WO2025116035A1 - Acquisition method for information pertaining to size of nucleic acid included in virus-derived capsid by using nanopore device, and nanopore device and acquisition device used for said acquisition method - Google Patents

Abstract

Provided are: an acquisition method for information pertaining to the size of a nucleic acid included in a virus-derived capsid by using a nanopore device; and a nanopore device and an acquisition device used for said acquisition method.　The nanopore device includes: a substrate that has a first surface and a second surface; a nanopore that penetrates from the first surface toward the second surface and through which the capsid passes; a first chamber member; and a second chamber member. The first chamber member forms, using the first surface, a first chamber filled with a first electrolytic solution. The second chamber member forms, using the second surface, a second chamber filled with a second electrolytic solution.　The acquisition method includes: a capsid passing step for causing the capsid, which is included in the first electrolytic solution or the second electrolytic solution, to pass through the nanopore; and an ion current measurement step for measuring the change in an ion current when the capsid passes through the nanopore.

Description

Method for obtaining information related to the size of nucleic acid contained in a virus-derived capsid using a nanopore device, and nanopore device and obtaining apparatus used in said method

The disclosure in this application relates to a method for obtaining information related to the size of nucleic acids contained in virus-derived capsids using a nanopore device, an apparatus for obtaining information related to the size of nucleic acids contained in virus-derived capsids, and a nanopore device used in the apparatus.

Gene therapy is known to treat disease by repairing and correcting defects in cells that have become dysfunctional due to genetic abnormalities. One example of gene therapy is a method in which a retrovirus or other vector carrying a therapeutic gene is used to invade dysfunctional cells. With this method, the therapeutic gene is artificially incorporated into the retrovirus vector, so quality control is necessary when actually using it for treatment.

Incidentally, a device that forms a nanopore (a through-hole that penetrates the substrate) in a substrate and measures the change in ionic current as a sample passes through the nanopore is attracting attention as a device that can be widely applied to sensing bacteria, DNA, proteins, etc.

A related technique is known that involves, for example, analyzing the shape distribution of exosomes by measuring the change in ion current when exosomes pass through nanopores formed in a substrate (see Patent Document 1).

JP 2017-156168 A

As described in Patent Document 1 above, it is known that nanopore devices can be used to measure the shape, etc., of minute samples such as exosomes. However, no method is known that uses nanopore devices in terms of quality control of viral vectors incorporating therapeutic genes.

As a result of intensive research, the inventors have newly discovered that (1) the larger the nucleic acid contained within a virus-derived capsid, the larger the capsid itself becomes, (2) by using a nanopore device, minute differences in the size of the capsid can be measured as changes in ion current, and (3) the change in ion current of a virus-derived capsid incorporating therapeutic nucleic acid (hereinafter, a capsid incorporating therapeutic nucleic acid may be referred to as a "viral preparation") reflects information about the size of the nucleic acid incorporated into the capsid.

In other words, the disclosure of this application is to provide a method for obtaining information related to the size of nucleic acids contained in virus-derived capsids using a nanopore device, and a nanopore device and an obtaining apparatus for use in said method.

The present application discloses a method for obtaining information related to the size of nucleic acids contained in virus-derived capsids using a nanopore device, as well as a nanopore device and an obtaining apparatus for use in the method, as described below.

(1) A method for obtaining information related to the size of a nucleic acid contained in a virus-derived capsid using a nanopore device, comprising:
The nanopore device comprises:
a substrate having a first side and a second side;
a nanopore extending from the first surface to the second surface through which the capsid passes;
A first chamber member;
A second chamber member;
Including,
The first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface including at least the first opening of the nanopore;
The second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface including at least the second opening of the nanopore;
The acquisition method includes:
a capsid passing step in which the capsid contained in the first electrolytic solution or the second electrolytic solution passes through the nanopore;
an ion current measuring step of measuring a change in ion current when the capsid passes through the nanopore;
Including,
The capsid passage step comprises:
A voltage is applied to the first electrolytic solution filled in the first chamber and the second electrolytic solution filled in the second chamber,
A method for obtaining a capsid, the capsid contained in the first chamber being passed through the nanopore in a direction toward the second chamber, or the capsid contained in the second chamber being passed through the nanopore in a direction toward the first chamber.
(2) The method according to (1) above, wherein the virus is any one of adeno-associated virus, human bocavirus, adenovirus, retrovirus, vaccinia virus, poxvirus, herpes virus, lentivirus, and Sendai virus.
(3) The method according to (1) or (2) above, wherein the capsid becomes larger in size as the size of the nucleic acid encapsulated therein increases.
(4) comprising an analysis step following the measurement step,
The method for obtaining a nucleic acid according to the above (3), wherein the analyzing step analyzes the presence or absence of a nucleic acid to be encapsulated in the capsid based on the amount of change in the ion current measured in the measuring step.
(5) comprising an analysis step following the measurement step,
The method for obtaining nucleic acid according to (3) above, wherein the analyzing step calculates a size of the nucleic acid encapsulated in the capsid based on the amount of change in the ion current measured in the measuring step.
(6) The method according to any one of (1) to (5) above, wherein the thickness of the substrate is greater than the size of the capsid.
(7) The method according to any one of (1) to (6) above, wherein the size of the nanopore is 1.2 times or more the average particle diameter of the capsid.
(8) The method according to any one of (1) to (7) above, wherein a substance having a higher viscosity than water is added to the first electrolytic solution and/or the second electrolytic solution.
(9) A nanopore device used in an apparatus for acquiring information related to the size of a nucleic acid contained in a virus-derived capsid, comprising:
The nanopore device comprises:
a substrate having a first side and a second side;
a nanopore extending from the first surface to the second surface through which the capsid passes;
A first chamber member;
A second chamber member;
Including,
The first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface including at least the first opening of the nanopore;
The second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface including at least the second opening of the nanopore;
A nanopore device, wherein the thickness of the substrate is greater than the size of the capsid.
(10) The nanopore device described in (9) above, wherein the size of the nanopore is 1.2 times or more the average particle diameter of the capsid.
(11) An apparatus for acquiring information related to the size of a nucleic acid contained in a virus-derived capsid, comprising:
The acquisition device includes:
A nanopore device.
A measurement unit;
An analysis unit;
Including,
The nanopore device comprises:
a substrate having a first side and a second side;
a nanopore extending from the first surface to the second surface through which the capsid passes;
A first chamber member;
A second chamber member;
Including,
The first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface including at least the first opening of the nanopore;
The second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface including at least the second opening of the nanopore;
The measurement unit measures a change in ion current when the capsid passes through the nanopore,
The analysis unit, based on the amount of change in the ion current measured by the measurement unit,
Analyzing the presence or absence of a nucleic acid to be encapsulated in the capsid, and/or
An acquisition device for calculating the size of the nucleic acid encapsulated in the capsid.
(12) The acquisition apparatus according to (11) above, wherein the nanopore device is the nanopore device according to (8) or (9) above.

