WO2022080023A1 - Method for measuring viral nucleic acid, device for measuring viral nucleic acid, program, sensor, layered electrode, and substrate with electrode - Google Patents

Method for measuring viral nucleic acid, device for measuring viral nucleic acid, program, sensor, layered electrode, and substrate with electrode Download PDF

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Publication number
WO2022080023A1
WO2022080023A1 PCT/JP2021/031778 JP2021031778W WO2022080023A1 WO 2022080023 A1 WO2022080023 A1 WO 2022080023A1 JP 2021031778 W JP2021031778 W JP 2021031778W WO 2022080023 A1 WO2022080023 A1 WO 2022080023A1
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electrode
nucleic acid
virus
boron
viral nucleic
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PCT/JP2021/031778
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French (fr)
Japanese (ja)
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章玄 岡本
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国立研究開発法人物質・材料研究機構
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Priority to JP2022557250A priority Critical patent/JP7455431B2/en
Publication of WO2022080023A1 publication Critical patent/WO2022080023A1/en

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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • 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
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • 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
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • 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
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Definitions

  • the present invention relates to a method for measuring viral nucleic acid, a virus nucleic acid measuring device, a program, a sensor, a laminated electrode, and a substrate with an electrode.
  • an object of the present invention is to provide a method for measuring viral nucleic acid, which can rapidly measure viral nucleic acid in a sample collected from an aerosol or the like.
  • Another object of the present invention is to provide a program, a virus nucleic acid measuring device, a sensor, a laminated electrode, and a substrate with an electrode.
  • a sample containing a virus is brought into contact with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode.
  • a constant potential is applied to the laminated electrode to elute the copper coating layer, generate copper ions, release viral nucleic acid from the virus, and expose the boron-doped diamond electrode to the sample.
  • a method for measuring a viral nucleic acid comprising sweeping the potential of the boron-doped diamond electrode and measuring an electrochemical response derived from the viral nucleic acid.
  • [2] The description in [1], which comprises comparing the response current when the constant potential is applied with a predetermined threshold value and continuing the application of the constant potential until the response current becomes equal to or less than the threshold value.
  • Method for measuring viral nucleic acid [3] The electrochemical response derived from the viral nucleic acid is described in [1] or [2], which is at least one value selected from the group consisting of the magnitude of the peak current and the current peak area. How to measure viral nucleic acid. [4] The method for measuring viral nucleic acid according to [3], wherein the peak potential is more positive than the constant potential.
  • the sweeping of the potential is performed by any one of [1] to [4] selected from the group consisting of a linear sweep voltammetry method, a differential pulse voltammetry method, and a cyclic voltammetry method.
  • An aerosol containing a virus is sorbed on the surface of a solid electrolyte, a sample is prepared on the surface, and the surface is brought into contact with the combination electrode to bring the sample into contact with the combination electrode.
  • the method for measuring a viral nucleic acid according to any one of [1] to [6], which comprises.
  • [8] A sensor for contacting a sample containing a virus with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode.
  • a constant potential application part that applies a constant potential to the part and the laminated electrode to elute the copper coating layer to generate copper ions, and expose the boron-doped diamond electrode to the sample, and the boron-doped diamond.
  • a virus nucleic acid measuring apparatus having a sweep potential application unit that applies a sweep potential to an electrode and measures an electrochemical response derived from the virus.
  • the viral nucleic acid measuring apparatus has a comparison unit that compares the response current due to the application of the constant potential with a predetermined threshold value, and the constant potential application unit is described until the response current becomes equal to or less than the threshold value as a result of the comparison.
  • the viral nucleic acid measuring apparatus according to [8], which continues to apply a constant potential.
  • the sensor unit includes a substrate, the combination electrode arranged on the substrate, and a solid electrolyte for adhering the sample to the surface and bringing the surface to which the sample is attached into contact with the combination electrode.
  • the viral nucleic acid measuring apparatus which comprises a connector for electrically connecting the combination electrode of the sensor including the above-mentioned constant potential application unit and the above-mentioned sweep potential application unit. .. [11] Any one of [8] to [10], wherein the sensor unit includes a cell for accommodating the sample and the combination electrode arranged in the cell so as to be in contact with the sample.
  • the viral nucleic acid measuring device according to. [12] A sensor for contacting a sample containing a virus with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode by a computer.
  • the constant potential application part which applies a constant potential to the laminated electrode to elute the copper coating layer to generate copper ions, and exposes the boron-doped diamond electrode to the sample, and the boron-doped diamond electrode.
  • the above-mentioned stacking The procedure of applying a constant potential to the electrode and the application of the constant potential elutes the copper coating layer, sweeps the potential of the boron-doped diamond electrode exposed to the sample, and conducts electricity derived from the viral nucleic acid of the virus.
  • the combined electrode is a sensor including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the surface of the boron-doped diamond electrode, and a counter electrode.
  • the sensor according to [14], wherein the combination electrode further has a reference electrode.
  • viral nucleic acid in a sample collected from an aerosol or the like can be rapidly measured.
  • a program, a virus nucleic acid measuring device, a sensor, a laminated electrode, and a substrate with an electrode can also be provided.
  • FIG. 1 It is a flowchart which shows the measuring method of the viral nucleic acid by Example 1 of this invention. It is an exploded perspective view of the sensor used for the measurement of the viral nucleic acid by Example 1 of this invention. It is a perspective view of the sensor used for the measurement of the viral nucleic acid by Example 1 of this invention.
  • 2A is a cross-sectional view taken along the line XY of the substrate 201 with electrodes of FIG. 2A.
  • FIG. 6B is a sectional view taken along line VW of FIG. 6B.
  • FIG. 6B is a sectional view taken along line VW of FIG. 6B.
  • FIG. 6B is a sectional view taken along line VW of FIG. 6B.
  • It is a hardware block diagram of the virus nucleic acid measuring apparatus according to Example 3 of this invention.
  • the term "aerosol” means a mixture of fine particles and a gas, and the diameter of the fine particles is generally not particularly limited, but is preferably 1 nm to 100 ⁇ m, more preferably 1 to 100 nm.
  • These fine particles contain fine particles, the surroundings of which are covered with water, and have a particle diameter of 5 ⁇ m or more as a whole, called “droplets”, and typically, the above-mentioned water is removed by drying or the like. It includes any of the fine particles themselves called “droplet nuclei” having a diameter of less than 5 ⁇ m. Among them, droplet nuclei having a smaller sedimentation rate in the air are considered to be one of the causes of virus infection by aerosols, and it is preferable to collect a sample from the aerosol containing the droplet nuclei.
  • FIG. 1 is a flowchart showing a method for measuring viral nucleic acid according to the first embodiment of the present invention.
  • step S11 an aerosol containing a virus is collected on the surface of the solid electrolyte, the virus is accumulated on the surface of the solid electrolyte, and this is used as a sample.
  • the solid electrolyte preferably contains a water-swelled polymer compound containing the electrolyte.
  • the polymer compound may be, for example, agarose or the like.
  • the solid electrolyte may be a hydrogel containing agar, agar, gelatin and the like, an electrolyte and water.
  • Hydrogel is preferable as the solid electrolyte.
  • the hydrogel include an agar gel and a gelatin gel.
  • the solid electrolyte one having high ionic conductivity is preferable, and more specifically, one in which ions (for example, hydrogen ion, sulfate ion, etc.) can move inside thereof is preferable.
  • the solid electrolyte may contain a sulfate or the like in that it has more excellent ionic conductivity.
  • Example 1 the aerosol is adsorbed on the solid electrolyte, and the virus is concentrated on the surface of the solid electrolyte to prepare a sample.
  • the sample may be prepared without using a solid electrolyte.
  • viral nucleic acid derived from a virus contained in other than aerosol may be detected.
  • a liquid containing a virus may be used as a sample.
  • the virus may be transferred from the solid surface onto the solid electrolyte (the solid electrolyte is brought into contact with the solid surface) to prepare a sample.
  • step S12 the sample containing the virus is brought into contact with the combination electrode.
  • the combination electrode includes a laminated electrode and a counter electrode.
  • the laminated electrode has a boron-doped Diamond (BDD electrode) and a copper coating layer arranged on the BDD electrode. Therefore, in this step, the copper coating layer and the sample come into contact with each other.
  • the solid electrolyte in which the sample is prepared may be pressed against the combination electrode on the surface thereof.
  • FIG. 2A is an exploded perspective view of a sensor used for collecting a sample and measuring viral nucleic acid in Example 1, and FIG. 2B is a perspective view of the sensor. A method of performing steps S11 and S12 using this sensor will be described.
  • the senor 20 is roughly classified into three parts.
  • the three components are a substrate 201 with electrodes, a spacer 26, and a cover 202 with a solid electrolyte.
  • Preparation of the sample in step S11 is performed using the cover 202 with a solid electrolyte.
  • the cover 202 with a solid electrolyte has a cover 27 and a solid electrolyte 25 arranged on the surface of one side of the cover 27, and can be removed from the sensor 20.
  • the sample can be prepared by removing the cover 202 with the solid electrolyte from the sensor 20 and accommodating the aerosol on the solid electrolyte 25. Since the cover 202 with the solid electrolyte has the cover 27, the virus in the aerosol can be collected without touching the solid electrolyte 25, so that unintended contaminants can be suppressed from being mixed into the sample.
  • the contact between the sample and the combination electrode in step S12 can be realized by pressing the solid electrolyte 25 against the combination electrode of the electrode-attached substrate 201.
  • the substrate 201 with an electrode has a substrate 21, a laminated electrode 22 which is a working electrode arranged on the substrate 21, a counter electrode 23, and a reference electrode 24 (reference electrode). are doing.
  • the solid electrolyte 25 may be brought into contact with the laminated electrode 22, the counter electrode 23, and the reference electrode 24 (combination electrode; combination electrode) via the notch of the spacer 26.
  • the spacer 26 has a function of a guide for aligning the solid electrolyte 25 with the combination electrode, which has an advantage that it is easy to reattach the cover 202 with the solid electrolyte once removed.
  • the sensor does not have to include the spacer 26.
  • FIG. 2C is a cross-sectional view taken along the line XY of the sensor 20.
  • a laminated electrode 22, a counter electrode 23, and a reference electrode 24 are arranged on the substrate 21.
  • a sealing member (for example, epoxy resin) represented by "SL" in the figure is arranged between these electrodes.
  • the laminated electrode 22 has a BDD electrode 28 and a copper coating layer 29 arranged on the BDD electrode 28.
  • the laminated electrode 22 is higher than the counter electrode 23 and the reference electrode 24 in the thickness direction, but the height of each electrode is not limited to the above.
  • the laminated electrode 22 has no step on the surface of the counter electrode 23 and the reference electrode 24, and one or both of the counter electrode 23 and the reference electrode 24 are higher than the laminated electrode 22. But it may be. From the viewpoint of obtaining a more excellent effect of the present invention, it is preferable that the surfaces of the BDD electrode 28, the counter electrode 23, and the reference electrode 24 constituting the laminated electrode 22 have substantially no step.
  • the method for manufacturing the BDD electrode 28 is not particularly limited, and a known method can be used. Above all, it is preferable to produce by a chemical vapor deposition (CVD) method.
  • CVD chemical vapor deposition
  • a thermal filament, microwave, high frequency, DC glow discharge, DC arc discharge, combustion flame and the like can be used. Further, a plurality of these can be combined to adjust the nucleation density, increase the area, or make the nucleation uniform.
  • As a raw material many kinds of compounds containing carbon can be used. For example, CH 4 , C 2 H 2 , C 2 H 4 , C 10 H 16 , CO, CF 4 , etc. as gas; CH 3 OH, C 2 H 5 OH, and (CH 3 ) 2 CO as liquid. Etc .; Examples of the solid include graphite, fullerene and the like.
  • Examples of the addition of boron include a method of introducing a substance containing boron into the system to introduce boron into the carbon vapor phase.
  • Examples of such a substance containing boron include diborane, trimethylborane, and trimethoxyborane, but trimethoxyborane is preferable because it is easier to handle.
  • trimethoxyborane is used as the boron source and acetone is used as the solvent for dissolving the boron, since acetone also serves as a carbon source, it is easier to handle.
  • the BDD electrode is formed by a plasma CVD method using microwaves in that the growth rate of diamond is faster and the obtained diamond film is more uniform.
  • a diamond film can be formed by generating hydrogen plasma by microwaves and introducing a raw material gas into it.
  • the amount of boron added is not particularly limited, but 10 to 12,000 ppm is preferable, and 1,000 to 10, 000 ppm is more preferable.
  • the method of laminating the laminated electrode 22, the counter electrode 23, and the reference electrode 24 on the substrate 21 is not particularly limited, and a known method can be used. Examples of such a method include the methods described in paragraphs 0037 to 0050 of JP-A-2006-10357, the methods described in paragraphs 0034 to 0057 of JP-A-2020-33199, and the like.
  • the sensor 20 has a laminated electrode 22, a counter electrode 23, and a reference electrode 24, but the sensor that can be used in the method for measuring viral nucleic acid of Example 1 is not limited to the above, and the laminated electrode is not limited to the above. And a counter electrode. Further, the arrangement of the laminated electrode 22, the counter electrode 23, and the reference electrode 24 in the sensor 20 may be replaced.
  • the substrate 21 may be a conductive substrate or an insulating substrate, but an insulating substrate is preferable.
  • the substrate include tungsten, silicon, silicon oxide, silicon nitride, silicon carbide, niobium and the like.
  • the substrate 21 may be quartz glass or the like.
  • the thickness and size of the substrate may be appropriately selected from the viewpoint of handleability and the like, and is typically 1 ⁇ m to 5 mm.
  • the thickness of the BDD electrode 28 can be adjusted by the film formation time.
  • the thickness of the BDD electrode 28 is typically 0.1 ⁇ m or more, more preferably 1 ⁇ m or more, further preferably 5 ⁇ m or more, and particularly preferably 10 ⁇ m or more.
  • the upper limit is not particularly limited, but is generally preferably 1 mm or less. The above thickness range is the same for the counter electrode 23 and the reference electrode 24.
  • the material of the counter electrode 23 is not particularly limited, and a known material for the counter electrode can be used. Examples of such a material include platinum, carbon material, stainless steel, SnO 2 , and the like.
  • the reference electrode 24 may be, for example, a calomel electrode, a silver / silver chloride electrode, or the like.
  • step S13 a constant potential is applied to the laminated electrode 22, whereby the copper coating layer 29 is eluted (electrolyzed) to the solid electrolyte 25 side, and copper ions are generated.
  • the potential applied at this time is not particularly limited, but is generally preferably +0.4 to 0.5V.
  • Copper ions destroy the viral envelope and at least part of the capsid by direct involvement and / or indirect involvement such as catalyzing the generation of reactive oxygen species (ROS). Therefore, viral nucleic acid is released from the inside of the virus.
