WO2024075637A1 - Procédé et dispositif de mesure de courant ionique - Google Patents

Procédé et dispositif de mesure de courant ionique Download PDF

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Publication number
WO2024075637A1
WO2024075637A1 PCT/JP2023/035451 JP2023035451W WO2024075637A1 WO 2024075637 A1 WO2024075637 A1 WO 2024075637A1 JP 2023035451 W JP2023035451 W JP 2023035451W WO 2024075637 A1 WO2024075637 A1 WO 2024075637A1
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chamber
electrolytic solution
ion current
dielectric constant
filled
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PCT/JP2023/035451
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English (en)
Japanese (ja)
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真楠 筒井
知二 川合
一道 横田
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国立大学法人大阪大学
国立研究開発法人産業技術総合研究所
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Publication of WO2024075637A1 publication Critical patent/WO2024075637A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • 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).
  • 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.
  • 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.
  • 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;
  • 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
  • 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 dielectric constant of the first electrolytic solution is higher than the dielectric constant of the second electrolytic solution.
  • the dielectric constant of the first electrolytic solution is lower than the dielectric constant of the second electrolytic solution.
  • the charged sample is DNA or RNA.
  • 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 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
  • the first chamber is filled with a first electrolyte;
  • the second chamber is filled with a second electrolyte.
  • 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.
  • 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 organic solvent is at least one selected from the group consisting of monohydric alcohols, dihydric alcohols, and trihydric alcohols.
  • 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.
  • FIG. 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.
  • a numerical range expressed using “ ⁇ ” means a range including the numerical values before and after “ ⁇ ” as the lower and upper limits
  • Numerical values, numerical ranges, and qualitative expressions indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field.
  • 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.
  • 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.
  • 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.
  • 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 substrate 2 in which the through-hole 3 is formed is thin.
  • examples include 5 ⁇ m or less, 1 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the size may be such that the cell can pass through, for example, about 10 ⁇ m. As described above, the smaller the volume of the through-hole 3, the higher the sensitivity.
  • the lower limit of the through-hole 3 may be, for example, 10 ⁇ m or less, 7.5 ⁇ m or less, 5 ⁇ m or less, 2.5 ⁇ m or less, 1 ⁇ m 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.
  • the size of the through-hole 3 means the diameter.
  • the size of the through-hole 3 means the diameter of the inscribed circle of the cross section.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Fig. 2 is a schematic cross-sectional view showing a configuration example of the device 1a
  • Fig. 3 is a flowchart of the measurement method according to the embodiment.
  • the second electrode 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.
  • 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.
  • 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.
  • the first electrode 52 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.
  • the first electrode 52 is disposed on the inner surface of the first chamber member 51 via a lead 53.
  • 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.
  • 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.
  • 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.
  • the second electrode 62 is disposed on the inner surface of the second chamber member 61 via a lead 63.
  • 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.
  • 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.
  • 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.
  • 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.
  • the power supply 54 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.
  • the ammeter 7 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • charged biological substances such as bacteria, cells, viruses, DNA, RNA, proteins, and liposomes
  • charged non-biological substances such as polymer microparticles and metal microparticles.
  • the charged biological substances vary depending on the type, many bacteria, cells, and viruses are negatively charged.
  • DNA and RNA are also negatively charged.
  • 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.
  • 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).
  • organic solvents with a lower dielectric constant than water include monohydric alcohols, dihydric alcohols, trihydric alcohols, and other organic solvents.
  • 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.
  • Monohydric alcohols examples 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.
  • natural alcohols and synthetic alcohols e.g., Ziegler alcohols or oxo alcohols
  • 2-ethylbutanol 2-methylpentanol
  • 4-methylpentanol 1-hexanol
  • 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 ⁇ -terpineol.
  • Aromatic alcohols include phenethyl alcohol and salicyl alcohol.
  • dihydric alcohols examples include ethylene glycol (37.7), diethylene glycol (31.7), triethylene glycol (23.7), and propylene glycol (32.0).
  • Trihydric alcohols examples include 1,2,4-butanetriol (38) and glycerin (glycerol: 44).
  • Other organic solvents examples 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).
  • acetic acid (6.15), pyridine (12.3), tetrahydrofuran (THF) (7.5), acetone (20.7), methyl ethyl ketone (MEK) (15.45), ethyl a
  • the organic solvents exemplified in (1) to (4) above may be used in combination of two or more kinds.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • the first electrolyte solution and the second electrolyte solution may be filled in containers such as bottles and provided together with the device 1a.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • PDMS polydimethylsiloxane
  • 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.
  • 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.
  • Figure 4 shows the measurement results of Example 2, Example 3, and Comparative Example 1.
  • the electrolyte filled in the first chamber and the second chamber had the same composition as shown in Comparative Example 1.
  • the change in ion current when DNA passed through the nanopore was 0.4 nA from the baseline.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 4 0.69M NaCl was used as the first electrolytic solution.
  • 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
  • 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.
  • 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.
  • 10% ethanol, which is an electrolyte with a low dielectric constant is described as "10% Eth”
  • 20% ethanol is described as "20% Eth”
  • 30% ethanol is described as "10% Eth”
  • the electrolyte with a high dielectric constant is described as "5xPBS”.