The method of obtaining information related to the size of nucleic acid contained in a virus-derived capsid using the nanopore device disclosed in this application allows information about the size of the nucleic acid contained in the capsid to be obtained by measuring the amount of change in ion current caused by a change in the size of the capsid. Therefore, since size information about the nucleic acid contained in the capsid can be easily obtained, this information can be used for quality control of virus preparations, etc.

FIG. 1 is a schematic cross-sectional view of a device 1 according to an embodiment. FIG. 2 is a schematic cross-sectional view showing an example of the configuration of the acquisition device 1a. FIG. 3 is a flowchart of an acquisition method according to an embodiment. FIG. 4 is a graph showing the change in ionic current when an empty capsid containing no nucleic acid, a capsid encapsulating 1,451-base DNA, and a capsid encapsulating 2,599-base DNA pass through nanopore 3 in Example 2. FIG. 5 is a plot of Ip, which is the change in ionic current when individual capsids pass through the nanopore in Example 2. FIG. 6 is a graph showing the average value of the amount of change Ip in the ion current obtained from the plots in FIG. Figure 7 shows the results of measuring the capsid size in Example 2, and Figures 7a and 7b are transmission electron microscope photographs of an empty capsid and a capsid encapsulating 2599-base DNA, respectively. Figure 7c is a graph showing the distribution of dvec obtained from 2052 Empty images and 1576 Full images. FIG. 8 is a graph in which the length of the encapsulated nucleic acid is plotted on the horizontal axis and the average change in the measured ion current (Ip) is plotted on the vertical axis in Example 2. FIG. 9A is a graph showing the measurement results obtained in Example 2 (glycerol not added), and FIG. 9B is a graph showing the measurement results obtained in Example 3 (glycerol added).

Below, a method for acquiring information related to the size of nucleic acid contained in a virus-derived capsid using a nanopore device (hereinafter, sometimes simply referred to as the "acquisition method"), an apparatus for acquiring information related to the size of nucleic acid contained in a virus-derived capsid (hereinafter, sometimes simply referred to as the "acquisition apparatus"), and a nanopore device used in the acquisition apparatus (hereinafter, sometimes simply referred to as the "device") are described in detail. Note that in this specification, components having the same type of function are given the same or similar reference symbols. Furthermore, repeated explanations of components given the same or similar reference symbols may be omitted.

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

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

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

The device 1 includes a substrate 2, a nanopore 3 formed in the substrate 2, a first chamber member 51, and a second chamber member 61. The substrate 2 has a first surface 21 and a second surface 22, and the nanopore 3 penetrates the substrate 2 from the first surface 21 to the second surface 22. When the acquisition method is carried out, the capsid S passes through the nanopore 3.

The first chamber member 51, together with the surface of the first surface 21 that includes at least the first opening 31 of the nanopore 3, forms a first chamber 5 that is filled with a first electrolyte. The second chamber member 61, together with the surface of the second surface 22 that includes at least the second opening 32 of the nanopore 3, forms a second chamber 6 that is filled with a second electrolyte.

In this specification, "capsid" refers to the protein shell of a virus strain that has lost or partially lost its replication and proliferation capabilities, and refers to a carrier (vector) into which a nucleic acid to be introduced for therapeutic purposes is incorporated, allowing the nucleic acid to be efficiently introduced into cells and expressed. In addition, in this specification, nucleic acid refers to DNA and RNA. DNA and RNA may be single-stranded or double-stranded.

There are no particular limitations on the capsid, so long as the size of the capsid itself increases with the size of the nucleic acid encapsulated within. Viruses from which capsids are derived include, but are not limited to, enveloped viruses (viruses in which the capsid is covered by an envelope, a membrane mainly made of lipids), such as retroviruses, lentiviruses, herpes viruses, and Sendai viruses, and non-enveloped viruses (viruses that do not have an envelope), such as adenoviruses and adeno-associated viruses (AAV). Among these, AAV is used in gene therapy aimed at treating various diseases, because it can infect many types of cells, is not pathogenic to humans, and the virus particles are physically stable.

Specific examples of viruses include, but are not limited to, enveloped viruses such as DNA viruses, such as herpes viruses, pox viruses, hepadna viruses, vaccinia viruses, and lentiviruses, and RNA viruses, such as flaviviruses, togaviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, retroviruses, and Sendai viruses. Specific examples of viruses include, but are not limited to, DNA viruses, such as adenoviruses, adeno-associated viruses (AAV), papilloma viruses, and human bocaviruses, and RNA viruses, such as picornaviruses, caliciviruses, noroviruses, and rotaviruses. Note that adeno-associated viruses and human bocaviruses belong to the Parvoviridae family.

