WO2024075637A1 - Ionic current measurement method, and ionic current measurement device - Google Patents

Abstract

 Provided are an ionic current measurement method and an ionic current measurement device capable of increasing an S/N ratio of a measurement result of a change in an ionic current. The ionic current measurement device comprises a board having a first surface and a second surface, a through-hole that penetrates through from the first surface toward the second surface to allow a charged sample to pass through, a first chamber member which, together with a surface of the first surface including a first opening of the through hole forms a first chamber that is filled with a first electrolytic solution, and a second chamber member which, together with a surface of the second surface including a second opening of the through hole forms a second chamber that is filled with a second electrolytic solution, and the ionic current measurement method includes a step for applying a voltage across the first electrolytic solution and the second electrolytic solution to cause the charged sample contained in one of the chambers to pass through the through-hole in a direction toward the other chamber, and a step for measuring a change in an ionic current when the charged sample passes through the through-hole. Furthermore, the first electrolytic solution and the second electrolytic solution have different dielectric constants.

Description

Ion current measuring method and device for measuring ion current

The disclosure in this application relates to a device for measuring ion current and a method for measuring the ion current of a sample using the device for measuring ion current.

A device that forms nanopores in a substrate and measures the change in ionic current as a sample passes through the nanopores is attracting attention as a device with broad applications for sensing bacteria, viruses, DNA, proteins, etc.

Known 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), forming molecules in through-holes formed in a substrate that interact with the passing sample, thereby lengthening the time it takes for a sample to pass through the through-hole (see Patent Document 2), and forming two or more through-holes in a substrate (see Patent Document 3).

JP 2017-156168 A International Publication No. 2017/183716 International Publication No. 2020/138021

As described in the above Patent Documents 1 to 3, it is well known to measure the change in ion current when a sample passes through a through hole formed in a substrate. However, it is desirable for the measurement results of the change in ion current to have a high S/N ratio.

The inventors conducted further research to increase the S/N ratio and discovered that (1) for a first electrolyte filled in a first chamber on the first surface side of a substrate forming a through hole and a second electrolyte filled in a second chamber on the second surface side of the substrate, (2) by making the dielectric constant of the first electrolyte different from that of the second electrolyte, (3) it is possible to increase the S/N ratio of the measurement results of the change in ion current when a charged sample passes through a through hole.

In other words, the disclosure of this application is to provide an ion current measurement method that can increase the S/N ratio of the measurement results of changes in ion current, and an ion current measurement device for use in said ion current measurement method.

The disclosure in this application relates to a device for measuring ion current and a device for measuring ion current, as shown below.

(1) A method for measuring an ion current of a charged sample using a device for measuring an ion current, comprising the steps of:
The ion current measuring device comprises:
a substrate having a first side and a second side;
a through hole extending from the first surface to the second surface through which the charged sample passes;
A first chamber member;
A second chamber member;
Including,
the first chamber member forms a first chamber filled with a first electrolytic solution together with the first surface including at least a surface of the first opening of the through hole;
the second chamber member forms a second chamber filled with a second electrolytic solution together with at least a surface of the second surface including the second opening of the through hole;
The method for measuring the ion current comprises:
A charged sample passing step;
an ion current measuring step;
Including,
The charged sample passing step includes:
A voltage is applied to the first electrolytic solution filled in the first chamber and the second electrolytic solution filled in the second chamber,
Passing the charged sample contained in the first chamber through the through hole toward the second chamber, or passing the charged sample contained in the second chamber through the through hole toward the first chamber;
The ion current measuring step includes:
measuring a change in ionic current as the charged sample passes through the through-hole;
The dielectric constant of the first electrolytic solution is different from the dielectric constant of the second electrolytic solution.
Ion current measurement method.
(2) An organic solvent having a dielectric constant lower than that of water is dissolved in one of the first electrolytic solution or the second electrolytic solution.
The ion current measuring method according to (1) above.
(3) The organic solvent is at least one selected from the group consisting of monohydric alcohols, dihydric alcohols, and trihydric alcohols.
The ion current measuring method according to (2) above.
(4) When the higher of the dielectric constants of the first electrolytic solution and the second electrolytic solution is taken as 1, the lower one has a dielectric constant of 0.1 or more and 0.9 or less.
The ion current measuring method according to (1) above.
(5) The organic solvent is selected from solvents that can increase the viscosity of the first electrolytic solution or the second electrolytic solution.
The ion current measuring method according to (2) above.
(6) The dielectric constant of the first electrolytic solution is higher than the dielectric constant of the second electrolytic solution.
The ion current measuring method according to any one of (1) to (5) above.
(7) The dielectric constant of the first electrolytic solution is lower than the dielectric constant of the second electrolytic solution.
The ion current measuring method according to any one of (1) to (5) above.
(8) The charged sample is DNA or RNA.
The ion current measuring method according to any one of (1) to (5) above.
(9) A device for measuring an ion current, comprising:
a substrate having a first side and a second side;
a through hole extending from the first surface to the second surface through which the charged sample passes;
A first chamber member;
A second chamber member;
Including,
the first chamber member forms a first chamber filled with a first electrolytic solution together with the first surface including at least a surface of the first opening of the through hole;
the second chamber member forms a second chamber filled with a second electrolytic solution together with at least a surface of the second surface including the second opening of the through hole;
The dielectric constant of the first electrolytic solution filled in the first chamber is different from the dielectric constant of the second electrolytic solution filled in the second chamber.
A device for measuring ion current.
(10) The first chamber is filled with a first electrolyte;
The second chamber is filled with a second electrolyte.
The device for measuring ion current according to (9) above.
(11) The first electrolytic solution filled in the first chamber and the second electrolytic solution filled in the second chamber are each contained in a container.
The device for measuring ion current according to (9) above.
(12) An organic solvent having a dielectric constant lower than that of water is dissolved in one of the first electrolytic solution or the second electrolytic solution.
The device for measuring ion current according to (10) or (11) above.
(13) The organic solvent is at least one selected from the group consisting of monohydric alcohols, dihydric alcohols, and trihydric alcohols.
The device for measuring ion current according to (12) above.
(14) When the higher of the dielectric constants of the first electrolytic solution and the second electrolytic solution is taken as 1, the lower one has a dielectric constant of 0.1 or more and 0.9 or less.
The device for measuring ion current according to (10) or (11) above.
(15) The organic solvent is selected from solvents that can increase the viscosity of the first electrolytic solution or the second electrolytic solution.
The device for measuring ion current according to (12) above.

