Device, method for manufacturing said device, and ion current measurement method

Abstract

The present invention addresses the problem of providing: a method for producing a device using a new principle; a device; and a method for measuring ion current using a device.　This problem can be solved by a device comprising: a substrate having a first face and a second face; a first through hole that passes through the substrate from the first face towards the second face; and a deposit of a metal compound which is at least one type selected from the group consisting of a divalent metal hydroxide, a trivalent metal hydroxide, a divalent metal phosphate and a trivalent metal phosphate and which is formed on a wall surface of the first through hole.

Description

Device, method for manufacturing the device, and method for measuring ion current.

[0001] Devices that form through-holes (nanopores) in a substrate and measure the change in ion current as a sample passes through these holes are attracting attention as devices with a wide range of applications in sensing bacteria, viruses, DNA, proteins, and other microorganisms.

[0002] Related technologies include, for example, analyzing the shape distribution of exosomes by measuring the ion current when exosomes pass through through-holes formed in a substrate (see Patent Document 1), increasing the time it takes for a sample to pass through through-holes by forming molecules that interact with the sample passing through the through-holes formed in the substrate (see Patent Document 2), and forming two or more through-holes in the substrate (see Patent Document 3).

[0003] Furthermore, in order to accurately fabricate a desired nanopore diameter, it is also known to backfill nanopores formed on a substrate at the atomic layer level using ALD (see Patent Document 4).

[0004] Japanese Patent Publication No. 2017-156168, International Publication No. 2017 / 183716, International Publication No. 2020 / 138021, Japanese Patent Publication No. 2014-210245

[0005] As described in Patent Document 4 above, it is known that the diameter of through-holes formed in a substrate can be adjusted retrospectively. However, the manufacturing method described in Patent Document 4 uses an ALD to adjust the size of the nanopore diameter, which presents a problem in that it requires a different device than the one needed to measure the ion current in order to adjust the size of the nanopore diameter. Therefore, the development of a method that can adjust the diameter size of through-holes based on a new principle is desired.

[0006] The inventors have conducted diligent studies and have newly discovered that (1) by placing a first aqueous solution containing divalent and / or trivalent metal ions on one of the first and second surfaces of the device substrate, and a second aqueous solution containing monovalent metal ions on the other side, (2) by applying a voltage to the first and second aqueous solutions so that the divalent and / or trivalent metal ions contained in the first aqueous solution move in the direction of the through-hole, (3) in the through-hole portion, the divalent and / or trivalent metal ions contained in the first aqueous solution change into metal hydroxides and / or metal phosphorides and deposit on the wall surface of the through-hole, (4) the diameter size of the through-hole can be adjusted.

[0007] In other words, the disclosure in this application provides a method for manufacturing a device based on a new principle, the device itself, and a method for measuring ion current using the device.

[0008] The disclosures in this application relate to the following: a device, a method for manufacturing the device, and a method for measuring ion current.

[0009] (1) A device comprising: a substrate having a first surface and a second surface; a first through-hole penetrating from the first surface to the second surface; and a deposit of a metal compound formed on the wall surface of the first through-hole, the deposit of a metal compound being at least one selected from the group consisting of divalent metal hydroxide, trivalent metal hydroxide, divalent metal phosphorus oxide, and trivalent metal phosphorus oxide. (2) The device according to (1) above, wherein the deposit of the metal compound is formed only on the wall surface of the first through-hole and not on the first surface and the second surface. (3) The device according to (1) or (2) above, wherein a second through-hole is formed in the layer formed of the deposit of the metal compound. (4) A method for manufacturing the device described in (1) above, the manufacturing method comprising: a preparation step and a metal compound deposition step, the preparation step comprising: preparing a substrate having a first surface and a second surface and including a first through-hole penetrating from the first surface toward the second surface; placing a first aqueous solution containing divalent metal ions and / or trivalent metal ions on one of the first surface or the second surface of the substrate; placing a second aqueous solution containing monovalent metal ions on the other of the first surface or the second surface of the substrate; bringing the first aqueous solution and the second aqueous solution into liquid junction through the first through-hole; and bringing a first electrode into contact with the first aqueous solution and a second electrode into contact with the second aqueous solution, the metal compound deposition step comprising: a first voltage application step of applying a voltage to the first electrode and the second electrode so that the metal ions contained in the first aqueous solution move toward the first through-hole, A manufacturing method comprising the step of: in the first through-hole portion, metal ions contained in the first aqueous solution are changed to metal hydroxides by a hydroxylation reaction, or metal ions contained in the first aqueous solution are changed to metal phosphorides by a phosphorylation reaction, and deposited on the wall surface of the first through-hole as a metal compound. (5) The manufacturing method according to (4) above, wherein the pH of the second aqueous solution is 7.0 or higher.(6) The manufacturing method according to (4) or (5) above, wherein if the second aqueous solution contains a phosphate buffer, a metal phosphate compound is obtained as the metal compound, and if the second aqueous solution does not contain a phosphate buffer, a metal hydroxide is obtained as the metal compound. (7) The manufacturing method according to any one of (4) to (6) above, further comprising a metal compound dissolution step of dissolving the deposited metal compound after the metal compound deposition step, wherein the metal compound dissolution step includes a second voltage application step of applying a voltage to the first electrode and the second electrode such that the positive and negative charges are reversed compared to the first voltage application step. (8) The manufacturing method according to (7) above, wherein the metal compound deposition step and the metal compound dissolution step are repeated. (9) A method for measuring the ion current of a charged sample using an ion current measuring device, wherein the ion current measuring device includes the device described in (3) above, a first chamber member, and a second chamber member, wherein the first chamber member forms a first chamber filled with a first electrolyte solution with at least the surface of the first surface of the substrate of the device including a second through-hole, and the second chamber member forms a second chamber filled with a second electrolyte solution with at least the surface of the second surface of the substrate of the device including a second through-hole, and the method for measuring the ion current includes a charged sample passing step and an ion current measurement step, wherein the charged sample passing step involves applying a voltage to the first electrolyte solution filled in the first chamber and the second electrolyte solution filled in the second chamber, thereby causing the charged sample contained in the first chamber to pass through the second through-hole in the direction of the second chamber, or causing the charged sample contained in the second chamber to pass through the second through-hole in the direction of the first chamber, and the ion current measurement step is a method for measuring the change in ion current when the charged sample passes through the second through-hole. (10) The ion current measurement method according to (9) above, wherein in the charged sample passing step, the polarity of the voltage applied to the first electrolyte and the second electrolyte is the same as the polarity when the metal compound is deposited in the first through hole, and the ion current measurement step is performed in a state in which the formation and disappearance of the second through hole is repeated.

[0010] The device manufacturing method disclosed in this application allows for adjustment of the diameter size of the through-hole using a novel principle. Furthermore, the device manufacturing method disclosed in this application can be implemented using components of an ion current measuring device used in an ion current measurement method. Therefore, when implemented using components of an ion current measuring device, a separate device such as an ALD is not required to adjust the diameter size of the through-hole.

