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

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 acids contained in virus-derived capsids using a nanopore device, and nanopore device and obtaining apparatus used in the method

[0001] 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.

[0002] Gene therapy is known to treat diseases 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. In this method, the therapeutic gene is artificially incorporated into the retrovirus vector, so quality control is required when actually using it for treatment.

[0003] Incidentally, devices that form nanopores (through-holes that penetrate the substrate) in a substrate and measure changes in ionic current as a sample passes through the nanopore are attracting attention as devices that can be widely applied to sensing bacteria, DNA, proteins, etc.

[0004] A related technique is known, for example, to analyze the shape distribution of exosomes by measuring changes in ionic current when exosomes pass through nanopores formed in a substrate (see Patent Document 1).

[0005] Japanese Patent Application Laid-Open No. 2017-156168

[0006] As described in Patent Document 1, it is known that nanopore devices can be used to measure the shape and other properties of minute samples such as exosomes. However, no method using nanopore devices is known from the perspective of quality control of viral vectors incorporating therapeutic genes.

[0007] As a result of extensive research, the inventors have newly discovered that (1) the larger the size of a viral capsid is, the larger the capsid itself becomes, (2) by using a nanopore device, minute differences in capsid size can be measured as changes in ionic current, and (3) the change in ionic current of a viral capsid incorporating a therapeutic nucleic acid (hereinafter, a capsid incorporating a therapeutic nucleic acid may be referred to as a "viral preparation") reflects information about the size of the nucleic acid incorporated into the capsid.

[0008] In other words, the disclosure of the present 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 to be used in the obtaining method.

[0009] 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 used in the method, as described below.

[0010] (1) A method for obtaining information related to the size of nucleic acid contained in a virus-derived capsid using a nanopore device, the nanopore device comprising: a substrate having a first surface and a second surface; a nanopore penetrating from the first surface to the second surface through which the capsid passes; a first chamber member; and a second chamber member, wherein the first chamber member forms a first chamber filled with a first electrolyte solution between the first surface and a surface of the first surface including at least the first opening of the nanopore; and the second chamber member forms a second chamber filled with a second electrolyte solution between the second surface and a surface of the second surface including at least the second opening of the nanopore, the obtaining method comprising: a capsid passing step in which the capsid contained in the first electrolyte solution or the second electrolyte solution passes through the nanopore; and an ion current measuring step in which a change in ion current when the capsid passes through the nanopore is measured by applying a voltage to the first electrolyte solution filled in the first chamber and the second electrolyte solution filled in the second chamber, An acquisition method comprising passing the capsid contained in the first chamber through the nanopore toward the second chamber, or passing the capsid contained in the second chamber through the nanopore toward the first chamber. (2) The acquisition method according to (1) above, wherein the virus is any one of adeno-associated virus, human bocavirus, adenovirus, retrovirus, vaccinia virus, poxvirus, herpesvirus, lentivirus, and Sendai virus. (3) The acquisition method according to (1) or (2) above, wherein the capsid increases in size as the size of the nucleic acid encapsulated therein increases. (4) The acquisition method according to (3) above, comprising an analysis step following the measurement step, in which the analysis step analyzes the presence or absence of the nucleic acid to be encapsulated in the capsid based on the change in ion current measured in the measurement step. (5) The acquisition method according to (3) above, comprising an analysis step following the measurement step, in which the analysis step calculates the size of the nucleic acid encapsulated in the capsid based on the change in ion current measured in the measurement step.(6) The method according to any one of (1) to (5), 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), 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), wherein a substance having a viscosity greater than that of 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 nucleic acid contained in a virus-derived capsid, the nanopore device comprising: a substrate having a first surface and a second surface, a nanopore penetrating from the first surface to the second surface through which the capsid passes, a first chamber member, and a second chamber member, wherein the first chamber member forms a first chamber filled with a first electrolyte solution together with a surface of the first surface including at least the first opening of the nanopore, and the second chamber member forms a second chamber filled with a second electrolyte solution together with a surface of the second surface including at least the second opening of the nanopore, and the thickness of the substrate is greater than the size of the capsid. (10) The nanopore device according to (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 nucleic acid contained in a virus-derived capsid, the apparatus comprising: a nanopore device; a measurement unit; and an analysis unit; the nanopore device comprising: a substrate having a first surface and a second surface; a nanopore that penetrates from the first surface to the second surface and through which the capsid passes; a first chamber member; and a second chamber member; the first chamber member forms a first chamber filled with a first electrolyte solution between itself and a surface of the first surface that includes at least the first opening of the nanopore; the second chamber member forms a second chamber filled with a second electrolyte solution between itself and a surface of the second surface that includes at least the second opening of the nanopore; the measurement unit measures the change in ionic current when the capsid passes through the nanopore; and the analysis unit analyzes the presence or absence of nucleic acid to be encapsulated in the capsid based on the change in ionic current measured by the measurement unit, and / or calculates the size of the nucleic acid encapsulated in the capsid. (12) The acquisition apparatus according to (11), wherein the nanopore device is the nanopore device according to (8) or (9).

