JP7455431B2 - Virus nucleic acid measurement method, virus nucleic acid measurement device, program, sensor, laminated electrode, and substrate with electrode - Google Patents

Description

The present invention relates to a method for measuring viral nucleic acid, a device for measuring viral nucleic acid, a program, a sensor, a laminated electrode, and a substrate with an electrode.

Due to the rapid spread of the novel coronavirus (SARS-CoV-2), it has been pointed out that there is a possibility of the virus being transmitted to medical workers through aerosols and surgical smoke in operating rooms.
For example, the Japanese Society of Surgical Medicine's "Recommendations for operating room management and operation under the new coronavirus infection epidemic" (http://jaom.kenkyuukai.jp/information/information_detail.asp?id=102978, July 22, 2020) ``There is a certain level of agreement that aerosol generation is a factor in the spread of the new coronavirus, and that the risk of aerosol generation increases during surgical operations and intubation.'' , points out the possibility of virus transmission through aerosols.

“Recommendations for operating room management and operation under the new coronavirus infection epidemic”, April 24, 2020, Japanese Society of Surgical Medicine, retrieved July 22, 2020, Internet, <URL: http://jaom. kenkyuukai. jp/information/information_detail. asp? id=102978>

A known method for detecting viruses in aerosol is to capture the virus in the aerosol with a filter or the like and detect it using quantitative PCR (Polymerase Chain Reaction), but this method takes time to detect and is difficult to operate. The problem was that it was complicated and required specialized knowledge.

Therefore, an object of the present invention is to provide a method for measuring viral nucleic acid that can rapidly measure viral nucleic acid in a specimen 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.

As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that the above-mentioned problems can be achieved by the following configuration.

[1] Bringing a sample containing a virus into contact with a combination electrode that includes at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on the boron-doped diamond electrode, and a counter electrode; Applying a constant potential to the laminated electrode to elute the copper coating layer and generate copper ions to release viral nucleic acid from the virus, and exposing the boron-doped diamond electrode to the specimen; A method for measuring 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 method described in [1], comprising comparing a response current when applying the constant potential with a predetermined threshold value, and continuing to apply the constant potential until the response current becomes equal to or less than the threshold value. A method for measuring viral nucleic acids.
[3] The electrochemical response derived from the viral nucleic acid described in [1] or [2] is at least one value selected from the group consisting of peak current magnitude and current peak area. A method for measuring viral nucleic acids.
[4] The method for measuring viral nucleic acid according to [3], wherein the peak potential is more positive than the constant potential.
[5] Any one of [1] to [4], wherein the potential sweep is performed by at least one method selected from the group consisting of linear sweep voltammetry, differential pulse voltammetry, and cyclic voltammetry. A method for measuring viral nucleic acid described in .
[6] The method for measuring viral nucleic acid according to any one of [1] to [5], further comprising collecting aerosol and preparing the specimen.
[7] Adsorbing an aerosol containing a virus on the surface of a solid electrolyte, preparing a specimen on the surface, and bringing the surface into contact with the combination electrode, thereby bringing the specimen into contact with the combination electrode. The method for measuring viral nucleic acid according to any one of [1] to [6], comprising:
[8] A sensor for bringing a sample containing a virus into contact with a combination electrode, which includes at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on the boron-doped diamond electrode, and a counter electrode. a constant potential application section that applies a constant potential to the laminated electrode to elute the copper coating layer and generate copper ions, and expose the boron-doped diamond electrode to the specimen; A viral nucleic acid measuring device, comprising a sweep potential application section that applies a sweep potential to an electrode and measures an electrochemical response derived from the virus.
[9] A comparison unit that compares a response current resulting from application of the constant potential with a predetermined threshold value, and the constant potential application unit operates as described above until the response current becomes equal to or less than the threshold value as a result of the comparison. The virus nucleic acid measuring device according to [8], which continues to apply a constant potential.
[10] The sensor section includes a substrate, the combination electrode disposed on the substrate, and a solid electrolyte for attaching the analyte to the surface and bringing the surface to which the analyte has adhered into contact with the combination electrode. The virus nucleic acid measuring device according to [8] or [9], comprising a connecting tool for electrically connecting the combined electrode of the sensor including the constant potential application section and the sweep potential application section. .
[11] Any one of [8] to [10], wherein the sensor section includes a cell for accommodating the specimen, and the combination electrode arranged in the cell so as to be in contact with the specimen. Viral nucleic acid measuring device described in.
[12]
A sensor for bringing a virus-containing specimen into contact with a combination electrode, which comprises at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on the boron-doped diamond electrode, and a counter electrode. a constant potential application section that applies a constant potential to the laminated electrode to elute the copper coating layer and generate copper ions, and exposes the boron-doped diamond electrode to the specimen; and the boron-doped diamond electrode. A virus nucleic acid measuring device having a sweep potential application unit that applies a sweep potential to the sample and measures an electrochemical response derived from the virus is placed in a state in which the sample containing the virus is in contact with the combination electrode. The procedure includes applying a constant potential to the electrode, eluating the copper coating layer by applying the constant potential, sweeping the potential of the boron-doped diamond electrode exposed to the sample, and detecting the electricity derived from the viral nucleic acid of the virus. A procedure to measure a chemical response and a program to run it.
[13] Furthermore, a procedure for comparing a response current resulting from 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. The program according to [12], which causes the program to execute the following.
[14] A sensor comprising a substrate, a combination electrode disposed on the substrate, and a solid electrolyte for attaching a sample containing a virus to the surface and bringing the surface to which the sample has adhered into contact with the combination electrode. The combination electrode includes at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on a surface of the boron-doped diamond electrode, and a counter electrode.
[15] The sensor according to [14], wherein the combined electrode further includes a reference electrode.
[16] A laminated electrode comprising a boron-doped diamond electrode and a copper coating layer disposed over the entire surface of the boron-doped diamond electrode.
[17] A substrate with electrodes, comprising a substrate and the laminated electrode according to [16] arranged on the substrate.

