Nucleic acid detection method, nucleic acid detection system, and substrate

Abstract

To provide a nucleic acid detection method, a nucleic acid detection system, and a substrate that allow high-sensitivity detection of a target nucleic acid included in a vesicle.SOLUTION: In one embodiment, a nucleic acid detection method includes four steps. A first step comprises preparing a dispersion by converting a sample solution, which contains saline, vesicles, an amplification reagent, and an amplification detection reagent, into droplets and dispersing them in oil. A second step comprises applying the dispersion to a substrate having lipase adhered to surfaces of a plurality of electrodes of a base body provided with a support and the electrodes on the surface of the support, and capturing the droplets at the electrodes by dielectrophoresis through application of an AC voltage to the electrodes, thereby decomposing a lipid membrane by the lipase that has entered into the captured droplets. A third step comprises amplifying a target nucleic acid within the droplets. A fourth step comprises identifying droplets that contained vesicles enclosing the target nucleic acid, based on detection results of the amplification detection reagent.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] SUMMARY OF THE INVENTION Embodiments of the present invention relate to a nucleic acid detection method, a nucleic acid detection system, and a substrate. [Background technology]

[0002] Extracellular vesicles (EVs) have attracted attention as biomarkers for use in early detection of diseases and recurrence monitoring. Highly sensitive detection of target nucleic acids contained in vesicles such as EVs is required. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2020-046284 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 detection method, a nucleic acid detection system, and a substrate that are capable of detecting a target nucleic acid contained in a vesicle with high sensitivity. [Means for solving the problem]

[0005] According to an embodiment of the present invention, a nucleic acid detection method is a method for detecting a target nucleic acid contained inside a vesicle covered with a lipid membrane. The nucleic acid detection method includes a first step, a second step, a third step, and a fourth step. In the first step, a dispersion liquid is prepared. The dispersion liquid includes oil and a sample liquid dispersed as droplets in the oil. The sample liquid includes salt water, the vesicles, an amplification reagent capable of amplifying the target nucleic acid, and an amplification detection reagent capable of detecting the amplification of the target nucleic acid. In the second step, the dispersion liquid is applied to a substrate. The substrate includes a base and lipase. The base includes a support and a plurality of electrodes provided on the surface of the support. The lipase is attached to the surfaces of the plurality of electrodes. In the second step, an AC voltage is applied to the plurality of electrodes to capture the droplets on the plurality of electrodes by dielectrophoresis. In the third step, the lipid membrane is decomposed by the lipase that has penetrated into the droplets captured on the plurality of electrodes. In the third step, the target nucleic acid is amplified inside the droplet, and in the fourth step, the droplet containing the vesicle encapsulating the target nucleic acid is identified based on the detection result of the amplification detection reagent.

[0006] According to an embodiment of the present invention, a nucleic acid detection system is a system for detecting target nucleic acids contained inside vesicles covered with a lipid membrane. The nucleic acid detection system includes a substrate preparation device, a dispersion preparation device, and a nucleic acid detection device. The substrate preparation device prepares a substrate having a support and a plurality of electrodes provided on the surface of the support by applying a solution containing lipase to the surfaces of the plurality of electrodes, thereby preparing a substrate having lipase attached to the surfaces of the plurality of electrodes. The dispersion preparation device prepares a dispersion by dispersing droplets of a sample liquid in oil. The sample liquid includes salt water, the vesicles, an amplification reagent capable of amplifying the target nucleic acid, and an amplification detection reagent capable of detecting the amplification of the target nucleic acid. The nucleic acid detection device applies the dispersion to the substrate. The nucleic acid detection device applies an AC voltage to the plurality of electrodes to capture the droplets on the plurality of electrodes by dielectrophoresis. The nucleic acid detection device decomposes the lipid membrane using the lipase that has penetrated into the droplets captured on the plurality of electrodes. The nucleic acid detection device amplifies the target nucleic acid inside the droplets. The nucleic acid detection device identifies the droplets containing the vesicles encapsulating the target nucleic acid based on the detection results of the amplification detection reagent.

[0007] According to an embodiment of the present invention, the substrate includes a substrate and a lipase. The substrate has a support and a plurality of electrodes provided on the surface of the support. The lipase is attached to the surfaces of the plurality of electrodes. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a flowchart showing an example of a nucleic acid detection method according to an embodiment. [Figure 2] FIG. 2 is an explanatory diagram showing the first step of the nucleic acid detection method according to the embodiment. [Figure 3] FIG. 2 is an explanatory diagram showing a substrate used in the second step of the nucleic acid detection method according to the embodiment. [Figure 4] 4(a) to 4(d) are explanatory views showing the second to fourth steps of the nucleic acid detection method according to the embodiment. [Figure 5]1 is a block diagram schematically illustrating a nucleic acid detection system according to an embodiment. [Figure 6] FIG. 2 is an explanatory diagram illustrating a substrate preparation device of the nucleic acid detection system according to the embodiment. [Figure 7] FIG. 2 is an explanatory diagram illustrating a dispersion liquid preparation device of the nucleic acid detection system according to the embodiment. [Figure 8] 8(a) and 8(b) are explanatory diagrams showing a nucleic acid detection device of a nucleic acid detection system according to an embodiment. [Figure 9] 9(a) and 9(b) are explanatory diagrams showing a nucleic acid detection device of a nucleic acid detection system according to an embodiment. [Figure 10] 10(a) and 10(b) are explanatory diagrams showing a nucleic acid detection device of a nucleic acid detection system according to an embodiment. [Figure 11] FIG. 2 is an explanatory diagram illustrating a droplet recovery device of the nucleic acid detection system according to the embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0009] 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 may be different depending on the drawing. In this specification and in each drawing, elements similar to those previously described with reference to the previous drawings are designated by the same reference numerals, and detailed descriptions thereof will be omitted where appropriate.