[0011] The method for obtaining information related to the size of nucleic acids contained in virus-derived capsids using the nanopore device disclosed in the present application allows for the measurement of changes in ion current resulting from changes in capsid size, thereby obtaining information about the size of nucleic acids encapsulated in capsids. Therefore, since size information about nucleic acids encapsulated in capsids can be easily obtained, this information can be used for quality control of virus formulations, etc.

[0012] 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 configuration of an acquisition device 1a. FIG. 3 is a flowchart of an acquisition method according to an embodiment. FIG. 4 is a graph showing changes in ion current when empty capsids containing no nucleic acid, capsids encapsulating 1,451-base DNA, and capsids encapsulating 2,599-base DNA pass through a nanopore 3 in Example 2. FIG. 5 is a plot of Ip, which is the change in ion current when individual capsids pass through a nanopore in Example 2. FIG. 6 is a graph showing the average value of the change in ion current Ip calculated from the plot in FIG. 5 in Example 2. FIG. 7 shows the results of measuring capsid size in Example 2, and FIGS. 7a and 7b are photographs of empty capsids and capsids encapsulating 2,599-base DNA, respectively, taken with a transmission electron microscope. Figure 7c is a graph showing the distribution of dvec obtained from 2052 empty images and 1576 full images. Figure 8 is a graph plotting the length of the encapsulated nucleic acid on the horizontal axis and the average value of the measured change in ionic current (Ip) on the vertical axis in Example 2. Figure 9A is a graph showing the measurement results obtained in Example 2 (glycerol not added), and Figure 9B is a graph showing the measurement results obtained in Example 3 (glycerol added).

[0013] Below, we will explain in detail a method for acquiring information related to the size of nucleic acids contained in virus-derived capsids using a nanopore device (hereinafter, sometimes simply referred to as the "acquisition method"), an apparatus for acquiring information related to the size of nucleic acids contained in virus-derived capsids (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"). Note that in this specification, components having similar functions are assigned the same or similar symbols. Furthermore, repeated explanations of components assigned the same or similar symbols may be omitted.

[0014] Furthermore, for ease of understanding, the position, size, range, etc. of each component shown in the drawings may not represent the actual position, size, range, etc. Therefore, the disclosure in this application is not necessarily limited to the position, size, range, etc. disclosed in the drawings.

[0015] Furthermore, in this specification, (1) a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" 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 that are generally acceptable in the technical field, and (3) when it is written "approximately ____-shaped," it is interpreted as including not only the exact ____-shaped, but also a shape that is understood to be roughly ____-shaped.

[0016] (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.

[0017] 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 capture method is performed, the capsid S passes through the nanopore 3.

[0018] 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 solution. 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 solution.

[0019] As used herein, "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) that incorporates a nucleic acid to be introduced for therapeutic purposes and efficiently introduces and expresses the nucleic acid into cells. Furthermore, as used herein, nucleic acid refers to DNA and RNA. DNA and RNA may be single-stranded or double-stranded.

[0020] The capsid is not particularly limited as long as the size of the capsid itself increases with the size of the nucleic acid encapsulated therein. Viruses from which capsids are derived include, but are not limited to, enveloped viruses (viruses in which the capsid is surrounded by an envelope, which is a membrane mainly composed of lipids) such as retroviruses, lentiviruses, herpesviruses, and Sendai viruses, and non-enveloped viruses (viruses without an envelope) such as adenoviruses and adeno-associated viruses (AAVs). Among these, AAVs are used in gene therapy for the treatment of various diseases because they can infect many types of cells, are non-pathogenic to humans, and their viral particles are physically stable.

[0021] More specific examples of viruses include, but are not limited to, enveloped viruses such as DNA viruses, 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. Non-enveloped viruses include, but are not limited to, DNA viruses, such as adenoviruses, adeno-associated viruses (AAVs), papillomaviruses, and human bocaviruses, and RNA viruses, such as picornaviruses, caliciviruses, noroviruses, and rotaviruses. Adeno-associated viruses and human bocaviruses belong to the Parvoviridae family.

[0022] In this specification, the viruses from which capsids are derived include not only wild-type viruses, but also inactivated viruses (for example, inactivated vaccine antigens), virus-like particles (VLPs) that do not have genetic information, and viruses that carry foreign genes and are used as vectors (also referred to as viral vectors).