According to the present invention, viral nucleic acid in a specimen 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.

1 is a flowchart showing a method for measuring viral nucleic acid according to Example 1 of the present invention. FIG. 1 is an exploded perspective view of a sensor used for measuring viral nucleic acids according to Example 1 of the present invention. FIG. 2 is a perspective view of a sensor used for measuring viral nucleic acid according to Example 1 of the present invention. 2A is an XY cross-sectional view of the electrode-equipped substrate 201 in FIG. 2A. FIG. FIG. 1 is a schematic diagram showing the principle of a method for measuring viral nucleic acid according to Example 1 of the present invention. FIG. 1 is a schematic diagram showing the principle of a method for measuring viral nucleic acid according to Example 1 of the present invention. FIG. 2 is a hardware configuration diagram of a virus nucleic acid measuring device according to Example 2 of the present invention. FIG. 2 is a perspective view of a virus nucleic acid measuring device according to Example 2 of the present invention. FIG. 2 is a perspective view of a virus nucleic acid measuring device according to Example 2 of the present invention. 6B is a sectional view taken along the line VW in FIG. 6B. FIG. 2 is a functional block diagram of a virus nucleic acid measuring device according to Example 2 of the present invention. 2 is a flowchart showing the operation of the control section of the virus nucleic acid measuring device according to Example 2 of the present invention. FIG. 3 is a hardware configuration diagram of a virus nucleic acid measuring device according to Example 3 of the present invention. FIG. 3 is a schematic diagram of a virus nucleic acid measuring device according to Example 3 of the present invention. FIG. 4 is an exploded perspective view of a sensor according to Example 4 of the present invention. FIG. 4 is a perspective view of a sensor according to Example 4 of the present invention.

Embodiments of the present invention will be described below with reference to the drawings.
In this specification, a numerical range expressed using "~" means a range that includes the numerical values written before and after "~" as the lower limit and upper limit.

(Explanation of terms)
The term "aerosol" as used herein means a mixture of fine particles and 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 include those called "droplets," which contain fine particles, are surrounded by moisture, and have an overall particle size of 5 μm or more, and particles, which are typically particles from which the moisture has been removed by drying, etc. It includes both microparticles themselves called "droplet nuclei" with a diameter of less than 5 μm. Among them, droplet nuclei, which have a lower sedimentation speed in the air, are considered to be one of the causes of virus infection by aerosol, and it is preferable to collect the specimen from aerosol containing droplet nuclei.

FIG. 1 is a flowchart showing a method for measuring viral nucleic acid according to Example 1 of the present invention.

In step S11, an aerosol containing a virus is adsorbed on the surface of the solid electrolyte, and the virus is accumulated on the surface of the solid electrolyte, and this is used as a sample. In this specification, the above is referred to as "preparing a specimen on the surface of a solid electrolyte."
The solid electrolyte preferably includes a water-swollen polymer compound that typically contains an electrolyte. The polymer compound may be, for example, agarose. Further, the solid electrolyte may be a hydrogel containing agar, agar, gelatin, etc., 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 viral nucleic acids.

