 20, and in the second step, the second nucleic acid 32 is paired with the first nucleic acid 31 fixed on the surface of the sensor element 20. In the first step, the second nucleic acid 32 may be fixed on the surface of the sensor element 20, and in the second step, the first nucleic acid 31 may be paired with the second nucleic acid 32 fixed on the surface of the sensor element 20.

[0049] (Reference example) FIG. 7 is a table showing the simulation results of Reference Examples 1 to 8. In Reference Examples 1 to 8, it was assumed that the first base sequence 31a in the first nucleic acid 31 shown in FIG. 2 was as shown in FIG. 7, and the eighth base sequence 32a in the second nucleic acid 32 shown in FIG. 2 was as shown in FIG. 7. In Reference Examples 1 to 8, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 10 μM, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 1 nM, and the melting temperature (Tm) of the structure in which the first base sequence 31a and the second nucleic acid 32 (the eighth base sequence 32a) are paired after the formation of a higher-order structure were calculated by simulation, and the results are shown in FIG. 7. The melting temperature (Tm) of the first nucleic acid 31 in the second state is 52.3° C.

[0050] 7, when the number of bases in the eighth base sequence 32a was increased, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 10 μM and the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 1 nM were higher than when the number of bases in the eighth base sequence 32a was smaller. This suggests that when the number of bases in the eighth base sequence 32a was increased, the nucleic acid probe 30 can maintain the first state (i.e., a state in which the target substance 1 is not captured) more stably than when the number of bases in the eighth base sequence 32a was smaller.

[0051] More specifically, as shown in FIG. 7, in Reference Examples 1 to 3, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 10 μM was higher than the melting temperature of the first nucleic acid 31 in the second state. This suggests that the nucleic acid probe 30 can maintain the first state (i.e., the state in which the target substance 1 is not captured) more stably in Reference Examples 1 to 3 than in Reference Examples 4 to 8. Furthermore, in Reference Examples 1 to 3, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 1 nM was 30° C. or higher, which is above room temperature. This suggests that the nucleic acid probe 30 can maintain the first state (i.e., the state in which the target substance 1 is not captured) stably in Reference Examples 1 to 3 even when the concentration of the second nucleic acid 32 was 1 nM. However, if the formation of higher-order structures of the first nucleic acid 31 competes, a strand exchange reaction may occur, causing a situation in which the first state is not stably maintained. When the 7th A and the 21st A from the 5' side are brought closer together by the binding of the target substance 1 to the first nucleic acid 31, the two Gs forming the spacer sequence between the third base sequence 31c and the second base sequence 31b form GA mismatches with these two A. As a result, as shown in FIG. 4, when the second stem portion 31y side of the first stem portion 31x starts to be continuously formed from the second stem portion 31y via the two GA mismatches, the first stem portion 31x is formed as it is by replacing the base pair with the 8th base sequence 32a.

[0052] FIG. 8 is a table showing the simulation results of Reference Examples 9 to 13. As shown in FIG. 8, in Reference Examples 9 to 13, it was assumed that in the first nucleic acid 31 shown in FIG. 2, the first base sequence 31a was as shown in FIG. 8, and in the second nucleic acid 32 shown in FIG. 2, the eighth base sequence 32a was as shown in FIG. 8. In Reference Examples 9 to 13, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 is 10 μM, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 is 1 nM, and the melting temperature (Tm) of the structure in which the first base sequence 31a and the second nucleic acid 32 (the eighth base sequence 32a) are paired after the formation of the higher-order structure were calculated by simulation, and the results are shown in FIG. 8. The melting temperature (Tm) of the first nucleic acid 31 in the second state is 52.3° C.

[0053] 8, when the number of bases in the eighth base sequence 32a was increased, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 10 μM and the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 1 nM were higher than when the number of bases in the eighth base sequence 32a was smaller. This suggests that when the number of bases in the eighth base sequence 32a was increased, the nucleic acid probe 30 could maintain the first state (i.e., a state in which the target substance 1 is not captured) more stably than when the number of bases in the eighth base sequence 32a was smaller.