[0023] The material forming the substrate 2 is not particularly limited as long as it can form the nanopore 3 and measure changes in the ionic current of the capsid passing through the nanopore 3. Examples of materials forming the substrate 2 include insulating materials commonly used in the field of semiconductor manufacturing technology. Examples of insulating materials include Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, and SiN. The substrate 2 may also be formed in the form of a thin film called a solid membrane using materials such as SiN, SiO 2 , and HfO 2 , or in the form of a sheet called a two-dimensional material using materials such as graphene, graphene oxide, molybdenum dioxide (MoS 2 ), and boron nitride (BN). The substrate 2 may also 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, for example, JP-A-2011-527191 and JP-A-2020-000056. The contents of JP-A-2011-527191 and JP-A-2020-000056 are incorporated herein by reference. Furthermore, a commercially available product may be used as a measurement device using a 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.

[0024] Note that 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 fabricate a substrate 2 with a thickness of 1 nm or less. However, if the thickness of the substrate 2 is very thin, it may be difficult to handle without damaging it. Therefore, 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. In the case of a laminated structure, the solid membrane or two-dimensional material is laminated on a support plate having a hole larger than the nanopore 3, and the nanopore 3 is formed in the solid membrane or two-dimensional material.

[0025] 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, which changes depending on the size of the nucleic acid encapsulated when a capsid passes through the nanopore 3. In the technical field of measuring the ionic current when a target substance 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 target substance. This is because the thinner the substrate 2 is compared to the size of the target substance, the more information about the target substance's passage through the nanopore 3 is reflected in the change in ionic current. As a result, the measured change in ionic current reflects, for example, information about the target substance's orientation when it enters the nanopore 3, in addition to size information about the target substance. From this perspective, the thickness of the substrate 2 can be 0.3 nm or more.

[0026] On the other hand, the acquisition method disclosed in the present application only requires obtaining 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 due to the encapsulation of nucleic acid passes through the nanopore 3. Therefore, in the device disclosed in the present application, the thickness of the substrate 2 may be smaller than or larger than the size of the capsid. By making the thickness of the 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, sensitivity can be increased by making the thickness of the substrate 2 larger than the size of the capsid. Furthermore, the thicker the substrate 2, the more convenient its manufacture and handling become.

[0027] Although capsids vary depending on the type, they 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 capsid size, sensitivity decreases. Therefore, the upper limit of the thickness of the substrate 2 may 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.

[0028] The thickness of substrate 2 may be determined in relation to the size of the capsid to be measured. While not limited thereto, when the size of a capsid is taken as 1, the lower limit of the thickness of substrate 2 is 0.015 times or more, and from the viewpoint of measuring the size information of the nucleic acid encapsulated 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 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.

[0029] The nanopore 3 is formed to penetrate the substrate 2 from the first surface 21 of the substrate 2 toward the second surface 22, which is the surface opposite the first surface 21. As described above, the device disclosed in the present application is only required to acquire information regarding the size change of the capsid due to the encapsulation of nucleic acid. Therefore, the size of the nanopore 3 is larger than the capsid, but may be appropriately adjusted so as not to be too large. When the capsid size is 1, the lower limit may be, but is not limited to, 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 may 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.

[0030] As described above, capsids vary in size depending on the type, but are generally about 20 nm to about 100 nm. 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.

[0031] Note that when the cross-sectional shape of the nanopore 3 parallel to the first surface 21 is circular, the size of the nanopore 3 refers to its 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 refers to the diameter of the inscribed circle of the cross-section. When a material other than a lipid bilayer membrane is used as the material of the substrate 2, the nanopore 3 may be formed by etching or the like, as shown in the examples described below. Furthermore, the nanopore 3 may 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 sizes. For example, the nanopore 3 may be formed so that it widens from the first surface 21 to the second surface 22 within the substrate 2. In this case, the size of the nanopore 3 refers to the size of the first opening 31 formed in the first surface 21 (the size of the smaller opening).

[0032] 1 shows an example in which one nanopore 3 is formed in the substrate 2, but 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 accuracy of capsid measurement. The extent to which the distance between adjacent nanopores 3 is set is described in detail in WO 2020 / 138021, and therefore a detailed description thereof will be omitted in the disclosure of this application. The matters described in WO 2020 / 138021 are incorporated herein by reference.

[0033] 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, and Al2O3.