In this specification, the viruses from which the capsids are derived include not only wild-type viruses, but also inactivated viruses (e.g., inactivated vaccine antigens, etc.), virus-like particles (VLPs) that do not contain genetic information, and viruses carrying foreign genes used as vectors (also called viral vectors).

The material for forming the substrate 2 is not particularly limited as long as it can form the nanopore 3 and can measure the change in the ion current of the capsid passing through the nanopore 3. Examples of materials for forming the substrate 2 include insulating materials that are commonly used in the field of semiconductor manufacturing technology. Examples of insulating materials include Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, SiN, and the like. The substrate 2 may be formed in a thin film shape called a solid membrane using materials such as SiN, SiO ₂ , and HfO ₂ , or in a sheet shape called a two-dimensional material using materials such as graphene, graphene oxide, molybdenum dioxide (MoS ₂ ), and boron nitride (BN). The substrate 2 may be formed using an artificial membrane such as a lipid bilayer membrane or a naturally occurring membrane. Measurement devices using lipid bilayer membranes are described in JP-A-2011-527191 and JP-A-2020-000056, etc. The matters described in JP-T-2011-527191 and JP-A-2020-000056 are incorporated herein by reference. In addition, a commercially available product may be used as the measurement device using the lipid bilayer membrane. Examples of commercially available products that can perform nanopore analysis using a lipid bilayer membrane include MinION, GridIONX5, SmidgION, and PromethION manufactured by Oxford Nanopore Technologies.

When a solid membrane or two-dimensional material is used as the substrate 2, the thickness can be made very thin; for example, graphene can be used to create a substrate 2 with a thickness of 1 nm or less. However, if the substrate 2 is very thin, it may be difficult to handle it without damaging it. For this reason, the substrate 2 may have a laminated structure in which a solid membrane or two-dimensional material is laminated on a support plate formed from the insulating material described above. When using a laminated structure, the solid membrane or two-dimensional material is laminated on a support plate in which a hole larger than the nanopore 3 is formed, and the nanopore 3 is formed in the solid membrane or two-dimensional material.

The thickness of the substrate 2 forming the nanopore 3 is not particularly limited as long as it is within a range that allows measurement of the amount of change in ionic current that changes depending on the size of the nucleic acid contained within the capsid when the capsid passes through the nanopore 3. In the technical field of measuring the ionic current when the object to be measured passes through the nanopore 3, it is generally considered that the smaller the volume of the nanopore 3, the more preferable it is; in other words, it is preferable for the substrate to be thinner than the size of the object to be measured. This is because the thinner the substrate 2 is compared to the size of the object to be measured, the more various pieces of information about the object to be measured when passing through the nanopore 3 are reflected in the change in ionic current. As a result, the measured change in ionic current reflects, for example, information about the orientation of the object to be measured when it enters the nanopore 3, in addition to size information about the object to be measured. From this perspective, the thickness of the substrate 2 can be 0.3 nm or more.

On the other hand, in the acquisition method disclosed in the present application, it is sufficient to acquire the amount of change in ion current that reflects the size information of the encapsulated nucleic acid, in other words, the magnitude of the change in ion current when a capsid whose size has increased by encapsulating nucleic acid passes through nanopore 3. Therefore, in the device disclosed in the present application, the thickness of substrate 2 may be smaller than the size of the capsid, or it may be larger. By making the thickness of substrate 2 larger than the size of the capsid, the size information of the capsid is more strongly reflected in the change in ion current. Therefore, from the perspective of measuring the size of the capsid, the sensitivity can be increased by making the thickness of substrate 2 larger than the size of the capsid. Also, the thicker the substrate 2, the more convenient it is to manufacture and handle.

Capsids vary depending on the type, but have a size (average particle diameter) of about 20 nm to about 100 nm. Therefore, the thickness of substrate 2 is not limited, but examples include 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 110 nm or more, 120 nm or more, and 130 nm or more. On the other hand, if substrate 2 is made too thick compared to the size of the capsid, the sensitivity decreases. Therefore, the upper limit of the thickness of the substrate 2 can be 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, or 100 nm or less.

The thickness of the substrate 2 may be determined in relation to the size of the capsid to be measured. Although not limited thereto, when the size of the capsid is taken as 1, the lower limit of the thickness of the substrate 2 is 0.015 times or more, and from the viewpoint of measuring the size information of the nucleic acid contained in the capsid with higher sensitivity, examples of the thickness include 1.0 times or more, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, etc. On the other hand, the upper limit of the thickness of the substrate 2 is 5 times or less, 4.5 times or less, 4 times or less, 3.5 times or less, 3 times or less, 2.75 times or less, 2.5 times or less, 2.25 times or less, and 2 times or less.

The nanopore 3 is formed so as to penetrate the substrate 2 from the first surface 21 of the substrate 2 in the direction of the second surface 22, which is the surface opposite to the first surface 21. As described above, the device disclosed in this application only needs to acquire information regarding the size change of the capsid due to the inclusion of nucleic acid. Therefore, the size of the nanopore 3 is larger than the capsid, but can be appropriately adjusted so as not to be too large. Without being limited thereto, when the size of the capsid is 1, the lower limit can be 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2.0 times or more. On the other hand, the upper limit can be 4 times or less, 3.8 times or less, 3.6 times or less, 3.4 times or less, 3.2 times or less, 3.0 times or less, 2.8 times or less, 2.6 times or less, 2.4 times or less, or 2.2 times or less.

As mentioned above, capsids vary depending on the type, but are about 20 nm to about 100 nm in size. Therefore, the lower limit of the size of the nanopore 3 can be 24 nm, 26 nm or more, 28 nm or more, 30 nm or more, 32 nm or more, 34 nm or more, 36 nm or more, 38 nm or more, or 40 nm or more. On the other hand, the upper limit of the size of the nanopore 3 can be 400 nm or less, 380 nm or less, 360 nm or less, 340 nm or less, 320 nm or less, 300 nm or less, 280 nm or less, 260 nm or less, 240 nm or less, or 220 nm or less.