  • ROS reactive oxygen species
  • FIG. 3 is a schematic diagram showing the principle of the method for measuring viral nucleic acid of Example 1.
  • the detection system 30 is composed of a BDD electrode 28, a copper coating layer 29 formed on the BDD electrode 28, and a solid electrolyte 25, and a coronavirus 31 to be detected is arranged on the surface of the solid electrolyte 25. ..
  • FIG. 3 the structure of the coronavirus 31 is schematically shown, and the diameter thereof (the length corresponding to L1 in FIG. 3), the BDD electrode 28, and the copper coating layer 29 (together, laminated).
  • the relationship between the electrode 22) and the thickness of the solid electrolyte 25 does not actually correspond. Since the size, thickness, etc. of each part have already been described, the description thereof will be omitted here. The above is the same for L2 in FIG. 4, which will be described later.
  • the surface of the coronavirus 31 is covered with a lipid membrane 32, in which a plus-stranded single-stranded RNA genome 33 wrapped around a nucleocapsid (N) protein is arranged.
  • a Spike (S) protein, an envelope (E) protein, and a Membrane (M) protein are arranged on the surface of the coronavirus 31, and the shape resembles a crown.
  • S Spike
  • E envelope
  • M Membrane
  • the copper coating layer 29 elutes to the solid electrolyte 25 side, and copper ions are generated. Copper ions destroy the envelope of the virus and at least part of the capsid by direct and / or indirect involvement, thereby causing viral nucleic acid (one for coronavirus 31) from within the virus. The strand RNA genome 33) is released.
  • step S14 the current obtained as a response when a constant potential is applied to the laminated electrode 22 (hereinafter, also referred to as “response current”) is compared with a predetermined threshold value.
  • This response current reflects the elution process of the copper coating layer 29. Since the method for measuring viral nucleic acid of Example 1 requires exposing at least a part (preferably all) of the BDD electrode 28 to the sample, the response current is monitored and the copper coating layer 29 elutes to a desired degree. It is preferable to confirm that it has been done (disassembled).
  • the elution amount (decomposition amount) of the copper coating layer 29 may be controlled by a method other than monitoring the response current, for example, by applying a constant potential. In that case, steps S14 and S15 may be omitted.
  • the threshold value may be, for example, a value in which a predetermined margin is taken into consideration for the response current when all the copper coating layer 29 is eluted.
  • the response current is equal to or less than the above threshold value (a state in which the threshold value is not exceeded) when the potential is applied to the laminated electrode 22, it can be determined that the copper coating layer 29 has eluted to a desired degree (for example, all) (for example, all).
  • the response current exceeds the threshold value (step S15: True) since more copper coating layer 29 remains than desired, it is sufficient to continue applying the constant potential and monitoring the response current (step S15: True). Steps S13 to S15).
  • FIG. 4 is a schematic diagram showing a detection system after a constant potential is applied to the laminated electrodes and the copper coating layer is eluted. Since the copper coating layer 29 was completely eluted (decomposed), the detection system 40 was composed of a BDD electrode 28 and a solid electrolyte 25, and the coronavirus 41 on the surface of the solid electrolyte 25 had an envelope and a capsid of copper. It is destroyed by the direct and indirect involvement of ions, and the viral nucleic acid is released from the inside.
  • the electrochemical response derived from the viral nucleic acid is obtained by sweeping the potential of the BDD electrode 28. Can be done (step S16).
  • the electrochemical response derived from this viral nucleic acid is preferably the oxidation current of the viral nucleic acid, and more preferably a peak-shaped response (there is one or more maximum values in the potential vs. current measurement result). preferable.
  • the maximum current value of this peak is referred to as "peak current”
  • the potential giving this "peak current” is referred to as "peak potential”
  • the area of this peak is referred to as "current peak area”.
  • the method of sweeping the potential of the BDD electrode is at least one method selected from the group consisting of the linear sweep voltammetry method, the differential pulse voltammetry method, and the cyclic voltammetry method, the response of the peak shape as described above is performed. Is easy to obtain.
  • the peak potential of the oxidation current of viral nucleic acid is often detected in the range of +1.0 to 1.5V (vs Ag / AgCl).
  • the copper coating layer 29 is eluted, the BDD electrode 28 is exposed to the sample, and then the potential of the BDD electrode 28 is swept. Measurement becomes possible.
  • each peak current or each peak area may be totaled as a measured value, or a single peak current or a peak area may be used as a measured value.
  • each peak current or the total value of each peak area it is preferable to use each peak current or the total value of each peak area as the measured value. According to the method of Example 1, the viral nucleic acid in the sample collected from the aerosol can be rapidly measured.
  • FIG. 5 is a hardware configuration diagram of the virus nucleic acid measuring device according to the second embodiment of the present invention.
  • the virus nucleic acid measuring device 50 includes a processor 51, a storage device 52, a display device 53, an input device 54, an electrochemical measuring device 55, and a connector 56.
  • the processor 51, the storage device 52, the display device 53, the input device 54, and the electrochemical measurement device 55 can exchange data with each other via a bus (denoted as “BUS” in the figure).
  • a sensor 20 is connected to the virus nucleic acid measuring device 50 via a connector 56. Although the structure will be described later, the sensor 20 is removable with respect to the virus nucleic acid measuring device 50, and for example, the sensor 20 can be replaced for each sample. Since the sensor 20 is as described with reference to FIGS. 2A, 2B, and 2C, the description thereof will be omitted here.
  • the processor 51 controls each part of the virus nucleic acid measuring device 50 to realize the function of the virus nucleic acid measuring device.
  • the processor 51 may be, for example, a microprocessor, a processor core, a multi-processor, an ASIC (application-specific integrated circuit), an FPGA (field programgable gate array), a GPGPU (general-purpose processing), etc.
  • the storage device 52 has a function of temporarily and / or non-temporarily storing programs and data, and provides a work area of the processor 51.
  • the storage device 52 may be, for example, a ROM (Read Only Memory), a RAM (Random Access Memory), an HDD (Hard Disk Drive), a flash memory, an SSD (Solid State Drive), or the like.
  • the display device 53 can display the measurement result, the voltage application status to the laminated electrode (or BBD electrode), the sample name, the operation procedure, and the like.
  • the display device 53 may be a liquid crystal display, an organic EL (Electroluminescence) display, or the like. Further, the display device 53 may be configured integrally with the input device 54. In this case, the display device 53 is a touch panel display, and a form of providing a GUI (Graphical User Interface) can be mentioned.
  • GUI Graphic User Interface
  • the input device 54 can input voltage application conditions, measurement conditions, sample names, etc. to the combination electrodes.
  • the input device 54 may be a keyboard, a mouse, a scanner, a touch panel, or the like.
  • the electrochemical measurement device 55 can measure the response current by applying a constant potential to the laminated electrode 22 of the sensor 20 connected via the connector 56. In addition, the electrochemical measurement device 55 can apply a sweep potential to the BDD electrode 28 after the copper coating layer 29 has been eluted to measure the electrochemical response derived from the viral nucleic acid.
  • the electrochemical measurement device 55 may be, for example, a potentiostat or the like.
  • the connector 56 electrically connects the electrochemical measurement device 55 and the combination electrode (stacked electrode 22, counter electrode 23, and reference electrode 24) of the sensor 20.
  • the connector 56 may be, for example, a combination of a terminal and a conducting wire.
  • FIG. 6A is a perspective view of the virus nucleic acid measuring device of Example 2.
  • the virus nucleic acid measuring device 60 has a main body 61 and a touch panel display 62 arranged in the central portion of the main body 61, and each hardware (processor 51 and an electrochemical measuring device) already described is contained in the main body 61.
  • a circuit board on which 55 etc.) is mounted is arranged.
  • the virus nucleic acid measuring device 60 has an insertion port 63 for inserting the sensor 20.
  • FIG. 6B is a perspective view of the virus nucleic acid measuring device 60 with the sensor 20 inserted.
  • FIG. 6C is a sectional view taken along line VW of FIG. 6B.
  • the sensor 20 inserted into the insertion slot 63 with the substrate 21 facing upward is sandwiched between the main body 61 supported by the filling member 69 and the spring member 64 arranged inside the insertion slot 63.
  • the combination electrode (reference electrode 24 in the VW cross section) arranged on the surface of the substrate 21 is pressed against the spring member 64 from the opposite surface of the substrate 21 and comes into close contact with the terminal 65.
  • the terminal 65 is connected to the terminal 67 of the circuit board 68 via the lead wire 66.
  • the terminal 67 is connected to the electrochemical measurement device 55 arranged on the circuit board 68. Thereby, the electrochemical measurement device 55 is connected to the combination electrode.
  • FIG. 7 is a functional block diagram of the virus nucleic acid measuring device of Example 2.
  • the virus nucleic acid measuring device 70 includes a control unit 71, a storage unit 72, a display unit 73, an input unit 74, a comparison unit 75, a constant potential application unit 76, a sweep potential application unit 77, and a sensor unit 78. Has.
  • the control unit 71 includes a processor 51.
  • the control unit 71 controls each of the storage unit 72, the display unit 73, the input unit 74, the comparison unit 75, the constant potential application unit 76, and the sweep potential application unit 77 to realize the function of the virus nucleic acid measuring device 70. do.
  • the storage unit 72 includes a storage device 52.
  • the storage unit 72 stores the program, the threshold value, and the like, and stores the measurement data and the like.
  • the display unit 73 includes a display device 53. Further, the input unit 74 includes an input device 54. By controlling these, the control unit 71 receives the input from the user of the virus nucleic acid measuring device 70 and stores it in the storage unit 72, or the constant potential application unit 76 and the constant potential application unit 76 stored in the storage unit 72. , The measurement data (response current and electrochemical response) obtained by the sweep potential application unit 77 can be displayed to the user of the apparatus.
  • the comparison unit 75 is a function realized by the control unit 71 executing the program stored in the storage unit 72.
  • the comparison unit 75 compares the response current due to the application of a constant potential to the laminated electrode 22 with a predetermined threshold value.
  • the threshold value is stored in the storage unit 72.
  • the response current is obtained by the constant potential application unit 76 described later.
  • the constant potential application unit 76 is configured to include the electrochemical measurement device 55, and is a function realized by the control unit 71 executing a program stored in the storage unit 72 and controlling the electrochemical measurement device 55.
  • the constant potential application unit 76 applies a constant voltage to the laminated electrode 22 of the sensor connected to the sensor unit 78, and measures the response current thereof.
  • the sweep potential application unit 77 is configured to include the electrochemical measurement device 55, and is a function realized by the control unit 71 executing a program stored in the storage unit 72 and controlling the electrochemical measurement device 55.
  • the sweep potential application unit 77 is connected to the sensor unit 78, applies a sweep voltage to the BDD electrode 28 exposed to the sample, and measures the electrochemical response derived from the viral nucleic acid.
  • the sensor unit 78 is configured to include a connector 56, and has a function of electrically connecting each electrode of the sensor 20 and the electrochemical measurement device 55.
  • FIG. 8 is a flowchart showing the operation of the control unit 71 of the virus nucleic acid measuring device according to the second embodiment.
  • step S81 the control unit 71 controls the constant potential application unit 76 to apply a constant voltage to the laminated electrode 22 in contact with the sample containing the virus and measure the response current.
  • the laminated electrode 22 is included in the sensor 20, and the sensor 20 is controlled by the constant potential application unit 76 connected via the sensor unit 78.
  • step S82 the control unit 71 controls the comparison unit 75 to compare the response current obtained from the laminated electrode 22 with the threshold value stored in advance in the storage unit 72.
  • This threshold is predetermined and stored as corresponding to the preferred elution state of the copper coating layer 29.
  • step S83 True
  • the copper coating layer 29 arranged on the BDD electrode 28 is not completely eluted to a desired degree.
  • step S84 the control unit 71 controls the constant potential application unit 76 to continue applying the constant potential to the laminated electrode 22.
  • the threshold value determination (steps S82 to S83) is executed again by the comparison unit 75, and the process is repeated until the response current becomes equal to or less than the threshold value.
  • step S83 False
  • the control unit 71 controls the sweep potential application unit 77 and is exposed to the sample.
  • a sweep voltage is applied to the BDD electrode 28, and the obtained electrochemical response is measured (step S85).
  • This electrochemical response was typically released by the action of copper ions generated by the application of a constant potential to the laminated electrode 22 to eliminate and / or at least part of the viral capsid and / or envelope. It is the oxidation current of viral nucleic acid.
  • the oxidation current of the viral nucleic acid is typically obtained as a curve having a peak in the curve of potential vs. current. This peak current and the current peak area are proportional to the amount of viral nucleic acid in the sample.
  • the peak current and the current peak area are stored in the storage unit 72 by the sweep potential application unit 77 and displayed in the input unit 74.
  • the virus nucleic acid measuring device of Example 2 the virus nucleic acid derived from the virus contained in the sample collected from the aerosol can be rapidly measured.
  • FIG. 9 is a hardware configuration diagram of the virus nucleic acid measuring device according to the third embodiment of the present invention.
  • the virus nucleic acid measuring device 90 has a processor 51, a storage device 52, a display device 53, an input device 54, an electrochemical measuring device 55, and a cell 91 connected to the electrochemical measuring device 55.
  • the virus nucleic acid measuring device 90 has a cell 91 connected to the electrochemical measuring device 55 and has a hardware configuration similar to that of the virus nucleic acid measuring device of Example 2 except that it does not have a connector. Many, the differences will be mainly explained below.
  • the cell 91 is a container that can accommodate a sample, and has a laminated electrode 22, a counter electrode 23, and a reference electrode 24 inside. Typically, when a liquid sample is housed in the cell 91, the housed sample comes into contact with each of the electrodes constituting the combination electrode.
  • the laminated electrode 22, the counter electrode 23, and the reference electrode 24 are electrically connected to the electrochemical measuring device 55, respectively, and can control and measure the potential and the current.
  • FIG. 10 is a schematic diagram of the virus nucleic acid measuring device according to the third embodiment.
  • the virus nucleic acid measuring apparatus 100 has a computer 101, a potentiostat 102 connected to the computer 101, a laminated electrode 22, a counter electrode 23, and a reference electrode 24 connected to the potentiostat 102, respectively. ..
  • the laminated electrode 22, the counter electrode 23, and the reference electrode 24 are housed in the cell 91 and can come into contact with the sample also housed in the cell 91.
  • the computer 101 includes a processor 51, a storage device 52, a display device 53, and an input device 54, and the potentiostat 102 includes an electrochemical measurement device 55.
  • the virus nucleic acid measuring device of Example 3 has a built-in cell including a combination electrode, whereas the virus nucleic acid measuring device of Example 2 has a detachable sensor, and has a simpler configuration. Are better. Since the functions and operations of the virus nucleic acid measuring device of Example 3 are the same as those of the virus nucleic acid measuring device of Example 2, the description thereof will be omitted.