Hydrogel is preferred as the solid electrolyte. Examples of the hydrogel include agar gel and gelatin gel. As the solid electrolyte, one having high ionic conductivity is preferable, and more specifically, one in which ions (eg, hydrogen ions, sulfate ions, etc.) can move is preferable. The solid electrolyte may contain sulfate or the like since it has better ionic conductivity.

In Example 1, the aerosol is adsorbed on a solid electrolyte and the virus is concentrated on the surface of the solid electrolyte to be used as a sample. However, the specimen may be prepared without using a solid electrolyte. Such a method includes, for example, a method in which a virus in an aerosol is captured with a filter or the like and used as a sample.
Furthermore, the method for measuring viral nucleic acids of the present invention may detect viral nucleic acids derived from viruses contained other than aerosols. For example, a liquid containing a virus may be used as the sample.
Alternatively, the virus may be transferred onto a solid electrolyte from the solid surface (by bringing the solid electrolyte into contact with the solid surface) and used as a sample.

In step S12, a sample containing a virus is brought into contact with the combination electrode. The combination electrode includes a stacked electrode and a counter electrode. The laminated electrode includes a boron-doped diamond (BDD electrode) and a copper coating layer disposed on the BDD electrode. Therefore, in this step, the copper coating layer and the specimen come into contact.
In order to bring the specimen into contact with the combination electrode, a solid electrolyte with the specimen prepared on its surface may be pressed against the combination electrode.

FIG. 2A is an exploded perspective view of a sensor used for sample collection and viral nucleic acid measurement in Example 1, and FIG. 2B is a perspective view of the sensor. A method of implementing step S11 and step S12 using this sensor will be explained.

First, as shown in FIG. 2A, the sensor 20 is roughly divided into three parts. The three parts are a substrate 201 with electrodes, a spacer 26, and a cover 202 with solid electrolyte.

Preparation of the sample in step S11 is performed using the cover 202 with solid electrolyte. The cover 202 with solid electrolyte includes a cover 27 and a solid electrolyte 25 disposed on one surface of the cover 27, and can be removed from the sensor 20.
A sample can be prepared by removing the solid electrolyte cover 202 from the sensor 20 and adsorbing the aerosol onto the solid electrolyte 25.
Since the solid electrolyte-equipped cover 202 includes the cover 27, it is possible to collect viruses in the aerosol without touching the solid electrolyte 25, thereby suppressing unintended contaminants from being mixed into the sample.

Contact between the specimen and the combined electrode in step S12 can be achieved by pressing the solid electrolyte 25 against the combined electrode of the electrode-attached substrate 201.
The electrode-equipped substrate 201 includes a substrate 21, a laminated electrode 22 which is a working electrode disposed on the substrate 21, a counter electrode 23, and a reference electrode 24. 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) through the notch of the spacer 26.

The spacer 26 has a guide function for aligning the solid electrolyte 25 with the combined electrode, and has the advantage that it becomes easy to reattach the solid electrolyte cover 202 once removed. However, the sensor may not include spacer 26.

FIG. 2C is an XY cross-sectional view 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) indicated by "SL" in the figure is arranged between these electrodes. The laminated electrode 22 includes a BDD electrode 28 and a copper coating layer 29 disposed on the BDD electrode 28.
Note that 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 a state in which there is no step on the surfaces of the counter electrode 23 and the reference electrode 24, and a state in which one or both of the counter electrode 23 and the reference electrode 24 is higher than the laminated electrode 22. But that's fine.
In order to obtain better effects 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 level difference.

The method for manufacturing the BDD electrode 28 is not particularly limited, and any known method can be used. Among these, it is preferable to manufacture by chemical vapor deposition (CVD) method. As the excitation source for the CVD method, a hot filament, microwave, high frequency, direct current glow discharge, direct current arc discharge, combustion flame, etc. can be used. Furthermore, by combining a plurality of these, the nucleation density can be adjusted, the area can be increased, and the nucleation density can be made uniform.
Many types of compounds containing carbon can be used as raw materials. For example, gases include CH ₄ , C ₂ H ₂ , C ₂ H ₄ , C ₁₀ H ₁₆ , CO, and CF ₄ ; liquids include CH ₃ OH, C ₂ H ₅ OH, and (CH ₃ ) ₂ CO. etc.; Examples of the solid include graphite and fullerene.

Examples of the addition of boron include a method of introducing a substance containing boron into the system and introducing boron into the carbon gas phase. Examples of such boron-containing substances include diborane, trimethylborane, and trimethoxyborane, and trimethoxyborane is preferred because it is easier to handle.
When trimethoxyborane is used as a boron source and acetone is used as a solvent for dissolving it, handling becomes easier because acetone also serves as a carbon source.