[0054] More specifically, as shown in FIG. 8, in Reference Examples 9 and 10, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 10 μM was higher than the melting temperature of the first nucleic acid 31 in the second state. This suggests that the nucleic acid probe 30 can maintain the first state (i.e., the state in which the target substance 1 is not captured) more stably in Reference Examples 9 and 10 compared to Reference Examples 11 to 13. Also, in Reference Examples 9 and 10, the melting temperature (Tm) of the nucleic acid probe 30 in the first state when the concentration of the second nucleic acid 32 was 1 nM was 30° C. or higher. This suggests that the nucleic acid probe 30 can maintain the first state (i.e., the state in which the target substance 1 is not captured) stably in Reference Examples 9 and 10 even when the concentration of the second nucleic acid 32 was 1 nM. However, if the formation of higher-order structures of the first nucleic acid 31 competes, a strand exchange reaction may occur, causing a situation in which the first state is not stably maintained. When the 7th A and the 21st A from the 5' side are brought closer together by the binding of the target substance 1 to the first nucleic acid 31, the two Gs forming the spacer sequence between the third base sequence 31c and the second base sequence 31b form GA mismatches with these two A. As a result, as shown in FIG. 4, when the second stem portion 31y side of the first stem portion 31x starts to be continuously formed from the second stem portion 31y via the two GA mismatches, the first stem portion 31x is formed as it is by replacing the base pair with the 8th base sequence 32a.

[0055] The embodiment may include the following features.

[0056] (Configuration 1) a first nucleic acid having a binding site for capturing a target substance; A second nucleic acid that pairs with a portion of the first nucleic acid; Equipped with The first nucleic acid is A first base sequence; a second base sequence bound to the first base sequence; a third base sequence bound to the second base sequence; a fourth base sequence complementary to the third base sequence; a fifth base sequence complementary to the second base sequence; a sixth base sequence bound to the fifth base sequence; a seventh base sequence complementary to the sixth base sequence; having the binding site is located at least between the fourth base sequence and the seventh base sequence; the second nucleic acid has an eighth base sequence complementary to at least a portion of the first base sequence and at least a portion of the second base sequence; A nucleic acid probe, wherein the number of bases in the eighth base sequence is greater than the number of bases in the second base sequence.

[0057] (Configuration 2) The nucleic acid probe comprises: a first state in which the second nucleic acid is paired with the first nucleic acid; a second state in which the second nucleic acid dissociates from the first nucleic acid and the first nucleic acid forms a higher-order structure having a first stem portion in which the second base sequence and the fifth base sequence are paired, a second stem portion in which the third base sequence and the fourth base sequence are paired, and a third stem portion in which the sixth base sequence and the seventh base sequence are paired; having The nucleic acid probe according to claim 1, wherein the nucleic acid probe is in the first state and when the binding site captures the target substance, the nucleic acid probe is in the second state.

[0058] (Configuration 3) 3. The nucleic acid probe according to claim 2, wherein the melting temperature of the first nucleic acid in the second state is 25° C. or higher.

[0059] (Configuration 4) 4. The nucleic acid probe according to any one of configurations 1 to 3, wherein the first nucleic acid is a cocaine aptamer.

[0060] (Configuration 5) 5. The nucleic acid probe according to any one of configurations 1 to 4, wherein the fourth sequence is connected to the seventh sequence via a spacer sequence.

[0061] (Configuration 6) the third base sequence binds to the fourth base sequence, The nucleic acid probe according to any one of configurations 1 to 5, wherein the sixth base sequence is bound to the seventh base sequence.

[0062] (Configuration 7) The first nucleic acid is a chain including the first base sequence, the second base sequence, and the third base sequence; A chain including the fourth base sequence and the seventh base sequence; A chain including the fifth base sequence and the sixth base sequence; 6. The nucleic acid probe according to any one of configurations 1 to 5, comprising:

[0063] (Configuration 8) A nucleic acid probe according to any one of configurations 1 to 7, A sensor element; A first electrode electrically connected to the sensor element; A second electrode electrically connected to the sensor element; Equipped with The nucleic acid probe is located on a surface of the sensor element.