  • the virus nucleic acid derived from the virus typically contained in the liquid sample can be rapidly measured.
  • FIG. 11A is an exploded perspective view of the sensor 110 according to the fourth embodiment, and FIG. 11B is a perspective view of the sensor.
  • the sensor 110 has a substrate 21, a laminated electrode 22 arranged on the substrate 21, a counter electrode 23, and a reference electrode 24. It also has a spacer 26 and a cover 27 that covers the spacer 26.
  • the cover 27 has a recessed portion 111.
  • a liquid sample is introduced from the recess 111 and wets the space hidden by the upper surface of the substrate 21, the lower surface of the cover 27, and the side surface of the notch of the spacer 26.
  • the sample and the combination electrode come into contact with each other, and measurement can be performed.
  • the sensor 110 can be used by inserting it into the virus nucleic acid measuring device of Example 2 instead of the sensor 20 already described. Since the method of use is the same as that of the sensor 20, the description thereof will be omitted.
  • Example 4 Since the sensor of Example 4 is removable from the virus nucleic acid measuring device, it is possible to prevent the samples from being mixed with each other and causing an error in the measurement result by replacing each sample.

Abstract

The method for measuring viral nucleic acid according to the present invention comprises: bringing a specimen containing a virus (31) into contact with a combined electrode including at least a counter electrode (25) and a layered electrode (22) having a boron-doped diamond electrode (28) and a copper-coating layer (29) disposed on the electrode; applying a constant potential to the layered electrode to cause elution of the copper-coating layer, to generate copper ions, to cause the virus (41) to release viral nucleic acid (33), and to expose the boron-doped diamond electrode to the specimen; and sweeping the potential of the boron-doped diamond electrode and measuring an electrochemical response derived from the viral nucleic acid. The method enables quick measurement of viral nucleic acid in a specimen collected from aerosol or the like.

Description

ウィルス核酸の測定方法、ウィルス核酸測定装置、プログラム、センサ、積層電極、及び、電極付き基板Viral nucleic acid measuring method, viral nucleic acid measuring device, program, sensor, laminated electrode, and substrate with electrode
　本発明は、ウィルス核酸の測定方法、ウィルス核酸測定装置、プログラム、センサ、積層電極、及び、電極付き基板に関する。 The present invention relates to a method for measuring viral nucleic acid, a virus nucleic acid measuring device, a program, a sensor, a laminated electrode, and a substrate with an electrode.
　新型コロナウィルス（ＳＡＲＳ－ＣｏＶ－２）感染の急速拡大を受け、手術室におけるエアロゾル、及び、サージカルスモーク等を介した医療従事者へのウィルス感染の可能性が指摘されている。
　例えば、日本手術医学会の「新型コロナウイルス感染流行下での手術室管理・運営に関する提言」（ｈｔｔｐ：／／ｊａｏｍ．ｋｅｎｋｙｕｕｋａｉ．ｊｐ／ｉｎｆｏｒｍａｔｉｏｎ／ｉｎｆｏｒｍａｔｉｏｎ＿ｄｅｔａｉｌ．ａｓｐ？ｉｄ＝１０２９７８、２０２０年７月２２日インターネット検索）では、「新型コロナウイルスの感染拡大においてエアロゾル発生がひとつの因子となっており、手術操作や挿管においてその発生リスクが高まることは、一定の合意が得られている。」等とし、エアロゾルによるウィルス感染の可能性について指摘している。 Following the rapid spread of the new coronavirus (SARS-CoV-2) infection, it has been pointed out that there is a possibility of virus infection to medical staff via aerosols in the operating room and surgical smoke.
For example, "Recommendations for operating room management and operation under the epidemic of new coronavirus infection" (http://jaom.kenkyukai.jp/information/information_detail.asp? Id = 102978, July 22, 2020) of the Japanese Society of Surgical Medicine. According to the Japanese Internet search), "A certain agreement has been reached that the outbreak of aerosol is one of the factors in the spread of the new coronavirus, and the risk of its outbreak increases in surgical operations and intubation." , Points out the possibility of virus infection by aerosols.
　エアロゾル中のウィルスを検知する方法としては、エアロゾル中のウィルスをフィルター等で補足し、定量ＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ）により検知する方法が知られているが、検出に時間が掛ること、及び、操作が煩雑で専門知識を必要とする点に課題があった。 As a method of detecting a virus in an aerosol, a method of supplementing the virus in the aerosol with a filter or the like and detecting it by quantitative PCR (Polymerase Chain Reaction) is known, but it takes a long time to detect and an operation. However, there was a problem in that it was complicated and required specialized knowledge.
　そこで、本発明は、エアロゾル等から捕集した検体中のウィルス核酸を迅速に測定できる、ウィルス核酸の測定方法の提供を課題とする。
　また、本発明は、プログラム、ウィルス核酸測定装置、センサ、積層電極、及び、電極付き基板の提供も課題とする。 Therefore, an object of the present invention is to provide a method for measuring viral nucleic acid, which can rapidly measure viral nucleic acid in a sample collected from an aerosol or the like.
Another object of the present invention is to provide a program, a virus nucleic acid measuring device, a sensor, a laminated electrode, and a substrate with an electrode.
　本発明者らは、上記課題を達成すべく鋭意検討した結果、以下の構成により上記課題を達成することができることを見出した。 As a result of diligent studies to achieve the above-mentioned problems, the present inventors have found that the above-mentioned problems can be achieved by the following configurations.
　［１］　ホウ素ドープダイヤモンド電極、及び、上記ホウ素ドープダイヤモンド電極上に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、組合せ電極にウィルスを含む検体を接触させることと、上記積層電極に定電位を印加して、上記銅被覆層を溶出させ、銅イオンを発生させて、上記ウィルスから、ウィルス核酸を放出させるとともに、上記ホウ素ドープダイヤモンド電極を上記検体に晒すことと、上記ホウ素ドープダイヤモンド電極の電位を掃引し、上記ウィルス核酸に由来する電気化学的応答を測定することと、を含む、ウィルス核酸の測定方法。
　［２］　上記定電位を印加した際の応答電流を予め定めた閾値と比較し、上記応答電流が上記閾値以下となるまで、上記定電位の印加を継続することを含む、［１］に記載のウィルス核酸の測定方法。
　［３］　上記ウィルス核酸に由来する電気化学的応答は、ピーク電流の大きさ、及び、電流ピーク面積からなる群より選択される少なくとも１種の値である、［１］又は［２］に記載のウィルス核酸の測定方法。
　［４］　ピーク電位が上記定電位よりも正である、［３］に記載のウィルス核酸の測定方法。
　［５］　上記電位の掃引は、リニアスイープボルタンメトリー法、微分パルスボルタンメトリー法、及び、サイクリックボルタンメトリー法からなる群より選択される少なくとも１種の方法により行われる、［１］～［４］のいずれかに記載のウィルス核酸の測定方法。
　［６］　更に、エアロゾルを捕集して上記検体を準備することを含む、［１］～［５］のいずれかに記載のウィルス核酸の測定方法。
　［７］　固体電解質の表面にウィルスを含むエアロゾルを収着させ、上記表面上に検体を準備して、上記表面を上記組合せ電極と接触させることにより、上記検体と上記組合せ電極とを接触させることを含む、［１］～［６］のいずれかに記載のウィルス核酸の測定方法。
　［８］　ホウ素ドープダイヤモンド電極、及び、上記ホウ素ドープダイヤモンド電極上に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、組合せ電極にウィルスを含む検体を接触させるためのセンサ部と、上記積層電極に定電位を印加し、上記銅被覆層を溶出させ、銅イオンを発生させるとともに、上記ホウ素ドープダイヤモンド電極を上記検体に対して晒す定電位印加部と、上記ホウ素ドープダイヤモンド電極に掃引電位を印加し、上記ウィルスに由来する電気化学的応答を測定する掃引電位印加部とを有する、ウィルス核酸測定装置。
　［９］　上記定電位の印加による応答電流を予め定められた閾値と比較する比較部を有し、上記比較の結果、上記応答電流が、上記閾値以下となるまで、上記定電位印加部が上記定電位の印加を継続する、［８］に記載のウィルス核酸測定装置。
　［１０］　上記センサ部は、基板と、上記基板上に配置された上記組合せ電極と、上記検体を表面に付着させ、上記検体が付着した上記表面を上記組合せ電極と接触させるための固体電解質と、を含むセンサの上記組合せ電極を、上記定電位印加部、及び、上記掃引電位印加部と電気的に接続させるための接続具を含む、［８］又は［９］に記載のウィルス核酸測定装置。
　［１１］　上記センサ部は、上記検体を収容するためのセルと、上記検体と接触するように上記セル内に配置された上記組合せ電極と、を含む、［８］～［１０］のいずれかに記載のウィルス核酸測定装置。
　［１２］
　コンピュータにより、ホウ素ドープダイヤモンド電極、及び、上記ホウ素ドープダイヤモンド電極上に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、組合せ電極にウィルスを含む検体を接触させるためのセンサ部、上記積層電極に定電位を印加し、上記銅被覆層を溶出させ、銅イオンを発生させるとともに、上記ホウ素ドープダイヤモンド電極を上記検体に対して晒す定電位印加部と、上記ホウ素ドープダイヤモンド電極に掃引電位を印加し、上記ウィルスに由来する電気化学的応答を測定する掃引電位印加部とを有するウィルス核酸測定装置に、上記組合せ電極に対してウィルスを含む検体が接触した状態で、上記積層電極に定電位を印加する手順と、上記定電位の印加によって上記銅被覆層が溶出し、上記検体に晒された上記ホウ素ドープダイヤモンド電極の電位を掃引し、上記ウィルスのウィルス核酸に由来する電気化学的応答を測定する手順と、を実行させるプログラム。
　［１３］　更に、上記定電位の印加による応答電流を予め定められた閾値と比較する手順と、上記比較の結果、上記応答電流が上記閾値以下となるまで、上記定電位の印加を継続する手順と、を実行させる、［１２］に記載のプログラム。
　［１４］　基板と、上記基板上に配置された組合せ電極と、ウィルスを含む検体を表面に付着させ、上記検体が付着した上記表面を上記組合せ電極と接触させるための固体電解質と、を含むセンサであって、上記組合せ電極は、ホウ素ドープダイヤモンド電極、及び、上記ホウ素ドープダイヤモンド電極の表面に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、センサ。
　［１５］　上記組合せ電極が参照電極を更に有する、［１４］に記載のセンサ。
　［１６］　ホウ素ドープダイヤモンド電極と、上記ホウ素ドープダイヤモンド電極の表面の全体に配置された銅被覆層と、を有する積層電極。
　［１７］　基板と、上記基板上に配置された［１６］に記載の積層電極とを有する電極付き基板。 [1] A sample containing a virus is brought into contact with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode. A constant potential is applied to the laminated electrode to elute the copper coating layer, generate copper ions, release viral nucleic acid from the virus, and expose the boron-doped diamond electrode to the sample. A method for measuring a viral nucleic acid, comprising sweeping the potential of the boron-doped diamond electrode and measuring an electrochemical response derived from the viral nucleic acid.
[2] The description in [1], which comprises comparing the response current when the constant potential is applied with a predetermined threshold value and continuing the application of the constant potential until the response current becomes equal to or less than the threshold value. Method for measuring viral nucleic acid.
[3] The electrochemical response derived from the viral nucleic acid is described in [1] or [2], which is at least one value selected from the group consisting of the magnitude of the peak current and the current peak area. How to measure viral nucleic acid.
[4] The method for measuring viral nucleic acid according to [3], wherein the peak potential is more positive than the constant potential.
[5] The sweeping of the potential is performed by any one of [1] to [4] selected from the group consisting of a linear sweep voltammetry method, a differential pulse voltammetry method, and a cyclic voltammetry method. The method for measuring viral nucleic acid described in the above.
[6] The method for measuring a viral nucleic acid according to any one of [1] to [5], which further comprises collecting an aerosol and preparing the above-mentioned sample.
[7] An aerosol containing a virus is sorbed on the surface of a solid electrolyte, a sample is prepared on the surface, and the surface is brought into contact with the combination electrode to bring the sample into contact with the combination electrode. The method for measuring a viral nucleic acid according to any one of [1] to [6], which comprises.
[8] A sensor for contacting a sample containing a virus with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode. A constant potential application part that applies a constant potential to the part and the laminated electrode to elute the copper coating layer to generate copper ions, and expose the boron-doped diamond electrode to the sample, and the boron-doped diamond. A virus nucleic acid measuring apparatus having a sweep potential application unit that applies a sweep potential to an electrode and measures an electrochemical response derived from the virus.
[9] It has a comparison unit that compares the response current due to the application of the constant potential with a predetermined threshold value, and the constant potential application unit is described until the response current becomes equal to or less than the threshold value as a result of the comparison. The viral nucleic acid measuring apparatus according to [8], which continues to apply a constant potential.
[10] The sensor unit includes a substrate, the combination electrode arranged on the substrate, and a solid electrolyte for adhering the sample to the surface and bringing the surface to which the sample is attached into contact with the combination electrode. The viral nucleic acid measuring apparatus according to [8] or [9], which comprises a connector for electrically connecting the combination electrode of the sensor including the above-mentioned constant potential application unit and the above-mentioned sweep potential application unit. ..
[11] Any one of [8] to [10], wherein the sensor unit includes a cell for accommodating the sample and the combination electrode arranged in the cell so as to be in contact with the sample. The viral nucleic acid measuring device according to.
[12]
A sensor for contacting a sample containing a virus with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode by a computer. The constant potential application part, which applies a constant potential to the laminated electrode to elute the copper coating layer to generate copper ions, and exposes the boron-doped diamond electrode to the sample, and the boron-doped diamond electrode. In a state where the sample containing the virus is in contact with the combination electrode to the viral nucleic acid measuring device having the sweep potential application unit for applying the sweep potential to the above-mentioned virus and measuring the electrochemical response derived from the virus, the above-mentioned stacking The procedure of applying a constant potential to the electrode and the application of the constant potential elutes the copper coating layer, sweeps the potential of the boron-doped diamond electrode exposed to the sample, and conducts electricity derived from the viral nucleic acid of the virus. A procedure to measure the chemical response and a program to execute.
[13] Further, a procedure for comparing the response current due to the application of the constant potential with a predetermined threshold value, and a procedure for continuing the application of the constant potential until the response current becomes equal to or less than the threshold value as a result of the comparison. And, the program described in [12] to execute.
[14] A sensor including a substrate, a combination electrode arranged on the substrate, and a solid electrolyte for adhering a sample containing a virus to a surface and bringing the surface to which the sample is attached into contact with the combination electrode. The combined electrode is a sensor including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the surface of the boron-doped diamond electrode, and a counter electrode.