[0034] The first chamber 5 and the second chamber 6 are formed to sandwich the nanopore 3, and there are no particular limitations as long as they are formed so that capsids introduced into the first chamber 5 can pass through the nanopore 3 to the second chamber 6, or so that capsids introduced into the second chamber 6 can pass through the nanopore 3 to the first chamber 5. For example, the first chamber member 51 and the second chamber member 61 can be fabricated separately and then adhered to the substrate 2 in a liquid-tight manner. Alternatively, a roughly rectangular box member with one open side 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 this case, the first chamber member 51 and the second chamber member 61 do not mean separate components, but rather refer to parts of a box member separated by the substrate 2. Although not shown, the first chamber member 51 and the second chamber member 61 may be formed with holes, as necessary, for filling and discharging the electrolyte and capsid solution and for inserting electrodes and / or leads.

[0035] (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 the configuration of the acquisition device 1a according to the embodiment.

[0036] 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 above "Embodiment of Device 1." Therefore, to avoid redundancy, a detailed description of the device 1 will be omitted.

[0037] 2 shows a first electrode 52 formed in contact with the first electrolytic solution in the first chamber 5, a second electrode 62 formed in contact with the second electrolytic solution in the second chamber 6, and a power supply 54 that applies a voltage between the first electrode 52 and the second electrode 62, but the first electrode 52, the second electrode 62, and the power supply 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 supply 54 are optional components of the acquisition device 1a.

[0038] Additionally, the acquisition device 1a may optionally include a display unit 9 for displaying the results of the analysis by the analysis unit 8, a program memory 10 in which programs for operating the analysis unit 8 and the display unit 9 are stored in advance, and a control unit 11 for reading and executing the programs stored in the program memory 10. The programs may be stored in advance in the program memory 10, or may be recorded on a recording medium and stored in the program memory 10 using an installation means.

[0039] The first electrode 52 and the second electrode 62 can be formed from a known conductive metal such as aluminum, copper, platinum, gold, silver, silver / silver chloride, or titanium. Figure 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 serving as a negative pole and the second electrode 62 side serving as a positive pole. Alternatively, the first electrode 52 side may serve as a positive pole and the second electrode 62 side as a negative pole. Which side of the first electrode 52 or the second electrode 62 is to be positive can be determined appropriately depending on the charge possessed by the capsid, which will be described later.

[0040] There are no particular limitations on the first electrode 52 as long as it is formed in a location that contacts the first electrolytic solution 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 the lead 53. Still alternatively, 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.

[0041] Like 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 electrolytic solution 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 the lead 63. Still alternatively, the second electrode 62 may be disposed so as to penetrate the second chamber member 61 through a hole formed in the second chamber member 61.

[0042] 2, the first electrode 52 is connected to a power supply 54 and a ground 55 via a lead 53. The second electrode 62 is connected to a measurement unit 7 and a ground 64 via a lead 63. In the example shown in FIG. 2, the power supply 54 is connected to the first electrode 52 side and the measurement unit 7 is connected to the second electrode 62 side, but the power supply 54 and the measurement unit 7 may be provided on the same electrode side.

[0043] There are no particular limitations on the power supply 54 as 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 as long as it can measure over time the ion current generated when 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.

[0044] The measuring unit 7 is not particularly limited as long as it can measure the amount of change in ion current when the capsid S passes through the nanopore 3, and examples thereof include a known ammeter.

[0045] When the capsid S passes through the nanopore 3, the ionic current flowing through the nanopore 3 is blocked by the capsid S, causing a change in the ionic current flowing through the nanopore 3. The analysis unit 8 analyzes the change in the ionic current measured by the measurement unit 7. Therefore, by performing data analysis in the analysis unit 8 based on the measured change in the ionic current, information regarding the size of the nucleic acid encapsulated in the capsid S can be analyzed. As described above, the larger the size of the encapsulated nucleic acid (the longer the nucleic acid), the larger the size of the capsid S. Therefore, (1) the change in the ionic current when the capsid S before introducing the nucleic acid passes through the nanopore 3 is measured in advance, (2) the change in the ionic current when the capsid S, which is the object to be measured, passes through the nanopore 3 is measured, and (3) by comparing the change in the ionic current measured in advance with the change in the ionic current of the object to be measured, (4) it is possible to analyze the presence or absence of the nucleic acid to be encapsulated in the capsid S, in other words, whether the intended nucleic acid has been introduced into the capsid S.

[0046] In addition, (1) the change in ionic current when capsid S before nucleic acid introduction 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, the object to be measured, passes through nanopore 3, and (3) by comparing the change in ionic current measured in advance 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.

[0047] 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 nucleic acid introduction passes through the nanopore 3, or (b) the amount of change in ion current when the capsid S before nucleic acid introduction and capsids 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. Use of machine learning is expected to improve the accuracy of analysis.

[0048] The display unit 9 may be any known display device such as a liquid crystal display, plasma display, or organic EL display as long as it can display the measured change in ion current and the results of analysis by the analysis unit 8. The program memory 10 is not particularly limited as long as it can store programs for operating the analysis unit 8 and 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.