[0011] Figure 1 is a schematic cross-sectional view of device 1 according to the embodiment. Figure 1A shows device 1 before the formation of deposit 4, and Figure 1B is a schematic cross-sectional view of device 1 with deposit 4 formed in the first through-hole 3. Figure 2 is a top view of device 1 according to the embodiment. Figure 3 is a flowchart of the manufacturing method according to the embodiment. Figure 4 is a schematic diagram showing an example of the apparatus used when carrying out the manufacturing method. Figure 5 is a photograph used as a substitute for a drawing, and is an SEM image of the substrate 2 in which the first through-hole 3 was formed in Example 1. Figure 6A is a graph showing the measurement results of the ion current measured when device 1 was manufactured in Example 1. Figure 6B is a graph showing the measured value of the ion current in Figure 6A when the applied voltage is near 0V, changed from the order of μA to the order of nA. Figure 7 is a photograph used as a substitute for a drawing, and shows the metal phosphorus oxide (Mn(PO) deposited in the first through-hole 3 manufactured in Example 1. ４ ) ｘFigure 8 is an SEM image of substrate 2 completely sealed by the first through-hole 3. Figure 9 is a diagram showing the results of method 1 for evaluating the reaction heat in the first through-hole 3. Figure 10 is a graph showing the measurement results of the ion current measured when the device 1 was fabricated in Example 2. Figure 11 is a graph showing the measurement results of the ion current measured when the device 1 was fabricated in Example 3. Figure 12 is a graph showing the measurement results of the ion current measured when the device 1 was fabricated in Comparative Example 1. Figure 13 is a graph showing the results of measuring the ion current using plasmid DNA as a sample with the fabricated device 1 in Example 4. Figure 14 is a graph showing the measurement results of the ion current when a pulsed voltage was applied in Example 5. Figure 15 is a graph showing the results of measuring the ion current while maintaining the applied voltage at -1.1V in Example 6. Figure 16 is a graph showing the results of measuring the ion current while maintaining the applied voltage at -1.1V in Example 7. Figures 17A to 17C show the results of measuring the ion current using nucleic acid as a sample while maintaining an applied voltage of -1.0V in Example 8. Figure 18A shows the results of measuring the ion current using amino acids as a sample while maintaining an applied voltage of -1.0V in Example 8. Figure 18B shows the results of measuring the ion current using amino acids as a sample while maintaining an applied voltage of -1.0V in Example 8.

[0012] The following describes in detail the device disclosed in this application, the method for manufacturing the device (hereinafter sometimes simply referred to as the "manufacturing method"), and the method for measuring ion current using the device (hereinafter sometimes simply referred to as the "measurement method"). In this specification, components having the same or similar functions are denoted by the same or similar reference numerals. In some cases, redundant descriptions of components denoted by the same or similar reference numerals may be omitted.

[0013] Furthermore, the position, size, and extent of each component shown in the drawings may not represent the actual position, size, and extent in order to facilitate understanding. Therefore, the disclosures in this application are not necessarily limited to the positions, sizes, and extents disclosed in the drawings.

[0014] Furthermore, in this specification, (1) a numerical range expressed using "~" means a range that includes the numerical values ​​written before and after "~" as the lower and upper limits, (2) numerical values, numerical ranges, and qualitative expressions (for example, expressions such as "identical" and "same") indicate numerical values, numerical ranges, and properties that include errors that are generally acceptable in the relevant technical field, and (3) when it is written as "approximately ○○ shape", it is interpreted to include not only the exact ○○ shape but also a shape that is understood to be approximately ○○ shape.

[0015] (Embodiment of Device 1) Device 1 according to an embodiment will be described with reference to Figures 1 and 2. Figure 1 is a schematic cross-sectional view of Device 1 according to an embodiment, where Figure 1A is a view of Device 1 before the formation of the deposit 4, and Figure 1B is a schematic cross-sectional view of Device 1 after the formation of the deposit 4 in the first through hole 3. Figure 2 is a top view of Device 1 according to an embodiment.

[0016] Device 1 includes a substrate 2 having a first surface 21 and a second surface 22, a first through-hole 3 penetrating from the first surface 21 to the second surface 22, and a deposit 4 formed on the wall surface 31 of the first through-hole 3, which is at least one selected from the group consisting of divalent metal hydroxide, divalent metal phosphorus oxide, trivalent metal hydroxide, and trivalent metal phosphorus oxide (hereinafter sometimes referred to as "metal compound"). In the example shown in Figures 1B and 2, a second through-hole 41 is formed in the layer formed by the metal compound deposit 4. As will be described in detail later, when performing the measurement method, the change in ion current when a charged sample passes through the second through-hole 41 is measured.

[0017] The material used to form the substrate 2 is not particularly limited as long as it can form the first through-hole 3. Examples of materials used to form the substrate 2 include insulating materials commonly used in the field of semiconductor manufacturing technology. Examples of insulating materials include Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, SiN, etc. The substrate 2 may also be formed as a thin film called a solid membrane using materials such as SiN, SiO2, HfO2, or as a sheet called a two-dimensional material using materials such as graphene, graphene oxide, molybdenum dioxide (MoS2), or boron nitride (BN). The substrate 2 may also be formed using an artificial film such as a lipid bilayer or a naturally occurring film. Measurement devices using lipid bilayers are described in Japanese Patent Publication No. 2011-527191 and Japanese Patent Application Publication No. 2020-000056, etc. Matters described in Japanese Patent Publication No. 2011-527191 and Japanese Patent Application Publication No. 2020-000056 are incorporated herein by reference. Furthermore, commercially available measurement devices using lipid bilayers may also be used. Examples of commercially available devices capable of nanopore analysis using lipid bilayers include the MinION and GridION from Oxford Nanopore Technologies. Ｘ５ Examples include SmidgION and PromethION.

[0018] Generally, the measurement sensitivity of the ion current increases as the volume of the second through-hole 41 through which the charged sample passes decreases. While not limited to these, the thickness of the substrate 2 can be 5 μm or less, 1 μm or less, 750 nm or less, 500 nm or less, 250 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, etc. For example, graphene allows for the fabrication of a substrate 2 with a film thickness of 1 nm or less. When a solid membrane or two-dimensional material is used as the substrate 2, the film thickness can be made very thin. However, if the film thickness of the substrate 2 is very thin, it may be difficult to handle without damage. Therefore, the substrate 2 may be a laminated structure in which a solid membrane or two-dimensional material is laminated on a support plate formed of the above-mentioned insulating material.

[0019] As described in the manufacturing method later, the device 1 according to this embodiment can form a second through-hole 41 having a smaller diameter than the first through-hole 3 by depositing a metal compound deposit 4 on the wall surface 31 of the first through-hole 3. Furthermore, by continuing to deposit the metal compound, a device without the second through-hole 41 can be formed, in other words, a device in which the first through-hole 3 is sealed with the metal compound deposit 4. When using the device 1 in a measurement method, since the charged sample needs to pass through the through-hole, it is sufficient to use a device 1 having a second through-hole 41 of a desired diameter.

[0020] Conventionally, the first through-hole 3 was formed in the substrate 2 using lithography technology or the like. Therefore, there were limitations in terms of accuracy, but in the device 1 according to this embodiment, the diameter can be adjusted by the manufacturing method described later. The diameter of the second through-hole 41 is not particularly limited as long as it is not sealed. Although not limited, examples of the diameter of the second through-hole 41 include 0.1 nm or more, 0.2 nm or more, 0.3 nm or more, 0.4 nm or more, 0.5 nm or more, 0.6 nm or more, 0.7 nm or more, 0.8 nm or more, 0.9 nm or more, 1 nm or more, 1.5 nm or more, 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 10 nm or more, etc. On the other hand, since sensitivity increases as the volume of the second through-hole 41 decreases, the diameter of the second through-hole 41 is not limited, but examples include 3 μm or less, 2 μm or less, 1 μm or less, 750 nm or less, 500 nm or less, 250 nm or less, 100 nm or less, etc. In this specification, the diameter of the second through-hole 41 refers to the diameter if the second through-hole 41 is a circle, and the diameter of the circumscribed circle if it is not a circle.