[15] The sensor according to [14], wherein the combination electrode further has a reference electrode.
[16] A laminated electrode having a boron-doped diamond electrode and a copper-coated layer arranged on the entire surface of the surface of the boron-doped diamond electrode.
[17] A substrate with electrodes having a substrate and the laminated electrode according to [16] arranged on the substrate.
　本発明によれば、エアロゾル等から捕集した検体中のウィルス核酸を迅速に測定できる。
　また、本発明によれば、プログラム、ウィルス核酸測定装置、センサ、積層電極、及び、電極付き基板も提供できる。 According to the present invention, viral nucleic acid in a sample collected from an aerosol or the like can be rapidly measured.
Further, according to the present invention, a program, a virus nucleic acid measuring device, a sensor, a laminated electrode, and a substrate with an electrode can also be provided.
本発明の実施例１によるウィルス核酸の測定方法を示すフローチャートである。It is a flowchart which shows the measuring method of the viral nucleic acid by Example 1 of this invention. 本発明の実施例１によるウィルス核酸の測定に用いるセンサの分解斜視図である。It is an exploded perspective view of the sensor used for the measurement of the viral nucleic acid by Example 1 of this invention. 本発明の実施例１によるウィルス核酸の測定に用いるセンサの斜視図である。It is a perspective view of the sensor used for the measurement of the viral nucleic acid by Example 1 of this invention. 図２Ａの電極付き基板２０１におけるＸ－Ｙ断面図である。2A is a cross-sectional view taken along the line XY of the substrate 201 with electrodes of FIG. 2A. 本発明の実施例１によるウィルス核酸の測定方法の原理を示す模式図である。It is a schematic diagram which shows the principle of the measuring method of a viral nucleic acid by Example 1 of this invention. 本発明の実施例１によるウィルス核酸の測定方法の原理を示す模式図である。It is a schematic diagram which shows the principle of the measuring method of a viral nucleic acid by Example 1 of this invention. 本発明の実施例２によるウィルス核酸の測定装置のハードウェア構成図である。It is a hardware block diagram of the measuring apparatus of viral nucleic acid according to Example 2 of this invention. 本発明の実施例２によるウィルス核酸測定装置の斜視図である。It is a perspective view of the virus nucleic acid measuring apparatus according to Example 2 of this invention. 本発明の実施例２によるウィルス核酸測定装置の斜視図である。It is a perspective view of the virus nucleic acid measuring apparatus according to Example 2 of this invention. 図６ＢのＶ－Ｗ断面図である。FIG. 6B is a sectional view taken along line VW of FIG. 6B. 本発明の実施例２によるウィルス核酸測定装置の機能ブロック図である。It is a functional block diagram of the virus nucleic acid measuring apparatus according to Example 2 of this invention. 本発明の実施例２によるウィルス核酸測定装置の制御部の動作を示すフローチャートである。It is a flowchart which shows the operation of the control part of the virus nucleic acid measuring apparatus according to Example 2 of this invention. 本発明の実施例３によるウィルス核酸測定装置のハードウェア構成図である。It is a hardware block diagram of the virus nucleic acid measuring apparatus according to Example 3 of this invention. 本発明の実施例３によるウィルス核酸測定装置の模式図である。It is a schematic diagram of the virus nucleic acid measuring apparatus according to Example 3 of this invention. 本発明の実施例４によるセンサの分解斜視図である。It is an exploded perspective view of the sensor according to Example 4 of this invention. 本発明の実施例４によるセンサの斜視図である。It is a perspective view of the sensor according to Example 4 of this invention.
　以下、図面を用いて本発明の実施例について説明する。
　なお、本明細書において、「～」を用いて表される数値範囲は、「～」の前後に記載される数値を下限値及び上限値として含む範囲を意味する。 Hereinafter, examples of the present invention will be described with reference to the drawings.
In the present specification, the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value.
（用語の説明）
　本明細書において「エアロゾル」とは、微粒子と気体との混合物を意味し、微粒子の直径としては、一般に、特に制限されないが、１ｎｍ～１００μｍが好ましく、１～１００ｎｍがより好ましい。この微粒子には、微粒子を含み、その周囲を水分が覆い、全体として粒子径が５μｍ以上である「飛沫」（ｄｒｏｐｌｅｔｓ）と呼ばれるものと、典型的には上記水分が乾燥等によって除去された粒子径が５μｍ未満である「飛沫核」（ｄｒｏｐｌｅｔ　ｎｕｃｌｅｉ）と呼ばれる微粒子そのものとのいずれをも含む。なかでも、空気中における沈降速度がより小さい飛沫核は、エアロゾルによるウィルス感染の原因の一つと考えられており、飛沫核を含むエアロゾルから検体を捕集することが好ましい。 (Explanation of terms)
As used herein, the term "aerosol" means a mixture of fine particles and a gas, and the diameter of the fine particles is generally not particularly limited, but is preferably 1 nm to 100 μm, more preferably 1 to 100 nm. These fine particles contain fine particles, the surroundings of which are covered with water, and have a particle diameter of 5 μm or more as a whole, called “droplets”, and typically, the above-mentioned water is removed by drying or the like. It includes any of the fine particles themselves called "droplet nuclei" having a diameter of less than 5 μm. Among them, droplet nuclei having a smaller sedimentation rate in the air are considered to be one of the causes of virus infection by aerosols, and it is preferable to collect a sample from the aerosol containing the droplet nuclei.
　図１は本発明の実施例１によるウィルス核酸の測定方法を示すフローチャートである。 FIG. 1 is a flowchart showing a method for measuring viral nucleic acid according to the first embodiment of the present invention.
　ステップＳ１１において、固体電解質の表面にウィルスを含むエアロゾルを収着させ、上記固体電解質の表面上にウィルスを集積させ、これを検体とする。本明細書では、上記を「固体電解質の表面上に検体を準備する」という。
　固体電解質は、典型的には電解質を含む水で膨潤した高分子化合物を含むことが好ましい。高分子化合物としては、例えば、アガロース等であってよい。また、固体電解質は、寒天、アガー、及び、ゼラチン等と、電解質と、水とを含むヒドロゲルであってもよい。
　固体電解質にエアロゾルが接触すると、固体電解質の表面上でエアロゾルの固形分が濃縮され、ウィルス核酸の検出がより容易になる。 In step S11, an aerosol containing a virus is collected on the surface of the solid electrolyte, the virus is accumulated on the surface of the solid electrolyte, and this is used as a sample. In the present specification, the above is referred to as "preparing a sample on the surface of a solid electrolyte".
The solid electrolyte preferably contains a water-swelled polymer compound containing the electrolyte. The polymer compound may be, for example, agarose or the like. Further, the solid electrolyte may be a hydrogel containing agar, agar, gelatin and the like, an electrolyte and water.
When the aerosol comes into contact with the solid electrolyte, the solid content of the aerosol is concentrated on the surface of the solid electrolyte, making it easier to detect the viral nucleic acid.
　固体電解質としてはヒドロゲルが好ましい。ヒドロゲルとしては、例えば、寒天ゲル、及び、ゼラチンゲル等が挙げられる。固体電解質としては、高いイオン電導性を有するものが好ましく、より具体的には、その内部をイオン（例えば、水素イオン、及び、硫酸イオン等）が移動できるものが好ましい。固体電解質はより優れたイオン電導性を有する点で、硫酸塩等を含有していてもよい。 Hydrogel is preferable as the solid electrolyte. Examples of the hydrogel include an agar gel and a gelatin gel. As the solid electrolyte, one having high ionic conductivity is preferable, and more specifically, one in which ions (for example, hydrogen ion, sulfate ion, etc.) can move inside thereof is preferable. The solid electrolyte may contain a sulfate or the like in that it has more excellent ionic conductivity.
　なお、実施例１では、エアロゾルを固体電解質に収着させて、固体電解質の表面上でウィルスを濃縮して検体とするが、エアロゾルに含まれるウィルスに由来するウィルス核酸を検出対象とする場合であっても、固体電解質を用いずに検体を準備してもよい。そのような方法としては、例えば、エアロゾル中のウィルスをフィルター等で補足して、それを検体とする方法等がある。
　また、本発明のウィルス核酸の測定方法は、エアロゾル以外に含まれるウィルスに由来するウィルス核酸を検出対象としてもよい。例えば、ウィルスを含む液体を検体としてもよい。
　また、固体電解質上に固体表面からウィルスを移し取って（固体電解質を固体表面と接触させて）検体としてもよい。 In Example 1, the aerosol is adsorbed on the solid electrolyte, and the virus is concentrated on the surface of the solid electrolyte to prepare a sample. However, in the case where the viral nucleic acid derived from the virus contained in the aerosol is targeted for detection. If so, the sample may be prepared without using a solid electrolyte. As such a method, for example, there is a method of supplementing a virus in an aerosol with a filter or the like and using it as a sample.
Further, in the method for measuring viral nucleic acid of the present invention, viral nucleic acid derived from a virus contained in other than aerosol may be detected. For example, a liquid containing a virus may be used as a sample.
Further, the virus may be transferred from the solid surface onto the solid electrolyte (the solid electrolyte is brought into contact with the solid surface) to prepare a sample.
　ステップＳ１２において、ウィルスを含む検体を組合せ電極と接触させる。組合せ電極は、積層電極と対電極とを含む。積層電極は、ホウ素ドープダイヤモンド（Ｂｏｒｏｎ－ｄｏｐｅｄ　Ｄｉａｍｏｎｄ；ＢＤＤ電極）とＢＤＤ電極上に配置された銅被覆層とを有している。このため、本ステップにおいて、銅被覆層と検体とが接触することになる。
　検体と組合せ電極とを接触させるには、その表面上に検体が準備された固体電解質を組合せ電極に押し付ければよい。 In step S12, the sample containing the virus is brought into contact with the combination electrode. The combination electrode includes a laminated electrode and a counter electrode. The laminated electrode has a boron-doped Diamond (BDD electrode) and a copper coating layer arranged on the BDD electrode. Therefore, in this step, the copper coating layer and the sample come into contact with each other.
In order to bring the sample into contact with the combination electrode, the solid electrolyte in which the sample is prepared may be pressed against the combination electrode on the surface thereof.
　図２Ａは、実施例１における検体の採取、及び、ウィルス核酸の測定に用いるセンサの分解斜視図であり、図２Ｂは同センサの斜視図である。このセンサを使ってステップＳ１１とステップＳ１２を実施する方法を説明する。 FIG. 2A is an exploded perspective view of a sensor used for collecting a sample and measuring viral nucleic acid in Example 1, and FIG. 2B is a perspective view of the sensor. A method of performing steps S11 and S12 using this sensor will be described.
　まず、図２Ａに示されるように、センサ２０は、大別して３つの部品から構成されている。３つの部品は、電極付き基板２０１、スペーサー２６、及び、固体電解質付きカバー２０２である。 First, as shown in FIG. 2A, the sensor 20 is roughly classified into three parts. The three components are a substrate 201 with electrodes, a spacer 26, and a cover 202 with a solid electrolyte.
　ステップＳ１１における検体の準備は、固体電解質付きカバー２０２を用いて行う。固体電解質付きカバー２０２は、カバー２７と、カバー２７の一方側の表面に配置された固体電解質２５とを有しており、センサ２０から取り外せる。
　センサ２０から固体電解質付きカバー２０２を取り外し、固体電解質２５上にエアロゾルを収着させることで、検体が準備できる。
　固体電解質付きカバー２０２はカバー２７を有しているため、固体電解質２５に触らずにエアロゾル中のウィルスを捕集できるので、意図しない汚染物質が検体中に混入するのを抑制できる。 Preparation of the sample in step S11 is performed using the cover 202 with a solid electrolyte. The cover 202 with a solid electrolyte has a cover 27 and a solid electrolyte 25 arranged on the surface of one side of the cover 27, and can be removed from the sensor 20.
The sample can be prepared by removing the cover 202 with the solid electrolyte from the sensor 20 and accommodating the aerosol on the solid electrolyte 25.
Since the cover 202 with the solid electrolyte has the cover 27, the virus in the aerosol can be collected without touching the solid electrolyte 25, so that unintended contaminants can be suppressed from being mixed into the sample.
　ステップＳ１２における、検体と組合せ電極との接触は、固体電解質２５を電極付き基板２０１の組合せ電極に押し付けることで実現できる。
　電極付き基板２０１は、基板２１と、上記基板２１上に配置された作用電極（ｗｏｒｋｉｎｇ　ｅｌｅｃｔｒｏｄｅ）である積層電極２２と、対電極２３（ｃｏｕｎｔｅｒ　ｅｌｅｃｔｒｏｄｅ）と、参照電極２４（ｒｅｆｅｒｅｎｃｅ　ｅｌｅｃｔｒｏｄｅ）とを有している。
　固体電解質２５を、スペーサー２６の切欠き部を介して積層電極２２、対電極２３、及び、参照電極２４（組合せ電極；ｃｏｍｂｉｎａｔｉｏｎ　ｅｌｅｃｔｒｏｄｅ）と接触させればよい。 The contact between the sample and the combination electrode in step S12 can be realized by pressing the solid electrolyte 25 against the combination electrode of the electrode-attached substrate 201.
The substrate 201 with an electrode has a substrate 21, a laminated electrode 22 which is a working electrode arranged on the substrate 21, a counter electrode 23, and a reference electrode 24 (reference electrode). are doing.
The solid electrolyte 25 may be brought into contact with the laminated electrode 22, the counter electrode 23, and the reference electrode 24 (combination electrode; combination electrode) via the notch of the spacer 26.
　スペーサー２６は、固体電解質２５を組合せ電極との位置合わせのためのガイドの機能を有しており、これにより、一旦取り外した固体電解質付きカバー２０２を再度取り付けるのが容易になるという利点がある。しかし、センサはスペーサー２６を含まなくてもよい。 The spacer 26 has a function of a guide for aligning the solid electrolyte 25 with the combination electrode, which has an advantage that it is easy to reattach the cover 202 with the solid electrolyte once removed. However, the sensor does not have to include the spacer 26.
　図２Ｃはセンサ２０におけるＸ－Ｙ断面図である。基板２１上には、積層電極２２、対電極２３、及び、参照電極２４が配置されている。これらの電極の間には、図中「ＳＬ」で表される封止部材（例えば、エポキシ樹脂）が配置されている。積層電極２２は、ＢＤＤ電極２８と、ＢＤＤ電極２８上に配置された銅被覆層２９とを有している。
　なお、センサ２０において、積層電極２２は、対電極２３、及び、参照電極２４よりも厚み方向に沿って高くなっているが、各電極の高さは上記に制限されない。積層電極２２は、対電極２３、及び、参照電極２４の表面に段差がない状態、及び、対電極２３、及び、参照電極２４のいずれか一方又は両方が、積層電極２２よりも高くなった状態でもよい。
　より優れた本発明の効果が得られる点では、積層電極２２を構成するＢＤＤ電極２８と、対電極２３と、参照電極２４との表面にそれぞれ略段差がないことが好ましい。 FIG. 2C is a cross-sectional view taken along the line XY of the sensor 20. A laminated electrode 22, a counter electrode 23, and a reference electrode 24 are arranged on the substrate 21. A sealing member (for example, epoxy resin) represented by "SL" in the figure is arranged between these electrodes. The laminated electrode 22 has a BDD electrode 28 and a copper coating layer 29 arranged on the BDD electrode 28.