The ion current measurement device and ion current measurement method disclosed in this application can increase the signal-to-noise ratio of the measurement results of changes in ion current.

1 is a schematic cross-sectional view of a device 1 according to an embodiment. FIG. 2 is a schematic cross-sectional view showing a configuration example of a device 1a. 3 is a flowchart of a measurement method according to an embodiment. FIG. 1 is a diagram showing the measurement results of the ion current I _ion measured in Example 2, Example 3, and Comparative Example 1. FIG. 13 is a diagram showing the measurement results of the ion current I _ion measured in Example 4. FIG. 13 is a diagram showing the measurement results of the ion current I _ion measured in Comparative Example 2. FIG. 13 is a diagram showing combinations of the electrolytes filled in the first chamber and the second chamber in Example 5, and the measurement results of the ionic current I _ion .

The ion current measurement method (hereinafter, sometimes simply referred to as the "measurement method") and the ion current measurement device (hereinafter, sometimes simply referred to as the "device") are described in detail below. Note that in this specification, components having the same type of function are given the same or similar reference symbols. Furthermore, repeated descriptions of components given the same or similar reference symbols may be omitted.

Furthermore, in order to facilitate understanding, the position, size, range, etc. of each component shown in the drawings may not represent the actual position, size, range, etc. For this reason, the disclosure in this application is not necessarily limited to the position, size, range, etc. disclosed in the drawings.

In addition, in this specification,
(1) A numerical range expressed using "~" means a range including the numerical values before and after "~" as the lower and upper limits,
(2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field.
(3) When describing something as "approximately ____ shaped," this includes not only the exact ____ shape, but also a shape that can be understood to be roughly ____ shaped.
This is interpreted as:

(Embodiment of Device 1)
A device 1 according to an embodiment will be described with reference to Fig. 1. Fig. 1 is a schematic cross-sectional view of the device 1 according to an embodiment.

The device 1 includes a substrate 2, a through-hole 3, a first chamber member 51, and a second chamber member 61. The substrate 2 has a first surface 21 and a second surface 22, and the through-hole 3 penetrates the substrate 2 from the first surface 21 to the second surface 22. When performing the ion current measurement method, a charged sample passes through the through-hole 3.

The first chamber member 51, together with the surface of the first surface 21 including at least the first opening 31 of the through hole 3, forms a first chamber 5 filled with a first electrolyte solution. The second chamber member 61, together with the surface of the second surface 22 including at least the second opening 32 of the through hole 3, forms a second chamber 6 filled with a second electrolyte solution. The device 1 disclosed in this application is characterized in that the dielectric constant of the first electrolyte solution filled in the first chamber 5 is different from the dielectric constant of the second electrolyte solution filled in the second chamber 6. As shown in the examples and comparative examples described later, by making the dielectric constant of the first electrolyte solution different from the dielectric constant of the second electrolyte solution, the S/N ratio of the measurement results of the change in ionic current can be increased.

The material for forming the substrate 2 is not particularly limited as long as it can form the through-hole 3 and can measure the ion current of the charged sample passing through the through-hole 3. Examples of materials for forming the substrate 2 include insulating materials that are commonly used in the field of semiconductor manufacturing technology. Examples of insulating materials include Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, SiN, and the like. The substrate 2 may be formed in a thin film shape called a solid membrane using materials such as SiN, SiO ₂ , and HfO ₂ , or in a sheet shape called a two-dimensional material using materials such as graphene, graphene oxide, molybdenum dioxide (MoS ₂ ), and boron nitride (BN). The substrate 2 may be formed using an artificial membrane such as a lipid bilayer membrane or a naturally occurring membrane. Measurement devices using lipid bilayer membranes are described in JP-A-2011-527191 and JP-A-2020-000056, etc. The matters described in JP-T-2011-527191 and JP-A-2020-000056 are incorporated herein by reference. In addition, a commercially available product may be used as the measurement device using a lipid bilayer membrane. Examples of commercially available products that can perform nanopore analysis using a lipid bilayer membrane include MinION, GridION _X5 , SmidgION, and PromethION manufactured by Oxford Nanopore Technologies.

The smaller the volume of the through-hole 3, the higher the measurement sensitivity of the charged sample, so it is preferable that the substrate 2 in which the through-hole 3 is formed is thin. Although not limited thereto, examples include 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. In addition, when a solid membrane or a two-dimensional material is used as the substrate 2, the film thickness can be made very thin, for example, graphene can be used to fabricate a substrate 2 with a film thickness of 1 nm or less. However, if the film thickness of the substrate 2 is very thin, it may be difficult to handle it without breaking it. Therefore, the substrate 2 may have a laminated structure in which a solid membrane or a two-dimensional material is laminated on a support plate formed of the above-mentioned insulating material. To create a laminated structure, a solid membrane or two-dimensional material is laminated on a support plate with holes larger than the through-holes 3, and the through-holes 3 are formed in the solid membrane or two-dimensional material.

