Method and device for producing nanopores

Abstract

One embodiment of the present invention provides a method for processing a thin film, said method comprising: (a) provision of a thin film; (b) irradiation of the thin film with an electron beam; (c) observation of the thin film and determination of whether pores satisfying a prescribed condition have been formed therein; and (d) repetition of (b) and (c) if it is determined in (c) that the pores satisfying the prescribed condition have not been formed.

Description

Method and apparatus for fabricating nanopores

[0001] The present disclosure relates to methods and apparatus for fabricating nanopores.

[0002] Nanometer-sized pores (nanopores) formed in thin films of solid materials have been shown to be useful for sensing substances that pass through them, and are used in various applications. For example, nucleic acids (DNA, RNA) can be passed through a nanopore and their nucleotide sequences can be determined by measuring the change in the electrical signal that occurs during the passage of the nucleic acid.

[0003] When a thin film containing a nanopore is immersed in a conductive fluid and a voltage potential is applied across it, an ionic current passing through the nanopore can be observed. This ionic current is determined by the effective cross-sectional area of ​​the nanopore. When a nucleic acid molecule passes through the nanopore, the molecule partially blocks the nanopore, changing the magnitude of the current passing through the nanopore. Because each nucleotide has a unique shape and size, the ionic current changes depending on the nucleotide. By reading this change, the sequence of the nucleotide can be determined.

[0004] Nanopore sensing is therefore sensitive to the size (diameter) and shape of the nanopore, and therefore the process of fabricating nanopores in thin films must be controlled at the nanometer level.

[0005] The thickness of the membrane affects sensitivity. Nucleotides have a size on the nanometer level in the direction of movement. For a thick membrane, multiple nucleotides are present inside the nanopore at any given time along the membrane's thickness. Therefore, using a nanometer-sized membrane improves readout accuracy.

[0006] MoS with small and uniform thickness ２ It has also been reported that nanopores can be formed in a monolayer membrane using transmission electron microscopy (TEM) or electrochemical reaction (ECR) (Non-Patent Document 1).

[0007] However, thin films with nanometer-level thicknesses are sensitive to the effects of manufacturing processes. For example, the thin film can be deformed or destroyed by mechanical or thermal stress or impact due to lithography processes, electron beam irradiation, etc. For example, in the case of electron beam irradiation, the size of the nanopore can be controlled by the irradiation time, but there have been problems with controlling the size and shape.

[0008] To solve these problems, a technique is needed to form nanopores with high precision in solid thin films with nanometer-level thicknesses.

[0009] Graf, M. , Lihter, M. , Thakur, M. et al. , Fabrication and practical applications of molybdenum disulfide nanopores, Nat Protoc 14, 1130-1168 (2019)

[0010] According to one aspect of the present disclosure, there is provided a method for processing a thin film, comprising: (a) providing a thin film; (b) irradiating the thin film with an electron beam; (c) observing the thin film to determine whether pores satisfying predetermined conditions have been formed; and (d) repeating (b) and (c) if it is determined in (c) that pores satisfying the predetermined conditions have not been formed.

[0011]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0012] 1 shows a flowchart of a method for processing a thin film according to an embodiment of the present disclosure. FIG. 2 shows a flowchart of a method for processing a thin film according to an embodiment of the present disclosure. FIG. 3 shows a flowchart of a method for processing a thin film according to an embodiment of the present disclosure. FIG. 4 shows a schematic diagram of the vicinity of a pore to explain the modification of the pore shape according to an embodiment of the present disclosure. FIG. 5 shows a Ronchigram image indicating the formation of a nanopore in an example. FIG. 6 shows an electron microscope image of a nanopore fabricated in an example. FIG. 7 shows an electron microscope image of a nanopore fabricated in an example. FIG. 8 shows a schematic diagram to explain the configuration of a nanopore fabrication system according to an embodiment of the present disclosure.