In the sensor 20, the laminated electrode 22 is higher than the counter electrode 23 and the reference electrode 24 in the thickness direction, but the height of each electrode is not limited to the above. The laminated electrode 22 has no step on the surface of the counter electrode 23 and the reference electrode 24, and one or both of the counter electrode 23 and the reference electrode 24 are higher than the laminated electrode 22. But it may be.
From the viewpoint of obtaining a more excellent effect of the present invention, it is preferable that the surfaces of the BDD electrode 28, the counter electrode 23, and the reference electrode 24 constituting the laminated electrode 22 have substantially no step.
　ＢＤＤ電極２８の製造方法としては特に制限されず、公知の方法が使用できる。なかでも、化学気相（ＣＶＤ：Ｃｈｅｍｉｃａｌ　Ｖａｐｏｒ　Ｄｅｐｏｓｉｔｉｏｎ）法で製造することが好ましい。ＣＶＤ法の励起源としては、熱フィラメント、マイクロ波、高周波、直流グロー放電、直流アーク放電、及び、燃焼炎等が使用できる。また、これらを複数組み合わせて核生成密度を調整したり、大面積化したり、均一化したりすることもできる。
　原料は、炭素が含まれている多くの種類の化合物が使用できる。例えば、気体としてＣＨ４、Ｃ２Ｈ２、Ｃ２Ｈ４、Ｃ１０Ｈ１６、ＣＯ、及び、ＣＦ４等；液体としてＣＨ３ＯＨ、Ｃ２Ｈ５ＯＨ、及び、（ＣＨ３）２ＣＯ等；固体として黒鉛、及び、フラーレン等が挙げられる。 The method for manufacturing the BDD electrode 28 is not particularly limited, and a known method can be used. Above all, it is preferable to produce by a chemical vapor deposition (CVD) method. As the excitation source of the CVD method, a thermal filament, microwave, high frequency, DC glow discharge, DC arc discharge, combustion flame and the like can be used. Further, a plurality of these can be combined to adjust the nucleation density, increase the area, or make the nucleation uniform.
As a raw material, many kinds of compounds containing carbon can be used. For example, CH 4 , C 2 H 2 , C 2 H 4 , C 10 H 16 , CO, CF 4 , etc. as gas; CH 3 OH, C 2 H 5 OH, and (CH 3 ) 2 CO as liquid. Etc .; Examples of the solid include graphite, fullerene and the like.
　ホウ素の添加は、例えば、ホウ素を含む物質を系内に導入して炭素気相にホウ素を導入する方法等が挙げられる。このようなホウ素を含む物質としては、ジボラン、トリメチルボラン、及び、トリメトキシボラン等が挙げられるが、取り扱いがより容易である点で、トリメトキシボランが好ましい。
　ホウ素源としてトリメトキシボラン、これを溶解する溶媒としてアセトンを用いると、アセトンが炭素源も兼ねるため、更に取り扱いが容易となる。 Examples of the addition of boron include a method of introducing a substance containing boron into the system to introduce boron into the carbon vapor phase. Examples of such a substance containing boron include diborane, trimethylborane, and trimethoxyborane, but trimethoxyborane is preferable because it is easier to handle.
When trimethoxyborane is used as the boron source and acetone is used as the solvent for dissolving the boron, since acetone also serves as a carbon source, it is easier to handle.
　ダイヤモンドの成長速度がより速く、得られるダイヤモンド膜がより均一である点で、ＢＤＤ電極はマイクロ波によるプラズマＣＶＤ法により形成されることがより好ましい。マイクロ波によって水素プラズマを発生させ、ここに原料ガスを導入すればダイヤモンド膜が形成できる。 It is more preferable that the BDD electrode is formed by a plasma CVD method using microwaves in that the growth rate of diamond is faster and the obtained diamond film is more uniform. A diamond film can be formed by generating hydrogen plasma by microwaves and introducing a raw material gas into it.
　炭素源にホウ素を添加する場合、ホウ素の添加量としては、特に制限されないが、得られるＢＤＤ電極がより優れた導電性を有する点で、１０～１２，０００ｐｐｍが好ましく、１，０００～１０，０００ｐｐｍがより好ましい。 When boron is added to the carbon source, the amount of boron added is not particularly limited, but 10 to 12,000 ppm is preferable, and 1,000 to 10, 000 ppm is more preferable.
　基板２１上に積層電極２２、対電極２３、及び、参照電極２４を積層する方法としては特に制限されず、公知の方法が使用できる。このような方法としては、例えば、特開２００６－１０３５７号公報の００３７～００５０段落に記載の方法、及び、特開２０２０－３３１９９号公報の００３４～００５７段落に記載の方法等が挙げられる。 The method of laminating the laminated electrode 22, the counter electrode 23, and the reference electrode 24 on the substrate 21 is not particularly limited, and a known method can be used. Examples of such a method include the methods described in paragraphs 0037 to 0050 of JP-A-2006-10357, the methods described in paragraphs 0034 to 0057 of JP-A-2020-33199, and the like.
　なお、センサ２０は、積層電極２２と、対電極２３と、参照電極２４とを有しているが、実施例１のウィルス核酸の測定方法に使用可能なセンサは上記に制限されず、積層電極と、対電極とを有していればよい。
　また、センサ２０における積層電極２２、対電極２３、及び、参照電極２４の配置は、交換されてもよい。 The sensor 20 has a laminated electrode 22, a counter electrode 23, and a reference electrode 24, but the sensor that can be used in the method for measuring viral nucleic acid of Example 1 is not limited to the above, and the laminated electrode is not limited to the above. And a counter electrode.
Further, the arrangement of the laminated electrode 22, the counter electrode 23, and the reference electrode 24 in the sensor 20 may be replaced.
　基板２１は導電性基板でも絶縁性基板でもよいが、絶縁性基板が好ましい。基板としては、例えば、タングステン、ケイ素、酸化ケイ素、窒化ケイ素、炭化ケイ素、及び、ニオブ等が挙げられる。また、基板２１は石英ガラス等であってもよい。基板の厚み及び大きさは、取り扱い性等の観点から適宜選択されればよく、典型的には、１μｍ～５ｍｍが好ましい。 The substrate 21 may be a conductive substrate or an insulating substrate, but an insulating substrate is preferable. Examples of the substrate include tungsten, silicon, silicon oxide, silicon nitride, silicon carbide, niobium and the like. Further, the substrate 21 may be quartz glass or the like. The thickness and size of the substrate may be appropriately selected from the viewpoint of handleability and the like, and is typically 1 μm to 5 mm.
　ＢＤＤ電極２８の厚みは、成膜時間により調整できる。ＢＤＤ電極２８の厚みとしては、典型的には、０．１μｍ以上が好ましく、１μｍ以上がより好ましく、５μｍ以上が更に好ましく、１０μｍ以上が特に好ましい。なお、上限としては特に制限されないが、一般に、１ｍｍ以下が好ましい。
　上記の厚みの範囲は、対電極２３、及び、参照電極２４についても同様である。 The thickness of the BDD electrode 28 can be adjusted by the film formation time. The thickness of the BDD electrode 28 is typically 0.1 μm or more, more preferably 1 μm or more, further preferably 5 μm or more, and particularly preferably 10 μm or more. The upper limit is not particularly limited, but is generally preferably 1 mm or less.
The above thickness range is the same for the counter electrode 23 and the reference electrode 24.
　対電極２３の材料としては特に制限されず、対電極用として公知の材料が使用できる。このような材料としては、例えば、白金、炭素材料、ステンレス、及び、ＳｎＯ２等が挙げられる。参照電極２４は、例えば、カロメル電極、及び、銀／塩化銀電極等であってよい。 The material of the counter electrode 23 is not particularly limited, and a known material for the counter electrode can be used. Examples of such a material include platinum, carbon material, stainless steel, SnO 2 , and the like. The reference electrode 24 may be, for example, a calomel electrode, a silver / silver chloride electrode, or the like.
　図１のフローチャートに戻り、ステップ１３以降の手順を説明する。
　ステップＳ１３において、積層電極２２に対して定電位が印加され、これにより銅被覆層２９が固体電解質２５側に溶出し（電気分解され）、銅イオンが発生する。このとき印加される電位は、特に制限されないが、一般に＋０．４～０．５Ｖが好ましい。
　銅イオンは、直接的関与、及び／又は、活性酸素種（Ｒｅａｃｔｉｖｅ　Ｏｘｙｇｅｎ　Ｓｐｅｃｉｅｓ：ＲＯＳ）の発生を触媒する等の間接的関与により、ウィルスのエンベロープ、及び、カプシドの少なくとも一部を破壊する。そのため、ウィルスの内部からウィルス核酸が放出される。 Returning to the flowchart of FIG. 1, the procedure after step 13 will be described.
In step S13, a constant potential is applied to the laminated electrode 22, whereby the copper coating layer 29 is eluted (electrolyzed) to the solid electrolyte 25 side, and copper ions are generated. The potential applied at this time is not particularly limited, but is generally preferably +0.4 to 0.5V.
Copper ions destroy the viral envelope and at least part of the capsid by direct involvement and / or indirect involvement such as catalyzing the generation of reactive oxygen species (ROS). Therefore, viral nucleic acid is released from the inside of the virus.
　図３は、実施例１のウィルス核酸の測定方法の原理を表す模式図である。検出系３０は、ＢＤＤ電極２８と、ＢＤＤ電極２８上に形成された銅被覆層２９と、固体電解質２５からなり、固体電解質２５の表面には、検出対象であるコロナウィルス３１が配置されている。 FIG. 3 is a schematic diagram showing the principle of the method for measuring viral nucleic acid of Example 1. The detection system 30 is composed of a BDD electrode 28, a copper coating layer 29 formed on the BDD electrode 28, and a solid electrolyte 25, and a coronavirus 31 to be detected is arranged on the surface of the solid electrolyte 25. ..
　なお、図３においては、コロナウィルス３１の構造が模式的に示されており、その直径（図３中、Ｌ１に対応する長さ）と、ＢＤＤ電極２８、銅被覆層２９（合わせて、積層電極２２）及び、固体電解質２５の厚みとの関係は、実際に即していない。各部の大きさ、及び、厚み等はすでに説明したとおりであるため、ここでは説明を省略する。なお、上記は、後述する図４のＬ２についても同様である。 In addition, in FIG. 3, the structure of the coronavirus 31 is schematically shown, and the diameter thereof (the length corresponding to L1 in FIG. 3), the BDD electrode 28, and the copper coating layer 29 (together, laminated). The relationship between the electrode 22) and the thickness of the solid electrolyte 25 does not actually correspond. Since the size, thickness, etc. of each part have already been described, the description thereof will be omitted here. The above is the same for L2 in FIG. 4, which will be described later.
　コロナウィルス３１の表面は、脂質膜３２で覆われており、その中に、Ｎｕｃｌｅｏｃａｐｓｉｄ（Ｎ）蛋白に巻き付いたプラス鎖の一本鎖ＲＮＡゲノム３３が配置されている。コロナウィルス３１の表面には、Ｓｐｉｋｅ（Ｓ）蛋白、Ｅｎｖｅｌｏｐｅ（Ｅ）蛋白、Ｍｅｍｂｒａｎｅ（Ｍ）蛋白が配置され、形状が王冠に類似している。
　一般に、ウィルス核酸は膜（エンベロープ）、及び／又は、殻（カプシド）に包まれているため、ウィルス核酸を直接的に電気化学測定することは難しい。 The surface of the coronavirus 31 is covered with a lipid membrane 32, in which a plus-stranded single-stranded RNA genome 33 wrapped around a nucleocapsid (N) protein is arranged. A Spike (S) protein, an envelope (E) protein, and a Membrane (M) protein are arranged on the surface of the coronavirus 31, and the shape resembles a crown.
In general, since viral nucleic acid is wrapped in a membrane (envelope) and / or a shell (capsid), it is difficult to directly electrochemically measure viral nucleic acid.
　このとき、積層電極２２に対して定電位が印加されると、銅被覆層２９が固体電解質２５側に溶出し、銅イオンが発生する。
　銅イオンは、直接的関与、及び／又は、間接的関与によって、ウィルスのエンベロープ、及び、カプシドの少なくとも一部を破壊するため、これによってウィルスの内部からウィルス核酸（コロナウィルス３１については、一本鎖ＲＮＡゲノム３３）が放出される。 At this time, when a constant potential is applied to the laminated electrode 22, the copper coating layer 29 elutes to the solid electrolyte 25 side, and copper ions are generated.
Copper ions destroy the envelope of the virus and at least part of the capsid by direct and / or indirect involvement, thereby causing viral nucleic acid (one for coronavirus 31) from within the virus. The strand RNA genome 33) is released.
　次に、ステップＳ１４において、積層電極２２に定電位を印加した際の応答として得られた電流（以下「応答電流」ともいう。）を予め定めた閾値と比較する。この応答電流は銅被覆層２９の溶出経過を反映する。実施例１のウィルス核酸の測定方法は、検体に対してＢＤＤ電極２８の少なくとも一部（好ましくは全部）を晒す必要があるため、この応答電流をモニターして銅被覆層２９が所望の程度溶出したこと（分解されたこと）を確認することが好ましい。
　なお、応答電流のモニター以外の方法、例えば、定電位の印加時間によって、銅被覆層２９の溶出量（分解量）を管理してもよい。その場合は、ステップＳ１４及びステップＳ１５を省略してもよい。 Next, in step S14, the current obtained as a response when a constant potential is applied to the laminated electrode 22 (hereinafter, also referred to as “response current”) is compared with a predetermined threshold value. This response current reflects the elution process of the copper coating layer 29. Since the method for measuring viral nucleic acid of Example 1 requires exposing at least a part (preferably all) of the BDD electrode 28 to the sample, the response current is monitored and the copper coating layer 29 elutes to a desired degree. It is preferable to confirm that it has been done (disassembled).
The elution amount (decomposition amount) of the copper coating layer 29 may be controlled by a method other than monitoring the response current, for example, by applying a constant potential. In that case, steps S14 and S15 may be omitted.