The through-hole 3 is formed so as to penetrate the substrate 2 from the first surface 21 of the substrate 2 in the direction of the second surface 22, which is the surface opposite to the first surface 21. When measuring the ion current, the smaller the volume of the through-hole 3, the higher the sensitivity. Therefore, the substrate 2 is made thin, and the size of the through-hole 3 is adjusted appropriately so that it is larger than the charged sample but not too large. For example, when the charged sample is very small, such as DNA, the size of the through-hole 3 is not particularly limited as long as the charged sample can pass through it, but if the through-hole 3 is made too small, there is a risk that the error in making each through-hole 3 will be large and the measurement error between devices will be large. Although not limited, the lower limit of the through-hole 3 can be, for example, 0.8 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, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, etc. In addition, when the charged sample is a cell, the size may be such that the cell can pass through, for example, about 10 μm. As described above, the smaller the volume of the through-hole 3, the higher the sensitivity. Therefore, although not limited, the lower limit of the through-hole 3 may be, for example, 10 μm or less, 7.5 μm or less, 5 μm or less, 2.5 μm or less, 1 μm or less, 750 nm or less, 500 nm or less, 250 nm or less, 100 nm or less, etc., depending on the size of the charged sample. In addition, when the cross-sectional shape of the through-hole 3 parallel to the first surface 21 is circular, the size of the through-hole 3 means the diameter. When the cross-sectional shape of the through-hole 3 parallel to the first surface 21 is not circular, the size of the through-hole 3 means the diameter of the inscribed circle of the cross section. When the material of the substrate 2 is a material other than the lipid bilayer membrane, the through-hole 3 may be formed by etching or the like, as shown in the examples described later. The through-hole 3 may be formed so that the first opening 31 of the through-hole 3 on the first surface 21 side and the second opening 32 of the through-hole 3 on the second surface 22 side have the same shape. Alternatively, the first opening 31 and the second opening 32 may be different in size, for example, the through-hole 3 may be formed so as to expand from the first surface 21 to the second surface 22 in the base material 2. Although FIG. 1 shows an example in which one through-hole 3 is formed in the substrate 2, two or more through-holes 3 may be formed. When two or more through-holes 3 are formed in the substrate 2, the distance between adjacent through-holes 3 may be adjusted as necessary to increase the measurement accuracy of the charged sample. The extent to which the distance between adjacent through-holes 3 is set is described in detail in Patent Document 3, so a detailed description will be omitted in the disclosure of this application. The matters described in Patent Document 3 are incorporated herein by reference.

The first chamber member 51 and the second chamber member 61 are preferably formed from an electrically and chemically inert material, such as, but not limited to, glass, sapphire, ceramic, resin, rubber, elastomer, _SiO2 , SiN _{, Al2O3} , and the like.

The first chamber 5 and the second chamber 6 are formed to sandwich the through hole 3, and there is no particular restriction as long as the charged sample introduced into the first chamber 5 can move through the through hole 3 to the second chamber 6, or the charged sample introduced into the second chamber 6 can move through the through hole 3 to the first chamber 5. For example, the first chamber member 51 and the second chamber member 61 may be separately manufactured and bonded to the substrate 2 so as to be liquid-tight. Alternatively, a roughly rectangular box member with one side open may be formed, the substrate 2 may be inserted and fixed in the center of the box, and then the open side may be sealed liquid-tight. In that case, the first chamber member 51 and the second chamber member 61 do not mean separate members, but rather mean parts of the box member separated by the substrate 2. Although not shown in the figure, the first chamber member 51 and the second chamber member 61 may be formed with holes for filling and discharging the electrolyte and the charged sample liquid, and for inserting electrodes and/or leads, as necessary.

(Embodiment of the measurement method and configuration example of the device 1a when carrying out the measurement method)
An embodiment of the measurement method and a configuration example of the device 1a when the measurement method is performed will be described with reference to Fig. 2 and Fig. 3. Fig. 2 is a schematic cross-sectional view showing a configuration example of the device 1a, and Fig. 3 is a flowchart of the measurement method according to the embodiment.

2 includes at least a first electrode 52 formed at a location in contact with the first electrolyte in the first chamber 5, a second electrode 62 formed at a location in contact with the second electrolyte in the second chamber 6, a power source 54 for applying a voltage between the first electrode 52 and the second electrode 62, and an ammeter 7 for measuring the ion current when the charged sample S passes through the through hole 3, in addition to the device 1 according to the embodiment. The first electrode 52, the second electrode 62, the power source 54, and the ammeter 7 may be prepared separately from the device 1 and attached to the device 1 when performing the measurement method, or may be built into the device 1 from the beginning. In addition, the device 1a may optionally include 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 result of the analysis by the analysis unit 8, a program memory 10 in which programs for operating the analysis unit 8 and the display unit 9 are stored in advance, and a control unit 11 for reading and executing the program stored in the program memory 10. The program may be stored in advance in the program memory 10, or may be recorded on a recording medium and then stored in the program memory 10 using an installation means.

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. In FIG. 2, the first electrode 52 and the second electrode 62 are formed to sandwich the through-hole 3, and an example is shown in which a voltage is applied so that a direct current flows with the first electrode 52 side as a negative pole and the second electrode 62 side as a positive pole, but alternatively, the first electrode 52 side may be a positive pole and the second electrode 62 side as a negative pole. Depending on the charge of the charged sample described later, it is possible to appropriately determine which side of the first electrode 52 or the second electrode 62 is to be made positive.

There are no particular limitations on the first electrode 52, so long as it is formed in a location that contacts the first electrolyte in the first chamber 5. In the example shown in FIG. 2, the first electrode 52 is disposed on the inner surface of the first chamber member 51 via a lead 53. Alternatively, the first electrode 52 may be disposed on the first surface 21 of the substrate 2 or in the space within the first chamber 5 via a lead 53. As a further alternative, the first electrode 52 may be disposed so as to penetrate the first chamber member 51 from a hole formed in the first chamber member 51.