[0021] The diameter of the first through-hole 3 only needs to be larger than the diameter of the above-described second through-hole 41. However, if it is too large, the amount of the deposited material 4 will increase, and the manufacturing time will also become longer. Therefore, the diameter of the first through-hole 3 may be appropriately designed according to the size of the diameter of the designed second through-hole 41. Although not limited, when the size of the second through-hole 41 is set to 1, the size of the first through-hole 3 may be larger than 1 and 100 times or less. Further, the shape of the first through-hole 3 (the shape viewed from the direction from the first surface 21 to the second surface 22) is not particularly limited as long as the deposited material 4 can be deposited. It may be any of a circular shape, an elliptical shape, a regular polygon, etc. Even if the first through-hole 3 has a shape other than a circular shape, in the process of depositing the deposited material 4 by the manufacturing method described later, the second through-hole 41 gradually becomes a shape close to a circular shape. From the viewpoint that the deposited material 4 is deposited symmetrically, the shape of the first through-hole 3 is preferably a substantially circular shape.

[0022] The principle of depositing the deposited material 4 will be described in detail in the manufacturing method described later. The deposited material 4 is at least one selected from the group consisting of divalent metal hydroxides, trivalent metal hydroxides, divalent metal phosphates, and trivalent metal phosphates. As the metal forming the divalent metal hydroxide (M(OH) ２ ) or the divalent metal phosphate (M ３ (PO ４ ) ２ ), calcium (Ca), magnesium (Mg), barium (Ba), strontium (Sr), copper (Cu), zinc (Zn), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), etc. can be mentioned. As the metal forming the trivalent metal hydroxide (M(OH) ３ ) or the trivalent metal phosphate (M(PO ４ )), aluminum (Al), iron (Fe), chromium (Cr), gallium (Ga), indium (In), manganese (Mn), etc. can be mentioned. Note that the phosphates of Mn are divalent Mn ３ (PO ４ ) ２ and trivalent MnPO ４ are known. When combining divalent and trivalent, Mn(PO ４ ) ｘIt may be described as follows: Sediment 4 may be formed from only one type selected from the group consisting of divalent metal hydroxides, trivalent metal hydroxides, divalent metal phosphorides, and trivalent metal phosphorides, or it may be a combination of two or more types. In the case of a combination, at least two types selected from the group consisting of divalent metal hydroxides, trivalent metal hydroxides, divalent metal phosphorides, and trivalent metal phosphorides may be formed in a layered state, or they may be formed as randomly mixed layers.

[0023] As shown in the embodiment described later, the deposit 4 of the device 1 is formed only on the wall surface 31 of the first through hole 3, and not on the first surface 21 and the second surface 22.

[0024] The device 1 according to this embodiment can be suitably used as a component of an ion current measuring device for measuring ion current, but is not limited to this application. As shown in the examples described later, the metal compound deposited in the first through-hole 3 of the device 1 allows the effective size of the second through-hole 41 to be controlled by voltage through a deposition and dissolution reaction. As the electrical conductivity of the second through-hole 41 changes dynamically in response to the deposition and dissolution reaction, it exhibits functions as a diode or a low-power memristor. Therefore, the device 1 according to this embodiment can also be applied to diodes and memristors.

[0025] (Embodiment of the Manufacturing Method) The manufacturing method according to the embodiment will be described with reference to Figures 3 and 4. Figure 3 is a flowchart of the manufacturing method according to the embodiment. Figure 4 is a schematic diagram showing an example of the apparatus used when carrying out the manufacturing method.

[0026] An example of the apparatus used when carrying out the manufacturing method will be described. As described above, the manufacturing method according to the embodiment can use some of the components of the ion current measuring device 100 used in the ion current measurement method. Therefore, first, the outline of the ion current measuring device 100 will be described.

[0027] The ion current measuring device 100 includes a device 1, a first chamber member 51 that forms a first chamber 5 together with the first surface 21 of the device 1, a second chamber member 61 that forms a second chamber 6 together with the second surface 22 of the device 1, a first electrode 52 formed at a location in contact with the first aqueous solution in the first chamber 5, a second electrode 62 formed at a location in contact with the second aqueous solution in the second chamber 6, a power supply 54 that applies a voltage between the first electrode 52 and the second electrode 62, an ammeter 7 for measuring the ion current when a charged sample passes through the second through hole 41, an analysis unit 8 for analyzing the ion current measured by the ammeter 7, a display unit 9 for displaying the measured ion current value and / or the results of the analysis by the analysis unit 8, a program memory 10 that stores a program in advance for operating the analysis unit 8 and the display unit 9, and a control unit 11 for reading and executing the program stored in the program memory 10. The program may be stored in the program memory 10 in advance, or it may be recorded on a recording medium and stored in the program memory 10 using an installation means.

[0028] The first chamber member 51 and the second chamber member 61 are preferably formed from electrically and chemically inert materials. Examples of such materials, though not limited to, include glass, sapphire, ceramic, resin, rubber, elastomer, SiO2, SiN, and Al2O3.

[0029] The first electrode 52 and the second electrode 62 can be made of known conductive metals such as aluminum, copper, platinum, gold, silver, silver / silver chloride, and titanium. Figure 4 shows an example in which the first electrode 52 and the second electrode 62 are formed so as to straddle the second through hole 41, and a voltage is applied so that a DC current flows with the first electrode 52 side as the negative electrode and the second electrode 62 side as the positive electrode. Alternatively, the first electrode 52 side may be the positive electrode and the second electrode 62 side as the negative electrode. Depending on the charge of the charged sample described later, it can be appropriately decided which side of the first electrode 52 and the second electrode 62 is the positive electrode.

[0030] The first electrode 52 is not particularly limited as long as it is formed at a location in contact with the first aqueous solution in the first chamber 5. In the example shown in Figure 4, the first electrode 52 is arranged on the inner surface of the first chamber member 51 via a lead 53. Alternatively, the first electrode 52 may be arranged on the first surface 21 of the substrate 2 or in the space within the first chamber 5 via a lead 53. Further alternatively, the first electrode 52 may be arranged to penetrate the first chamber member 51 through a hole formed in the first chamber member 51.

[0031] Similar to the first electrode 52, there are no particular restrictions on the location of the second electrode 62 as long as it is formed in a location that is in contact with the second aqueous solution in the second chamber 6. In the example shown in Figure 4, the second electrode 62 is arranged on the inner surface of the second chamber member 61 via a lead 63. Alternatively, the second electrode 62 may be arranged on the second surface 22 of the substrate 2, or in the space within the second chamber 6 via a lead 63. Further alternatively, the second electrode 62 may be arranged to penetrate the second chamber member 61 through a hole formed in the second chamber member 61.

[0032] In the example shown in Figure 4, the first electrode 52 is connected to the power supply 54 and ground 55 via a lead 53. The second electrode 62 is connected to the ammeter 7 and ground 64 via a lead 63. In the example shown in Figure 4, the power supply 54 is connected to the first electrode 52 side and the ammeter 7 is connected to the second electrode 62 side, but the power supply 54 and the ammeter 7 may be provided on the same electrode side.

[0033] The power supply 54 is not particularly limited as long as it can supply DC current to the first electrode 52 and the second electrode 62. The ammeter 7 is not particularly limited as long as it can measure the ion current generated over time when current is supplied to the first electrode 52 and the second electrode 62. Although not shown in Figure 4, the ion current measuring device 100 may be equipped with a noise reduction circuit, a voltage stabilization circuit, etc., as needed.