　閾値は、例えば、銅被覆層２９がすべて溶出した際の応答電流に所定の裕度を考慮した値とすればよい。積層電極２２に電位を印加している際に、その応答電流が上記閾値以下（閾値を超えない状態）となれば、銅被覆層２９が所望の程度（例えば、すべて）溶出したと判断できる（ステップＳ１５：Ｆａｌｓｅ）。一方で、応答電流が閾値を超える場合（ステップＳ１５：Ｔｒｕｅ）、銅被覆層２９が所望の程度より多く残存しているため、定電位の印加、及び、応答電流のモニターを継続すればよい（ステップＳ１３～Ｓ１５）。 The threshold value may be, for example, a value in which a predetermined margin is taken into consideration for the response current when all the copper coating layer 29 is eluted. When the response current is equal to or less than the above threshold value (a state in which the threshold value is not exceeded) when the potential is applied to the laminated electrode 22, it can be determined that the copper coating layer 29 has eluted to a desired degree (for example, all) (for example, all). Step S15: False). On the other hand, when the response current exceeds the threshold value (step S15: True), since more copper coating layer 29 remains than desired, it is sufficient to continue applying the constant potential and monitoring the response current (step S15: True). Steps S13 to S15).
　図４は、積層電極に定電位を印加し、銅被覆層が溶出した後の検出系を表す模式図である。銅被覆層２９が全て溶出した（分解された）ため、検出系４０は、ＢＤＤ電極２８と、固体電解質２５とからなり、固体電解質２５の表面のコロナウィルス４１は、エンベロープ、及び、カプシドが銅イオンの直接的関与、及び、間接的関与により破壊され、その内部からウィルス核酸が放出された状態となっている。 FIG. 4 is a schematic diagram showing a detection system after a constant potential is applied to the laminated electrodes and the copper coating layer is eluted. Since the copper coating layer 29 was completely eluted (decomposed), the detection system 40 was composed of a BDD electrode 28 and a solid electrolyte 25, and the coronavirus 41 on the surface of the solid electrolyte 25 had an envelope and a capsid of copper. It is destroyed by the direct and indirect involvement of ions, and the viral nucleic acid is released from the inside.
　検出系４０においては、コロナウィルス４１に対してＢＤＤ電極２８が晒されている（暴露している）ため、このＢＤＤ電極２８の電位を掃引することで、ウィルス核酸由来の電気化学的応答を取得することができる（ステップＳ１６）。
　このウィルス核酸由来の電気化学的応答は、ウィルス核酸の酸化電流であることが好ましく、ピーク形状の応答であること（電位対電流の測定結果に１つ以上の極大値が存在すること）がより好ましい。なお、本明細書では、このピークの最大電流値を「ピーク電流」といい、この「ピーク電流」を与える電位を「ピーク電位」といい、このピークの面積を「電流ピーク面積」という。 In the detection system 40, since the BDD electrode 28 is exposed (exposed) to the coronavirus 41, the electrochemical response derived from the viral nucleic acid is obtained by sweeping the potential of the BDD electrode 28. Can be done (step S16).
The electrochemical response derived from this viral nucleic acid is preferably the oxidation current of the viral nucleic acid, and more preferably a peak-shaped response (there is one or more maximum values in the potential vs. current measurement result). preferable. In the present specification, the maximum current value of this peak is referred to as "peak current", the potential giving this "peak current" is referred to as "peak potential", and the area of this peak is referred to as "current peak area".
　ＢＤＤ電極の電位を掃引する方法が、リニアスイープボルタンメトリー法、微分パルスボルタンメトリー法、及び、サイクリックボルタンメトリー法からなる群より選択される少なくとも1種の方法であると、上記のようなピーク形状の応答が得られやすい。 When the method of sweeping the potential of the BDD electrode is at least one method selected from the group consisting of the linear sweep voltammetry method, the differential pulse voltammetry method, and the cyclic voltammetry method, the response of the peak shape as described above is performed. Is easy to obtain.
　ウィルス核酸の酸化電流のピーク電位は＋１．０～１．５Ｖ（ｖｓ　Ａｇ／ＡｇＣｌ）の範囲に検出されることが多い。実施例１のウィルス核酸測定方法においては、銅被覆層２９を溶出させて、ＢＤＤ電極２８を検体に対して晒したのちに、ＢＤＤ電極２８の電位を掃引するため、このウィルス核酸の酸化電流の測定が可能になる。 The peak potential of the oxidation current of viral nucleic acid is often detected in the range of +1.0 to 1.5V (vs Ag / AgCl). In the method for measuring viral nucleic acid of Example 1, the copper coating layer 29 is eluted, the BDD electrode 28 is exposed to the sample, and then the potential of the BDD electrode 28 is swept. Measurement becomes possible.
　一般的な電極部材、例えばグラッシーカーボン、金、及び、白金等を用いると、＋１．３Ｖ（ｖｓ　Ａｇ／ＡｇＣｌ）程度で水分子の酸化による酸素の発生に伴い、電流値の増大が検出されてしまい、ウィルス核酸の酸化電流を正確に検出することは難しい。一方、ＢＤＤ電極２８は、表面がｓｐ３炭素からなり、分子が吸着できるサイトが少ないため、上記一般的な電極部材と比較して電位窓が広く、ウィルス核酸の酸化電流を検出することができる。 When general electrode members such as glassy carbon, gold, and platinum are used, an increase in the current value is detected with the generation of oxygen due to the oxidation of water molecules at about +1.3 V (vs Ag / AgCl). Therefore, it is difficult to accurately detect the oxidation current of viral nucleic acid. On the other hand, since the surface of the BDD electrode 28 is made of sp3 carbon and there are few sites on which molecules can be adsorbed, the potential window is wider than that of the above general electrode member, and the oxidation current of viral nucleic acid can be detected. ..
　ウィルス核酸の酸化電流は、ウィルス核酸を構成する核酸塩基の種類に起因して複数のピークが検出されることがある。その場合、各ピーク電流、又は、各ピーク面積を合計して測定値としてもよいし、単一のピーク電流、又は、ピーク面積を測定値としてもよい。
　総量として評価する場合には、各ピーク電流、又は、各ピーク面積を合計した値を測定値とするのが好ましい。
　実施例１の方法によれば、エアロゾルから捕集した検体中のウィルス核酸を迅速に測定できる。 Multiple peaks may be detected in the oxidation current of viral nucleic acid due to the type of nucleobase constituting the viral nucleic acid. In that case, each peak current or each peak area may be totaled as a measured value, or a single peak current or a peak area may be used as a measured value.
When evaluating as a total amount, it is preferable to use each peak current or the total value of each peak area as the measured value.
According to the method of Example 1, the viral nucleic acid in the sample collected from the aerosol can be rapidly measured.
　図５は本発明の実施例２によるウィルス核酸測定装置のハードウェア構成図である。 FIG. 5 is a hardware configuration diagram of the virus nucleic acid measuring device according to the second embodiment of the present invention.
　ウィルス核酸測定装置５０は、プロセッサ５１、記憶デバイス５２、表示デバイス５３、入力デバイス５４、電気化学測定デバイス５５、及び、接続具５６を有している。プロセッサ５１、記憶デバイス５２、表示デバイス５３、入力デバイス５４、及び、電気化学測定デバイス５５はバス（図中「ＢＵＳ」と表記している）を介して相互にデータを交換できる。 The virus nucleic acid measuring device 50 includes a processor 51, a storage device 52, a display device 53, an input device 54, an electrochemical measuring device 55, and a connector 56. The processor 51, the storage device 52, the display device 53, the input device 54, and the electrochemical measurement device 55 can exchange data with each other via a bus (denoted as “BUS” in the figure).
　ウィルス核酸測定装置５０には、接続具５６を介してセンサ２０が接続されている。構造は後述するが、センサ２０はウィルス核酸測定装置５０に対して取り外し可能であり、例えば、検体毎にセンサ２０を交換できる。
　なお、センサ２０は図２Ａ、図２Ｂ、及び、図２Ｃで説明したとおりであるので、ここでは説明を省略する。 A sensor 20 is connected to the virus nucleic acid measuring device 50 via a connector 56. Although the structure will be described later, the sensor 20 is removable with respect to the virus nucleic acid measuring device 50, and for example, the sensor 20 can be replaced for each sample.
Since the sensor 20 is as described with reference to FIGS. 2A, 2B, and 2C, the description thereof will be omitted here.
　プロセッサ５１は、ウィルス核酸測定装置５０の各部を制御して、ウィルス核酸測定装置の機能を実現する。
　プロセッサ５１は、例えば、マイクロプロセッサ、プロセッサコア、マルチプロセッサ、ＡＳＩＣ（ａｐｐｌｉｃａｔｉｏｎ－ｓｐｅｃｉｆｉｃ　ｉｎｔｅｇｒａｔｅｄ　ｃｉｒｃｕｉｔ）、ＦＰＧＡ（ｆｉｅｌｄ　ｐｒｏｇｒａｍｍａｂｌｅ　ｇａｔｅ　ａｒｒａｙ）、及び、ＧＰＧＰＵ（Ｇｅｎｅｒａｌ－ｐｕｒｐｏｓｅ　ｃｏｍｐｕｔｉｎｇ　ｏｎ　ｇｒａｐｈｉｃｓ　ｐｒｏｃｅｓｓｉｎｇ　ｕｎｉｔｓ）等でよい。 The processor 51 controls each part of the virus nucleic acid measuring device 50 to realize the function of the virus nucleic acid measuring device.
The processor 51 may be, for example, a microprocessor, a processor core, a multi-processor, an ASIC (application-specific integrated circuit), an FPGA (field programgable gate array), a GPGPU (general-purpose processing), etc.
　記憶デバイス５２は、プログラム、及び、データを一時的に、及び／又は、非一時的に記憶する機能を有し、プロセッサ５１の作業エリアを提供する。
　記憶デバイス５２は、例えば、ＲＯＭ（Ｒｅａｄ　Ｏｎｌｙ　Ｍｅｍｏｒｙ）、ＲＡＭ（Ｒａｎｄｏｍ　Ａｃｃｅｓｓ　Ｍｅｍｏｒｙ）、ＨＤＤ（Ｈａｒｄ　Ｄｉｓｋ　Ｄｒｉｖｅ）、フラッシュメモリ、及び、ＳＳＤ（Ｓｏｌｉｄ　Ｓｔａｔｅ　Ｄｒｉｖｅ）等でよい。 The storage device 52 has a function of temporarily and / or non-temporarily storing programs and data, and provides a work area of the processor 51.
The storage device 52 may be, for example, a ROM (Read Only Memory), a RAM (Random Access Memory), an HDD (Hard Disk Drive), a flash memory, an SSD (Solid State Drive), or the like.
　表示デバイス５３は、測定結果、積層電極（又はＢＢＤ電極）への電圧印加状況、検体名、及び、操作手順等を表示できる。表示デバイス５３は、液晶ディスプレイ、及び、有機ＥＬ（Ｅｌｅｃｔｒｏ　Ｌｕｍｉｎｅｓｃｅｎｃｅ）ディスプレイ等でよい。
　また、表示デバイス５３は、入力デバイス５４と一体として構成されていてもよい。この場合、表示デバイス５３がタッチパネルディスプレイであって、ＧＵＩ（Ｇｒａｐｈｉｃａｌ　Ｕｓｅｒ　Ｉｎｔｅｒｆａｃｅ）を提供する形態が挙げられる。 The display device 53 can display the measurement result, the voltage application status to the laminated electrode (or BBD electrode), the sample name, the operation procedure, and the like. The display device 53 may be a liquid crystal display, an organic EL (Electroluminescence) display, or the like.
Further, the display device 53 may be configured integrally with the input device 54. In this case, the display device 53 is a touch panel display, and a form of providing a GUI (Graphical User Interface) can be mentioned.
　入力デバイス５４は、組合せ電極への電圧印加条件、測定条件、及び、検体名等を入力できる。入力デバイス５４は、キーボード、マウス、スキャナ、及び、タッチパネル等でよい。 The input device 54 can input voltage application conditions, measurement conditions, sample names, etc. to the combination electrodes. The input device 54 may be a keyboard, a mouse, a scanner, a touch panel, or the like.
　電気化学測定デバイス５５により、接続具５６を介して接続されたセンサ２０の積層電極２２に定電位を印加して、応答電流を測定できる。また、電気化学測定デバイス５５により、銅被覆層２９が溶出した後のＢＤＤ電極２８に掃引電位を印加して、ウィルス核酸に由来する電気化学的応答を測定できる。電気化学測定デバイス５５は、例えば、ポテンシオスタット等であってよい。 The electrochemical measurement device 55 can measure the response current by applying a constant potential to the laminated electrode 22 of the sensor 20 connected via the connector 56. In addition, the electrochemical measurement device 55 can apply a sweep potential to the BDD electrode 28 after the copper coating layer 29 has been eluted to measure the electrochemical response derived from the viral nucleic acid. The electrochemical measurement device 55 may be, for example, a potentiostat or the like.
　接続具５６は、電気化学測定デバイス５５と、センサ２０の組合せ電極（積層電極２２、対電極２３、及び、参照電極２４）とを電気的に接続する。接続具５６としては、例えば、端子と導線の組合せ等であってよい。 The connector 56 electrically connects the electrochemical measurement device 55 and the combination electrode (stacked electrode 22, counter electrode 23, and reference electrode 24) of the sensor 20. The connector 56 may be, for example, a combination of a terminal and a conducting wire.
　図６Ａは、実施例２のウィルス核酸測定装置の斜視図である。ウィルス核酸測定装置６０は、本体６１と、本体６１の中央部に配置されたタッチパネルディスプレイ６２とを有し、本体６１内には、すでに説明した各ハードウェア（プロセッサ５１、及び、電気化学測定デバイス５５等）が搭載された回路基板が配置されている。 FIG. 6A is a perspective view of the virus nucleic acid measuring device of Example 2. The virus nucleic acid measuring device 60 has a main body 61 and a touch panel display 62 arranged in the central portion of the main body 61, and each hardware (processor 51 and an electrochemical measuring device) already described is contained in the main body 61. A circuit board on which 55 etc.) is mounted is arranged.
　ウィルス核酸測定装置６０は、センサ２０を挿入するための挿入口６３を有している。図６Ｂは、センサ２０を挿入した状態のウィルス核酸測定装置６０の斜視図である。 The virus nucleic acid measuring device 60 has an insertion port 63 for inserting the sensor 20. FIG. 6B is a perspective view of the virus nucleic acid measuring device 60 with the sensor 20 inserted.