Similar to the first electrode 52, the second electrode 62 is not particularly limited as long as it is formed in a location that contacts the second electrolyte in the second chamber 6. In the example shown in FIG. 2, the second electrode 62 is disposed on the inner surface of the second chamber member 61 via a lead 63. Alternatively, the second electrode 62 may be disposed on the second surface 22 of the substrate 2 or in the space within the second chamber 6 via a lead 63. As a further alternative, the second electrode 62 may be disposed so as to penetrate the second chamber member 61 from a hole formed in the second chamber member 61.

In the example shown in FIG. 2, the first electrode 52 is connected to a power source 54 and earth 55 via a lead 53. The second electrode 62 is connected to an ammeter 7 and earth 64 via a lead 63. Note that in the example shown in FIG. 2, the power source 54 is connected to the first electrode 52 side and the ammeter 7 is connected to the second electrode 62 side, but the power source 54 and ammeter 7 may be provided on the same electrode side.

There are no particular limitations on the power supply 54, so long as it can pass a direct current through the first electrode 52 and the second electrode 62. There are no particular limitations on the ammeter 7, so long as it can measure over time the ion current generated when a current is passed through the first electrode 52 and the second electrode 62. Although not shown in FIG. 2, the device 1a may also include a noise removal circuit, a voltage stabilization circuit, etc., as necessary.

The analysis unit 8 analyzes the change in the ion current measured by the ammeter 7. Generally, in a device 1a having a through-hole (nanopore) 3, when a charged sample passes through the through-hole 3, the ion current flowing through the through-hole 3 is blocked by the charged sample, and the ion current decreases. Therefore, the charged sample can be identified by analyzing the data in the analysis unit 8 based on the change in the measured ion current (the peak value of the changed ion current, the waveform of the ion current, etc.).

As shown in the examples described later, in this application, even when a long and thin DNA was used as a charged sample, the change in ion current when the DNA passed through the through-hole 3 could be measured with a large S/N ratio. Furthermore, when the dielectric constant of the first electrolyte was higher than that of the second electrolyte, the direction of the change in ion current (positive direction, negative direction) was reversed. From this, it is presumed that the results measured by the measurement method disclosed in this application are not simply proportional to the size of the charged sample passing through the through-hole 3, but that the ion concentration in the through-hole 3 increases due to ions gathering around the charged sample. Therefore, it is also possible to identify unknown charged samples by measuring a large number of known charged samples in advance and measuring unknown charged samples under the same conditions (components and dielectric constant of the first electrolyte, components and dielectric constant of the second electrolyte, magnitude of applied voltage, etc.) as when the known charged samples were measured, and comparing the measurement results of the known charged samples with the measurement results of the unknown charged samples.

The display unit 9 may be any known display device capable of displaying the changes in the measured ion current and the results of the analysis performed by the analysis unit 8, such as a liquid crystal display, plasma display, or organic electroluminescence display. The program memory 10 is not particularly limited as long as it can store programs for operating the analysis unit 8 and the display unit 9, and examples of such memory 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 programs stored in the program memory 10, and examples of such memory include a processor (CPU) or a general-purpose computer equipped with a CPU.

An embodiment of the measurement method includes a charged sample passing step (ST1) and an ion current measuring step (ST2).

The charged sample passing step (ST1) applies a voltage to the first electrolyte filled in the first chamber 5 and the second electrolyte filled in the second chamber 6, causing the charged sample S contained in the first chamber 5 to pass through the through hole 3 in the direction of the second chamber 6, or causing the charged sample S contained in the second chamber 6 to pass through the through hole 3 in the direction of the second chamber 6.

In the measurement method disclosed in this application, the S/N ratio is improved by the difference between the dielectric constant of the first electrolyte and the dielectric constant of the second electrolyte. The reason for this is thought to be that, as described above, in addition to the volume of the charged sample S, the ion concentration in the through hole 3 increases due to the ions that gather around the charged sample S. Therefore, it is desirable that the surface of the charged sample S measured by the measurement method disclosed in this application is charged. There is no particular restriction on whether the surface of the charged sample S is positively or negatively charged. Examples of the charged sample S include, but are not limited to, charged biological substances such as bacteria, cells, viruses, DNA, RNA, proteins, and liposomes, or charged non-biological substances such as polymer microparticles and metal microparticles. Although the charged biological substances vary depending on the type, many bacteria, cells, and viruses are negatively charged. In addition, DNA and RNA are also negatively charged. On the other hand, proteins are amphoteric molecules that constitute proteins, and may be positively or negatively charged depending on the pH environment. Therefore, when measuring proteins, the measurement conditions should be set taking into account the pH of the first and second electrolyte solutions.

The first and second electrolyte solutions must be capable of conducting electricity between the first electrode 52 and the second electrode 62, and therefore typically use ion-containing solutions (electrolytes) such as TE buffer, PBS buffer, HEPES buffer, and KCl aqueous solution. To make the dielectric constants of the first and second electrolyte solutions different, it is possible, without any particular limitation, to dissolve an organic solvent with a lower dielectric constant than water in the solution (electrolyte) whose dielectric constant is desired to be lower. The dielectric constant varies depending on the temperature, but the dielectric constant of water is 80.4 (20°C), 79.6 (22°C), 78.6 (25°C), and 76.8 (30°C).

Specific examples of organic solvents with a lower dielectric constant than water include monohydric alcohols, dihydric alcohols, trihydric alcohols, and other organic solvents. Among the organic solvents listed below, the dielectric constant of representative ones is also listed in parentheses after the name of the organic solvent. Note that the dielectric constants listed below were measured at temperatures of around 20°C to 25°C, and may include some error depending on the measurement conditions, but it is clear that the values are much lower than the dielectric constant of water.