[0010] (Nucleic acid detection method) FIG. 1 is a flowchart showing an example of a nucleic acid detection method according to an embodiment. FIG. 2 is an explanatory diagram showing the first step of the nucleic acid detection method according to the embodiment. FIG. 3 is an explanatory diagram showing a substrate used in the second step of the nucleic acid detection method according to the embodiment. 4(a) to 4(d) are explanatory views showing the second to fourth steps of the nucleic acid detection method according to the embodiment. The nucleic acid detection method according to the embodiment is a method for detecting a target nucleic acid contained inside a vesicle covered with a lipid membrane. The vesicle is, for example, an extracellular vesicle. The vesicle is covered with a lipid membrane. The vesicle is, for example, covered with a lipid bilayer membrane. The target nucleic acid may be DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). The target nucleic acid is, for example, miRNA (microRNA).

[0011] As shown in FIG. 1, the nucleic acid detection method according to the embodiment includes a first step, a second step, a third step, and a fourth step.

[0012] In the first step, a dispersion liquid 25 is prepared. As shown in FIG. 2, the dispersion liquid 25 is prepared by dispersing droplets of a specimen liquid 26 in oil 27. The specimen liquid 26 contains, for example, a salt solution 26a, vesicles 26b, an amplification reagent 26c, and an amplification detection reagent 26d. The salt solution 26a contains water and a salt. Here, a salt is a compound consisting of a negative acid component and a positive base component. Examples of salts include sodium chloride, magnesium chloride, magnesium sulfate, and potassium chloride.

[0013] The amplification reagent 26c is a reagent capable of amplifying a target nucleic acid. When the target nucleic acid is DNA, the amplification reagent 26c is a DNA amplification reagent. When the target nucleic acid is RNA, the amplification reagent 26c is an RNA amplification reagent. The amplification reagent 26c includes, for example, nucleotides, a polymerase, and a primer.

[0014] The amplification detection reagent 26d is a reagent capable of detecting the amplification of a target nucleic acid. The amplification detection reagent 26d is, for example, a fluorescent probe such as SYBR (registered trademark) Green or TaqMan (registered trademark). The fluorescent probe emits fluorescence when the target nucleic acid is amplified, for example.

[0015] The specimen liquid 26 may further contain a surface marker detection reagent 26e. The surface marker detection reagent 26e is a reagent capable of detecting surface markers of vesicles. The surface marker detection reagent 26e is a reagent capable of detecting the presence or absence of vesicles. The specimen liquid 26 does not necessarily need to contain the surface marker detection reagent 26e.

[0016] Dispersion liquid 25 contains a plurality of droplets of specimen liquid 26. Preferably, one droplet contains one vesicle 26b. Some droplets may contain two or more vesicles 26b, or may not contain any vesicles 26b. If the density of droplets of specimen liquid 26 in dispersion liquid 25 is too high, the dispersion liquid 25 may be diluted with oil.

[0017] In the second step, the dispersion liquid 25 is applied to the substrate 15. In the second step, for example, the substrate 15 is first prepared. As shown in Fig. 3, the substrate 15 has a structure in which a lipase 17a is attached to the surface of a base 16. In Fig. 3, the substrate 15 has a structure in which a solution 17 containing lipase 17a and water 17b is attached to the surface of the base 16.

[0018] The base 16 has, for example, a support 16a, a plurality of electrodes 16b, and a protective film 16c. The support 16a is located at the bottom of the base 16. The plurality of electrodes 16b and the protective film 16c are each provided on the surface (upper surface) of the support 16a. The plurality of electrodes 16b are each exposed from the protective film 16c. The protective film 16c is hydrophobic. The support 16a is, for example, a printed wiring board or a ceramic wiring board. The plurality of electrodes 16b includes, for example, a metal. The protective film 16c includes, for example, a resin such as polyimide. The number of the plurality of electrodes 16b is, for example, 10,000 or more. The number of the plurality of electrodes 16b is, for example, approximately 1 million.

[0019] Lipase 17a (solution 17 containing lipase 17a) is attached to the surfaces of the plurality of electrodes 16b of substrate 16. Lipase 17a (solution 17 containing lipase 17a) may be attached to a part of the surface of protective film 16c in addition to the surfaces of the plurality of electrodes 16b.

[0020] In the second step, dispersion liquid 25 is applied to substrate 15, and an AC voltage is applied to the plurality of electrodes 16b, thereby capturing droplets of specimen liquid 26 on the plurality of electrodes 16b by dielectrophoresis. The second step is performed after the first step. As shown in FIGS. 4(a) and 4(b), when an AC voltage is applied to the plurality of electrodes 16b, droplets of specimen liquid 26, which has a higher dielectric constant than oil 27, are attracted to and captured by electrode 16b.

[0021] In the third step, lipase 17a penetrates into the droplets of sample liquid 26 captured by the electrodes 16b and decomposes the lipid membrane of vesicles 26b, amplifying the target nucleic acid within the droplets of sample liquid 26. The third step is performed after the second step. As shown in FIG. 4(c), when the droplets of sample liquid 26 are captured by electrodes 16b, lipase 17a attached to the electrodes 16b penetrates into the droplets of sample liquid 26 and decomposes the lipid membrane of vesicles 26b. The amplification of the target nucleic acid may be performed by isothermal amplification such as loop-mediated isothermal amplification (LAMP), or by a temperature cycling method such as polymerase chain reaction (PCR).