　図６Ｃは、図６ＢのＶ－Ｗ断面図である。挿入口６３に基板２１を上側にして挿入されたセンサ２０は、充填部材６９で支持された本体６１と、挿入口６３の内部に配置されたバネ部材６４によって挟持される。
　基板２１の表面に配置された組合せ電極（Ｖ－Ｗ断面では、参照電極２４）は、基板２１の反対側表面からバネ部材６４に押し付けられて、端子６５と密着する。端子６５は導線６６を介して、回路基板６８の端子６７と接続されている。端子６７は、回路基板６８上に配置された電気化学測定デバイス５５と接続されている。これによって、電気化学測定デバイス５５が、組合せ電極と接続される。 FIG. 6C is a sectional view taken along line VW of FIG. 6B. The sensor 20 inserted into the insertion slot 63 with the substrate 21 facing upward is sandwiched between the main body 61 supported by the filling member 69 and the spring member 64 arranged inside the insertion slot 63.
The combination electrode (reference electrode 24 in the VW cross section) arranged on the surface of the substrate 21 is pressed against the spring member 64 from the opposite surface of the substrate 21 and comes into close contact with the terminal 65. The terminal 65 is connected to the terminal 67 of the circuit board 68 via the lead wire 66. The terminal 67 is connected to the electrochemical measurement device 55 arranged on the circuit board 68. Thereby, the electrochemical measurement device 55 is connected to the combination electrode.
　図７は、実施例２のウィルス核酸測定装置の機能ブロック図である。ウィルス核酸測定装置７０は、制御部７１と、記憶部７２と、表示部７３と、入力部７４と、比較部７５と、定電位印加部７６と、掃引電位印加部７７と、センサ部７８とを有する。 FIG. 7 is a functional block diagram of the virus nucleic acid measuring device of Example 2. The virus nucleic acid measuring device 70 includes a control unit 71, a storage unit 72, a display unit 73, an input unit 74, a comparison unit 75, a constant potential application unit 76, a sweep potential application unit 77, and a sensor unit 78. Has.
　制御部７１は、プロセッサ５１を含んで構成される。制御部７１は、記憶部７２、表示部７３、入力部７４、比較部７５、定電位印加部７６、及び、掃引電位印加部７７のそれぞれを制御して、ウィルス核酸測定装置７０の機能を実現する。 The control unit 71 includes a processor 51. The control unit 71 controls each of the storage unit 72, the display unit 73, the input unit 74, the comparison unit 75, the constant potential application unit 76, and the sweep potential application unit 77 to realize the function of the virus nucleic acid measuring device 70. do.
　記憶部７２は、記憶デバイス５２を含んで構成される。記憶部７２により、プログラム、及び、閾値等が記憶され、測定データ等が記憶される。 The storage unit 72 includes a storage device 52. The storage unit 72 stores the program, the threshold value, and the like, and stores the measurement data and the like.
　表示部７３は、表示デバイス５３を含んで構成される。また、入力部７４は入力デバイス５４を含んで構成される。制御部７１が、これらを制御することで、ウィルス核酸測定装置７０の使用者からの入力を受け付けてそれを記憶部７２に記憶させたり、記憶部７２に記憶された定電位印加部７６、及び、掃引電位印加部７７によって得られた測定データ（応答電流、及び、電気化学的応答）を装置の使用者に対して表示したりできる。 The display unit 73 includes a display device 53. Further, the input unit 74 includes an input device 54. By controlling these, the control unit 71 receives the input from the user of the virus nucleic acid measuring device 70 and stores it in the storage unit 72, or the constant potential application unit 76 and the constant potential application unit 76 stored in the storage unit 72. , The measurement data (response current and electrochemical response) obtained by the sweep potential application unit 77 can be displayed to the user of the apparatus.
　比較部７５は、記憶部７２に記憶されたプログラムを制御部７１が実行することによって実現される機能である。比較部７５は、積層電極２２への定電位の印加による応答電流と予め定められた閾値とを比較する。
　なお、閾値は記憶部７２に記憶されている。応答電流は、後述する定電位印加部７６によって得られる。 The comparison unit 75 is a function realized by the control unit 71 executing the program stored in the storage unit 72. The comparison unit 75 compares the response current due to the application of a constant potential to the laminated electrode 22 with a predetermined threshold value.
The threshold value is stored in the storage unit 72. The response current is obtained by the constant potential application unit 76 described later.
　定電位印加部７６は、電気化学測定デバイス５５を含んで構成され、記憶部７２に記憶されたプログラムを制御部７１が実行し、電気化学測定デバイス５５が制御されて実現される機能である。
　定電位印加部７６は、センサ部７８に接続されたセンサの積層電極２２に定電圧を印加し、その応答電流を測定する。 The constant potential application unit 76 is configured to include the electrochemical measurement device 55, and is a function realized by the control unit 71 executing a program stored in the storage unit 72 and controlling the electrochemical measurement device 55.
The constant potential application unit 76 applies a constant voltage to the laminated electrode 22 of the sensor connected to the sensor unit 78, and measures the response current thereof.
　掃引電位印加部７７は、電気化学測定デバイス５５を含んで構成され、記憶部７２に記憶されたプログラムを制御部７１が実行し、電気化学測定デバイス５５が制御されて実現される機能である。
　掃引電位印加部７７は、センサ部７８に接続され、検体に晒されたＢＤＤ電極２８に掃引電圧を印加し、ウィルス核酸に由来する電気化学的応答を測定する。 The sweep potential application unit 77 is configured to include the electrochemical measurement device 55, and is a function realized by the control unit 71 executing a program stored in the storage unit 72 and controlling the electrochemical measurement device 55.
The sweep potential application unit 77 is connected to the sensor unit 78, applies a sweep voltage to the BDD electrode 28 exposed to the sample, and measures the electrochemical response derived from the viral nucleic acid.
　センサ部７８は接続具５６を含んで構成され、センサ２０の各電極と、電気化学測定デバイス５５とを電気的に接続する機能である。 The sensor unit 78 is configured to include a connector 56, and has a function of electrically connecting each electrode of the sensor 20 and the electrochemical measurement device 55.
　図８は、実施例２によるウィルス核酸測定装置の制御部７１の動作を示すフローチャートである。 FIG. 8 is a flowchart showing the operation of the control unit 71 of the virus nucleic acid measuring device according to the second embodiment.
　ステップＳ８１において、制御部７１は定電位印加部７６を制御して、ウィルスを含む検体と接触させた積層電極２２に定電圧を印加させ、応答電流を測定させる。なお、積層電極２２は、センサ２０に含まれており、センサ２０は、センサ部７８を介して接続された定電位印加部７６により制御される。 In step S81, the control unit 71 controls the constant potential application unit 76 to apply a constant voltage to the laminated electrode 22 in contact with the sample containing the virus and measure the response current. The laminated electrode 22 is included in the sensor 20, and the sensor 20 is controlled by the constant potential application unit 76 connected via the sensor unit 78.
　ステップＳ８２において、制御部７１は、比較部７５を制御して、積層電極２２から得られる応答電流と、記憶部７２に予め記憶されている閾値とを比較する。
　この閾値は、銅被覆層２９の好ましい溶出状態に対応するものとして予め定められ、記憶されている。実施例１のウィルス核酸の測定方法で説明したが、銅イオンによりウィルスのカプシド、及び、エンベロープを破壊した後は、ウィルス核酸の酸化電流をＢＤＤ電極で測定する必要がある。そのため、銅被覆層２９が溶出し、検体に対してＢＤＤ電極が十分に露出していることが好ましく、銅被覆層２９がすべて溶出していることがより好ましい。 In step S82, the control unit 71 controls the comparison unit 75 to compare the response current obtained from the laminated electrode 22 with the threshold value stored in advance in the storage unit 72.
This threshold is predetermined and stored as corresponding to the preferred elution state of the copper coating layer 29. Although described in the method for measuring viral nucleic acid in Example 1, it is necessary to measure the oxidation current of viral nucleic acid with a BDD electrode after the virus capsid and envelope are destroyed by copper ions. Therefore, it is preferable that the copper coating layer 29 is eluted and the BDD electrode is sufficiently exposed to the sample, and it is more preferable that all the copper coating layer 29 is eluted.
　このような観点で定められた閾値について、応答電流がそれを超える場合（ステップＳ８３：Ｔｒｕｅ）、ＢＤＤ電極２８上に配置された銅被覆層２９は所望の程度溶出しきれていないことになる。 When the response current exceeds the threshold value determined from such a viewpoint (step S83: True), the copper coating layer 29 arranged on the BDD electrode 28 is not completely eluted to a desired degree.
　この場合、ステップＳ８４として、制御部７１は定電位印加部７６を制御して、積層電極２２に定電位の印加を継続させる。
　その後、再度、比較部７５によって閾値の判定（ステップＳ８２～ステップＳ８３）が実行され、応答電流が閾値以下となるまで繰り返される。 In this case, as step S84, the control unit 71 controls the constant potential application unit 76 to continue applying the constant potential to the laminated electrode 22.
After that, the threshold value determination (steps S82 to S83) is executed again by the comparison unit 75, and the process is repeated until the response current becomes equal to or less than the threshold value.
　一方、比較部７５による応答電流と閾値との比較の結果、応答電流が閾値以下であった場合（ステップＳ８３：Ｆａｌｓｅ）、制御部７１は掃引電位印加部７７を制御して、検体に晒されたＢＤＤ電極２８に掃引電圧を印加させ、得られる電気化学的応答を測定させる（ステップＳ８５）。
　この電気化学的応答は、典型的には、積層電極２２への定電位の印加によって生じた銅イオンの働きにより、ウィルスのカプシド、及び／又は、エンベロープの少なくとも一部が消滅し、放出されたウィルス核酸の酸化電流である。 On the other hand, when the response current is equal to or less than the threshold value as a result of comparison between the response current and the threshold value by the comparison unit 75 (step S83: False), the control unit 71 controls the sweep potential application unit 77 and is exposed to the sample. A sweep voltage is applied to the BDD electrode 28, and the obtained electrochemical response is measured (step S85).
This electrochemical response was typically released by the action of copper ions generated by the application of a constant potential to the laminated electrode 22 to eliminate and / or at least part of the viral capsid and / or envelope. It is the oxidation current of viral nucleic acid.
　ウィルス核酸の酸化電流は、典型的には、電位対電流の曲線において、ピークを有する曲線として得られることが好ましい。このピーク電流、及び、電流ピーク面積は検体中のウィルス核酸の量に比例する。ピーク電流、及び、電流ピーク面積は、掃引電位印加部７７によって記憶部７２に格納され、入力部７４に表示される。 It is preferable that the oxidation current of the viral nucleic acid is typically obtained as a curve having a peak in the curve of potential vs. current. This peak current and the current peak area are proportional to the amount of viral nucleic acid in the sample. The peak current and the current peak area are stored in the storage unit 72 by the sweep potential application unit 77 and displayed in the input unit 74.
　実施例２のウィルス核酸の測定装置によれば、エアロゾルから捕集した検体に含まれるウィルスに由来するウィルス核酸を迅速に測定できる。 According to the virus nucleic acid measuring device of Example 2, the virus nucleic acid derived from the virus contained in the sample collected from the aerosol can be rapidly measured.
　図９は、本発明の実施例３によるウィルス核酸測定装置のハードウェア構成図である。
　ウィルス核酸測定装置９０は、プロセッサ５１、記憶デバイス５２、表示デバイス５３、入力デバイス５４、電気化学測定デバイス５５、及び、電気化学測定デバイス５５に接続されたセル９１を有している。 FIG. 9 is a hardware configuration diagram of the virus nucleic acid measuring device according to the third embodiment of the present invention.
The virus nucleic acid measuring device 90 has a processor 51, a storage device 52, a display device 53, an input device 54, an electrochemical measuring device 55, and a cell 91 connected to the electrochemical measuring device 55.
　ウィルス核酸測定装置９０は、電気化学測定デバイス５５と接続されたセル９１を有し、接続具を有しないことを除いては、ハードウェア構成として実施例２のウィルス核酸測定装置と類似の点が多く、以下、相違点を中心に説明する。 The virus nucleic acid measuring device 90 has a cell 91 connected to the electrochemical measuring device 55 and has a hardware configuration similar to that of the virus nucleic acid measuring device of Example 2 except that it does not have a connector. Many, the differences will be mainly explained below.
　セル９１は、検体を収容できる容器であり、内部に積層電極２２、対電極２３、及び、参照電極２４を有している。典型的には液状の検体をセル９１に収容すると、収容された検体が、組合せ電極を構成する上記各電極と接触する。
　積層電極２２、対電極２３、及び、参照電極２４は、それぞれ電気化学測定デバイス５５と電気的に接続され、電位、及び、電流を制御、及び、測定できる。 The cell 91 is a container that can accommodate a sample, and has a laminated electrode 22, a counter electrode 23, and a reference electrode 24 inside. Typically, when a liquid sample is housed in the cell 91, the housed sample comes into contact with each of the electrodes constituting the combination electrode.
The laminated electrode 22, the counter electrode 23, and the reference electrode 24 are electrically connected to the electrochemical measuring device 55, respectively, and can control and measure the potential and the current.
　図１０は、実施例３によるウィルス核酸測定装置の模式図である。
　ウィルス核酸測定装置１００は、コンピュータ１０１と、コンピュータ１０１に接続されたポテンシオスタット１０２と、ポテンシオスタット１０２にそれぞれ接続された積層電極２２、対電極２３、及び、参照電極２４を有している。積層電極２２、対電極２３、及び、参照電極２４は、セル９１内に収容され、セル９１に同じく収容される検体と接触できる。なお、コンピュータ１０１はプロセッサ５１、記憶デバイス５２、表示デバイス５３、及び、入力デバイス５４を含み、ポテンシオスタット１０２は電気化学測定デバイス５５を含む。 FIG. 10 is a schematic diagram of the virus nucleic acid measuring device according to the third embodiment.
The virus nucleic acid measuring apparatus 100 has a computer 101, a potentiostat 102 connected to the computer 101, a laminated electrode 22, a counter electrode 23, and a reference electrode 24 connected to the potentiostat 102, respectively. .. The laminated electrode 22, the counter electrode 23, and the reference electrode 24 are housed in the cell 91 and can come into contact with the sample also housed in the cell 91. The computer 101 includes a processor 51, a storage device 52, a display device 53, and an input device 54, and the potentiostat 102 includes an electrochemical measurement device 55.
　実施例３のウィルス核酸測定装置は、実施例２のウィルス核酸測定装置において、センサが着脱式であるのに対して、組合せ電極を含むセルを内蔵しており、構成がより単純である点で優れている。なお、実施例３のウィルス核酸測定装置の機能、及び、動作は、実施例２のウィルス核酸測定装置と同様なので、説明を省略する。 The virus nucleic acid measuring device of Example 3 has a built-in cell including a combination electrode, whereas the virus nucleic acid measuring device of Example 2 has a detachable sensor, and has a simpler configuration. Are better. Since the functions and operations of the virus nucleic acid measuring device of Example 3 are the same as those of the virus nucleic acid measuring device of Example 2, the description thereof will be omitted.