(1) Monohydric alcohols Examples of monohydric alcohols include methanol (33.0), ethanol (24.0), 1-propanol (20.0), 2-propanol (18.0), 1-butanol (17.5), 2-butanol (16.6), isobutyl alcohol (17.9), isopentyl alcohol (15.2), cyclohexanol (15.0), and the like, as well as aliphatic saturated monohydric alcohols, aliphatic unsaturated alcohols, alicyclic alcohols, and aromatic alcohols.
The aliphatic saturated monohydric alcohols include, for example, straight-chain and branched alcohols such as natural alcohols and synthetic alcohols (e.g., Ziegler alcohols or oxo alcohols), specifically 2-ethylbutanol, 2-methylpentanol, 4-methylpentanol, 1-hexanol, 2-ethylpentanol, 2-methylhexanol, 1-heptanol, 2-heptanol, 3-heptanol, 2-ethylhexanol, 1-octanol, 2-octanol, 1-nonanol, decanol, undecanol, dodecanol, and tridecanol.
The aliphatic unsaturated alcohols include, for example, alkenols and alkadienols, and specific examples thereof include 2-propylallyl alcohol, 2-methyl-4-pentenol, 1-hexenol, 2-ethyl-4-pentenol, 2-methyl-5-hexenol, 1-heptenol, 2-ethyl-5-hexenol, 1-octenol, 1-nonenol, undecenol, dodecenol, and geraniol.
Alicyclic alcohols include, for example, cycloalkanols and cycloalkenols, and specific examples thereof include methylcyclohexanol and α-terpineol.
Aromatic alcohols include phenethyl alcohol and salicyl alcohol.

(2) Dihydric alcohols Examples of dihydric alcohols include ethylene glycol (37.7), diethylene glycol (31.7), triethylene glycol (23.7), and propylene glycol (32.0).

(3) Trihydric alcohols Examples of trihydric alcohols include 1,2,4-butanetriol (38) and glycerin (glycerol: 44).
(4) Other organic solvents Examples of organic solvents include acetic acid (6.15), pyridine (12.3), tetrahydrofuran (THF) (7.5), acetone (20.7), methyl ethyl ketone (MEK) (15.45), ethyl acetate (6.4), aniline (6.89), N-methyl-2-pyrrolidone (NMP) (32.2), dimethyl sulfoxide (DMSO) (45), N,N-dimethylformamide (DMF) (38), hexane (1.8), toluene (2.4), diethyl ether (4.3), and chloroform (4.8).

The organic solvents exemplified in (1) to (4) above may be used in combination of two or more kinds. In addition, the organic solvents exemplified in (1) to (4) above may be added only to the electrolyte solution for which the dielectric constant is desired to be reduced, but organic solvents with different dielectric constants may be added to both the first electrolyte solution and the second electrolyte solution.

In the above example, the dielectric constant of the first electrolyte solution is made different from that of the second electrolyte solution by adding an organic solvent with a lower dielectric constant than water, but the dielectric constants may also be made different by adding an organic solvent with a higher dielectric constant than water to one of the electrolyte solutions. Examples of organic solvents with a higher dielectric constant than water include ethylene carbonate (89.8), formamide (111.0), N-methylformamide (182.4), and N-methylacetamide (191.3).

Furthermore, among the organic solvents exemplified above, for example, an electrolyte solution to which a highly viscous organic solvent such as glycerin or DMSO has been added has a high viscosity. Therefore, when carrying out the measurement method, the time it takes for the charged sample S to pass through the through-hole 3 is lengthened, which has the effect of enabling more detailed information about the charged sample S to be obtained. In other words, when carrying out the measurement method, an organic solvent with a high viscosity performs both the function of adjusting the dielectric constant of the electrolyte solution and the completely different function of lengthening the time it takes for the charged sample S to pass through the through-hole 3.

In the measurement method and device 1a disclosed in this application, the S/N ratio of the change in the measured ion current is increased by making the dielectric constant of the first electrolyte solution different from that of the second electrolyte solution. Therefore, even a small difference in the dielectric constant will produce the effect disclosed in this application. Although not limited to this, when the higher of the dielectric constants of the first electrolyte solution and the second electrolyte solution is set to 1, the lower dielectric constant may be 0.99 or less, 0.98 or less, 0.97 or less, 0.96 or less, 0.95 or less, 0.94 or less, 0.93 or less, 0.92 or less, 0.91 or less, 0.9 or less, 0.85 or less, 0.8 or less, 0.75 or less, 0.7 or less, etc. On the other hand, increasing the difference between the dielectric constants of the first electrolyte solution and the second electrolyte solution means that a larger amount of organic solvent is added to the electrolyte solution if the type (dielectric constant) of the organic solvent added is the same. And the more the amount of organic solvent added, the more the composition of the electrolyte contained in the first electrolyte solution and the second electrolyte solution changes. Although it is possible to carry out the measurement method even if the amount of electrolyte contained in the first electrolytic solution and the second electrolytic solution is different, from the viewpoint of not significantly changing the measurement conditions other than the dielectric constant, the dielectric constant of the lower one may be 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.45 or more, 0.5 or more, 0.55 or more, 0.6 or more, or 0.65 or more, when the higher of the dielectric constants of the first electrolytic solution and the second electrolytic solution is set to 1, but is not limited thereto. Note that although it takes time and effort to prepare the first electrolytic solution and the second electrolytic solution, it is also possible to prepare the first electrolytic solution and the second electrolytic solution so that the dielectric constants are different but the salt concentrations are the same by preparing a high-concentration electrolytic solution, an organic solvent, and ultrapure water and mixing them in appropriate amounts to prepare the first electrolytic solution and the second electrolytic solution.

If the first chamber 5 is already filled with the first electrolyte and the second chamber 6 is already filled with the second electrolyte, the charged sample is placed in the first chamber 5 or the second chamber 6, and the charged sample passing step (ST1) is carried out.