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Abstract

 L'invention concerne un procédé et un dispositif de mesure de courant ionique permettant d'augmenter le rapport S/B d'un résultat de mesure d'une variation d'un courant ionique. Le dispositif de mesure de courant ionique comprend une carte présentant une première surface et une seconde surface, un trou débouchant de la première surface vers la seconde surface pour permettre le passage d'un échantillon chargé, un premier élément de chambre qui, conjointement avec une surface de la première surface comportant une première ouverture du trou débouchant, forme une première chambre remplie d'une première solution électrolytique, et un second élément de chambre qui, conjointement avec une surface de la seconde surface comportant une seconde ouverture du trou débouchant, forme une seconde chambre remplie d'une seconde solution électrolytique, et le procédé de mesure de courant ionique comprend une étape d'application de tension entre la première solution électrolytique et la seconde solution électrolytique pour faire passer l'échantillon chargé contenu dans l'une des chambres à travers le trou débouchant en direction de l'autre chambre, et une étape de mesure d'un changement dans un courant ionique lorsque l'échantillon chargé traverse le trou débouchant. En outre, la première solution électrolytique et la seconde solution électrolytique ont des constantes diélectriques différentes.
PCT/JP2023/035451 2022-10-04 2023-09-28 Procédé et dispositif de mesure de courant ionique WO2024075637A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040120854A1 (en) * 2002-04-04 2004-06-24 Heath James R Silicon-wafer based devices and methods for analyzing biological material
JP2011501806A (ja) * 2007-10-02 2011-01-13 プレジデント アンド フェロウズ オブ ハーバード カレッジ ナノポアによる分子の捕捉、再捕捉およびトラッピング
JP2020524271A (ja) * 2017-06-20 2020-08-13 イラミーナ インコーポレーテッド ナノポアシーケンサー

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040120854A1 (en) * 2002-04-04 2004-06-24 Heath James R Silicon-wafer based devices and methods for analyzing biological material
JP2011501806A (ja) * 2007-10-02 2011-01-13 プレジデント アンド フェロウズ オブ ハーバード カレッジ ナノポアによる分子の捕捉、再捕捉およびトラッピング
JP2020524271A (ja) * 2017-06-20 2020-08-13 イラミーナ インコーポレーテッド ナノポアシーケンサー

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALESSIO FRAGASSO: "Comparing Current Noise in Biological and Solid-State Nanopores", ACS NANO, AMERICAN CHEMICAL SOCIETY, US, vol. 14, no. 2, 25 February 2020 (2020-02-25), US , pages 1338 - 1349, XP093156694, ISSN: 1936-0851, DOI: 10.1021/acsnano.9b09353 *

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