[0022] In the fourth step, droplets containing vesicles 26b encapsulating target nucleic acids are identified based on the detection results of the amplification detection reagent contained in the specimen liquid 26. The fourth step is performed after the third step. As shown in FIG. 4(d), droplets of the specimen liquid 26 in which the target nucleic acid has been amplified emit fluorescence due to the amplification detection reagent 26d. This allows the droplets containing vesicles 26b encapsulating target nucleic acids to be identified.

[0023] In the first step, if the dispersion 25 is prepared using a specimen liquid 26 containing the surface marker detection reagent 26e in addition to the amplification detection reagent 26d, then in the fourth step, droplets containing vesicles 26b encapsulating the target nucleic acid, droplets containing vesicles 26b not encapsulating the target nucleic acid, and droplets not containing vesicles 26b are identified based on the detection results of the amplification detection reagent 26d and the surface marker detection reagent 26e. For example, whether or not the droplet contained the target nucleic acid can be determined based on the detection results of the amplification detection reagent 26d. For example, whether or not the droplet contained vesicles 26b can be determined based on the detection results of the surface marker detection reagent 26e.

[0024] The detection results of the surface marker detection reagent 26e are obtained, for example, by droplet digital ELISA (enzyme-linked immunosorbent assay). The detection results of the surface marker detection reagent 26e by droplet digital ELISA are obtained, for example, by the following procedure. First, beads carrying antibodies capable of binding to the vesicles 26b are bound to the vesicles 26b. At this time, the concentration of the beads is set to be higher than the concentration of the vesicles 26b. Next, an enzyme such as HRP (horseradish peroxidase) is bound to the vesicles 26b. At this time, the concentration of the enzyme is set to be higher than the concentration of the vesicles 26b. Next, the beads are immobilized and excess enzyme is washed away. This results in a structure in which the enzyme binds to the vesicles 26b and the vesicles 26b are bound to the beads. A dispersion liquid 25 is prepared using a sample liquid 26 containing this structure and a color-developing reagent that develops color in response to the enzyme. The enzyme and color-developing reagent correspond to the surface marker detection reagent 26e. In droplets containing structures (i.e., droplets containing vesicles 26b), the enzyme contained in the structures causes the coloring reagent to develop a color. In contrast, in droplets not containing structures (i.e., droplets not containing vesicles 26b), the coloring reagent does not develop a color. This makes it possible to distinguish between droplets containing vesicles 26b and droplets not containing vesicles 26b.

[0025] The fluorescence observation in step 4 may be performed simultaneously with step 3. In this case, if the amplification of the target nucleic acid in step 3 is performed by PCR, it is possible to estimate the number of target nucleic acids contained in the droplets from the number of temperature cycles at which the fluorescence intensity exceeds a threshold. In this case, if the number of target nucleic acids encapsulated in vesicles 26b is known in advance, it is possible to identify and exclude from the sample droplets containing multiple vesicles 26b or droplets in which target nucleic acids have been mixed outside the vesicles 26b.

[0026] The nucleic acid detection method according to the embodiment may further include a fifth step. In the fifth step, droplets containing vesicles 26b encapsulating target nucleic acids are collected. The fifth step is performed after the fourth step. When the lipid membrane is decomposed in the third step, the contents of the vesicles 26b diffuse into the interior of the droplets but do not diffuse to the exterior of the droplets. Therefore, by collecting the entire droplets containing vesicles 26b encapsulating target nucleic acids, the contents of the vesicles 26b encapsulating target nucleic acids can be collected. The fifth step is performed as needed and can be omitted.

[0027] (Nucleic Acid Detection System) FIG. 5 is a block diagram schematically showing a nucleic acid detection system according to an embodiment. FIG. 6 is an explanatory diagram illustrating a substrate preparation device of a nucleic acid detection system according to an embodiment. FIG. 7 is an explanatory diagram illustrating a dispersion liquid preparation device of a nucleic acid detection system according to an embodiment. 8(a) and 8(b) are explanatory diagrams showing a nucleic acid detection device of a nucleic acid detection system according to an embodiment. 9(a) and 9(b) are explanatory diagrams showing a nucleic acid detection device of a nucleic acid detection system according to an embodiment. 10(a) and 10(b) are explanatory diagrams showing a nucleic acid detection device of a nucleic acid detection system according to an embodiment. FIG. 11 is an explanatory diagram illustrating a droplet recovery device of a nucleic acid detection system according to an embodiment.

[0028] The nucleic acid detection system 100 according to the embodiment is a system for detecting a target nucleic acid contained inside a vesicle covered with a lipid membrane. The nucleic acid detection method according to the embodiment is performed, for example, by the nucleic acid detection system 100 according to the embodiment.

[0029] 5, a nucleic acid detection system 100 according to the embodiment includes a substrate preparation apparatus 10, a dispersion liquid preparation apparatus 20, a nucleic acid detection apparatus 30, and a droplet recovery apparatus 40. The substrate 15 used in the second step described above is prepared by, for example, the substrate preparation apparatus 10. The first step described above is performed by, for example, the dispersion liquid preparation apparatus 20. The second to fourth steps described above are performed by, for example, the nucleic acid detection apparatus 30. The fifth step described above is performed by, for example, the droplet recovery apparatus 40.