　実施例３のウィルス核酸測定装置によれば、典型的には液体状の検体に含まれるウィルスに由来するウィルス核酸を迅速に測定できる。 According to the virus nucleic acid measuring apparatus of Example 3, the virus nucleic acid derived from the virus typically contained in the liquid sample can be rapidly measured.
　図１１Ａは実施例４によるセンサ１１０の分解斜視図であり、図１１Ｂは同センサの斜視図である。センサ１１０は、基板２１と、上記基板２１上に配置された積層電極２２と、対電極２３と、参照電極２４とを有している。
　また、スペーサー２６と、スペーサー２６を覆うカバー２７とを有している。
　なお、カバー２７は凹欠部１１１を有している。 11A is an exploded perspective view of the sensor 110 according to the fourth embodiment, and FIG. 11B is a perspective view of the sensor. The sensor 110 has a substrate 21, a laminated electrode 22 arranged on the substrate 21, a counter electrode 23, and a reference electrode 24.
It also has a spacer 26 and a cover 27 that covers the spacer 26.
The cover 27 has a recessed portion 111.
　典型的には液状の検体が、凹欠部１１１から導入され、基板２１の上面、カバー２７の下面、及び、スペーサー２６の切欠き部の側面で隠される空間を濡らす。これにより、検体と組合せ電極とが接触し、測定ができる。 Typically, a liquid sample is introduced from the recess 111 and wets the space hidden by the upper surface of the substrate 21, the lower surface of the cover 27, and the side surface of the notch of the spacer 26. As a result, the sample and the combination electrode come into contact with each other, and measurement can be performed.
　なお、本センサ１１０は、すでに説明したセンサ２０に代えて実施例２のウィルス核酸測定装置に挿入して使用することができる。使用方法は、センサ２０と同様であるので、説明を省略する。 The sensor 110 can be used by inserting it into the virus nucleic acid measuring device of Example 2 instead of the sensor 20 already described. Since the method of use is the same as that of the sensor 20, the description thereof will be omitted.
　実施例４のセンサは、ウィルス核酸測定装置に対して着脱可能なので、検体毎に取り替えることで、検体が互いに混入して測定結果に誤差の生ずるのを防ぐことができる。 Since the sensor of Example 4 is removable from the virus nucleic acid measuring device, it is possible to prevent the samples from being mixed with each other and causing an error in the measurement result by replacing each sample.
２０：センサ、２１：基板、２２：積層電極、２３：対電極、２４：参照電極、２５：固体電解質、２６：スペーサー、２７：カバー、２８：ＢＤＤ電極、２９：銅被覆層、３１、４１：コロナウィルス、３２：脂質膜、３３：一本鎖ＲＮＡゲノム、５０：ウィルス核酸測定装置、５１：プロセッサ、５２：記憶デバイス、５３：表示デバイス、５４：入力デバイス、５５：電気化学測定デバイス、５６：接続具、６０：ウィルス核酸測定装置、６１：本体、６２：タッチパネルディスプレイ、６３：挿入口、６４：バネ部材、６５：端子、６６：導線、６７：端子、６８：回路基板、６９：充填部材、７０：ウィルス核酸測定装置、７１：制御部、７２：記憶部、７３：表示部、７４：入力部、７５：比較部、７６：定電位印加部、７７：掃引電位印加部、７８：センサ部、９０：ウィルス核酸測定装置、９１：セル、１００：ウィルス核酸測定装置、１０１：コンピュータ、１０２：ポテンシオスタット、１１０：センサ、１１１：凹欠部、２０１：電極付き基板、２０２：固体電解質付きカバー 20: Sensor, 21: Substrate, 22: Laminated electrode, 23: Counter electrode, 24: Reference electrode, 25: Solid electrolyte, 26: Spacer, 27: Cover, 28: BDD electrode, 29: Copper coating layer, 31, 41 : Corona virus, 32: Lipid membrane, 33: Single-stranded RNA genome, 50: Viral nucleic acid measuring device, 51: Processor, 52: Storage device, 53: Display device, 54: Input device, 55: Electrochemical measuring device, 56: Connector, 60: Virus nucleic acid measuring device, 61: Main body, 62: Touch panel display, 63: Insertion port, 64: Spring member, 65: Terminal, 66: Lead wire, 67: Terminal, 68: Circuit board, 69: Filling member, 70: Virus nucleic acid measuring device, 71: Control unit, 72: Storage unit, 73: Display unit, 74: Input unit, 75: Comparison unit, 76: Constant potential application unit, 77: Sweep potential application unit, 78 : Sensor unit, 90: Viral nucleic acid measuring device, 91: Cell, 100: Viral nucleic acid measuring device, 101: Computer, 102: Potentiostat, 110: Sensor, 111: Notch, 201: Substrate with electrode, 202: Cover with solid electrolyte

Claims (17)

  1. 　ホウ素ドープダイヤモンド電極、及び、前記ホウ素ドープダイヤモンド電極上に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、組合せ電極にウィルスを含む検体を接触させることと、
    　前記積層電極に定電位を印加して、前記銅被覆層を溶出させ、銅イオンを発生させて、前記ウィルスから、ウィルス核酸を放出させるとともに、前記ホウ素ドープダイヤモンド電極を前記検体に晒すことと、
    　前記ホウ素ドープダイヤモンド電極の電位を掃引し、前記ウィルス核酸に由来する電気化学的応答を測定することと、を含む、ウィルス核酸の測定方法。     A sample containing a virus is brought into contact with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode.
    A constant potential is applied to the laminated electrode to elute the copper coating layer, generate copper ions, release viral nucleic acid from the virus, and expose the boron-doped diamond electrode to the sample.
    A method for measuring a viral nucleic acid, comprising sweeping the potential of the boron-doped diamond electrode and measuring an electrochemical response derived from the viral nucleic acid.
  2. 　前記定電位を印加した際の応答電流を予め定めた閾値と比較し、前記応答電流が前記閾値以下となるまで、前記定電位の印加を継続することを含む、請求項１に記載のウィルス核酸の測定方法。 The viral nucleic acid according to claim 1, wherein the response current when the constant potential is applied is compared with a predetermined threshold value, and the application of the constant potential is continued until the response current becomes equal to or less than the threshold value. Measurement method.
  3. 　前記ウィルス核酸に由来する電気化学的応答は、ピーク電流の大きさ、及び、電流ピーク面積からなる群より選択される少なくとも１種の値である、請求項１又は２に記載のウィルス核酸の測定方法。 The measurement of the viral nucleic acid according to claim 1 or 2, wherein the electrochemical response derived from the viral nucleic acid is at least one value selected from the group consisting of the magnitude of the peak current and the current peak area. Method.
  4. 　ピーク電位が前記定電位よりも正である、請求項３に記載のウィルス核酸の測定方法。 The method for measuring viral nucleic acid according to claim 3, wherein the peak potential is more positive than the constant potential.
  5. 　前記電位の掃引は、リニアスイープボルタンメトリー法、微分パルスボルタンメトリー法、及び、サイクリックボルタンメトリー法からなる群より選択される少なくとも１種の方法により行われる、請求項１～４のいずれか１項に記載のウィルス核酸の測定方法。 The sweeping of the potential is performed by at least one method selected from the group consisting of a linear sweep voltammetry method, a differential pulse voltammetry method, and a cyclic voltammetry method, according to any one of claims 1 to 4. Method for measuring viral nucleic acid.
  6. 　更に、エアロゾルを捕集して前記検体を準備することを含む、請求項１～５のいずれか１項に記載のウィルス核酸の測定方法。 The method for measuring viral nucleic acid according to any one of claims 1 to 5, further comprising collecting an aerosol and preparing the sample.
  7. 　固体電解質の表面にウィルスを含むエアロゾルを収着させ、前記表面上に検体を準備して、前記表面を前記組合せ電極と接触させることにより、前記検体と前記組合せ電極とを接触させることを含む、請求項１～６のいずれか１項に記載のウィルス核酸の測定方法。 The present invention comprises contacting the sample with the combination electrode by accommodating an aerosol containing a virus on the surface of the solid electrolyte, preparing a sample on the surface, and contacting the surface with the combination electrode. The method for measuring a viral nucleic acid according to any one of claims 1 to 6.
  8. 　ホウ素ドープダイヤモンド電極、及び、前記ホウ素ドープダイヤモンド電極上に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、組合せ電極にウィルスを含む検体を接触させるためのセンサ部と、
    　前記積層電極に定電位を印加し、前記銅被覆層を溶出させ、銅イオンを発生させるとともに、前記ホウ素ドープダイヤモンド電極を前記検体に対して晒す定電位印加部と、
    　前記ホウ素ドープダイヤモンド電極に掃引電位を印加し、前記ウィルスに由来する電気化学的応答を測定する掃引電位印加部と、を有する、ウィルス核酸測定装置。     A sensor unit for contacting a sample containing a virus with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode.
    A constant potential application unit that applies a constant potential to the laminated electrode to elute the copper coating layer to generate copper ions and expose the boron-doped diamond electrode to the sample.
    A virus nucleic acid measuring apparatus comprising a sweep potential applying unit for applying a sweep potential to the boron-doped diamond electrode and measuring an electrochemical response derived from the virus.
  9. 　前記定電位の印加による応答電流を予め定められた閾値と比較する比較部を有し、
    　前記比較の結果、前記応答電流が、前記閾値以下となるまで、前記定電位印加部が前記定電位の印加を継続する、請求項８に記載のウィルス核酸測定装置。     It has a comparison unit that compares the response current due to the application of the constant potential with a predetermined threshold value.
    The virus nucleic acid measuring apparatus according to claim 8, wherein the constant potential application unit continues to apply the constant potential until the response current becomes equal to or less than the threshold value as a result of the comparison.
  10. 　前記センサ部は、
    　基板と、前記基板上に配置された前記組合せ電極と、
    　前記検体を表面に付着させ、前記検体が付着した前記表面を前記組合せ電極と接触させるための固体電解質と、
    を含むセンサの前記組合せ電極を、前記定電位印加部、及び、前記掃引電位印加部と電気的に接続させるための接続具を含む、請求項８又は９に記載のウィルス核酸測定装置。     The sensor unit is
    The substrate and the combination electrode arranged on the substrate,
    A solid electrolyte for adhering the sample to the surface and contacting the surface to which the sample is attached with the combination electrode,
    The virus nucleic acid measuring apparatus according to claim 8 or 9, further comprising a connector for electrically connecting the combination electrode of the sensor including the constant potential application unit and the sweep potential application unit.
  11. 　前記センサ部は、
    　前記検体を収容するためのセルと、
    　前記検体と接触するように前記セル内に配置された前記組合せ電極と、を含む、請求項８～１０のいずれか１項に記載のウィルス核酸測定装置。     The sensor unit is
    A cell for accommodating the sample and
    The viral nucleic acid measuring apparatus according to any one of claims 8 to 10, further comprising the combination electrode arranged in the cell so as to be in contact with the sample.
  12. 　コンピュータにより、
    　ホウ素ドープダイヤモンド電極、及び、前記ホウ素ドープダイヤモンド電極上に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、組合せ電極にウィルスを含む検体を接触させるためのセンサ部と、前記積層電極に定電位を印加し、前記銅被覆層を溶出させ、銅イオンを発生させるとともに、前記ホウ素ドープダイヤモンド電極を前記検体に対して晒す定電位印加部と、前記ホウ素ドープダイヤモンド電極に掃引電位を印加し、前記ウィルスに由来する電気化学的応答を測定する掃引電位印加部とを有するウィルス核酸測定装置に、
    　前記組合せ電極に対してウィルスを含む検体が接触した状態で、前記積層電極に定電位を印加する手順と、
    　前記定電位の印加によって前記銅被覆層が溶出し、前記検体に晒された前記ホウ素ドープダイヤモンド電極の電位を掃引し、前記ウィルスのウィルス核酸に由来する電気化学的応答を測定する手順と、を実行させるプログラム。     By computer
    A sensor unit for contacting a sample containing a virus with a combination electrode including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the boron-doped diamond electrode, and a counter electrode. A constant potential is applied to the laminated electrode to elute the copper coating layer to generate copper ions, and the constant potential application portion that exposes the boron-doped diamond electrode to the sample and the boron-doped diamond electrode are swept. A virus nucleic acid measuring device having a sweep potential applying unit for applying a potential and measuring an electrochemical response derived from the virus.
    A procedure for applying a constant potential to the laminated electrode with a sample containing a virus in contact with the combination electrode, and a procedure for applying a constant potential to the laminated electrode.
    The procedure of elution of the copper coating layer by application of the constant potential, sweeping the potential of the boron-doped diamond electrode exposed to the sample, and measuring the electrochemical response derived from the viral nucleic acid of the virus. The program to be executed.
  13. 　更に、前記定電位の印加による応答電流を予め定められた閾値と比較する手順と、
    　前記比較の結果、前記応答電流が前記閾値以下となるまで、前記定電位の印加を継続する手順と、を実行させる、請求項１２に記載のプログラム。     Further, a procedure for comparing the response current due to the application of the constant potential with a predetermined threshold value, and
    The program according to claim 12, wherein the procedure of continuing the application of the constant potential until the response current becomes equal to or less than the threshold value as a result of the comparison is executed.
  14. 　基板と、
    　前記基板上に配置された組合せ電極と、
    　ウィルスを含む検体を表面に付着させ、前記検体が付着した前記表面を前記組合せ電極と接触させるための固体電解質と、
    を含むセンサであって、
    　前記組合せ電極は、ホウ素ドープダイヤモンド電極、及び、前記ホウ素ドープダイヤモンド電極の表面に配置された銅被覆層を有する積層電極と、対電極と、を少なくとも含む、センサ。     With the board
    With the combination electrode arranged on the substrate,
    A solid electrolyte for adhering a sample containing a virus to a surface and bringing the surface to which the sample is attached into contact with the combination electrode.
    Is a sensor that includes
    The combination electrode is a sensor including at least a boron-doped diamond electrode, a laminated electrode having a copper-coated layer arranged on the surface of the boron-doped diamond electrode, and a counter electrode.
  15. 　前記組合せ電極が参照電極を更に有する、請求項１４に記載のセンサ。 The sensor according to claim 14, wherein the combination electrode further has a reference electrode.
  16. 　ホウ素ドープダイヤモンド電極と、前記ホウ素ドープダイヤモンド電極の表面の全体に配置された銅被覆層と、を有する積層電極。 A laminated electrode having a boron-doped diamond electrode and a copper-coated layer arranged on the entire surface of the boron-doped diamond electrode.
  17. 　基板と、前記基板上に配置された請求項１６に記載の積層電極とを有する電極付き基板。 A substrate with electrodes having a substrate and the laminated electrode according to claim 16 arranged on the substrate.
PCT/JP2021/031778 2020-10-13 2021-08-30 Method for measuring viral nucleic acid, device for measuring viral nucleic acid, program, sensor, layered electrode, and substrate with electrode WO2022080023A1 (en)

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