It is more preferable that the BDD electrode be formed by plasma CVD using microwaves, since 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 using microwaves and introducing raw material gas into it.

When boron is added to the carbon source, the amount of boron added is not particularly limited, but is preferably 10 to 12,000 ppm, and 1,000 to 10,000 ppm, since the resulting BDD electrode has better conductivity. 000 ppm is more preferable.

The method of laminating the laminated electrode 22, counter electrode 23, and reference electrode 24 on the substrate 21 is not particularly limited, and any known method can be used. Examples of such a method include the method described in paragraphs 0037 to 0050 of JP-A No. 2006-10357, and the method described in paragraphs 0034 to 0057 of JP-A No. 2020-33199.

Note that although the sensor 20 has a laminated electrode 22, a counter electrode 23, and a reference electrode 24, the sensors that can be used in the method for measuring viral nucleic acid of Example 1 are not limited to the above, and include a laminated electrode 22, a counter electrode 23, and a reference electrode 24. and a counter electrode.
Furthermore, the arrangement of the laminated electrode 22, counter electrode 23, and reference electrode 24 in the sensor 20 may be exchanged.

Although the substrate 21 may be a conductive substrate or an insulating substrate, an insulating substrate is preferable. Examples of the substrate include tungsten, silicon, silicon oxide, silicon nitride, silicon carbide, and niobium. Furthermore, the substrate 21 may be made of quartz glass or the like. The thickness and size of the substrate may be appropriately selected from the viewpoint of handleability, etc., and are typically preferably 1 μm to 5 mm.

The thickness of the BDD electrode 28 can be adjusted by changing the film formation time. Typically, the thickness of the BDD electrode 28 is preferably 0.1 μm or more, more preferably 1 μm or more, even more 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 also applies to the counter electrode 23 and the reference electrode 24.

The material for the counter electrode 23 is not particularly limited, and known materials for counter electrodes can be used. Examples of such materials include platinum, carbon materials, stainless steel, and SnO ₂ . The reference electrode 24 may be, for example, a calomel electrode, a silver/silver chloride electrode, or the like.

Returning to the flowchart of FIG. 1, the procedure from step 13 onwards will be explained.
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 at least a portion of the viral envelope and capsid through direct involvement and/or indirect involvement, such as by catalyzing the generation of reactive oxygen species (ROS). Therefore, viral nucleic acid is released from inside the virus.

FIG. 3 is a schematic diagram showing the principle of the method for measuring viral nucleic acids of Example 1. The detection system 30 consists 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 placed on the surface of the solid electrolyte 25. .

In addition, in FIG. 3, the structure of the coronavirus 31 is schematically shown, and its diameter (the length corresponding to L1 in FIG. 3), the BDD electrode 28, the copper coating layer 29 (together with the laminated The relationship between the thickness of the electrode 22) and the solid electrolyte 25 does not correspond to reality. The size, thickness, etc. of each part are as already explained, so the explanation will be omitted here. Note that the above also applies to L2 in FIG. 4, which will be described later.

The surface of the coronavirus 31 is covered with a lipid membrane 32, within which a positive-strand single-stranded RNA genome 33 wrapped around a nucleocapsid (N) protein is arranged. Spike (S) protein, Envelope (E) protein, and Membrane (M) protein are arranged on the surface of coronavirus 31, and its shape resembles a crown.
Generally, viral nucleic acids are enclosed in a membrane (envelope) and/or a shell (capsid), so it is difficult to directly electrochemically measure viral nucleic acids.

At this time, when a constant potential is applied to the laminated electrode 22, the copper coating layer 29 is eluted toward the solid electrolyte 25 side, and copper ions are generated.
Copper ions destroy at least a portion of the viral envelope and capsid through direct and/or indirect involvement, thereby destroying viral nucleic acids (for coronavirus 31, one molecule) from the inside of the virus. The stranded RNA genome 33) is released.

Next, in step S14, a 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 progress of the copper coating layer 29. In the method for measuring viral nucleic acid in Example 1, it is necessary to expose at least a portion (preferably all) of the BDD electrode 28 to the sample, so this response current is monitored to ensure that the copper coating layer 29 is eluted to a desired extent. It is preferable to confirm that it has been done (decomposed).
Note that the amount of elution (decomposition amount) of the copper coating layer 29 may be managed by a method other than monitoring the response current, for example, by the application time of a constant potential. In that case, step S14 and step S15 may be omitted.