[0030] As shown in FIG. 6, the substrate preparation apparatus 10 applies a solution 17 containing lipase 17a to the surface of a substrate 16, thereby preparing a substrate 15 having lipase 17a (solution 17 containing lipase 17a) attached to the surface of the substrate 16. As described above, the substrate 16 has, for example, a support 16a, a plurality of electrodes 16b, and a protective film 16c. The solution 17 contains, for example, lipase 17a and water 17b. The concentration of lipase 17a in the solution 17 is preferably, for example, about 1 mM. Alternatively, lipase 17a may be mixed with water 17b using an organic solvent of about 10% by mass. Examples of the organic solvent that can be used include ethanol, DMSO (dimethyl sulfoxide), and DMF (N,N-dimethylformamide).

[0031] The substrate preparation apparatus 10 includes, for example, a stage 11 and a coating device 12. A substrate 16 is placed on the stage 11. The coating device 12 coats a solution 17 on the surface (upper surface) of the substrate 16 placed on the stage 11. When the coating device 12 coats the lipase 17a (the solution 17 containing the lipase 17a), the lipase 17a (the solution 17 containing the lipase 17a) may be coated locally on the surfaces of the electrodes 16b of the substrate 16 by, for example, printing or dispensing, or the lipase 17a (the solution 17 containing the lipase 17a) may be coated on the entire surface of the substrate 16, and then the lipase 17a (the solution 17 containing the lipase 17a) may be locally removed from the surface of the hydrophobic protective film 16c by air blowing. As a result, a substrate 15 is produced in which lipase 17a (solution 17 containing lipase 17a) is attached to the surfaces of the plurality of electrodes 16b of the base 16.

[0032] As shown in FIG. 7, dispersion liquid preparation device 20 prepares dispersion liquid 25 by dispersing droplets of specimen liquid 26 in oil 27. As described above, specimen liquid 26 contains, for example, salt water 26a, vesicles 26b, amplification reagent 26c, and amplification detection reagent 26d. Specimen liquid 26 may further contain surface marker detection reagent 26e. The concentration of specimen liquid 26 used to prepare dispersion liquid 25 is preferably adjusted in advance so that one droplet contains one vesicle 26b.

[0033] The dispersion liquid preparation device 20 has, for example, a first flow path 21, a second flow path 22, and a third flow path 23. The upstream end of the second flow path 22 is connected to the downstream end of the first flow path 21. The downstream end of the third flow path 23 is connected to a connection between the downstream end of the first flow path 21 and the upstream end of the second flow path 22. The dispersion liquid preparation device 20 flows a specimen liquid 26 from the first flow path 21 to the second flow path 22, while flowing oil 27 from the third flow path 23 to the second flow path 22. This prepares a dispersion liquid 25 in which the specimen liquid 26 is dispersed in the oil 27 as droplets. The number of third flow paths 23 may be one or more, but is preferably two. The dispersion liquid preparation device 20 shown in FIG. 7 is provided with two third flow paths 23.

[0034] 8(a), 8(b), 9(a), 9(b), 10(a), and 10(b), the nucleic acid detection device 30 uses dielectrophoresis to capture droplets of the specimen liquid 26 contained in the dispersion liquid 25 on the plurality of electrodes 16b of the substrate 15. The nucleic acid detection device 30 amplifies the target nucleic acid inside the captured droplets, and identifies the droplets that contained vesicles 26b encapsulating the target nucleic acid.

[0035] The nucleic acid detection device 30 includes, for example, a stage 31, a liquid supply unit 32, a probe 33, a temperature adjustment unit 34, a detection unit 35, and a control unit 36. A substrate 15 is placed on the stage 31. The liquid supply unit 32 is disposed above the substrate 15 placed on the stage 31, and supplies a liquid such as a dispersion liquid 25 or a cleaning liquid 37 (e.g., oil) to the surface (upper surface) of the substrate 15.

[0036] The liquid supply unit 32 includes, for example, a cap 32a and a packing 32b. The packing 32b is provided below the cap 32a. The packing 32b contacts the surface (top surface) of the substrate 15 and seals the gap between the cap 32a and the substrate 15. The cap 32a includes a plate portion 32a1, an inlet 32a2, and an outlet 32a3. The plate portion 32a1 is held in a position facing the substrate 15 with the packing 32b in between, thereby forming a flow path between the plate portion 32a1 and the substrate 15. The liquid flows from the inlet 32a2 through this flow path to the outlet 32a3. The inlet 32a2 and the outlet 32a3 are provided, for example, at the top of the plate portion 32a1 and open upward. The plate portion 32a1 has through-holes that penetrate in the vertical direction at positions overlapping with the inlet 32a2 and the outlet 32a3. The cap 32a is made of, for example, a transparent material. The liquid supply unit 32 is not limited to this, and may be any material that can supply liquid to the surface of the substrate 15.

[0037] The probe 33 applies an AC voltage to the plurality of electrodes 16b of the substrate 15. The probe 33 is connected to, for example, an external AC power source. The probe 33 is provided, for example, on the side of the liquid supply unit 32 and connected to the end of the substrate 15.

[0038] The temperature adjustment unit 34 adjusts the temperature of the stage 31 by heating or cooling the stage 31. The temperature adjustment unit 34 adjusts the temperature of the stage 31, thereby adjusting the temperature of the substrate 15. The temperature adjustment unit 34 has, for example, a Peltier element. The temperature adjustment unit 34 is provided, for example, below the stage 31.