When the cross-sectional shape of the nanopore 3 parallel to the first surface 21 is circular, the size of the nanopore 3 means the diameter. When the cross-sectional shape of the nanopore 3 parallel to the first surface 21 is not circular, the size of the nanopore 3 means the diameter of the inscribed circle of the cross-section. When the material of the substrate 2 is an example material other than a lipid bilayer membrane, the nanopore 3 may be formed by etching or the like, as shown in the examples described later. The nanopore 3 may also be formed so that the first opening 31 of the nanopore 3 on the first surface 21 side and the second opening 32 of the nanopore 3 on the second surface 22 side have the same shape. Alternatively, the first opening 31 and the second opening 32 may be different in size, for example, the nanopore 3 may be formed so that it spreads from the first surface 21 to the second surface 22 in the substrate 2. In that case, the size of the nanopore 3 means the size of the first opening 31 formed in the first surface 21 (the size of the smaller opening).

In addition, although FIG. 1 shows an example in which one nanopore 3 is formed in the substrate 2, two or more nanopores 3 may be formed. When two or more nanopores 3 are formed in the substrate 2, the distance between adjacent nanopores 3 may be adjusted as necessary to improve the measurement accuracy of the capsid. The extent to which the distance between adjacent nanopores 3 should be set is described in detail in WO 2020/138021, and therefore will not be described in detail in the disclosure of this application. The matters described in WO 2020/138021 are incorporated herein by reference.

The first chamber member 51 and the second chamber member 61 are preferably formed from an electrically and chemically inert material, such as, but not limited to, glass, sapphire, ceramic, resin, rubber, elastomer, _SiO2 , SiN, _Al2O3 , and _the like.

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

(Embodiment of Acquisition Device 1a)
An embodiment of the acquisition device 1a will be described with reference to Fig. 2. Fig. 2 is a schematic cross-sectional view showing an example of a configuration of the acquisition device 1a according to the embodiment.

The acquisition device 1a shown in FIG. 2 includes at least a measurement unit 7 and an analysis unit 8 in addition to the device 1 according to the embodiment. The device 1 has already been described in the "Embodiment of Device 1" above. Therefore, to avoid redundancy, a detailed description of the device 1 will be omitted.

In the example shown in FIG. 2, a first electrode 52 formed at a location in contact with the first electrolyte in the first chamber 5, a second electrode 62 formed at a location in contact with the second electrolyte in the second chamber 6, and a power source 54 that applies a voltage between the first electrode 52 and the second electrode 62 are shown, but the first electrode 52, the second electrode 62, and the power source 54 may be prepared separately from the acquisition device 1a and attached to the acquisition device 1a when carrying out the acquisition method. In other words, the first electrode 52, the second electrode 62, and the power source 54 are optional additional components in the acquisition device 1a.

The acquisition device 1a may also optionally include a display unit 9 for displaying the results of the analysis performed by the analysis unit 8, a program memory 10 that stores programs for operating the analysis unit 8 and the display unit 9, and a control unit 11 for reading and executing the programs stored in the program memory 10. The programs may be stored in the program memory 10 in advance, or may be recorded on a recording medium and stored in the program memory 10 using an installation means.

The first electrode 52 and the second electrode 62 can be formed of known conductive metals such as aluminum, copper, platinum, gold, silver, silver/silver chloride, and titanium. FIG. 2 shows an example in which the first electrode 52 and the second electrode 62 are formed to sandwich the nanopore 3, and a voltage is applied so that a direct current flows with the first electrode 52 side as a negative pole and the second electrode 62 side as a positive pole, but alternatively, the first electrode 52 side may be a positive pole and the second electrode 62 side as a negative pole. It is sufficient to appropriately determine which side of the first electrode 52 or the second electrode 62 is to be made positive depending on the charge possessed by the capsid described below.

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

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

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

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

There are no particular limitations on the measuring unit 7 as long as it can measure the amount of change in ion current when the capsid S passes through the nanopore 3, and examples of such a measuring unit include a known ammeter.

When the capsid S passes through the nanopore 3, the ion current flowing through the nanopore 3 is blocked by the capsid S, and the ion current flowing through the nanopore 3 changes. The analysis unit 8 analyzes the amount of change in the ion current measured by the measurement unit 7. Therefore, by performing data analysis in the analysis unit 8 based on the amount of change in the measured ion current, information regarding the size of the nucleic acid contained in the capsid S can be analyzed. As described above, the larger the size of the nucleic acid contained in the capsid S (the longer the nucleic acid), the larger the size of the capsid S becomes. Therefore, (1) the amount of change in the ion current when the capsid S before introducing the nucleic acid passes through the nanopore 3 is measured in advance, (2) the change in the ion current when the capsid S to be measured passes through the nanopore 3 is measured, and (3) the amount of change in the ion current measured in advance is compared with the amount of change in the ion current of the object to be measured, thereby (4) analyzing the presence or absence of the nucleic acid to be contained in the capsid S, in other words, whether or not the intended nucleic acid has been introduced into the capsid S.

In addition, (1) the change in ionic current when capsid S before the introduction of nucleic acid and capsid S encapsulating nucleic acids of different lengths pass through nanopore 3 is measured in advance, (2) the change in ionic current when capsid S, which is the object to be measured, passes through nanopore 3 is measured, and (3) the change in ionic current measured in advance is compared with the change in ionic current of the object to be measured, (4) the size of the nucleic acid encapsulated in the capsid can be calculated.

The analysis unit 8 may be provided with a memory unit that stores pre-measured data on (a) the amount of change in ion current when the capsid S before the introduction of nucleic acid passes through the nanopore 3, or (b) the amount of change in ion current when the capsid S before the introduction of nucleic acid and the capsid S encapsulating nucleic acids of different lengths pass through the nanopore 3. Furthermore, when performing analysis with the analysis unit 8, known machine learning may be used. By using machine learning, it is expected that the accuracy of the analysis will be improved.