[0034] The analysis unit 8 analyzes the change in ion current measured by the ammeter 7. Generally, in an ion current measuring device 100 having a second through-hole 41, when a charged sample passes through the second through-hole 41, the ion current flowing through the second through-hole 41 is interrupted by the charged sample, and the ion current decreases. Therefore, the charged sample can be identified by performing data analysis in the analysis unit 8 based on the measured change in ion current (such as the peak value of the changed ion current or the waveform of the ion current).

[0035] The display unit 9 only needs to be able to display the measured ion current change and the results of the analysis performed by the analysis unit 8, and any known display device such as a liquid crystal display, plasma display, or organic EL display may be used. The program memory 10 is not particularly limited as long as it can store the program for making the analysis unit 8 and the display unit 9 function, and examples include ROMs such as mask ROM, PROM, EPROM, and EEPROM. The control unit 11 is not particularly limited as long as it can read and execute the program stored in the program memory 10, and examples include a processor (CPU) or a general-purpose computer equipped with a CPU.

[0036] The manufacturing method according to this embodiment can use the first chamber member 51, power supply 54, second chamber member 61, first electrode 52, and second electrode 62 from among the components that make up the ion current measuring device 100 described above. Although the manufacturing method according to this embodiment can be carried out using the components that make up the ion current measuring device 100, it may also be carried out using components other than the ion current measuring device 100, as long as each step of the manufacturing method described later can be carried out.

[0037] Next, with reference to Figures 3 and 4, details of the manufacturing method according to the embodiment using the ion current measuring device 100 will be described. The manufacturing method according to the embodiment includes a preparation step (ST1) and a metal compound deposition step (ST2). Optionally, a metal compound dissolution step (ST3) may also be included.

[0038] The preparation step (ST1) includes the following sub-steps: ・ST1a: A step of preparing a substrate 2 having a first surface 21 and a second surface 22, and including a first through-hole 3 penetrating from the first surface 21 to the second surface 22. ・ST1b: A step of placing a first aqueous solution containing divalent metal ions and / or trivalent metal ions on one of the first surface 21 or the second surface 22 of the substrate 2. In the example shown in Figure 4, the first aqueous solution may be filled into the first chamber 5. ・ST1c: A step of placing a second aqueous solution containing monovalent metal ions on the other of the first surface 21 or the second surface 22 of the substrate 2. In the example shown in Figure 4, the second aqueous solution may be filled into the second chamber 6. ・ST1d: A step of creating a liquid junction between the first aqueous solution and the second aqueous solution via the first through-hole 3. By filling the first chamber 5 with the first aqueous solution and the second chamber 6 with the second aqueous solution, the first aqueous solution and the second aqueous solution will be liquid junctioned by capillary force. ST1e: A step of bringing the first electrode 52 into contact with the first aqueous solution and the second electrode 62 into contact with the second aqueous solution.

[0039] The substrate 2 including the first through-hole 3 may be a substrate 2 in which the first through-hole 3 has been formed in advance, or the first through-hole 3 may be formed in the substrate 2 using lithography techniques. Since methods for forming the first through-hole 3 in the substrate 2 are described in Patent Documents 1 to 3, a detailed description is omitted. Matters described in Patent Documents 1 to 3 are incorporated herein by reference.

[0040] The divalent metal ions dissolved in the first aqueous solution are calcium ions (Ca ２＋ ), magnesium ions (Mg ２＋ ), barium ions (Ba ２＋ ), strontium ions (Sr ２＋ ), copper ions (Cu ２＋ ), zinc ions (Zn ２＋ ), iron ions (Fe ２＋ ), manganese ions (Mn ２＋ ), nickel ions (Ni ２＋ ), lead ions (Pb ２＋ Examples include the divalent metal ions (M) mentioned above. ２＋ ) is MCl ２ , M (NO ３ ) ２, MSO ４ MCO ３ You can dissolve metal salts such as these in water. Trivalent metal ions include aluminum ions (Al ３＋ ), iron ions (Fe ３＋ ), chromium ions (Cr ３＋ ), gallium ion (Ga ３＋ ), indium ions (In ３＋ Examples include the trivalent metal ions (M) mentioned above. ３＋ ) is MCl ３ , M (NO ３ ) ３ M ２ (SO ４ ) ３ The metal salts of the above can be dissolved in water. The metal ions contained in the first aqueous solution may consist only of divalent metal ions, only of trivalent metal ions, or a mixture of divalent and trivalent metal ions. Alternatively, the first aqueous solution may be replaced with an aqueous solution containing other metal ions during the manufacturing process. In that case, a deposit 4 in which different types of metal compounds are layered is formed.

[0041] The monovalent metal ions contained in the second aqueous solution are sodium iodine (Na). ＋ ), potassium ions (K ＋ ), lithium ion (Li ＋ ), silver ions (Ag ＋ Examples include the monovalent metal ions (M) mentioned above. ＋ The second aqueous solution can be prepared by dissolving a metal salt such as MCl in water. It is preferable that the second aqueous solution be prepared with a pH of approximately 7.0 or higher. To make the pH alkaline, generally sodium hydroxide (NaOH), potassium hydroxide (KOH), or sodium carbonate (Na) can be used. ２ CO ３ ), sodium bicarbonate (NaHCO) ３Alkaline reagents such as ) are used. When using alkaline reagents, the second aqueous solution does not need to contain metal salts such as MCl mentioned above, or it may be used in combination with metal salts such as MCl. Furthermore, the second aqueous solution may be formed with phosphate buffer alone, or it may be mixed with the monovalent metal ions mentioned above. When phosphate buffer is included in the second aqueous solution, metal phosphorides are deposited by phosphorylating the metal ions contained in the first aqueous solution. Note that the second aqueous solution described above is merely an example, and there are no particular restrictions on the components of the second aqueous solution as long as metal compounds can be formed from the metal ions contained in the first aqueous solution. For example, the second aqueous solution may contain divalent and trivalent metal ions as long as the deposition and accumulation of metal compounds proceeds within the first through-hole 3.

[0042] The metal compound deposition process (ST2) includes the following sub-processes: • ST2a: A first voltage application process in which a voltage is applied to the first electrode 52 and the second electrode 62 so that metal ions contained in the first aqueous solution move toward the first through-hole 3. • ST2b: A process in which, in the portion of the first through-hole 3, metal ions contained in the first aqueous solution are converted into metal hydroxides by a hydroxylation reaction, or metal ions contained in the first aqueous solution are converted into metal phosphorides by a phosphorylation reaction, and deposited on the wall surface 31 of the first through-hole 3.

[0043] In the example shown in Figure 4, when the first aqueous solution is filled into the first chamber 5 and the second aqueous solution is filled into the second chamber 6, the first voltage application step is to apply a voltage such that the second electrode 62 becomes the negative electrode and the first electrode 52 becomes the positive electrode. By making the second electrode 62 the negative electrode, the divalent metal ions and / or trivalent metal ions contained in the first chamber move toward the first through hole 3.

[0044] As shown in the examples and comparative examples described later, in the manufacturing method disclosed in this application, the first through-hole 3 serves as the reaction site for material synthesis. More specifically, in the first through-hole 3, migrated divalent and trivalent metal ions precipitate as metal hydroxides by a hydroxylation reaction, or divalent and trivalent metal ions precipitate as metal phosphorides by a phosphorylation reaction, and accumulate on the wall surface 31 of the first through-hole 3. As shown in the comparative examples described later, when the second aqueous solution was acidic, no metal hydroxides accumulated in the first through-hole 3. On the other hand, when a phosphate buffer solution was used as the second aqueous solution, metal phosphoride accumulation was observed even when the pH of the second aqueous solution was near neutral. Therefore, it is desirable to set the pH of the second aqueous solution to neutral to alkaline depending on the components constituting the second aqueous solution. Similarly, when the first aqueous solution was strongly acidic, no metal hydroxides or metal phosphorides accumulated in the first through-hole 3. Therefore, it is desirable to set the pH of the first aqueous solution to weakly acidic to neutral depending on the components constituting the first aqueous solution.