[0039] The detection unit 35 acquires the detection result of the amplification detection reagent 26d. When the amplification detection reagent 26d is a fluorescent probe, the detection unit 35 is, for example, a fluorescent camera. The detection unit 35 has a function of irradiating light with the excitation wavelength of the fluorescent dye of the amplification detection reagent 26d. The detection unit 35 detects fluorescence emitted by the amplification detection reagent 26d when the target nucleic acid is amplified. The detection unit 35 can be switched, for example, between a state where it is positioned so as to overlap the stage 31 in the vertical direction and a state where it is not positioned so as to overlap the stage 31 in the vertical direction. When it is positioned so as to overlap the stage 31 in the vertical direction, the detection unit 35 is located, for example, above the liquid supply unit 32. The detection unit 35 detects, for example, fluorescence transmitted through the cap 32a. The detection unit 35 is electrically connected to the control unit 36. The detection unit 35 outputs the detection result to the control unit 36.

[0040] The control unit 36 identifies droplets that contained vesicles 26b encapsulating the target nucleic acid based on the detection results input from the detection unit 35. The control unit 36 determines, for example, that droplets that emit fluorescence are droplets that contained vesicles 26b encapsulating the target nucleic acid.

[0041] When using the nucleic acid detection device 30, first, the substrate 15 is placed on the stage 31 as shown in Fig. 8(a). Then, a liquid supply unit 32 is placed on the substrate 15 to form a flow path. Furthermore, a probe 33 is placed on the substrate 15. At this time, the detection unit 35 is placed at a position that does not overlap with the stage 31 in the vertical direction.

[0042] Next, as shown in Fig. 8(b), the dispersion liquid 25 is injected from the inlet 32a2 of the liquid supply unit 32, thereby applying the dispersion liquid 25 to the surface of the substrate 15. In Fig. 8(b), the inside of the flow path is filled with the dispersion liquid 25. With the dispersion liquid 25 applied to the surface of the substrate 15, an AC voltage is applied to the multiple electrodes 16b of the substrate 15 by the probe 33, thereby causing dielectrophoresis of droplets of the specimen liquid 26 contained in the dispersion liquid 25 and capturing them on the multiple electrodes 16b.

[0043] Next, as shown in Fig. 9(a), cleaning liquid 37 is injected from inlet 32a2 of liquid supply unit 32, and dispersion liquid 25 in the flow path is removed from outlet 32a3. This removes droplets of specimen liquid 26 that are not captured by the multiple electrodes 16b in the flow path. In Fig. 9(a), the flow path is filled with cleaning liquid 37.

[0044] 9(b), the substrate 15 is heated by the temperature adjustment unit 34, whereby the lipid membranes of the vesicles 26b are decomposed by the lipase that has penetrated into the droplets captured by the multiple electrodes 16b. The heating temperature is preferably set to, for example, 35°C or higher and 65°C or lower.

[0045] Next, as shown in FIG. 10( a), the detection unit 35 is positioned so as to overlap the stage 31 in the vertical direction. The temperature of the substrate 15 is then adjusted by the temperature adjustment unit 34, causing the amplification reagent 26c contained in the droplet (sample liquid 26) to amplify the target nucleic acid inside the droplet. For example, when amplifying the target nucleic acid by isothermal amplification such as the LAMP method, the substrate 15 is heated to a predetermined temperature. For example, when amplifying the target nucleic acid by a temperature cycling method such as the PCR method, the substrate 15 is heated and cooled multiple times. The detection unit 35 obtains the detection results of the amplification detection reagent 26d from the start to the end of amplification. The control unit 36 identifies the droplet containing the vesicles 26b encapsulating the target nucleic acid based on the detection results of the amplification detection reagent 26d.

[0046] If the sample liquid 26 contains the surface marker detection reagent 26e in addition to the amplification detection reagent 26d, the detection unit 35 acquires the detection results of the surface marker detection reagent 26e in addition to the detection results of the amplification detection reagent 26d. In this case, the detection unit 35 further includes a normal optical microscope function, i.e., a white light irradiation function and a visible light detection function, in order to detect enzyme color development. Furthermore, based on the detection results of the amplification detection reagent 26d and the surface marker detection reagent 26e, the control unit 36 identifies droplets containing vesicles 26b encapsulating target nucleic acid, droplets containing vesicles 26b not encapsulating target nucleic acid, and droplets containing no vesicles 26b. The control unit 36 determines whether the droplets contained target nucleic acid based on, for example, the detection results of the amplification detection reagent 26d. The control unit 36 determines whether the droplets contained vesicles 26b based on, for example, the detection results of the surface marker detection reagent 26e.

[0047] 10(b), the detection unit 35 is placed at a position where it does not overlap with the stage 31 in the vertical direction. Then, air is injected from the inlet 32a2 of the liquid supply unit 32, and the cleaning liquid 37 in the flow path is removed from the outlet 32a3.

[0048] 11, the droplet recovery device 40 recovers droplets that contained vesicles 26b that encapsulate target nucleic acids. That is, the droplet recovery device 40 recovers droplets identified by the control unit 36.

[0049] The droplet recovery device 40 includes, for example, a stage 41, a well 42, and a pipette 43. The substrate 15 is placed on the stage 41.

[0050] The well 42 is placed on the substrate 15 placed on the stage 41. The well 42 has, for example, a plate portion 42a and a packing 42b. The packing 42b is provided below the plate portion 42a. The packing 42b contacts the surface (top surface) of the substrate 15 and seals the gap between the plate portion 42a and the substrate 15. The plate portion 42a has a through-hole that penetrates in the vertical direction. Oil 45 is poured into the area surrounded by the plate portion 42a and the packing 42b. As a result, the oil 45 is applied to the surface (top surface) of the substrate 15.