The display unit 9 may be any known display device capable of displaying the amount of change in the measured ion current and the results of the analysis performed by the analysis unit 8, such as a liquid crystal display, plasma display, or organic electroluminescence display. The program memory 10 is not particularly limited as long as it can store programs for operating the analysis unit 8 and the display unit 9, and examples of such memory include ROMs such as mask ROM, PROM, EPROM, and EEPROM. The control unit 11 is not particularly limited as long as it can read and execute the programs stored in the program memory 10, and examples of such memory include a processor (CPU) or a general-purpose computer equipped with a CPU.

Note that the device 1 and acquisition device 1a described above are merely examples of embodiments and are not intended to be limiting. Any combination selected from the various exemplified embodiments and optional additional items may be used within the scope of the technical ideas disclosed in this application.

(Embodiment of Acquisition Method)
An embodiment of the acquisition method will be described with reference to Fig. 3. Fig. 3 is a flowchart of the acquisition method according to the embodiment. The acquisition method according to the embodiment includes a capsid passing step (ST1) and an ion current measuring step (ST2) as essential steps. Note that, although Fig. 3 shows an analysis step (ST3), the analysis step (ST3) is not an essential component in the acquisition method according to the embodiment, but is an optional additional component.

In the capsid passage step (ST1), a voltage is applied to the first electrolyte filled in the first chamber 5 and the second electrolyte filled in the second chamber 6, thereby causing the capsid S contained in the first chamber 5 to pass through the nanopore 3 in the direction of the second chamber 6, or causing the capsid S contained in the second chamber 6 to pass through the nanopore 3 in the direction of the second chamber 6. The first electrolyte and the second electrolyte can be any solution that can conduct electricity between the first electrode 52 and the second electrode 62, and can be any solution (electrolyte) containing ions such as TE buffer, PBS buffer, HEPES buffer, or KCl aqueous solution known in the art.

When carrying out the capsid passing step, a substance having a higher viscosity than water may be added to increase the viscosity of the first electrolyte solution and/or the second electrolyte solution. By increasing the viscosity of the first electrolyte solution and/or the second electrolyte solution, the time for the capsid S to pass through the nanopore 3 can be increased. The viscosities of the first electrolyte solution and the second electrolyte solution may be the same or different as long as the time for the capsid S to pass through the nanopore 3 can be increased. When the viscosities of the first electrolyte solution and the second electrolyte solution are the same, the conditions that the capsid S receives from the electrolyte before and after passing through the nanopore 3 (for example, the resistance of the electrolyte that the nanopore 3 receives) are the same. Therefore, the viscosities of the first electrolyte solution and the second electrolyte solution may be different, but it is preferable that the difference in viscosity is not too large. It is more preferable that the viscosities of the first electrolyte solution and the second electrolyte solution are the same.

Examples of substances with a higher viscosity than water include glycerin, DMSO, polyethylene glycol, hydrogel, and xanthan gum. The viscosity of water varies depending on the temperature, but is approximately 1 mPa·s at about 20°C. Although not limited thereto, the lower limit of the viscosity of the first electrolyte solution and/or the second electrolyte solution after the addition of the substance includes, at approximately 20°C, 2 mPa·s or more, 4 mPa·s or more, 6 mPa·s or more, 8 mPa·s or more, 10 mPa·s or more, 15 mPa·s or more, 20 mPa·s or more, 25 mPa·s or more, 30 mPa·s or more, 35 mPa·s or more, 40 mPa·s or more, 45 mPa·s or more, 50 mPa·s or more, etc. On the other hand, the upper limit can be 1000 mPa·s or less, 900 mPa·s or less, 800 mPa·s or less, 700 mPa·s or less, 600 mPa·s or less, 500 mPa·s or less, 450 mPa·s or less, 400 mPa·s or less, 350 mPa·s or less, 300 mPa·s or less, 250 mPa·s or less, 200 mPa·s or less, 150 mPa·s or less, 100 mPa·s or less, etc. By increasing the viscosity of the first electrolyte solution and/or the second electrolyte solution, the time it takes for the capsid S to pass through the nanopore 3 when the capsid passing step is performed is increased. Therefore, the effect is achieved that information regarding the size of the capsid S (information regarding the size of the nucleic acid encapsulated in the capsid) can be obtained with higher accuracy.

If the first chamber 5 is already filled with the first electrolyte and the second chamber 6 is already filled with the second electrolyte, capsids are introduced into the first chamber 5 or the second chamber 6, and the capsid passage step (ST1) is carried out.

When the first chamber 5 of the acquisition device 1a is not filled with the first electrolytic solution and the second chamber 6 is not filled with the second electrolytic solution, a preparation step may be performed before the capsid passing step (ST1) is performed. The preparation step may be performed by the following procedure.
(1) The first chamber 5 is filled with a first electrolytic solution, and the second chamber 6 is filled with a second electrolytic solution. A liquid junction is established between the first chamber 5 and the second chamber 6 via the nanopore 3.
(2) Capsid S is introduced into the first chamber 5 or the second chamber 6.
The above steps (1) and (2) may be carried out separately, or an electrolyte solution already containing capsid S may be introduced into the first chamber 5 or the second chamber 6.

In the capsid passage step (ST1) shown in FIG. 3, a current is applied to the first electrode 52 arranged in the first chamber 5 and the second electrode 62 arranged in the second chamber 6, and in addition to normal diffusion, the capsid S passes through the nanopore 3 formed in the substrate 2 by electrophoresis.

In the ion current measurement process (ST2), the change in the ion current caused by the passage of electricity is measured over time by the measurement unit 7. Therefore, when the capsid S passes through the nanopore 3, a large change in the ion current according to the size of the capsid S can be measured.