In the case where the first chamber 5 of the device 1a is not filled with the first electrolytic solution and the second chamber 6 is not filled with the second electrolytic solution, a preparation step may be carried out before carrying out the charged sample passing step (ST1). The preparation step may be carried out in the following manner.
(1) The first chamber 5 is filled with a first electrolytic solution, and the second chamber 6 is filled with a second electrolytic solution. A liquid junction is established between the first chamber 5 and the second chamber 6 via the through hole 3.
(2) The charged sample S is placed into the first chamber 5 or the second chamber 6 .
The above steps (1) and (2) may be carried out separately, or the electrolyte solution already containing the charged sample S may be introduced into the first chamber 5 or the second chamber 6 .

In addition, if the first chamber 5 of the device 1a is not filled with the first electrolyte solution and the second chamber 6 is not filled with the second electrolyte solution, the first electrolyte solution and the second electrolyte solution, whose dielectric constants have been adjusted in advance, may be filled in containers such as bottles and provided together with the device 1a. When measuring the obtained charged sample S and performing analysis such as identifying the charged sample S based on the measurement results, it is desirable to set the dielectric constant of the first electrolyte solution and the dielectric constant of the second electrolyte solution to predetermined conditions. When the first electrolyte solution and the second electrolyte solution, whose dielectric constants have been adjusted in advance, are provided filled in containers, the reproducibility of the measurement method is increased.

In the charged sample passing step (ST1) shown in FIG. 3, a current is passed through the first electrode 52 arranged in the first chamber 5 and the second electrode 62 arranged in the second chamber 6, and in addition to normal diffusion, the charged sample S passes through the through-hole 3 formed in the substrate 2 by electrophoresis.

In the ion current measurement step (ST2), the change in the ion current caused by the current flow is measured over time by the ammeter 7. In the measurement method disclosed in the present application, the dielectric constant of the first electrolyte solution is different from the dielectric constant of the second electrolyte solution, so that when the charged sample S passes through the through-hole 3, a measurement result with a large S/N ratio is obtained. The measurement method may optionally include an analysis step (ST3) in which information about the obtained charged sample is analyzed from the change in the ion current measured in the ion current measurement step (ST2). As shown in the examples described later, in the measurement method disclosed in the present application, the measurement result reflects the increase in the ion concentration in the through-hole 3 due to the volume of the charged sample and the ions gathering around the charged sample, so that the information about the charged sample can be analyzed in more detail compared to the analysis of only the volume information of the sample. Examples of information that can be analyzed include types of bacteria, cells, viruses, exosomes, etc., nucleic acid sequences such as DNA and RNA, and amino acid sequences of proteins.

The following examples are provided to specifically explain the embodiments disclosed in this application, but these examples are merely for the purpose of explaining the embodiments. They are not intended to limit or represent a restriction on the scope of the disclosure in this application.

Example 1
[Fabrication of Device 1]
A 4-inch silicon wafer coated on both sides with a 50 nm thick SiNx layer was diced into 30 mm x 30 mm chips. One side of the SiNx was partially removed by reactive ion etching (Samco) with _CHF3 etching gas through a metal mask. The silicon layer was then wet etched in KOH aq. (Wako) at 50 °C through the exposed 1 mm x 1 mm square area. As a result, a 50 nm thick SiNx film was formed. An electron beam resist (ZEP520A, Zeon) was spin-coated on the formed film and baked at 180 °C. Subsequently, a circle with a diameter of 100 nm was drawn by electron beam lithography (125 kV, Elionix). After development, the SiNx was removed via reactive etching through a resist mask to open the nanopores. Finally, the remaining resist layer was completely dissolved in N,N-dimethylformamide, followed by rinsing with ethanol and acetone to produce a nanopore chip with nanopores (through holes) formed in the SiNx substrate.

The fabricated nanopore chip was sealed with two polymer blocks (first and second chamber members) made of polydimethylsiloxane (PDMS) to create the first and second chambers. These blocks were fabricated by polymerizing a PDMS precursor (Sylgard 184, Dow) on a SU-8 mold at 80°C. The mold had an I-shaped pattern with sub-millimeter width and height to form trenches on the polymer block that act as channels for the flow of charged sample solutions into the nanopore. Three holes were punched in the block before sealing. The nanopore chip and the polymer blocks (first and second chamber members) were then exposed to oxygen plasma for surface activation, and the nanopore chip and the polymer blocks were then bonded to create device 1.

[Preparation of device 1a for carrying out the measurement method]
Ag/AgCl rods were used as the first and second electrodes, inserted into the first and second chambers through holes on either side of the polymer block. The ionic current through the nanopore was measured by pre-amplifying the output current through one of the rods using a custom-designed amplifier, then digitizing it using a high-speed digitizer (PXI-5922, NI) and accumulating it on a solid-state drive (PXI-8267, NI) at a sampling rate of 1 MHz under an applied voltage Vb.

[Implementation of the measurement method]
Example 2
(1) Preparation of electrolyte and charged sample Electrolyte with low dielectric constant: An equal amount of glycerol (manufactured by Aldrich: CAS 56-81-5) was added to 1.37 M NaCl (manufactured by Nippon Gene Co., Ltd.: 10x PBS Buffer (-), model number: 314-90185) to prepare a first electrolyte with a salt concentration of 0.69 M NaCl (5x PBS) and a glycerol concentration of 50%. The dielectric constant of the prepared first electrolyte at 20°C was 63.5.
Electrolyte solution with high dielectric constant: A second electrolytic solution with a salt concentration of 0.69 M NaCl (5xPBS) was prepared by adding the same amount of ultrapure water to the above 10xPBS Buffer. The dielectric constant of the prepared second electrolytic solution at 20°C was 80.
Charged sample: Double-stranded DNA (48.5 kbp) was used as the charged sample.

A first electrolyte solution having a low dielectric constant was filled into the first chamber through a hole formed in the block. Double-stranded DNA was also filled into the first chamber. A second electrolyte solution having a high dielectric constant was filled into the second chamber through a hole formed in the block. A voltage of 0.3 V was applied so that the first electrode 52 was the negative electrode and the second electrode 62 was the positive electrode, and the ionic current I _ion was measured.