[0045] The voltage applied in the first voltage application step is not particularly limited as long as a metal compound can be deposited on the wall surface 31 of the first through-hole 3. It should be set appropriately considering the concentrations of the first and second aqueous solutions, the size of the first through-hole 3, etc.

[0046] The manufacturing method according to the embodiment may optionally include a metal compound dissolution step (ST3) in which the deposited metal compound is dissolved after the metal compound deposition step (ST2) described above is performed. The metal compound dissolution step (ST3) includes a second voltage application step in which a voltage is applied to the first electrode 52 and the second electrode 62 such that the positive and negative terminals are reversed compared to the first voltage application step. In the example shown in Figure 4, since the second electrode 62 was the negative electrode and the first electrode 52 was the positive electrode in the first voltage application step described above, in the second voltage application step, a voltage should be applied such that the second electrode 62 is the positive electrode and the first electrode 52 is the negative electrode.

[0047] If the metal compound dissolution step (ST3) is performed, the metal compound deposited in the first through-hole 3 can be dissolved. Therefore, for example, when forming the second through-hole 41 by performing the metal compound deposition step (ST2), if the diameter of the second through-hole 41 becomes smaller than the desired size, the metal compound can be dissolved by performing the metal compound dissolution step (ST3) to increase the diameter size. The metal compound deposition step (ST2) and the metal compound dissolution step (ST3) may be repeated as needed.

[0048] The manufacturing method disclosed in this application and the devices manufactured by this method have the following effects: (1) Unlike the conventional manufacturing method using ALD described in Patent Document 4, the diameter size of the through-hole can be adjusted by a new principle. (2) The manufacturing method disclosed in this application adjusts the size of the second through-hole 41 by depositing a metal compound. Although Patent Documents 2 and 4 also disclose the formation of a layer on the wall surface of the first through-hole 3, that layer is metal. Therefore, the manufacturing method disclosed in this application for forming deposits 4 of metal hydroxide and / or metal phosphorus oxide on the wall surface of the first through-hole 3 is a novel method, and the device 1 manufactured by this method is also a novel device. (3) When a device is manufactured by the manufacturing method according to the embodiment, as shown in the examples described later, the deposits 4 of the metal compound are formed only on the wall surface 31 of the first through-hole 3, and not on the first surface 21 and the second surface. When forming a metal layer using conventional ALD or the like, it is difficult to form it only on the wall surface 31 of the first through-hole 3, and the metal layer also accumulates near the opening of the first through-hole 3, which may result in a change in the shape around the first through-hole 3. When using device 1 to measure ion current, a change in the shape of the first through-hole 3 is undesirable as it affects the measurement accuracy. Furthermore, Patent Document 2 describes forming a metal layer only on the wall surface of the through-hole. However, the method described in Patent Document 2 first coats a resist on the surface of the substrate, then coats a metal layer on the through-hole and the surface of the resist by sputtering or the like, and then lifts off the resist to form a metal layer only on the wall surface of the through-hole. Therefore, even with the method described in Patent Document 2, forming a metal layer only on the wall surface of the through-hole requires a sputtering apparatus and the procedure is complicated. On the other hand, the manufacturing method disclosed in this application has the remarkable effect of forming a deposit 4 of a metal compound only on the first through-hole 3 without using other equipment, by utilizing the electrodes and power supply used for measuring ion current. (4) When the manufacturing method includes a metal compound dissolution step (ST3), the diameter size of the second through hole 41 can be finely adjusted. (5) As shown in the examples described later, the device manufactured by the manufacturing method according to the embodiment is subject to the voltage conditions during manufacturing (polarity of the voltage applied to the first electrode 52 and the second electrode 62).The magnitude of the applied voltage may be the same as or different from the conditions under which the metal oxide is deposited. Maintaining this condition results in the unexpected effect of the metal compound deposited in the first through-hole 3 spontaneously opening and closing.

[0049] (Embodiment of Measurement Method) Next, a measurement method according to the embodiment will be described with reference to Figure 4. The assumed method according to the embodiment is carried out using the ion current measuring device described above. Since the ion current measuring device has already been described, a description will be omitted to avoid repetition.

[0050] The measurement method includes a charged sample passage step and an ion current measurement step. In the charged sample passage step, a voltage is applied to the first electrolyte filled in the first chamber 5 and the second electrolyte filled in the second chamber 6, causing the charged sample contained in the first chamber 5 to pass through the second through-hole 41 toward the second chamber 6, or the charged sample contained in the second chamber 6 to pass through the second through-hole 41 toward the first chamber 5. In the ion current measurement step, the change in ion current when the charged sample passes through the second through-hole 41 is measured.

[0051] The charged sample is not particularly limited as long as it can be moved from one chamber to the other by applying a voltage to the first electrode 52 and the second electrode 62. Examples of charged samples include nucleic acids, proteins, viruses, glycans, viral vectors, ribosomes, etc. Unlike conventional devices, the second through-hole 41 of the device 1 disclosed in this application can be made to a very small size. Therefore, even a sample at the single-molecule level can have its ion current measured.

[0052] The first and second electrolytes are not particularly limited as long as they allow current to pass through the first electrode 52 and the second electrode 62, and may be TE buffer, PBS buffer, HEPES buffer, NaCl, KCl aqueous solution, etc.

[0053] The ion current measurement method according to the embodiment provides the following advantages: (1) The device 1 disclosed in this application allows the diameter of the second through-hole 41 to be adjusted to a desired size. Therefore, the size of the second through-hole 41 can be adjusted to match the size of the charged sample to be measured, thereby improving the accuracy of measuring the ion current of the charged sample. (2) The device of this application can be manufactured using an ion current measuring device. For example, in the example shown in Figure 4, first, a substrate 2 having the first through-hole 3 is set, and the device 1 having the second through-hole 41 is manufactured according to the procedure described above. Next, the first aqueous solution and the second aqueous solution are replaced with the first electrolyte and the second electrolyte, and the charged sample is administered to either the first chamber 5 or the second chamber 6, so that the ion current measurement method can be immediately performed using the manufactured device 1.

[0054] Examples are provided below to specifically illustrate the embodiments disclosed in this application. These examples are solely for illustrative purposes and are not intended to limit or restrict the scope of disclosure in this application.