[0049] The device 1 and the 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 features may be used within the scope of the technical concept disclosed in this application.

[0050] (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 of the acquisition method according to the embodiment, but is an optional additional component.

[0051] In the capsid passage step (ST1), a voltage is applied to the first electrolytic solution filled in the first chamber 5 and the second electrolytic solution filled in the second chamber 6, thereby causing the capsid S contained in the first chamber 5 to pass through the nanopore 3 toward the second chamber 6, or causing the capsid S contained in the second chamber 6 to pass through the nanopore 3 toward the second chamber 6. The first electrolytic solution and the second electrolytic solution may be any solution that allows current to flow between the first electrode 52 and the second electrode 62, and may be a solution containing ions (electrolyte solution) such as TE buffer, PBS buffer, HEPES buffer, or KCl aqueous solution known in the art.

[0052] When performing the capsid passage step, a substance with a viscosity greater than that of water may be added to increase the viscosity of the first electrolytic solution and / or the second electrolytic solution. Increasing the viscosity of the first electrolytic solution and / or the second electrolytic solution can lengthen the time it takes for the capsid S to pass through the nanopore 3. The viscosities of the first electrolytic solution and the second electrolytic solution may be the same or different, as long as they can lengthen the time it takes for the capsid S to pass through the nanopore 3. When the viscosities of the first electrolytic solution and the second electrolytic solution are the same, the conditions that the capsid S receives from the electrolytic solution before and after passing through the nanopore 3 (e.g., the resistance of the electrolytic solution that the nanopore 3 receives) are the same. Therefore, while the viscosities of the first electrolytic solution and the second electrolytic solution may be different, it is preferable that the difference in viscosity is not too large. It is more preferable that the viscosities of the first electrolytic solution and the second electrolytic solution are the same.

[0053] 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. Examples of lower limits of the viscosity of the first electrolytic solution and / or the second electrolytic solution after adding the substance include, but are not limited to, 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, and 50 mPa·s or more at about 20°C. On the other hand, examples of the upper limit include 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, and 100 mPa·s or less. By increasing the viscosity of the first electrolytic solution and / or the second electrolytic solution, the time it takes for the capsid S to pass through the nanopore 3 when the capsid passing step is carried out is extended. Therefore, there is an effect 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.

[0054] When the first chamber 5 is already filled with the first electrolyte solution and the second chamber 6 is already filled with the second electrolyte solution, capsids are introduced into the first chamber 5 or the second chamber 6, and the capsid passage step (ST1) is carried out.

[0055] If the first chamber 5 of the acquisition device 1a is not filled with the first electrolyte solution and the second chamber 6 is not filled with the second electrolyte solution, a preparation step can be performed before carrying out the capsid passing step (ST1). The preparation step can be performed by the following procedure. (1) Fill the first chamber 5 with the first electrolyte solution and fill the second chamber 6 with the second electrolyte solution. A liquid junction is established between the first chamber 5 and the second chamber 6 via the nanopore 3. (2) Place the capsid S into the first chamber 5 or the second chamber 6. Note that the steps (1) and (2) above may be performed separately, or an electrolyte solution already containing the capsid S may be placed into the first chamber 5 or the second chamber 6.

[0056] In the capsid passage step (ST1) shown in Figure 3, by passing current through the first electrode 52 placed in the first chamber 5 and the second electrode 62 placed in the second chamber 6, the capsid S passes through the nanopore 3 formed in the substrate 2 by electrophoresis in addition to normal diffusion.

[0057] In the ion current measurement step (ST2), changes in the ion current generated by the application of current are measured over time by the measurement unit 7. Therefore, when the capsid S passes through the nanopore 3, large changes in the ion current corresponding to the size of the capsid S can be measured.

[0058] The 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 encapsulated in capsid S. Therefore, the acquisition method disclosed in the present application has the following effects.

[0059] (1) By measuring the size of the capsid, information regarding the size of the nucleic acid encapsulated in the capsid can be obtained. Therefore, information regarding the size of the encapsulated nucleic acid can be obtained without destroying the capsid. (2) When producing a viral formulation, 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 formulation will not only be substantially ineffective, but there is also concern about side effects caused by immune responses. Information regarding the size of the nucleic acid encapsulated in the capsid is very useful for determining whether a viral formulation used in gene therapy has been properly manufactured. Therefore, the acquisition method of the present application enables non-destructive quality control of viral formulations. (3) The acquisition method disclosed in the present application does not require large amounts of samples because it can measure ion current for each capsid. Therefore, it is useful not only for quality control after the production of a viral formulation, but also for spot checks during production.