Example 3
The ionic current I _ion was measured in the same manner as in Example 2, except that the electrolyte solution having a higher dielectric constant was used as the first electrolyte solution, and the electrolyte solution having a lower dielectric constant was used as the second electrolyte solution.

<Comparative Example 1>
The ionic current I _ion was measured in the same manner as in Example 2, except that the first and second electrolytic solutions both had high dielectric constants.

Figure 4 shows the measurement results of Example 2, Example 3, and Comparative Example 1. Conventionally, when measuring the change in ion current when a sample passes through a nanopore, the electrolyte filled in the first chamber and the second chamber had the same composition as shown in Comparative Example 1. In Comparative Example 1, the change in ion current when DNA passed through the nanopore was 0.4 nA from the baseline. On the other hand, by making the dielectric constant of the electrolyte filled in the first chamber and the second chamber different, the following remarkable effects were obtained: (1) in Example 2, the peak value of the change in ion current when DNA passed through the nanopore was 3.5 nA from the baseline, which was about 8 times stronger than in Comparative Example 1; and (2) in Example 3, the peak value of the change in ion current when DNA passed through the nanopore was 7.5 nA from the baseline, which was about 20 times stronger than in Comparative Example 1. In addition, the S/N ratio (calculated using the wave heights of multiple signals and the rms noise of the ion current) was approximately 1.1 in Comparative Example 1, whereas it was approximately 4.3 in Example 2 and approximately 5.0 in Example 3, which shows a significantly higher S/N ratio.

Also, as shown in FIG. 4, in Example 2, in which DNA migrates from the electrolyte with a low dielectric constant to the electrolyte with a high dielectric constant (positive dielectric constant gradient), the direction in which the ion current measurement signal was obtained was reversed from Comparative Example 1, even though the sample was the same. On the other hand, in Example 3, in which DNA migrates from the electrolyte with a high dielectric constant to the electrolyte with a low dielectric constant (negative dielectric constant gradient), the direction in which the ion current measurement signal was obtained was the same as in Comparative Example 1, but the change in ion current increased significantly. The reason why the change in ion current is significantly different as shown in Examples 2, 3, and Comparative Example 1 by changing the dielectric constant of the electrolyte is unclear, but it is thought that this is because DNA is negatively charged, but positive ions are less likely to gather around DNA when the dielectric constant is low, and therefore the ion concentration in the through hole 3 increases due to the difference in dielectric constant and the ions that gather around the DNA when the DNA passes through the through hole.

Example 4
0.69M NaCl was used as the first electrolytic solution. In addition, 1.37M NaCl (10xPBS), glycerol (manufactured by Aldrich: CAS 56-81-5), and ultrapure water were mixed in appropriate amounts to prepare three types of second electrolytic solutions with a salt concentration of 0.69M NaCl and glycerol concentrations of 10%, 30%, and 50%. I _ion was measured in the same manner as in Example 3, except that the prepared first and second electrolytic solutions were used. The viscosity of the second electrolytic solution when 10% glycerol was added was 1.5 mPas, and the viscosity of the second electrolytic solution when 50% glycerol was added was 14 mPas. The dielectric constant of the first electrolytic solution was 80, the dielectric constant of the second electrolytic solution when 10% glycerol was added was 76.7, and the dielectric constant of the second electrolytic solution when 50% glycerol was added was 63.5.

The results are shown in Figure 5. As is clear from Figure 5, the more the proportion of glycerol in the second electrolyte was increased, in other words, the lower the dielectric constant of the second electrolyte relative to the first electrolyte and the higher the viscosity of the second electrolyte, the longer the time it took for the DNA to pass through the nanopore and the greater the change in ionic current. As is clear from Figure 5, when comparing 10% and 50% glycerol, the longer the time it took for the DNA to pass through the nanopore and the greater the change in ionic current, so that information when the DNA passed through the nanopore could be obtained in more detail as the waveform of the measurement signal. As described above, it was confirmed that when the dielectric constant of the electrolyte is adjusted using an organic solvent that can increase the viscosity of the electrolyte, the remarkable effect of increasing the amount of change in ionic current and lengthening the measurement time is achieved, allowing the information contained in the charged sample to be measured in more detail.

<Comparative Example 2>
The amount of glycerol added to the first and second electrolytic solutions was varied (10%, 20%, 30%, 40%, 50%, the salt concentration was the same at 0.69 M NaCl), but the ionic current I _ion was measured in the same manner as in Comparative Example 1, except that the dielectric constant of the first electrolytic solution filled in the first chamber and the dielectric constant of the second electrolytic solution filled in the second chamber were the same.

The results are shown in Figure 6. As is clear from Figure 6, when the dielectric constant of the first electrolyte filled in the first chamber and the amount of organic solvent in the second electrolyte filled in the second chamber were the same, i.e., when they were made to have the same dielectric constant, the change in ionic current did not increase. From the above results, it was confirmed that the effect disclosed in this application can be obtained by making the dielectric constants of the first electrolyte filled in the first chamber and the second electrolyte filled in the second chamber different (making a difference).

Example 5
Electrolyte with low dielectric constant: Three types of electrolyte solutions with salt concentration of 0.69 M NaCl and ethanol concentration of 10%, 20%, and 30% were prepared by mixing appropriate amounts of 1.37 M NaCl (10x PBS), ethanol (Kishida Chemical Co., Ltd.: 000-28553), and ultrapure water. The dielectric constants of the prepared electrolyte solutions at 20°C were 74.4 (10% ethanol), 68.8 (20% ethanol), and 63.2 (30% ethanol).
High dielectric constant electrolyte: An electrolyte similar to the "high dielectric constant electrolyte" in Example 2 was used.