[0064] (Configuration 9) A method for detecting a target substance using the nucleic acid probe according to any one of configurations 1 to 7, a first step of immobilizing one of the first nucleic acid and the second nucleic acid on a surface of a sensor element; a second step of contacting a first solution having a first concentration of the other of the first nucleic acid and the second nucleic acid with the surface of the sensor element, thereby pairing the other with the one immobilized on the surface of the sensor element; a third step of replacing the surface of the sensor element with a second solution having a second concentration, the second concentration being lower than the first concentration; a fourth step of detecting the target substance while covering a surface of the sensor element with the second solution; The detection method comprising:

[0065] As described above, according to the embodiments, it is possible to provide a nucleic acid probe, a chemical sensor, and a detection method capable of detecting a target substance with high sensitivity.

[0066] Although some 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 implemented 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 in the scope and spirit of the invention, and are included in the scope of the invention and its equivalents described in the claims. [Explanation of symbols]

[0067] 1: Target substance 10: Substrate 20: Sensor element 25: Linker 30: Nucleic acid probe 31: 1st nucleic acid 31a: First base sequence 31b: Second base sequence 31c: Third base sequence 31d: 4th base sequence 31e: 5th base sequence 31f: 6th base sequence 31g: 7th base sequence 31p: First loop section 31q: Second loop section 31s: Binding site 31x: First stem section 31y: Second stem section 31z: 3rd stem section 32:Second nucleic acid 32a: 8th base sequence 41: 1st electrode 42:Second electrode 50: Solution 51: First solution 52: Second solution 100: Chemical sensor

Claims

1. a first nucleic acid having a binding site for capturing a target substance; a second nucleic acid that pairs with a portion of the first nucleic acid; Equipped with The first nucleic acid is A first base sequence; a second base sequence bound to the first base sequence; a third base sequence bound to the second base sequence; and a fourth base sequence complementary to the third base sequence; a fifth base sequence complementary to the second base sequence; a sixth base sequence bound to the fifth base sequence; a seventh base sequence complementary to the sixth base sequence; having the binding site is located at least between the fourth base sequence and the seventh base sequence; the second nucleic acid has an eighth base sequence complementary to at least a portion of the first base sequence and at least a portion of the second base sequence; A nucleic acid probe, wherein the number of bases in the eighth base sequence is greater than the number of bases in the second base sequence.

2. The nucleic acid probe comprises: a first state in which the second nucleic acid is paired with the first nucleic acid; a second state in which the second nucleic acid dissociates from the first nucleic acid and the first nucleic acid forms a higher-order structure having a first stem portion in which the second base sequence and the fifth base sequence are paired, a second stem portion in which the third base sequence and the fourth base sequence are paired, and a third stem portion in which the sixth base sequence and the seventh base sequence are paired; having The nucleic acid probe according to claim 1 , which is in the first state and when the binding site captures the target substance, the nucleic acid probe is in the second state.

3. The nucleic acid probe according to claim 2 , wherein the melting temperature of the first nucleic acid in the second state is 25° C. or higher.

4. The nucleic acid probe of claim 1 , wherein the first nucleic acid is a cocaine aptamer.

5. The nucleic acid probe of claim 1 , wherein the fourth sequence is connected to the seventh sequence via a spacer sequence.

6. the third base sequence binds to the fourth base sequence, The nucleic acid probe according to claim 5 , wherein the sixth base sequence is bound to the seventh base sequence.

7. The first nucleic acid is a chain including the first base sequence, the second base sequence, and the third base sequence; A chain including the fourth base sequence and the seventh base sequence; A chain including the fifth base sequence and the sixth base sequence; The nucleic acid probe according to claim 5 ,

8. A nucleic acid probe according to any one of claims 1 to 7, A sensor element; A first electrode electrically connected to the sensor element; A second electrode electrically connected to the sensor element; Equipped with The nucleic acid probe is located on a surface of the sensor element.

9. A method for detecting a target substance using the nucleic acid probe according to any one of claims 1 to 7, comprising: a first step of immobilizing one of the first nucleic acid and the second nucleic acid on a surface of a sensor element; a second step of contacting a first solution having a first concentration of the other of the first nucleic acid and the second nucleic acid with the surface of the sensor element, thereby pairing the other with the one immobilized on the surface of the sensor element; a third step of replacing the surface of the sensor element with a second solution having a second concentration, the second concentration being lower than the first concentration; a fourth step of detecting the target substance while covering a surface of the sensor element with the second solution; The detection method comprising:

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