The amount of change in ion current obtained by the acquisition method according to the embodiment includes information about the size of capsid S. The information about the size of capsid S includes information about the size of the nucleic acid contained within capsid S. Therefore, the acquisition method disclosed in this application has the following effects.

(1) By measuring the size of the capsid, information on the size of the nucleic acid encapsulated in the capsid can be obtained, thus making it possible to obtain information on the size of the encapsulated nucleic acid without destroying the capsid.
(2) When a viral preparation is produced, if the target nucleic acid is not contained in the capsid, or if the target nucleic acid is contained in the capsid in a cleaved state, the produced viral preparation will not only have no substantial therapeutic effect, but there is also concern about side effects caused by immune reactions. Information on the size of the nucleic acid contained in the capsid is very useful for checking whether a viral preparation used in gene therapy has been produced correctly. Therefore, the method of obtaining the viral preparation of the present application allows for non-destructive quality control of the viral preparation.
(3) The method disclosed in the present application does not require a large amount of samples because it is possible to measure the ion current for each capsid. Therefore, it is useful not only for quality control after production of a virus preparation, but also for spot checks during production.

Next, the analysis step (ST3), which is an optional additional configuration of the acquisition method, will be described. The analysis step (ST3) analyzes information on the nucleic acid contained in the capsid from the change in the ion current measured in the ion current measurement step (ST2). The contents of the analysis include (a) analyzing the presence or absence of the nucleic acid to be contained in the capsid S, in other words, whether or not the nucleic acid has been introduced into the capsid S, and/or (b) calculating the size of the nucleic acid contained in the capsid, as in the analysis unit 8 of the above-mentioned "embodiment of the acquisition device 1a."

The analysis step (ST3) may be performed manually or may be processed using a computer or the like. For example, when analyzing manually, it is sufficient to analyze whether or not a nucleic acid has been introduced into the capsid S to be measured based on the change in ion current (the difference between the baseline value of the ion current and the peak value of the change in ion current) by referring to (a) the change in ion current of a capsid not encapsulating a nucleic acid and the change in ion current of a capsid encapsulating a nucleic acid. In addition, (b) the change in ion current when the capsid S before the nucleic acid is introduced and the capsid S encapsulating nucleic acids of different lengths pass through the nanopore 3 may be measured in advance, and the length of the encapsulated nucleic acid and the change in ion current may be graphed based on the measurement results, and the length of the nucleic acid encapsulated in the capsid S may be calculated by referring to the graph and the change in ion current of the capsid to be measured.

When performing the analysis using a computer, the above analysis can be automated. The accuracy of the analysis can also be improved by using machine learning, etc.

When the acquisition method according to the embodiment includes the analysis step (ST3), the following effect is achieved in addition to the effects (1) to (3) described above.
(4) By carrying out the analysis process, the presence or absence of nucleic acid contained in the capsid and the length of the nucleic acid contained therein can be more accurately understood. Therefore, when used for quality control of a virus preparation, more detailed quality control can be performed. In particular, by measuring in advance the amount of change in ion current when capsid S containing nucleic acids of different lengths passes through the nanopore 3, it is possible to understand how the nucleic acid to be introduced is cleaved, which makes it possible to provide feedback to the manufacturing process.

The acquisition device 1a described above also achieves the effects of the acquisition method described in (1) to (4) above. And the device 1 achieves the effect that it can be used for the acquisition method and acquisition device 1a that achieve the effects described in (1) to (4) above.

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

Example 1
[Fabrication of Device 1]
A 4-inch silicon wafer coated on both sides with a 50 nm thick SiNx layer was diced into 30 mm x 30 mm chips. The silicon layer was partially dissolved by wet etching in a KOH aqueous solution to form a 30 nm thick SiNx film. Then, an electron beam resist (ZEP520A, zeon) was spin-coated on the SiNx film, pre-baked at 180 ° C. using a hot plate, and a circular pattern was drawn by electron beam lithography and developed. Using the residual resist layer thus obtained as a mask, a nanopore with a diameter of 66 nm was opened by reactive ion etching with CHF ₃ etching gas. Finally, a nanopore chip with a nanopore formed on the SiNx substrate was produced by immersing the substrate in N,N-dimethylformamide overnight and washing with ethanol/acetone.

Next, one surface of the fabricated nanopore chip was covered with a polyimide layer. This was to reduce the capacitance of the nanopore chip and reduce noise. More specifically, an imide precursor of photosensitive polyimide (PN-2010, Toray Industries, Inc.) was spin-coated onto the nanopore membrane. After baking, ultraviolet light was irradiated using LED lithography, and development was performed to dissolve the polyimide around the nanopore, which had a diameter and thickness of 5 μm.

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

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

[Implementation of acquisition method]
Example 2
(1) Preparation of AAV empty capsids Adenovirus E1a, adenovirus E1b, and 293EB cell line expressing Bcl-xL (Tomono T., et al., "Highly efficient ultracentrifugation-free chromatographic purification of recombinant AAV Empty Capsids") were cultured in a 550 mL multi-shelf flask (HYPERFlask, Corning, Corning, NY, USA) supplemented with Dulbecco's modified Eagle's medium (DMEM high sugar content, FUJIFILM Wako, Osaka, Japan) and 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA). serotype 9", Mol. Ther. Methods Clin. Dev. 11, 180-190 (2018)) were seeded at a density of 40,000 cells/ ^cm2 and cultured for 3 days. Then, transfection was performed with pCAX (CAG promoter backbone plasmid for generating empty capsids) (32.5 μg/flask) (Takara Bio, Kusatsu, Shiga, Japan), pR2C8 (serotype 8) or pR2C9 (serotype 9) (32.5 μg/flask) and helper plasmid (65 μg/flask) in DMEM (Nacalai Tesque, Nakagyo-ku, Kyoto) containing 2 mM L-alanyl-L-glutamine solution (100x) using polyethyleneimine max (520 μg/flask) (Polysciences, Warrington, PA, USA). Ten days after transfection, culture supernatants were collected and treated with 18.5 U/mL endonuclease (Kaneka, Minato-ku, Tokyo, Japan) and ₅ mM MgCl (Nacalai Tesque) for 30 min at 37° C. rAAV was then purified using an AAVpro® Concentrator kit (Takara, Japan) or a PhyTip® column CaptureSelect® AAVX (PhyNexus, USA).