[0060] Next, the analysis step (ST3), which is an optional additional configuration of the acquisition method, will be described. The analysis step (ST3) analyzes information about the nucleic acid encapsulated in the capsid from the change in ion current measured in the ion current measurement step (ST2). The analysis content, similar to the analysis unit 8 of the above-mentioned "embodiment of the acquisition device 1a," includes (a) analyzing the presence or absence of the nucleic acid to be encapsulated 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 encapsulated in the capsid.

[0061] The analysis step (ST3) may be performed manually or may be processed using a computer or the like. For example, when analyzing manually, based on the change in ion current (the difference between the baseline of the ion current and the peak value of the change in ion current), (a) the change in ion current of a capsid that does not encapsulate nucleic acid and the change in ion current of a capsid that encapsulates nucleic acid can be referenced to analyze whether or not nucleic acid has been introduced into the capsid S to be measured. In addition, (b) the change in ion current when capsid S before nucleic acid introduction and capsid S encapsulating nucleic acids of different lengths pass through the nanopore 3 can be measured in advance, and the length of the encapsulated nucleic acid and the change in ion current can be graphed based on the measurement results, and the change in ion current of the capsid to be measured can be referenced to the graph to calculate the length of the nucleic acid encapsulated in the capsid S.

[0062] When the analysis is performed using a computer, the above analysis may be automated. Furthermore, the accuracy of the analysis may be improved by using machine learning or the like.

[0063] When the acquisition method according to the embodiment includes an analysis step (ST3), in addition to the effects described above in (1) to (3), the following effect is achieved. (4) By performing the analysis step, the presence or absence of nucleic acid encapsulated in the capsid and the length of the encapsulated nucleic acid can be more accurately determined. Therefore, when used for quality control of a virus formulation, more detailed quality control can be achieved. In particular, by measuring in advance the amount of change in ionic current when capsids S encapsulating nucleic acids of different lengths pass through the nanopore 3, it is possible to determine how the nucleic acid to be introduced is cleaved, which enables feedback to the manufacturing process, etc.

[0064] The acquisition device 1a described above also achieves the effects (1) to (4) achieved by the acquisition method, and the device 1 achieves the effect of being usable for the acquisition method and acquisition device 1a that achieve the effects (1) to (4) achieved by the acquisition method.

[0065] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the disclosure of the present application.

[0066] 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. Subsequently, electron beam resist (ZEP520A, Zeon) was spin-coated onto the SiNx film, prebaked at 180°C using a hot plate, and then a circular pattern was written by electron beam lithography and developed. The resulting residual resist layer was used as a mask to form a 30-mm x 30-mm chip. ３ A nanopore with a diameter of 66 nm was opened by reactive ion etching using an etching gas. Finally, the substrate was immersed in N,N-dimethylformamide overnight and washed with ethanol / acetone to fabricate a nanopore chip with a nanopore formed in the SiNx substrate.

[0067] Next, one surface of the fabricated nanopore chip was coated with a polyimide layer. This was done to reduce the capacitance of the nanopore chip and reduce noise. More specifically, a photosensitive polyimide precursor (PN-2010, Toray Industries, Inc.) was spin-coated onto the nanopore membrane. After baking, the nanopore membrane was irradiated with ultraviolet light using LED lithography and developed, dissolving the 5 μm diameter and 5 μm thick polyimide around the nanopore.

[0068] 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 an SU-8 mold at 80°C. The mold had an I-shaped pattern with submillimeter width and height to form trenches in the polymer block that served as channels for the capsid solution to flow into the nanopore. Three holes were punched into the block prior to sealing. The nanopore chip and polymer blocks (first and second chamber members) were then exposed to oxygen plasma for surface activation, and then bonded together to fabricate Device 1.

[0069] Fabrication of Acquisition Device 1a for Performing the Acquisition Method: Ag / AgCl rods were used as the first and second electrodes, inserted into the first and second chambers through holes on both sides 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, digitizing it using a high-speed digitizer (PXI-5922, NI), and storing it on a solid-state drive (PXI-8267, NI) at a sampling rate of 1 MHz under an applied voltage Vb.