[0051] The pipette 43 collects, together with the oil 45, droplets that have adhered to the surface (upper surface) of the substrate 15 and that contain the vesicles 26b encapsulating the target nucleic acid.

[0052] (substrate) 3, the substrate 15 according to the embodiment includes a base 16 and a lipase 17a. The base 16 includes a support 16a, a plurality of electrodes 16b, and a protective film 16c. The lipase 17a is attached to the surfaces of the plurality of electrodes 16b of the base 16. A solution 17 containing the lipase 17a and water 17b may be attached to the surfaces of the plurality of electrodes 16b of the base 16. If the plurality of electrodes 16b are exposed from the hydrophobic protective film 16c, the lipase 17a (solution 17 containing the lipase 17a) is likely to adhere to the surfaces of the plurality of electrodes 16b but is less likely to adhere to the surface of the protective film 16c.

[0053] The effects of the nucleic acid detection method and the nucleic acid detection system 100 according to the embodiment will be described below.

[0054] Exosomes are a type of extracellular vesicle (EVV) found in living organisms. They are tiny vesicles with a diameter of approximately 100 nm. They have a lipid bilayer membrane structure containing nucleic acids and other molecules. They are known to move between distant organs, transmitting information and transporting substances. The lipid bilayer membrane contains proteins or glycoproteins, and observations suggest that cfDNA (cell-free DNA) is attached to the periphery. These EVVs are thought to contain information about the cell from which they were produced and the cells to which they will migrate. Furthermore, EVVs are known to contain miRNAs, single-stranded RNAs approximately 20–30 bases long. The miRNA sequences have been reported to be biomarkers for cancer. Furthermore, miRNAs are known to inhibit mRNA translation through a process known as RNA interference, thereby knocking down gene expression.

[0055] Extracellular vesicles have been reported to be deeply involved in cancer metastasis. They have also been reported to be involved in the transport of causative substances in non-cancer diseases, such as dementia (e.g., Alzheimer's disease, Parkinson's disease, and dementia with Lewy bodies), neurodegenerative diseases, and renal disorders. Therefore, extracellular vesicles have attracted attention as biomarkers for use in early detection of disease and recurrence monitoring. Furthermore, because much remains unknown about their structure and function, they have also attracted attention as a research subject, and the development of methods for detecting and isolating extracellular vesicles is desired.

[0056] One possible method for separating extracellular vesicles is to store them in submicron-sized microwells by dielectrophoresis. However, this method has problems such as storing multiple extracellular vesicles in one microwell, nonspecific adsorption of extracellular vesicles to the inner wall of the channel, and increased costs due to the formation of submicron-sized microwells.

[0057] In contrast, in the nucleic acid detection method and nucleic acid detection system 100 according to the embodiment, a dispersion liquid 25 in which a sample liquid 26 is dispersed in oil 27 is applied to a substrate 15, and the droplets are captured by dielectrophoresis on multiple electrodes 16b on the substrate 15. This makes it easy to separate and capture droplets containing vesicles 26b. At the electrode 16b where a droplet is captured, the electric field density decreases, making it difficult to capture other droplets, thereby preventing multiple droplets from being captured by a single electrode 16b. Furthermore, because the vesicles 26b are handled in the form of droplets, nonspecific adsorption of the vesicles 26b to the inner wall of the flow channel can be prevented.

[0058] Furthermore, in the nucleic acid detection method and nucleic acid detection system 100 according to the embodiment, lipase is used to decompose the lipid membrane of the vesicles 26b while the droplets are captured, and the target nucleic acid is amplified inside the captured droplets. Then, based on the detection results of the amplification detection reagent 26d, droplets containing vesicles 26b encapsulating the target nucleic acid are identified. This allows for highly sensitive detection of the target nucleic acid contained in the vesicles 26b. Furthermore, droplets can be separated based on the presence or absence and concentration of the target nucleic acid (e.g., miRNA) encapsulated in the vesicles 26b. The separated droplets can then be individually collected, and because the droplets contain the contents of the vesicles 26b, omics analysis such as mass spectrometry can be performed on the vesicles 26b encapsulating the target nucleic acid.

[0059] Furthermore, in the nucleic acid detection method and nucleic acid detection system 100 according to the embodiment, vesicles 26b (e.g., extracellular vesicles) with a diameter of approximately 100 nm can be treated as droplets with a diameter of approximately several tens of μm. Therefore, droplets containing vesicles 26b can be captured without forming submicron-sized microwells. This can suppress increases in costs and improve operability.

[0060] Furthermore, in the nucleic acid detection method and nucleic acid detection system 100 according to the embodiment, a dispersion liquid is prepared using a sample liquid 26 containing a surface marker detection reagent 26e in addition to the amplification detection reagent 26d. Therefore, based on the detection results of the amplification detection reagent 26d and the surface marker detection reagent 26e, droplets containing vesicles 26b encapsulating target nucleic acid, droplets containing vesicles 26b not encapsulating target nucleic acid, and droplets containing no vesicles 26b can be identified. This allows the proportion of vesicles 26b containing target nucleic acid to be determined. Furthermore, based on the detection results of the amplification detection reagent 26d and the surface marker detection reagent 26e, droplets containing vesicles 26b encapsulating target nucleic acid and droplets containing no vesicles 26b but containing target nucleic acid can be distinguished.

[0061] Furthermore, if the substrate 15 according to the embodiment is used, the nucleic acid detection method and nucleic acid detection system 100 according to the embodiment can be more easily realized.