The rAAV genome copy number was analyzed using the AAVpro® Titration Kit (for real-time PCR) Ver. 2 (Takara, Japan) with a QuantStudio 3 real-time PCR system (Applied Biosystems, Waltham, MA, USA).

(2) Preparation of AAV Capsids Encapsidating Nucleic Acids 293EB cell lines expressing adenovirus E1a, adenovirus E1b, and Bcl-xL were seeded at a density of 40,000 cells/cm2 into a 550 mL multi-shelf flask (HYPERFlask, Corning, Corning, NY, USA) supplemented with Dulbecco's modified Eagle's medium (DMEM high sugar content, FUJIFILM Wako, Osaka, Japan) and 10% fetal bovine serum (Thermo Fisher, ^Waltham , MA, USA), and cultured for 3 days. Thereafter, transfection was performed using pAAV-ZsGreen1 (2599 bases; Takara Bio #6231) or pAAV-ZsGreen1-short (1451 bases; pAAV-ZsGreen1 was digested with a restriction enzyme) (32.5 μg/flask), pR2C8 (serotype 8) or pR2C9 (serotype 9) (32.5 μg/flask), and a helper plasmid (65 μg/flask) in DMEM (Nacalai Tesque, Nakagyo-ku, Kyoto) containing 2 mM L-alanyl-L-glutamine solution (100x), using polyethyleneimine max (520 μg/flask) (Polysciences, Warrington, PA, USA). Ten days after transfection, culture supernatants were collected and treated with 18.5 U/mL endonuclease (Kaneka, Minato-ku, Tokyo, Japan) and ₅ mM MgCl (Nacalai Tesque) for 30 min at 37° C. rAAV was then purified using an AAVpro® Concentrator kit (Takara, Japan) or a PhyTip® column CaptureSelect® AAVX (PhyNexus, USA).

(3) Measurement Conditions For the first and second electrolyte solutions, 1.37 M NaCl (10x PBS Buffer (-), model number: 314-90185, manufactured by Nippon Gene Co., Ltd.) was used. The first electrolyte solution was filled into the first chamber through a hole formed in the block. Capsids encapsulating nucleic acids were also filled into the first chamber. The second electrolyte solution was filled into the second chamber through a hole formed in the block. A voltage of 0.3 V was applied so that the first electrode 52 was the positive pole and the second electrode 62 was the negative pole, and the ionic current Iion was measured.

Figure 4 shows the change in ionic current when an empty capsid containing no nucleic acid (labeled "AAV9/empty"), a capsid containing 1,451 bases of DNA (labeled "AAV9/1.5 kbDNA"), and a capsid containing 2,599 bases of DNA (labeled "AAV9/2.6 kbDNA") pass through nanopore 3. Note that the graph in Figure 4 is the 100-point average (adjacent average) of raw data measured at 1 MHz.

5 is a plot of Ip, which is the change in ion current when each capsid passes through the nanopore. Note that the display of "AAV9/1.5kb" is omitted in FIG. 5. In the example shown in FIG. 5, the change in ion current (Ip) is defined as the difference between the baseline (base), which is the value of the ion current measured when the capsid has not entered the nanopore 3, and the average value (Av) of the ion current value during the time td (the time from when the ion current suddenly drops from the baseline to just before it starts to suddenly return to the baseline) during which the entire capsid passes through the nanopore, but is not limited to this definition. The change in ion current (Ip) may be defined in other ways as long as the size information of the nucleic acid contained in the capsid is reflected. For example, the change in ion current (Ip) may be the difference between the average value of the minimum value of multiple peaks measured during the td period (the lower side of the waveform during the td period shown in FIG. 5) and the baseline. Alternatively, it may be the difference between the average value of the maximum value of multiple peaks measured during the td period (upper side of the waveform during the td period shown in FIG. 5) and the baseline. FIG. 6 shows the average value of the change in ion current Ip calculated from the plot in FIG. 5, which was 5.1 nA when the encapsulated DNA was 0, 6.1 nA when the encapsulated DNA was 1451 bases, and 6.7 nA when the encapsulated DNA was 2599 bases.

As shown in Figure 6, the change in the measured ion current varied depending on the size of the nucleic acid encapsulated, so the size of the capsid was examined. The results are shown in Figure 7. More specifically, empty capsids (Empty) and capsids encapsulating 2599 bases of DNA (Full) were photographed using a transmission electron microscope (Figures 7a and b). As shown by the arrows in Figures 7a and b, the vector diameter (dvec) indicated by the arrow for each capsid was measured using ImageJ software (scale bar is 20 nm). Figure 7c shows the distribution of dvec obtained from 2052 Empty images and 1576 Full images. The average diameter of Empty was 22.88 nm, and the average diameter of Full was 25.84 nm, with a size difference Δdvec of approximately 3 nm between the two (p<0.0001). From the above results, the capsid size increases as the nucleic acid contained therein increases, resulting in a difference in the amount of change in the measured ion current. Therefore, it was confirmed that the difference in the amount of change in the ion current reflects the size information of the nucleic acid contained therein.