[0055] <Example 1> [Preparation of Substrate 2 with First Through Hole 3 Formed] A 50 nm thick SiNx layer was coated on both sides of a 4-inch silicon wafer by low-pressure chemical vapor deposition. The wafer was diced into 25 mm square chips using a dicer (DISCO). Microelectrode patterns were formed on the silicon chips using photolithography with photoresist AZ5206 (AZ Electronic Materials). After development, a 50 nm thick Au layer was deposited on a 5 nm thick Cr adhesive layer by radio frequency magnetron sputtering (Samco). The chips were immersed overnight in N,N-dimethylformamide (Wako), and then ultrasonically cleaned for lift-off. Electron beam resist ZEP520A (Zeon) was spin-coated onto the microelectrode surface and pre-baked at 180°C using a hot plate. By electron beam lithography (Elionix), a diameter d was formed within the resist layer. ｐｏｒｅ A circle was drawn. After development, the resist layer was used as a mask for reactive ion etching (Samco), and HCF ３ Using an etchant gas, a diameter d is formed in the SiNx layer.ｐｏｒｅ A hole was drilled. Then, the chip was immersed in N,N-dimethylformamide overnight to remove the residual resist layer. A photoresist TSMR was spin-coated onto the back surface of the chip, and a 1 mm square area in the center was removed by photolithography using the microelectrode pattern on the front surface as a marking. Furthermore, a reactive ion etching process was performed to partially remove the SiNx layer on the back surface. Next, an aqueous solution of potassium hydroxide was applied to the back surface and the exposed Si layer was dissolved by anisotropic etching by heating at 90°C. As a result, a diameter d ｐｏｒｅ A 40 nm thick SiNx film with pores was fabricated. Note that the diameter d of the first through-hole 3 used in the example was... ｐｏｒｅ It had a roughly circular shape with a diameter of approximately 300 nm. Figure 5 shows an SEM image of the first through-hole 3 formed in the substrate 2.

[0056] [Fabrication of the First and Second Chambers] An I-shaped pattern was fabricated on a silicon wafer using photoresist SU-8 3000 by photolithography. Polydimethylsiloxane Sylgard 184 (Dow) was cured on this SU-8 mold at 90°C. A 15 mm PDMS block with an I-shaped groove on one side of the surface was cut out using a surgical knife. Three 1 mm diameter holes were punched into the block for inserting electrodes for ion current measurement and for allowing electrolyte solution to flow in and out of the holes. The perforated PDMS blocks (first chamber member, second chamber member) were exposed to oxygen plasma along with the fabricated substrate 2 to activate the surface. Subsequently, the first and second chambers were fabricated by bonding the first and second chamber members to the substrate 2.

[0057] [Fabrication of Ion Current Measurement Device (Device for Manufacturing Device 1)] Ag / AgCl rods were used as the first and second electrodes and inserted into the first and second chambers through holes on both sides of the PDMS block. The Ag / AgCl electrodes were connected to a picoammeter source unit (Keightley 6487, Keysight). The ion current generated by applying voltage to the first and second electrodes was recorded under GPIB control using a program coded in Visual Basic.

[0058] [Fabrication of a device in which a metal compound is deposited in the first through-hole 3] 2M MnCl as the first aqueous solution ２ An aqueous solution (pH 4.2) was prepared and filled into the first chamber. A phosphate buffer solution (pH 7.4) containing 1.37 M NaCl was prepared as a second aqueous solution and filled into the second chamber. Next, the applied voltage was gradually changed from 0V to -1.5V, with the first electrode acting as the positive electrode and the second electrode as the negative electrode. The measurement results of the ion current are shown to the left of the 0V voltage in Figure 6A. (MnCl) ２ The lines labeled / NaCl represent the measured results of the ion current. As is clear from the ion current measurements on the left side of Figure 6A, even when the applied negative voltage was increased, almost no ion current was observed. This is because, as shown in Figure 6A, Mn contained in the first aqueous solution... ２＋ When the substance flows into the first through hole 3, a phosphorylation reaction occurs, and Mn(PO) ４ ) ｘ Because Mn precipitated and accumulated in the first through-hole 3, ２＋ This is thought to be because the Mn contained in the first through-hole 3 did not pass through. Figure 6B is a graph showing the measured ion current when the applied voltage is near 0V, changed from the order of μA to the order of nA. As is clear from Figure 6B, when a negative voltage was applied from 0V, a positive ion current was measured first, and the measured ion current gradually shifted to the negative side. This is because the Mn contained in the first aqueous solution ２＋ Immediately after the precipitation begins, the Cl contained in the second aqueous solution － It was able to move towards the first chamber, but Mn(PO) to the wall surface of the first through hole 3 ４ ) ｘIt is thought that as the accumulation of sediment progressed, the first through-hole 3 became blocked by the sediment, making it difficult for ions to flow through the first through-hole 3.

[0059] Figure 7 shows a scanning electron microscope image of the substrate 2 from the first side after gradually changing the applied voltage from 0V to -1.5V with the first electrode acting as the positive electrode and the second electrode as the negative electrode. As is clear from Figure 7, the first circular through-hole 3 with a diameter of approximately 300 nm contains deposited metal phosphorus oxide (Mn(PO) ４ ) ｘ ) was completely blocked. On the other hand, the first surface of substrate 2 had Mn(PO ４ ) ｘ No deposits were observed. From these results, it was confirmed that metal phosphorus oxides are formed only inside the first through-hole 3.

[0060] Next, the substrate 2 (shown in Figure 7), which had been gradually changed from 0V to -1.5V with the first electrode as the positive electrode and the second electrode as the negative electrode, was then subjected to a similar procedure where the first electrode was the negative electrode and the second electrode as the positive electrode, with the applied voltage gradually changing from 0V to 1.5V. The measurement results of the ion current are shown to the right of the 0V voltage in Figure 6A. MnCl ２ The lines labeled / NaCl represent the measured ion current. As is clear from the ion current measurements to the right of 0V in Figure 6A, the measured ion current increased as the applied voltage value increased. The reason for this is that, as shown in the right-hand diagram of Figure 6A, the first through-hole 3 was blocked by Mn(PO) ４ ) ｘ This is thought to be because the dissolution progressed, allowing ions to pass through the first through-hole 3.

[0061] [Evaluation of the reaction heat of the first through-hole 3] From the results shown in Figures 6 and 7, it is clear that deposition of a metal compound occurred in the first through-hole 3. Furthermore, the reaction heat of the first through-hole 3 was evaluated. The experimental procedure is described below. (1) Preparation of evaluation substrate An evaluation substrate having a first through-hole 3 with a diameter of approximately 60 nm was prepared according to the procedure described in Example 1. Nano thermocouples (Au nanowires and Pt nanowires) were formed near the first through-hole 3 during the electron beam lithography and sputtering processes. A photograph of the prepared substrate 2 is shown in the upper left of Figure 8.

[0062] (2) Method for evaluating reaction heat and evaluation 1 The ion current measuring device (device for manufacturing device 1) described above was fabricated using the evaluation substrate that was prepared in place of the substrate of Example 1. Next, one end of the metal pin was connected to the microelectrode pads connected to the Au nanowire and Pt nanowire of the evaluation substrate. The other end of the metal pin was wired to a nanovoltmeter (Keithley 2182A, Keysight) and the thermovoltage was recorded. 2M CaCl was placed in the first chamber. ２ An aqueous solution was packed into the second chamber, and a phosphate buffer solution containing 1.37 M NaCl was added. A voltage was then applied. Simultaneous measurement of ion current and thermal voltage was performed using a Visual Basic program code under GPIB control, and the temperature of the first through-hole (nanopore) was measured when the voltage was changed.

[0063] - Rating 2: 2M CaCl (compared to Rating 1) ２ Instead of an aqueous solution, use 2M MgCl ２ The experiment was conducted using the same procedure as in Evaluation 1, except that the aqueous solution was filled into the first chamber.

[0064] The results of Evaluation 1 are shown in Figure 8, and the results of Evaluation 2 are shown in Figure 9. As shown in Figure 8, the configuration of the electrolyte solution that produces rectification (CaCl filled in the first chamber) ２ When ion current measurements were performed using the ion current, a significant rectification characteristic was observed. Furthermore, when the nanopore temperature was calculated from the thermoelectric voltage of the thermocouple measured simultaneously, it was observed that the temperature temporarily increased under a negative voltage, and then tended to become lower than room temperature at a certain point. This is because, during the voltage sweep process, the exothermic reaction of Mn(PO) ４ ) ｘIt is thought that precipitation occurred, and the heat of this reaction slightly increased the nanopore temperature, while the subsequent reverse voltage sweep caused a dissolution reaction and localized cooling of the nanopore.