[0062] Embodiments may include the following features.

[0063] (Configuration 1) A method for detecting a target nucleic acid contained inside a lipid membrane-enclosed vesicle, comprising: a first step of preparing a dispersion by dispersing droplets of a specimen solution containing salt water, the vesicles, an amplification reagent capable of amplifying the target nucleic acid, and an amplification detection reagent capable of detecting the amplification of the target nucleic acid in oil; a second step of applying the dispersion to a substrate having a support and a plurality of electrodes provided on the surface of the support, with lipase attached to the surfaces of the plurality of electrodes, and applying an AC voltage to the plurality of electrodes to capture the droplets on the plurality of electrodes by dielectrophoresis; a third step of decomposing the lipid membrane by the lipase that has penetrated into the droplets captured by the plurality of electrodes, and amplifying the target nucleic acid inside the droplets; a fourth step of identifying the droplets containing the vesicles encapsulating the target nucleic acid based on the detection result of the amplification detection reagent; A nucleic acid detection method comprising:

[0064] (Configuration 2) The nucleic acid detection method according to configuration 1, further comprising a fifth step of recovering the droplets containing the vesicles encapsulating the target nucleic acid.

[0065] (Configuration 3) In the first step, a sample liquid containing the vesicles, the amplification reagent, the amplification detection reagent, and a surface marker detection reagent capable of detecting the surface marker of the vesicles is formed into droplets and dispersed in oil to prepare a dispersion liquid; The nucleic acid detection method according to configuration 1 or 2, wherein in the fourth step, the droplets containing the vesicles encapsulating the target nucleic acid, the droplets containing the vesicles not encapsulating the target nucleic acid, and the droplets not containing the vesicles are identified based on the detection results of the amplification detection reagent and the detection results of the surface marker detection reagent.

[0066] (Configuration 4) 4. The nucleic acid detection method according to any one of aspects 1 to 3, wherein the target nucleic acid is miRNA.

[0067] (Configuration 5) 5. The nucleic acid detection method according to any one of aspects 1 to 4, wherein the vesicles are extracellular vesicles.

[0068] (Configuration 6) A system for detecting a target nucleic acid contained within a lipid membrane-enclosed vesicle, comprising: a substrate preparation device that prepares a substrate having a support and a plurality of electrodes provided on a surface of the support by applying a solution containing lipase to the surfaces of the plurality of electrodes of a base, thereby preparing a substrate having lipase attached to the surfaces of the plurality of electrodes; a dispersion liquid preparation device that prepares a dispersion liquid by dispersing droplets of a specimen liquid containing salt water, the vesicles, an amplification reagent capable of amplifying the target nucleic acid, and an amplification detection reagent capable of detecting the amplification of the target nucleic acid in oil; a nucleic acid detection device that applies the dispersion liquid to the substrate, applies an AC voltage to the plurality of electrodes, and captures the droplets on the plurality of electrodes by dielectrophoresis, decomposes the lipid membrane by the lipase that has penetrated into the droplets captured on the plurality of electrodes, amplifies the target nucleic acid inside the droplets, and identifies the droplets that contained the vesicles encapsulating the target nucleic acid based on the detection result of the amplification detection reagent; A nucleic acid detection system comprising:

[0069] (Configuration 7) The nucleic acid detection device includes: a stage on which the substrate is placed; a liquid supply unit that supplies a liquid to the surface of the substrate placed on the stage; a probe for applying an AC voltage to the plurality of electrodes; a temperature adjusting unit that adjusts the temperature of the stage; a detection unit that acquires the detection result of the amplification detection reagent; a control unit that identifies the droplets containing the vesicles encapsulating the target nucleic acid based on the detection results; 7. The nucleic acid detection system according to claim 6, comprising:

[0070] (Configuration 8) The nucleic acid detection system according to configuration 6 or 7, further comprising a droplet recovery device that recovers the droplets that contained the vesicles encapsulating the target nucleic acid.

[0071] (Configuration 9) the dispersion liquid preparation device prepares a dispersion liquid by dispersing droplets of a specimen liquid containing the vesicles, the amplification reagent, the amplification detection reagent, and a surface marker detection reagent capable of detecting the surface markers of the vesicles in oil; A nucleic acid detection system described in any one of configurations 6 to 8, wherein the nucleic acid detection device identifies the droplets that contained vesicles that encapsulate the target nucleic acid, the droplets that contained vesicles that do not encapsulate the target nucleic acid, and the droplets that did not contain the vesicles based on the detection results of the amplification detection reagent and the detection results of the surface marker detection reagent.

[0072] (Configuration 10) 10. The nucleic acid detection system according to any one of aspects 6 to 9, wherein the target nucleic acid is miRNA.

[0073] (Configuration 11) 11. The nucleic acid detection system according to any one of aspects 6 to 10, wherein the vesicles are extracellular vesicles.

[0074] (Configuration 12) a substrate having a support and a plurality of electrodes provided on a surface of the support; Lipase attached to the surfaces of the plurality of electrodes; A substrate comprising:

[0075] (Configuration 13) the substrate further has a hydrophobic protective film provided on the surface of the support, 13. The substrate of claim 12, wherein each of the plurality of electrodes is exposed from the protective film.

[0076] As described above, according to the embodiments, it is possible to provide a nucleic acid detection method, a nucleic acid detection system, and a substrate that are capable of detecting target nucleic acids contained in vesicles with high sensitivity.