Figure 8 shows a graph in which the length of the encapsulated nucleic acid is plotted on the horizontal axis and the average value of the measured change in ionic current (Ip) on the vertical axis. Note that, unlike Figures 5 and 6, the change in ionic current (Ip) shown in Figure 8 is the difference between the average value of the lowest value of multiple peaks measured during the td period and the baseline. In the example shown in Figure 8, there are three plots, but by increasing the number of plots, it is possible to create an approximation curve with higher accuracy. By creating a graph like that shown in Figure 8 in advance, the length of the nucleic acid encapsulated in the capsid can be calculated from the measured change in ionic current Ip.

Example 3
The ionic current of each capsid was measured in the same manner as in Example 2 (viscosity: 1 mPa·s), except that glycerol was added to the first and second electrolyte solutions to a concentration of 30 vol% (viscosity: approximately 3.6 mPa·s) and the NaCl concentration was set to 0.96 M.

FIG. 9A is a graph showing the measurement results obtained in Example 2 (glycerol not added), and FIG. 9B is a graph showing the measurement results obtained in Example 3 (glycerol added). As is clear from FIGS. 9A and 9B, by increasing the viscosity of the first and second electrolyte solutions, the accuracy of distinguishing capsids containing different sizes of nucleic acid was improved. In the example shown in FIG. 9A where glycerol was not added, the degree of separation between an empty capsid containing no nucleic acid (AAV9 empty) and a capsid containing 2599 bases of DNA (AAV9, 2.6 kbs DNA) was 0.4, while the degree of separation in the example shown in FIG. 9B where glycerol was added was 1.13. From the above results, it was confirmed that the accuracy of distinguishing capsids containing nucleic acids of different sizes was improved by adding a substance with a higher viscosity than water to the first and second electrolyte solutions.

The acquisition method disclosed in this application provides information on the size of the nucleic acid contained in the capsid. Therefore, it can be used for quality control of virus preparations, etc., and is therefore useful for the medical industry.

Reference Signs List 1, 1a...Ion current measuring device, 2...Substrate, 3...Nanopore, 5...First chamber, 6...Second chamber, 7...Ammeter, 8...Analysis unit, 9...Display unit, 10...Program memory, 11...Control unit, 21...First surface, 22...Second surface, 31...First opening, 32...Second opening, 51...First chamber member, 52...First electrode, 53...Lead, 54...Power supply, 55...Earth, 61...Second chamber member, 62...Second electrode, 63...Lead, 64...Earth, S...Sample

Claims (12)

A method for obtaining information related to the size of a nucleic acid contained in a virus-derived capsid using a nanopore device, comprising:
The nanopore device comprises:
a substrate having a first side and a second side;
a nanopore extending from the first surface to the second surface through which the capsid passes;
A first chamber member;
A second chamber member;
Including,
The first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface including at least the first opening of the nanopore;
The second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface including at least the second opening of the nanopore;
The acquisition method includes:
a capsid passing step in which the capsid contained in the first electrolytic solution or the second electrolytic solution passes through the nanopore;
an ion current measuring step of measuring a change in ion current when the capsid passes through the nanopore;
Including,
The capsid passage step comprises:
A voltage is applied to the first electrolytic solution filled in the first chamber and the second electrolytic solution filled in the second chamber,
A method for obtaining a capsid, the capsid contained in the first chamber being passed through the nanopore in a direction toward the second chamber, or the capsid contained in the second chamber being passed through the nanopore in a direction toward the first chamber. The method according to claim 1 , wherein the virus is any one of adeno-associated virus, human bocavirus, adenovirus, retrovirus, vaccinia virus, poxvirus, herpes virus, lentivirus, and Sendai virus. The method according to claim 1 , wherein the capsid has a larger size as the size of the nucleic acid encapsulated therein increases. An analysis step is included following the measurement step,
The method according to claim 3 , wherein the analyzing step analyzes the presence or absence of a nucleic acid to be encapsulated in the capsid based on the amount of change in ion current measured in the measuring step. An analysis step is included following the measurement step,
The method according to claim 3 , wherein the analyzing step calculates a size of the nucleic acid encapsulated in the capsid based on the amount of change in ion current measured in the measuring step. The method according to any one of claims 1 to 5, wherein the thickness of the substrate is greater than the size of the capsid. The method according to any one of claims 1 to 5, wherein the size of the nanopore is 1.2 times or more the average particle diameter of the capsid. The method according to any one of claims 1 to 5, wherein a substance having a higher viscosity than water is added to the first electrolytic solution and/or the second electrolytic solution. A nanopore device used in an apparatus for acquiring information related to the size of a nucleic acid contained in a virus-derived capsid,
The nanopore device comprises:
a substrate having a first side and a second side;
a nanopore extending from the first surface to the second surface through which the capsid passes;
A first chamber member;
A second chamber member;
Including,
The first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface including at least the first opening of the nanopore;
The second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface including at least the second opening of the nanopore;
A nanopore device, wherein the thickness of the substrate is greater than the size of the capsid. The nanopore device according to claim 9 , wherein the size of the nanopore is 1.2 times or more the average particle diameter of the capsid. An apparatus for obtaining information related to the size of a nucleic acid contained in a virus-derived capsid, comprising:
The acquisition device includes:
A nanopore device.
A measurement unit;
An analysis unit;
Including,
The nanopore device comprises:
a substrate having a first side and a second side;
a nanopore extending from the first surface to the second surface through which the capsid passes;
A first chamber member;
A second chamber member;
Including,
The first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface including at least the first opening of the nanopore;
The second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface including at least the second opening of the nanopore;
The measurement unit measures a change in ion current when the capsid passes through the nanopore,
The analysis unit, based on the amount of change in the ion current measured by the measurement unit,
Analyzing the presence or absence of a nucleic acid to be encapsulated in the capsid, and/or
An acquisition device for calculating the size of the nucleic acid encapsulated in the capsid. The acquisition apparatus according to claim 11 , wherein the nanopore device is a nanopore device according to claim 8 or 9 .