The threshold value may be, for example, a value that takes into account a predetermined margin for the response current when all of the copper coating layer 29 is eluted. When a potential is applied to the laminated electrode 22, if the response current is below the threshold (does not exceed the threshold), it can be determined that the copper coating layer 29 has been eluted to a desired extent (for example, all) ( Step S15: False). On the other hand, if the response current exceeds the threshold (step S15: True), the copper coating layer 29 remains to a greater extent than desired, so it is only necessary to continue applying the constant potential and monitoring the response current ( Steps S13 to S15).

FIG. 4 is a schematic diagram showing the detection system after the copper coating layer is eluted by applying a constant potential to the laminated electrode. Since the copper coating layer 29 has completely eluted (decomposed), the detection system 40 consists of the BDD electrode 28 and the solid electrolyte 25, and the coronavirus 41 on the surface of the solid electrolyte 25 has an envelope and a capsid made of copper. It is destroyed by direct and indirect involvement of ions, and the virus nucleic acid is released from inside.

In the detection system 40, since the BDD electrode 28 is exposed to the coronavirus 41, by sweeping the potential of this BDD electrode 28, an electrochemical response derived from the virus nucleic acid is obtained. (Step S16).
The electrochemical response derived from the viral nucleic acid is preferably an oxidation current of the viral nucleic acid, and more preferably a peak-shaped response (the presence of one or more local maximum values in the measurement results of potential versus current). preferable. In this specification, the maximum current value of this peak is referred to as a "peak current," the potential that provides this "peak current" is referred to as a "peak potential," and the area of this peak is referred to as a "current peak area."

When the method of sweeping the potential of the BDD electrode is at least one method selected from the group consisting of linear sweep voltammetry, differential pulse voltammetry, and cyclic voltammetry, the peak-shaped response as described above can be obtained. is easy to obtain.

The peak potential of the oxidation current of viral nucleic acids is often detected in the range of +1.0 to 1.5 V (vs Ag/AgCl). In the viral nucleic acid measuring method of Example 1, the potential of the BDD electrode 28 is swept after the copper coating layer 29 is eluted and the BDD electrode 28 is exposed to the sample, so that the oxidation current of the viral nucleic acid is Measurement becomes possible.

When using general electrode materials such as glassy carbon, gold, and platinum, an increase in current value is detected at around +1.3V (vs Ag/AgCl) due to the generation of oxygen due to oxidation of water molecules. Therefore, it is difficult to accurately detect the oxidation current of viral nucleic acids. On the other hand, the BDD electrode 28 has a surface made of ^sp3 carbon and has fewer sites where molecules can be adsorbed, so it has a wider potential window than the above-mentioned general electrode members and can detect the oxidation current of viral nucleic acids. .

Multiple peaks may be detected in the oxidation current of viral nucleic acids depending on the type of nucleobases that constitute the viral nucleic acids. In that case, each peak current or each peak area may be summed up as a measurement value, or a single peak current or peak area may be used as a measurement value.
When evaluating the total amount, it is preferable to use the sum of each peak current or each peak area as the measured value.
According to the method of Example 1, viral nucleic acids in specimens collected from aerosols can be rapidly measured.

FIG. 5 is a hardware configuration diagram of a virus nucleic acid measuring device according to Example 2 of the present invention.

The viral nucleic acid measurement device 50 has a processor 51, a memory device 52, a display device 53, an input device 54, an electrochemical measurement device 55, and a connector 56. The processor 51, the memory 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).

The 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 from the virus nucleic acid measuring device 50, and for example, the sensor 20 can be replaced for each sample.
Note that the sensor 20 is as described in FIGS. 2A, 2B, and 2C, so the description thereof will be omitted here.

The processor 51 controls each part of the virus nucleic acid measuring device 50 to realize the functions of the virus nucleic acid measuring device.
The processor 51 includes, for example, a microprocessor, a processor core, a multiprocessor, an application-specific integrated circuit (ASIC), a field programmable gate array (FPGA), and a general-purpose CPU (GPGPU). computing on graphics processing units), etc.

The storage device 52 has a function of temporarily and/or non-temporarily storing programs and data, and provides a work area for 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, or an SSD (Solid State Drive).

The display device 53 can display measurement results, voltage application status to the laminated electrode (or BBD electrode), sample name, operating procedure, and the like. The display device 53 may be a liquid crystal display, an organic EL (Electro Luminescence) 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 may be a touch panel display that provides a GUI (Graphical User Interface).