[0077] 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. [Explanation of symbols]

[0078] 10: Substrate manufacturing equipment 11: Stage 12: Coating device 15: Circuit board 16: Base 16a: Support 16b: Electrode 16c: Protective film 17: Solution 17a: Lipase 17b:Water 20: Dispersion liquid preparation device 21: First flow path 22: Second flow path 23: Third flow path 25:Dispersion liquid 26: Sample liquid 26a: Salt water 26b: vesicles 26c: Amplification reagents 26d: Amplification detection reagent 26e: Surface marker detection reagent 27: Oil 30: Nucleic acid detection device 31: Stage 32:Liquid supply section 32a: Cap 32a1: Board part 32a2:Entrance 32a3:Exit 32b: Packing 33: Probe 34: Temperature adjustment section 35:Detection unit 36: Control unit 37: Cleaning fluid 40: Droplet collection device 41: Stage 42: Well 42a: Board part 42b: Packing 43: Pipette 45: Oil 100: Nucleic acid detection system

Claims

1. A method for detecting a target nucleic acid contained inside a lipid membrane-enclosed vesicle, comprising: a first step of preparing a dispersion by dispersing droplets of a specimen solution containing salt water, the vesicles, an amplification reagent capable of amplifying the target nucleic acid, and an amplification detection reagent capable of detecting the amplification of the target nucleic acid in oil; a second step of applying the dispersion to a substrate having a support and a plurality of electrodes provided on the surface of the support, with lipase attached to the surfaces of the plurality of electrodes, and applying an AC voltage to the plurality of electrodes to capture the droplets on the plurality of electrodes by dielectrophoresis; a third step of decomposing the lipid membrane by the lipase that has penetrated into the droplets captured by the plurality of electrodes, and amplifying the target nucleic acid inside the droplets; a fourth step of identifying the droplets containing the vesicles encapsulating the target nucleic acid based on the detection result of the amplification detection reagent; A nucleic acid detection method comprising:

2. The nucleic acid detection method according to claim 1 , further comprising a fifth step of recovering the droplets containing the vesicles encapsulating the target nucleic acid.

3. In the first step, a dispersion is prepared by dispersing droplets of a sample liquid containing the vesicles, the amplification reagent, the amplification detection reagent, and a surface marker detection reagent capable of detecting the surface marker of the vesicles in oil; The nucleic acid detection method described in claim 1, wherein in the fourth step, based on the detection results of the amplification detection reagent and the surface marker detection reagent, the droplets containing the vesicles encapsulating the target nucleic acid, the droplets containing the vesicles not encapsulating the target nucleic acid, and the droplets not containing the vesicles are identified.

4. The nucleic acid detection method according to claim 1 , wherein the target nucleic acid is miRNA.

5. The nucleic acid detection method according to claim 1 , wherein the vesicles are extracellular vesicles.

6. A system for detecting a target nucleic acid contained within a lipid membrane-enclosed vesicle, comprising: a substrate preparation device that prepares a substrate having a support and a plurality of electrodes provided on a surface of the support by applying a solution containing lipase to the surfaces of the plurality of electrodes of a base, thereby preparing a substrate having lipase attached to the surfaces of the plurality of electrodes; a dispersion liquid preparation device that prepares a dispersion liquid by dispersing droplets of a specimen liquid containing salt water, the vesicles, an amplification reagent capable of amplifying the target nucleic acid, and an amplification detection reagent capable of detecting the amplification of the target nucleic acid in oil; a nucleic acid detection device that applies the dispersion liquid to the substrate, applies an AC voltage to the plurality of electrodes, and captures the droplets on the plurality of electrodes by dielectrophoresis, decomposes the lipid membrane by the lipase that has penetrated into the droplets captured on the plurality of electrodes, amplifies the target nucleic acid inside the droplets, and identifies the droplets that contained the vesicles encapsulating the target nucleic acid based on the detection result of the amplification detection reagent; A nucleic acid detection system comprising:

7. The nucleic acid detection device includes: a stage on which the substrate is placed; a liquid supply unit that supplies a liquid to the surface of the substrate placed on the stage; a probe for applying an AC voltage to the plurality of electrodes; a temperature adjusting unit that adjusts the temperature of the stage; a detection unit that acquires the detection result of the amplification detection reagent; a control unit that identifies the droplets containing the vesicles encapsulating the target nucleic acid based on the detection results; The nucleic acid detection system according to claim 6 , comprising:

8. The nucleic acid detection system according to claim 6 , further comprising a droplet recovery device that recovers the droplets that contained the vesicles encapsulating the target nucleic acid.

9. the dispersion liquid preparation device prepares a dispersion liquid by dispersing droplets of a specimen liquid containing the vesicles, the amplification reagent, the amplification detection reagent, and a surface marker detection reagent capable of detecting the surface markers of the vesicles in oil; The nucleic acid detection system described in claim 6, wherein the nucleic acid detection device identifies the droplets that contained vesicles that encapsulate the target nucleic acid, the droplets that contained vesicles that do not encapsulate the target nucleic acid, and the droplets that did not contain vesicles based on the detection results of the amplification detection reagent and the detection results of the surface marker detection reagent.

10. The nucleic acid detection system according to claim 6 , wherein the target nucleic acid is miRNA.

11. The nucleic acid detection system of claim 6 , wherein the vesicles are extracellular vesicles.

12. a substrate having a support and a plurality of electrodes provided on a surface of the support; Lipase attached to the surfaces of the plurality of electrodes; A substrate comprising:

13. the substrate further has a hydrophobic protective film provided on the surface of the support, The substrate according to claim 12 , wherein each of the plurality of electrodes is exposed from the protective film.

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