[0070] [Implementation of Acquisition Method] Example 2 (1) Preparation of AAV Empty Capsids A 293EB cell line (Tomono T., et al., "Highly efficient ultracentrifugation-free chromatographic purification of adenovirus E1a, E1b, and Bcl-xL) expressing adenovirus E1a, adenovirus E1b, and Bcl-xL was cultured in a 550 mL multi-shelf flask (HYPERFlask, Corning, Corning, NY, USA) containing Dulbecco's modified Eagle's medium (DMEM high sugar content, FUJIFILM Wako, Osaka, Japan) and 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA). recombinant AAV serotype 9, Mol. Ther. Methods Clin. Dev. 11, 180-190 (2018)) at a density of 40,000 cells / cm ２ The cells were seeded at a density of 100 μg / flask and cultured for 3 days. Subsequently, transfection was performed using pCAX (a 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 a helper plasmid (65 μg / flask) in DMEM (Nacalai Tesque, Nakagyo-ku, Kyoto) containing 2 mM L-alanyl-L-glutamine solution (100x). The cells were transfected with polyethyleneimine max (520 μg / flask) (Polysciences, Warrington, PA, USA). 10 days after transfection, the culture supernatant was collected and diluted with endonuclease (Kaneka, Minato-ku, Tokyo, Japan) at 18.5 U / mL and MgCl . ２ (Nacalai Tesque) was added to the mixture at 5 mM, and the mixture was treated at 37° C. for 30 minutes. Next, the rAAV was purified using an AAVpro® Concentrator Kit (Takara, Japan) or a PhyTip® column CaptureSelect® AAVX (PhyNexus, USA).

[0071] 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).

[0072] (2) Preparation of AAV capsids encapsulating nucleic acids 293EB cell lines expressing adenovirus E1a, adenovirus E1b, and Bcl-xL were cultured at 40,000 cells / cm 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). ２ The cells were seeded at a density of 100 μg / flask and cultured for 3 days. Subsequently, transfection was performed using pAAV-ZsGreen1 (2599 bases, Takara Bio #6231) or pAAV-ZsGreen1-short (1451 bases, pAAV-ZsGreen1 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). The cells were transfected with polyethyleneimine Max (520 μg / flask) (Polysciences, Warrington, PA, USA). Ten days after transfection, the culture supernatant was collected and treated with endonuclease (Kaneka, Minato-ku, Tokyo, Japan) at 18.5 U / mL and MgCl ２ (Nacalai Tesque) was added to the mixture at 5 mM, and the mixture was treated at 37° C. for 30 minutes. Next, the rAAV was purified using an AAVpro® Concentrator Kit (Takara, Japan) or a PhyTip® column CaptureSelect® AAVX (PhyNexus, USA).

[0073] (3) Measurement Conditions 1.37 M NaCl (10x PBS Buffer (-), model number 314-90185, manufactured by Nippon Gene Co., Ltd.) was used for the first and second electrolytic solutions. The first electrolytic 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 electrolytic 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 served as the positive pole and the second electrode 62 served as the negative pole, and the ionic current Iion was measured.

[0074] Figure 4 shows the change in ionic current when an empty capsid containing no nucleic acid (indicated as "AAV9 / empty"), a capsid encapsulating 1451 bases of DNA (indicated as "AAV9 / 1.5 kbDNA"), and a capsid encapsulating 2599 bases of DNA (indicated as "AAV9 / 2.6 kbDNA") pass through the nanopore 3. The graph in Figure 4 is an average of 100 points of raw data measured at 1 MHz (adjacent average).

[0075] FIG. 5 is a plot of Ip, which is the change in ionic current when individual capsids pass through the nanopore. Note that the label "AAV9 / 1.5kb" is omitted from FIG. 5. In the example shown in FIG. 5, the change in ionic current (Ip) is defined as the difference between the baseline (base), which is the value of the ionic current measured when no capsid has entered the nanopore 3, and the average value (Av) of the ionic current value during the time td (the time from when the ionic current has suddenly dropped from the baseline to just before it begins 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 ionic current (Ip) may be defined in other ways as long as it reflects the size information of the nucleic acid encapsulated in the capsid. For example, the change in ionic current (Ip) may be the difference between the average value of the lowest 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 baseline and the average value of the highest values ​​of multiple peaks measured during the td period (the upper side of the waveform for the td period shown in Figure 5). Figure 6 shows the average values ​​of the change in ionic current Ip calculated from the plot in Figure 5, which were 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.

[0076] As shown in Figure 6, the change in measured ionic current varied depending on the size of the nucleic acid encapsulated, so we investigated capsid size. The results are shown in Figure 7. More specifically, empty capsids (Empty) and capsids encapsulating 2,599 bases of DNA (Full) were photographed using a transmission electron microscope (Figures 7a and 7b). As shown by the arrows in Figures 7a and 7b, the vector diameter (dvec) indicated by the arrows for each capsid was measured using ImageJ software (scale bar: 20 nm). Figure 7c shows the distribution of dvec obtained from 2,052 Empty images and 1,576 Full images. The average diameter of the Empty capsids was 22.88 nm, while the average diameter of the Full capsids was 25.84 nm, revealing a size difference of approximately 3 nm, Δdvec, between the two (p<0.0001). These results indicate that the capsid size increases with increasing nucleic acid content, resulting in differences in the amount of change in the measured ionic current. Therefore, it was confirmed that the difference in the amount of change in ionic current reflects the size information of the encapsulated nucleic acid.