The input device 54 can input voltage application conditions to the combined electrodes, measurement conditions, sample name, etc. The input device 54 may be a keyboard, mouse, scanner, touch panel, or the like.

The electrochemical measurement device 55 can apply a constant potential to the laminated electrode 22 of the sensor 20 connected via the connector 56 and measure the response current. Further, by applying a sweep potential to the BDD electrode 28 after the copper coating layer 29 has been eluted, the electrochemical measurement device 55 can measure the electrochemical response derived from the viral nucleic acid. The electrochemical measurement device 55 may be, for example, a potentiostat.

The connector 56 electrically connects the electrochemical measurement device 55 and the combined electrodes (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 disposed in the center of the main body 61, and inside the main body 61 are each of the previously described hardware (the processor 51 and the electrochemical measuring device). 55 etc.) is mounted on the circuit board.

The virus nucleic acid measuring device 60 has an insertion port 63 into which the sensor 20 is inserted. FIG. 6B is a perspective view of the viral nucleic acid measuring device 60 with the sensor 20 inserted.

FIG. 6C is a sectional view taken along the line VW in FIG. 6B. The sensor 20 inserted into the insertion opening 63 with the substrate 21 facing upward is held between the main body 61 supported by the filling member 69 and the spring member 64 arranged inside the insertion opening 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 a terminal 67 of a circuit board 68 via a conductive wire 66. Terminal 67 is connected to electrochemical measurement device 55 placed on circuit board 68 . This connects the electrochemical measurement device 55 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 section 71, a storage section 72, a display section 73, an input section 74, a comparison section 75, a constant potential application section 76, a sweep potential application section 77, and a sensor section 78. has.

The control unit 71 is configured to include the 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 functions of the virus nucleic acid measuring device 70. do.

The storage unit 72 is configured to include the storage device 52. The storage unit 72 stores programs, threshold values, and the like, and stores measurement data and the like.

The display section 73 includes the display device 53. Further, the input section 74 is configured to include the input device 54. By controlling these, the control unit 71 receives input from the user of the viral nucleic acid measuring device 70 and stores it in the storage unit 72, and controls 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 section 77 can be displayed to the user of the device.

The comparison unit 75 is a function realized by the control unit 71 executing a program stored in the storage unit 72. The comparison unit 75 compares a response current generated by applying a constant potential to the laminated electrode 22 and a predetermined threshold value.
Note that the threshold value is stored in the storage unit 72. The response current is obtained by a constant potential applying section 76, which will be described later.

The constant potential application section 76 is configured to include the electrochemical measurement device 55, and is a function realized by the control section 71 executing a program stored in the storage section 72 and controlling the electrochemical measurement device 55.
The constant potential applying section 76 applies a constant voltage to the laminated electrode 22 of the sensor connected to the sensor section 78 and measures the response current.

The sweep potential application section 77 is configured to include the electrochemical measurement device 55, and is a function realized by the control section 71 executing a program stored in the storage section 72 and controlling the electrochemical measurement device 55.
The sweep potential application section 77 is connected to the sensor section 78 and applies a sweep voltage to the BDD electrode 28 exposed to the sample, thereby measuring the electrochemical response derived from the viral nucleic acid.

The sensor section 78 includes a connector 56, and has the 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.

In step S81, the control section 71 controls the constant potential application section 76 to apply a constant voltage to the laminated electrode 22 that has been brought into contact with the sample containing the virus, and to measure the response current. Note that the laminated electrode 22 is included in the sensor 20, and the sensor 20 is controlled by a constant potential application section 76 connected via a sensor section 78.

In step S82, the control unit 71 controls the comparison unit 75 to compare the response current obtained from the laminated electrode 22 with a threshold value stored in advance in the storage unit 72.
This threshold value is predetermined and stored as a value corresponding to a preferable elution state of the copper coating layer 29. As explained in the method for measuring viral nucleic acid in Example 1, after the capsid and envelope of the virus are destroyed by copper ions, it is necessary to measure the oxidation current of the viral nucleic acid using a BDD electrode. 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 the copper coating layer 29 is completely eluted.

If the response current exceeds the threshold determined from such a viewpoint (step S83: True), it means that the copper coating layer 29 disposed on the BDD electrode 28 has not been completely eluted to the desired extent.

In this case, in step S84, the control section 71 controls the constant potential application section 76 to continue applying the constant potential to the laminated electrode 22.
Thereafter, the comparator 75 executes the threshold value determination (steps S82 to S83) again, and repeats this until the response current becomes equal to or less than the threshold value.