The ion current I _ion was measured in the same manner as in Example 2, except that the combination of low-dielectric constant and high-dielectric constant electrolytes was changed and filled in the first and second chambers. Figure 7 shows the combinations of electrolytes filled in the first and second chambers and the measurement results. In Figure 7, 10% ethanol, which is an electrolyte with a low dielectric constant, is described as "10% Eth", 20% ethanol is described as "20% Eth", and 30% ethanol is described as "10% Eth", and the electrolyte with a high dielectric constant is described as "5xPBS".

As is clear from Figure 7, even when ethanol was used instead of glycerol, it was confirmed that the peak value of the change in ionic current when DNA passes through the nanopore was enhanced by making the dielectric constants of the electrolytes filled in the first and second chambers different. Furthermore, the direction in which the measurement signal of the ionic current was obtained was the same as when glycerol was used, even when ethanol was used as the organic solvent. The S/N ratio was 3.1 when the first chamber was filled with 30% ethanol and the second chamber was filled with 5xPBS. The S/N ratio was 2.5 when the first chamber was filled with 5xPBS and the second chamber was filled with 20% ethanol.

From the above results, it was confirmed that by making the dielectric constant of the electrolyte filled in the first chamber different from the dielectric constant of the electrolyte filled in the second chamber, the change in ion current is increased and the S/N ratio is increased.

When the method for measuring ion current is carried out using the device disclosed in this application, the value of the change in ion current is enhanced. Therefore, it is useful for the development of analytical equipment in the analytical instrument industry.

1, 1a...Ion current measuring device, 2...Substrate, 3...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...First opening, 32...Second opening, 51...First chamber member, 52...First electrode, 53...Lead, 54...Power supply, 55...Earth, 61...Second chamber member, 62...Second electrode, 63...Lead, 64...Earth, S...Sample

Claims (15)

1. A method for measuring an ion current of a charged sample using a device for measuring an ion current, comprising the steps of:
   The ion current measuring device comprises:
   a substrate having a first side and a second side;
   a through hole extending from the first surface to the second surface through which the charged sample passes;
   A first chamber member;
   A second chamber member;
   Including,
   the first chamber member forms a first chamber filled with a first electrolytic solution together with the first surface including at least a surface of the first opening of the through hole;
   the second chamber member forms a second chamber filled with a second electrolytic solution together with at least a surface of the second surface including the second opening of the through hole;
   The method for measuring the ion current comprises:
   A charged sample passing step;
   an ion current measuring step;
   Including,
   The charged sample passing step includes:
   A voltage is applied to the first electrolytic solution filled in the first chamber and the second electrolytic solution filled in the second chamber,
   Passing the charged sample contained in the first chamber through the through hole toward the second chamber, or passing the charged sample contained in the second chamber through the through hole toward the first chamber;
   The ion current measuring step includes:
   measuring a change in ionic current as the charged sample passes through the through-hole;
   The dielectric constant of the first electrolytic solution is different from the dielectric constant of the second electrolytic solution.
   Ion current measurement method.
2. An organic solvent having a dielectric constant lower than that of water is dissolved in one of the first electrolytic solution or the second electrolytic solution.
   The ion current measuring method according to claim 1 .
3. The organic solvent is at least one selected from the group consisting of monohydric alcohols, dihydric alcohols, and trihydric alcohols.
   The ion current measuring method according to claim 2 .
4. When the higher of the dielectric constants of the first electrolytic solution and the second electrolytic solution is taken as 1, the lower one has a dielectric constant of 0.1 or more and 0.9 or less.
   The ion current measuring method according to claim 1 .
5. The organic solvent is selected from solvents that can increase the viscosity of the first electrolytic solution or the second electrolytic solution.
   The ion current measuring method according to claim 2 .
6. The dielectric constant of the first electrolytic solution is higher than the dielectric constant of the second electrolytic solution.
   The ion current measuring method according to any one of claims 1 to 5.
7. The dielectric constant of the first electrolytic solution is lower than the dielectric constant of the second electrolytic solution.
   The ion current measuring method according to any one of claims 1 to 5.
8. The charged sample is DNA or RNA;
   The ion current measuring method according to any one of claims 1 to 5.
9. A device for measuring ion current, the device comprising:
   a substrate having a first side and a second side;
   a through hole extending from the first surface to the second surface through which the charged sample passes;
   A first chamber member;
   A second chamber member;
   Including,
   the first chamber member forms a first chamber filled with a first electrolytic solution together with the first surface including at least a surface of the first opening of the through hole;
   the second chamber member forms a second chamber filled with a second electrolytic solution together with at least a surface of the second surface including the second opening of the through hole;
   The dielectric constant of the first electrolytic solution filled in the first chamber is different from the dielectric constant of the second electrolytic solution filled in the second chamber.
   A device for measuring ion current.
10. The first chamber is filled with a first electrolyte;
    The second chamber is filled with a second electrolyte.
    The device for measuring ion current according to claim 9.
11. The first electrolytic solution filled in the first chamber and the second electrolytic solution filled in the second chamber are each contained in a container.
    The device for measuring ion current according to claim 9.
12. An organic solvent having a dielectric constant lower than that of water is dissolved in one of the first electrolytic solution or the second electrolytic solution.
    The device for measuring ion current according to claim 10 or 11.
13. The organic solvent is at least one selected from the group consisting of monohydric alcohols, dihydric alcohols, and trihydric alcohols.
    The device for measuring ion current according to claim 12.
14. When the higher of the dielectric constants of the first electrolytic solution and the second electrolytic solution is taken as 1, the lower one has a dielectric constant of 0.1 or more and 0.9 or less.
    The device for measuring ion current according to claim 10 or 11.
15. The organic solvent is selected from solvents that can increase the viscosity of the first electrolytic solution or the second electrolytic solution.
    The device for measuring ion current according to claim 12.