[0077] FIG. 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 ion current (Ip) on the vertical axis. Note that, unlike FIGS. 5 and 6, the change in ion current (Ip) shown in FIG. 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 FIG. 8, there are three plots, but by increasing the number of plots, it is possible to create a more accurate approximation curve. By creating a graph like that shown in FIG. 8 in advance, the length of the nucleic acid encapsulated in the capsid can be calculated from the measured change in ion current Ip.

[0078] 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 electrolytic solution and the second electrolytic solution to a concentration of 30 vol % (viscosity: approximately 3.6 mPa·s) and the NaCl concentration was set to 0.96 M.

[0079] 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, increasing the viscosity of the first and second electrolyte solutions improved the accuracy of distinguishing capsids containing different sizes of nucleic acid. In the example shown in FIG. 9A without glycerol added, the resolution between empty capsids containing no nucleic acid (AAV9 empty) and capsids containing 2599 bases of DNA (AAV9, 2.6 kb ss DNA) was 0.4, while the resolution in the example shown in FIG. 9B with glycerol added was 1.13. These results confirmed that adding a substance with a viscosity greater than water to the first and second electrolyte solutions improved the accuracy of distinguishing capsids containing nucleic acids of different sizes.

[0080] The method disclosed in the present application provides information about the size of the nucleic acid encapsulated in the capsid, which is useful for the medical industry as it can be used to control the quality of virus preparations, etc.

[0081] DESCRIPTION OF SYMBOLS 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

1. A method for obtaining information relating to the size of a nucleic acid contained in a virus-derived capsid using a nanopore device, the nanopore device comprising: a substrate having a first surface and a second surface; a nanopore penetrating from the first surface to the second surface through which the capsid passes; a first chamber member; and a second chamber member, the first chamber member forming a first chamber filled with a first electrolyte solution at a surface of the first surface including at least the first opening of the nanopore, and the second chamber member forming a second chamber filled with a second electrolyte solution at a surface of the second surface including at least the second opening of the nanopore, the obtaining method comprising: a capsid passing step in which the capsid contained in the first electrolyte solution or the second electrolyte solution passes through the nanopore; and an ion current measuring step in which a change in ion current when the capsid passes through the nanopore is measured by applying a voltage to the first electrolyte solution filled in the first chamber and the second electrolyte 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 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.

3. The method according to claim 1, wherein the capsid becomes larger in size as the size of the nucleic acid encapsulated therein increases.

4. The method according to claim 3, further comprising an analysis step following the measurement step, in which the presence or absence of a nucleic acid to be encapsulated in the capsid is analyzed based on the amount of change in ion current measured in the measurement step.

5. The method according to claim 3, further comprising an analysis step following the measurement step, in which the size of the nucleic acid encapsulated in the capsid is calculated based on the change in ion current measured in the measurement step.

6. 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.

7. 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.

8. 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.

9. A nanopore device used in an apparatus for obtaining information related to the size of nucleic acid contained in a virus-derived capsid, the nanopore device comprising: a substrate having a first surface and a second surface; a nanopore that penetrates from the first surface to the second surface and through which the capsid passes; a first chamber member; and a second chamber member, wherein the first chamber member forms a first chamber filled with a first electrolyte solution between a surface of the first surface including at least the first opening of the nanopore, and the second chamber member forms a second chamber filled with a second electrolyte solution between a surface of the second surface including at least the second opening of the nanopore, and the thickness of the substrate is greater than the size of the capsid.

10. The nanopore device according to claim 9, wherein the size of the nanopore is at least 1.2 times the average particle diameter of the capsid.

11. An apparatus for acquiring information related to the size of nucleic acid contained in a virus-derived capsid, the apparatus comprising: a nanopore device; a measurement unit; and an analysis unit; the nanopore device comprising: a substrate having a first surface and a second surface; a nanopore that penetrates from the first surface to the second surface and through which the capsid passes; a first chamber member; and a second chamber member; the first chamber member forms a first chamber filled with a first electrolyte solution between a surface of the first surface including at least the first opening of the nanopore, and the second chamber member forms a second chamber filled with a second electrolyte solution between a surface of the second surface including at least the second opening of the nanopore, the measurement unit measures the change in ion current when the capsid passes through the nanopore, and the analysis unit analyzes the presence or absence of nucleic acid to be encapsulated in the capsid based on the amount of change in ion current measured by the measurement unit and / or calculates the size of the nucleic acid encapsulated in the capsid.

12. The acquisition apparatus according to claim 11, wherein the nanopore device is a nanopore device according to claim 8 or 9.

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