On the other hand, as a result of the comparison between the response current and the threshold value by the comparison unit 75, if the response current is less than or equal to the threshold value (step S83: False), the control unit 71 controls the sweep potential application unit 77 so that the response current is not exposed to the specimen. A sweep voltage is applied to the BDD electrode 28, and the resulting electrochemical response is measured (step S85).
This electrochemical response typically occurs when at least a portion of the virus capsid and/or envelope is annihilated and released due to the action of copper ions generated by applying a constant potential to the laminated electrode 22. This is the oxidation current of viral nucleic acids.

It is preferable that the oxidation current of the viral nucleic acid is typically obtained as a curve having a peak in a potential versus current curve. This peak current and current peak area are proportional to the amount of viral nucleic acid in the sample. The peak current and current peak area are stored in the storage unit 72 by the sweep potential application unit 77 and displayed on the input unit 74.

According to the viral nucleic acid measuring device of Example 2, viral nucleic acid derived from a virus contained in a sample collected from an aerosol can be rapidly measured.

FIG. 9 is a hardware configuration diagram of a virus nucleic acid measuring device according to Example 3 of the present invention.
The virus nucleic acid measurement device 90 includes a processor 51 , a storage device 52 , a display device 53 , an input device 54 , an electrochemical measurement device 55 , and a cell 91 connected to the electrochemical measurement device 55 .

The virus nucleic acid measuring device 90 has a cell 91 connected to the electrochemical measuring device 55, and has a similar hardware configuration to the viral nucleic acid measuring device of Example 2, except that it does not have a connector. There are many, and the following will focus on the differences.

The cell 91 is a container that can contain a specimen, and has a stacked electrode 22, a counter electrode 23, and a reference electrode 24 inside. Typically, when a liquid specimen is contained in the cell 91, the contained specimen comes into contact with each of the above-mentioned electrodes forming the combination electrode.
The laminated electrode 22, counter electrode 23, and reference electrode 24 are each electrically connected to an electrochemical measurement device 55, and can control and measure potential and current.

FIG. 10 is a schematic diagram of a virus nucleic acid measuring device according to Example 3.
The virus nucleic acid measuring device 100 includes a computer 101, a potentiostat 102 connected to the computer 101, and a laminated electrode 22, a counter electrode 23, and a reference electrode 24 connected to the potentiostat 102, respectively. . The laminated electrode 22 , counter electrode 23 , and reference electrode 24 are housed within the cell 91 and can come into contact with the analyte also housed in the cell 91 . Note that 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 viral nucleic acid measuring device of Example 3 has the advantage that, unlike the viral nucleic acid measuring device of Example 2, in which the sensor is removable, it has a built-in cell containing a combination electrode, and has a simpler configuration. Are better. Note that 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, so a description thereof will be omitted.

According to the viral nucleic acid measuring device of Example 3, viral nucleic acid derived from a virus typically contained in a liquid sample can be rapidly measured.

FIG. 11A is an exploded perspective view of a sensor 110 according to Example 4, and FIG. 11B is a perspective view of the same sensor. The sensor 110 includes a substrate 21, a laminated electrode 22 disposed on the substrate 21, a counter electrode 23, and a reference electrode 24.
It also includes a spacer 26 and a cover 27 that covers the spacer 26.
Note that the cover 27 has a recessed notch 111.

Typically, a liquid sample is introduced through 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 surfaces of the notch of the spacer 26. Thereby, the specimen and the combination electrode come into contact and measurement can be performed.

Note that this sensor 110 can be used by being inserted into the virus nucleic acid measuring device of Example 2 in place of the sensor 20 already described. The method of use is the same as that of the sensor 20, so the explanation will be omitted.

Since the sensor of Example 4 is detachable from the virus nucleic acid measuring device, by replacing it for each sample, it is possible to prevent samples from mixing with each other and causing errors in measurement results.

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 : coronavirus, 32: lipid membrane, 33: single-stranded RNA genome, 50: virus nucleic acid measuring device, 51: processor, 52: storage device, 53: display device, 54: input device, 55: electrochemical measurement device, 56: Connector, 60: Viral nucleic acid measuring device, 61: Main body, 62: Touch panel display, 63: Insertion port, 64: Spring member, 65: Terminal, 66: Conductor, 67: Terminal, 68: Circuit board, 69: Filling member, 70: Viral nucleic acid measuring device, 71: Control section, 72: Storage section, 73: Display section, 74: Input section, 75: Comparison section, 76: Constant potential application section, 77: Sweep potential application section, 78 : Sensor part, 90: Viral nucleic acid measuring device, 91: Cell, 100: Viral nucleic acid measuring device, 101: Computer, 102: Potentiostat, 110: Sensor, 111: Recessed part, 201: Substrate with electrode, 202: Cover with solid electrolyte