[0065] On the other hand, as shown in Figure 9, CaCl ２ It is presumed that MgCl will produce only a small amount of reaction heat compared to the reaction of [another group]. ２ Regarding this, although significant rectification characteristics were observed, the nanopore temperature tended to remain almost unchanged even under negative voltage. From these results, it was confirmed that by manipulating the voltage applied to the electrode positioned across the first through-hole 3, the first through-hole 3 functions as a reaction field for depositing metal compounds from metal ions, and that this reaction field is controllable.

[0066] <Example 2> The first aqueous solution (MnCl) to be filled into the first chamber ２ The experiment was conducted using the same procedure as in Example 1, except that the pH of the solution was changed to pH 0.5 and pH 5.1. Figure 10A shows the results for pH 0.5, and Figure 10B shows the results for pH 5.1.

[0067] <Example 3> The first aqueous solution to be filled into the first chamber is 2M CaCl ２ The experiment was conducted in the same manner as in Example 1, except that the pH was set to 5.4, the second aqueous solution to be filled into the second chamber was an aqueous NaCl solution that did not contain phosphate buffer, and the pH of the aqueous NaCl solution was further adjusted to 2.4 using HCl and to 13.0 using NaOH. Figure 11A shows the results for pH 2.4, and Figure 11B shows the results for pH 13.0.

[0068] The results shown in Figures 10 and 11 confirm that metal compounds do not precipitate if the pH of the first aqueous solution is too low, and metal compounds do not precipitate if the pH of the second aqueous solution is too low. From these results, it was confirmed that the pH of the first aqueous solution and the pH of the second aqueous solution need to be appropriately adjusted within a range in which metal compounds do not precipitate in the first and second aqueous solutions.

[0069] <Comparative Example 1> The experiment was conducted in the same manner as in Example 1, except that the first and second chambers were filled only with an aqueous NaCl solution that did not contain phosphate buffer. The results are shown in Figure 12. As is clear from Figure 12, the first and second chambers were filled with monovalent metal ions, Na ＋ When filled with [the specified substance], no metal compound deposition reaction was observed. Also, although not shown in the diagram, Na [the specified substance] was used. ＋ Instead, Li ＋ _K ＋ Similarly, no deposition of metal compounds was observed when using [the other method]. From these results, it was confirmed that it is desirable to fill the first and second chambers with metal ions of different valencies.

[0070] [Measurement of Ion Current Using the Fabricated Device] <Example 4> Plasmid DNA colE1 (Nippon Gene) was used as the sample, and 1.37 M NaCl solution (buffer solution) was used as the electrolytic solution to fill the chamber. The upper part of Figure 13A shows the waveform of the ion current measured when buffer solution was filled in both the first and second chambers and a voltage of 0.2 V was applied. The lower part of Figure 13A shows the waveform of the ion current measured when colE1 was administered as a charged sample to the buffer solution in the first chamber, and buffer solution was filled in the second chamber and a voltage of 0.2 V was applied. Figure 13B is an enlarged graph of a part of Figure 13A, where the arrows indicate the peaks of plasmid DNA. As is clear from Figures 13A and 13B, it was confirmed that the ion current of the sample can be measured using the device fabricated by the manufacturing method disclosed in this application.

[0071] <Example 5> 2M CaCl as the first aqueous solution ２ An aqueous solution was prepared and packed into the first chamber. A phosphate buffer solution (pH 7.4) containing 1.37 M NaCl was prepared as a second aqueous solution and packed into the second chamber. A pulsed voltage (+0.4V or -0.4V; pulse application time 0.1 seconds: pulse interval 1 second) was applied. Figure 14 shows the applied pulse voltage and the measured ion current values. Note that the symbols (a) to (c) in Figure 14 indicate that (a) Ca was generated in the first through-hole (nanopore) by a negative voltage pulse. ３ (PO４ ) ２ indicates that the layer has grown, and (b) shows that Ca in the first through-hole (nanopore) due to a positive voltage pulse ３ (PO ４ ) ２ indicates that the layer has dissolved, and (c) shows that Ca in the first through-hole (nanopore) is proportional to the number of applied voltage pulses ３ (PO ４ ) ２ indicates that the layer has gradually dissolved and gradually become in a high conductivity state. As shown in FIG. 14, in the first through-hole 3 of the device disclosed in the present application, by reversing the ± of the applied voltage, a metal compound (Ca ３ (PO ４ ) ２ ) precipitation / dissolution reaction occurs, and as a result, the electrical conductivity of the first through-hole 3 changes dynamically. Therefore, it has been confirmed that the device disclosed in the present application can be used as a device for measuring an ionic current in a single molecule unit, and can also be applied as a diode with a rectification ratio exceeding 40000 and a low-power consumption memristor.

[0072] <Example 6> In the production of the [ionic current measuring device (device for manufacturing device 1)] of Example 1, after changing the applied voltage to 0 V to -1.1 V so that the first electrode is the positive electrode and the second electrode is the negative electrode, and then maintaining the state of -1.1 V, the device was produced and the ionic current was measured in the same procedure as in Example 1. The upper part of FIG. 15 is a graph showing the result of measuring the ionic current on the nA order, and the lower part is a graph obtained by enlarging the upper graph to the pA level.

[0073] <Example 7> An experiment was conducted in the same procedure as in Example 6, except that the first aqueous solution was changed from 2M MnCl ２ aqueous solution (pH 4.2) to 2M CaCl ２ / 10×PBS (pH 5.4). FIG. 16 shows the result of measuring the ionic current on the nA order.

[0074] As shown in Figures 15 and 16, pulsed ion currents were measured by maintaining the same voltage conditions even after the metal compound was deposited in the first through-hole 3. The measurement of pulsed ion currents means that opening and closing occurred repeatedly in the metal compound deposited in the first through-hole 3. From these results, it was confirmed that the device disclosed in this application has a function in which the metal compound deposited in the first through-hole 3 spontaneously opens and closes, in other words, a function in which the second through-hole 41 is repeatedly formed and disappears. In addition, it is thought that the following phenomena occurred in A to C shown in the ion current measurement values ​​in the lower part of Figure 15. ・A: The first through-hole 3 is completely closed due to the deposition of the metal compound. However, in this closed state, ion transport stops and the dissolution reaction proceeds. ・B: The dissolution reaction proceeds and an opening is created, and the electric field concentrates at the opening, causing ion transport. ・C: Ions from the first chamber are drawn into the opening by the electric field and the metal compound is deposited, causing the opening described in B to close.

[0075] <Example 8> [Measurement of Samples] Next, the samples (nucleic acids and amino acids) were measured using the device shown in Example 6, in which the metal compound deposited in the first through-hole 3 repeatedly opens and closes. (1) Samples used a: Nucleic acids - Adenosine monophosphate (AMP) - Thymidine monophosphate (TMP) - Guanosine monophosphate (GMP) - Cytidine monophosphate (CMP) b: Amino acids - Glycine - Leucine - Glutamic acid - Isoleucine - Histidine - Alanine - Aspartic acid

[0076] (2) Sample preparation In a phosphate buffer (pH 7.4) containing 1.37 M NaCl, which is the second aqueous solution of Example 1, the nucleic acids were dissolved in concentrations of 2 mM, 5 mM, 10 mM, 20 mM, and 50 mM for each sample, and the amino acids were dissolved in concentrations of 20 mM for each sample.

[0077] (3) Experimental procedure The diameter of the first through-hole 3 was set to 70 nm, and an aqueous solution containing the above-mentioned sample was used as the second aqueous solution. The applied voltage was changed from 0 V to -1.0 V, with the first electrode being the positive electrode and the second electrode being the negative electrode, and the -1.0 V state was maintained. Except for these changes, the device was fabricated and the ion current was measured in the same procedure as in Example 6.

[0078] (4) Measurement Results Figures 17A to 17C show the measurement results for nucleic acids, and Figures 18A to 18B show the measurement results for amino acids. Figure 17A shows the measurement results for each sample superimposed. Figure 17C shows the fitting curves obtained by fitting a Gaussian function to the distribution of the pulse height histograms of the measured AMP, GMP, TMP, and CMP. As shown in Figure 17A, the ion current signal was significantly smaller for TMP than for AMP in the measurement results for 50 mM AMP and TMP. Also, as shown in Figure 17B, a molecular-specific peak was observed when the molecular concentration was 10 mM or higher. Although not shown in the figure, a similar trend was observed for GMP and CMP. Furthermore, as shown in Figure 17C, it was confirmed that the fitting curves for AMP, GMP, TMP, and CMP can be clearly distinguished.

[0079] Figure 18A shows the measurement results for alanine, histidine, glycine, leucine, glutamic acid, isoleucine, and aspartic acid. Note that in Figure 18A, the vertical axis represents the measured ion current (I). Ｐ (A)), the horizontal axis is the time (t) the sample takes to pass through the hole. ｄ (s; dwell time) is a plot showing the measurement results of individual amino acids. As is clear from Figure 18A, the distribution patterns of each amino acid were different.

[0080] Figure 18B shows typical waveforms of the ion current values ​​of each amino acid measured. As is clear from Figure 18B, isoleucine was characterized by a very small signal amplitude. Leucine, glycine, histidine, and alanine were characterized by relatively high amplitudes and a relatively narrow distribution of wave widths. On the other hand, glutamic acid and aspartic acid were characterized by high amplitudes and wave widths.

[0081] From the above results, we confirmed that even when the metal compound deposited in the first through-hole 3 of the device spontaneously opens and closes (a state in which the second through-hole 41 is repeatedly formed and destroyed), the type of sample can be determined by measuring the ion current when the sample passes through the open second through-hole 41. It should be noted that the size of the second through-hole 41 when the metal compound is automatically opening and closing is considered to be very small, given the phenomenon described above. Furthermore, in the field of measuring ion current when a sample passes through a through-hole, it is generally known that the smaller the size of the through-hole, the higher the measurement sensitivity of the sample. Therefore, by measuring a sample in a state where the second through-hole 41 is repeatedly formed and destroyed, nucleic acids and amino acids can be measured with high sensitivity at the single-molecule level. For this reason, applications in sequencing analysis of long-chain nucleic acids and proteins, and in high-sensitivity sensors for small molecules such as PFAS, which have been linked to health hazards, can be expected.

[0082] The device manufacturing method disclosed in this application provides a device in which the size of the through-hole can be adjusted by a novel principle. Therefore, it is useful for the development of analytical instruments in the analytical instrument industry.

[0083] 1, 1a...Device, 2...Substrate, 3...First through hole, 4...Deposit, 41...Second through hole, 5...First chamber, 6...Second chamber, 7...Ammeter, 8...Analysis unit, 9...Display unit, 10...Program memory, 11...Control unit, 21...First surface, 22...Second surface, 31...Wall surface, 51...First chamber member, 52...First electrode, 53...Lead, 54...Power supply, 55...Ground, 61...Second chamber member, 62...Second electrode, 63...Lead, 64...Ground

Claims

1. A device comprising: a substrate having a first surface and a second surface; a first through-hole penetrating from the first surface to the second surface; and a deposit of a metal compound formed on the wall surface of the first through-hole, the deposit being at least one selected from the group consisting of divalent metal hydroxides, trivalent metal hydroxides, divalent metal phosphorides, and trivalent metal phosphorides.

2. The device according to claim 1, wherein the deposit of the metal compound is formed only on the wall surface of the first through hole, and not on the first and second surfaces.

3. The device according to claim 1, wherein a second through-hole is formed in the layer formed of the metal compound deposit.

4. A method for manufacturing the device according to claim 1, the manufacturing method comprising: a preparation step and a metal compound deposition step, the preparation step comprising: preparing a substrate having a first surface and a second surface and including a first through-hole penetrating from the first surface toward the second surface; placing a first aqueous solution containing divalent metal ions and / or trivalent metal ions on one of the first surface or the second surface of the substrate; placing a second aqueous solution containing monovalent metal ions on the other of the first surface or the second surface of the substrate; bringing the first aqueous solution and the second aqueous solution into liquid junction through the first through-hole; and bringing a first electrode into contact with the first aqueous solution and a second electrode into contact with the second aqueous solution, the metal compound deposition step comprising: a first voltage application step of applying a voltage to the first electrode and the second electrode so that the metal ions contained in the first aqueous solution move toward the first through-hole, A manufacturing method comprising the step of: in the first through-hole portion, metal ions contained in the first aqueous solution are changed to metal hydroxides by a hydroxylation reaction, or metal ions contained in the first aqueous solution are changed to metal phosphorides by a phosphorylation reaction, and deposited on the wall surface of the first through-hole as a metal compound.

5. The manufacturing method according to claim 4, wherein the pH of the second aqueous solution is 7.0 or higher.

6. The manufacturing method according to claim 4, wherein if the second aqueous solution contains a phosphate buffer, a metal phosphate compound is obtained as the metal compound, and if the second aqueous solution does not contain a phosphate buffer, a metal hydroxide is obtained as the metal compound.

7. A manufacturing method according to any one of claims 4 to 6, comprising a metal compound dissolution step of dissolving the deposited metal compound after carrying out the metal compound deposition step, wherein the metal compound dissolution step comprises a second voltage application step of applying a voltage to the first electrode and the second electrode such that the positive and negative charges are reversed compared to the first voltage application step.

8. The manufacturing method according to claim 7, wherein the metal compound deposition step and the metal compound dissolution step are carried out repeatedly.

9. A method for measuring the ion current of a charged sample using an ion current measuring device, wherein the ion current measuring device includes the device described in claim 3, a first chamber member, and a second chamber member, wherein the first chamber member forms a first chamber filled with a first electrolyte solution on at least the surface of the first surface of the substrate of the device including a second through-hole, and the second chamber member forms a second chamber filled with a second electrolyte solution on at least the surface of the second surface of the substrate of the device including a second through-hole, and the method for measuring the ion current includes a charged sample passing step and an ion current measurement step, wherein the charged sample passing step involves applying a voltage to the first electrolyte solution filled in the first chamber and the second electrolyte solution filled in the second chamber, thereby causing the charged sample contained in the first chamber to pass through the second through-hole in the direction of the second chamber, or causing the charged sample contained in the second chamber to pass through the second through-hole in the direction of the first chamber, and the ion current measurement step is a method for measuring the change in ion current when the charged sample passes through the second through-hole.

10. The ion current measurement method according to claim 9, wherein, in the charged sample passing step, the polarity of the voltage applied to the first electrolyte and the second electrolyte is the same as the polarity when the metal compound is deposited in the first through-hole, and the ion current measurement step is performed while the formation and disappearance of the second through-hole is repeatedly performed.

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