Claims (17)

Contacting a sample containing a virus with a combination electrode comprising at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on the boron-doped diamond electrode, and a counter electrode;
Applying a constant potential to the laminated electrode to elute the copper coating layer and generate copper ions to release viral nucleic acid from the virus, and exposing the boron-doped diamond electrode to the specimen;
A method for measuring viral nucleic acid, comprising sweeping the potential of the boron-doped diamond electrode and measuring an electrochemical response derived from the viral nucleic acid. The viral nucleic acid according to claim 1, comprising comparing a response current when applying the constant potential with a predetermined threshold value, and continuing to apply the constant potential until the response current becomes equal to or less than the threshold value. How to measure. Measurement of 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 peak current magnitude and current peak area. Method. The method for measuring viral nucleic acid according to claim 3, wherein the peak potential is more positive than the constant potential. 5. The sweeping of the potential is performed by at least one method selected from the group consisting of linear sweep voltammetry, differential pulse voltammetry, and cyclic voltammetry. A method for measuring viral nucleic acids. The method for measuring viral nucleic acid according to any one of claims 1 to 5, further comprising collecting an aerosol and preparing the specimen. contacting the analyte with the combination electrode by adsorbing an aerosol containing a virus on the surface of a solid electrolyte, providing an analyte on the surface, and contacting the surface with the combination electrode; The method for measuring viral nucleic acid according to any one of claims 1 to 6. A sensor unit for contacting a sample containing a virus with a combination electrode, the sensor unit including at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed 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 and generate copper ions, and exposes the boron-doped diamond electrode to the specimen;
A virus nucleic acid measuring device, comprising: a sweep potential application section that applies a sweep potential to the boron-doped diamond electrode and measures an electrochemical response derived from the virus. a comparison unit that compares a response current due to the application of the constant potential with a predetermined threshold;
The viral nucleic acid measuring device according to claim 8, wherein the constant potential applying section continues applying the constant potential until the response current becomes equal to or less than the threshold as a result of the comparison. The sensor section is
a substrate; and the combination electrode disposed on the substrate;
a solid electrolyte for attaching the analyte to a surface and contacting the surface to which the analyte is attached with the combination electrode;
The viral nucleic acid measuring device according to claim 8 or 9, further comprising a connector for electrically connecting the combined electrode of the sensor including the constant potential applying section and the sweep potential applying section. The sensor section is
a cell for accommodating the specimen;
The viral nucleic acid measuring device according to any one of claims 8 to 10, comprising the combination electrode arranged in the cell so as to be in contact with the specimen. By computer,
A sensor unit for contacting a sample containing a virus with a combination electrode, the sensor unit including at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on the boron-doped diamond electrode, and a counter electrode; A constant potential applying section applies a constant potential to the laminated electrode to elute the copper coating layer and generate copper ions, and exposes the boron-doped diamond electrode to the specimen, and sweeps the boron-doped diamond electrode. A viral nucleic acid measuring device having a sweep potential applying section that applies a potential and measures an electrochemical response derived from the virus,
a step of applying a constant potential to the laminated electrode while a sample containing a virus is in contact with the combined electrode;
The copper coating layer is eluted by the application of the constant potential, the potential of the boron-doped diamond electrode exposed to the specimen is swept, and an electrochemical response derived from the viral nucleic acid of the virus is measured. Program to be executed. Further, a step of comparing a response current due to the application of the constant potential with a predetermined threshold;
13. The program according to claim 12, further comprising: continuing to apply the constant potential until the response current becomes equal to or less than the threshold as a result of the comparison. A substrate and
a combination electrode disposed on the substrate;
a solid electrolyte for attaching a sample containing a virus to a surface and contacting the surface to which the sample is attached with the combination electrode;
A sensor comprising:
The combination electrode includes at least a boron-doped diamond electrode, a laminated electrode having a copper coating layer disposed on a surface of the boron-doped diamond electrode, and a counter electrode. 15. The sensor of claim 14, wherein the combination electrode further comprises a reference electrode. A laminated electrode for use in the method for measuring viral nucleic acid according to any one of claims 1 to 7,
boron-doped diamond electrode,
a copper coating layer disposed over the entire surface of the boron-doped diamond electrode. A substrate with an electrode, comprising a substrate and the laminated electrode according to claim 16 disposed on the substrate.

Images