[0013] 1 shows a flowchart S100 of a method for processing a thin film according to one embodiment of the present disclosure. In step S101, a thin film is provided. In step S111, the thin film is irradiated with an electron beam. In step S112, the region of the thin film irradiated with the electron beam is observed. In step S113, it is determined whether a nanopore satisfying predetermined conditions has been formed in the thin film. If it is determined that the predetermined nanopore has been formed, the process ends. If it is determined that the predetermined pore has not been formed, steps S111 and S112 are repeated. That is, substantially the same region is irradiated with the electron beam again (S111), the region irradiated with the electron beam is observed (S112), and it is determined whether a nanopore satisfying predetermined conditions has been formed in the thin film (S113). This process is repeated until it is determined that the predetermined nanopore has been formed.

[0014] FIG. 2 shows a flowchart S200 of a method for processing a thin film according to one embodiment of the present disclosure. In step S201, a thin film is provided. In step S210, the thin film is irradiated with a first electron beam to form a nanopore. This forms the first through-hole in the irradiated region. In step S221, a thin film region near the nanopore and a thin film region at the edge of the pore are irradiated with a second electron beam. In step S222, the shape of the nanopore is observed. In step S223, it is determined whether the shape of the nanopore satisfies predetermined conditions. If it is determined that the shape of the nanopore satisfies the predetermined conditions, the process ends. If it is determined that the shape of the nanopore does not satisfy the predetermined conditions, steps S221 and S222 are repeated. That is, the edge of the pore is again irradiated with the second electron beam (S221), the region irradiated by the electron beam is observed (S222), and it is determined whether the shape of the nanopore satisfies the predetermined conditions (S223). This process is repeated until it is determined that the shape of the nanopore satisfies the predetermined conditions. The illuminated area may be changed with each iteration, allowing for better control of the shape, for example.

[0015] 3 shows a flowchart S300 of a method for fabricating a thin film according to one embodiment of the present disclosure. In step S301, a thin film is provided.

[0016] In step S311, a first electron beam is irradiated onto the region of the thin film where a nanopore is to be formed. In step S312, the region of the thin film irradiated with the electron beam is observed. In step S313, it is determined whether a nanopore has been formed in the thin film. If it is determined that a nanopore has not been formed, steps S311 and S312 are repeated. That is, substantially the same region is irradiated again with the first electron beam (S311), the region irradiated with the electron beam is observed (S312), and it is determined whether a nanopore has been formed in the thin film (S313). This is repeated until it is determined that a predetermined nanopore has been formed. This forms a through-hole for the first time in the irradiated region. Moreover, the initially formed through-hole can have a good shape.

[0017] In some embodiments, the process may end if the formed nanopore meets predetermined conditions (step not shown), or the pore shape may be adjusted (steps S321-S323) as shown in FIG.

[0018] In step S321, the thin film region near the nanopore and the thin film region at the edge of the pore are irradiated with a second electron beam. In step S322, the shape of the nanopore is observed. In step S323, it is determined whether the shape of the nanopore satisfies predetermined conditions. If it is determined that the shape of the nanopore satisfies the predetermined conditions, the process ends. If it is determined that the shape of the nanopore does not satisfy the predetermined conditions, steps S321 and S322 are repeated. That is, the edge of the pore is again irradiated with the second electron beam (S321), the region irradiated by the electron beam is observed (S322), and it is determined whether the shape of the nanopore satisfies the predetermined conditions (S323). This is repeated until it is determined that the shape of the nanopore satisfies the predetermined conditions. The irradiation region may be changed each time. This allows, for example, better adjustment of the shape.

[0019] In some embodiments, the first electron beam used to form the initial pore and the second electron beam used to shape the pore may be electron beams with the same conditions, hi some embodiments, the first electron beam used to form the initial pore and the second electron beam used to shape the pore may be electron beams with different conditions.

[0020] <Thin Film> As used herein, the term "thin film" generally refers to a material or component that is formed into a film at least in the region including and adjacent to the pores, and has a film thickness that is sufficiently large relative to the size of the pores in the film plane direction. The thin film may be a so-called two-dimensional (2D) material. In some embodiments, the thin film may include an inorganic material. In some embodiments, the thin film may include an organic material. In some embodiments, the thin film may be made of substantially a single material. In some embodiments, the thin film may be made of multiple materials, layers, or components.

[0021] Thin film materials include, but are not limited to, graphene family, Xenes (group IV, V, VI graphene-like two-dimensional materials), chalcogenides, oxides, and the like.

[0022] The chalcogenide may be a transition metal chalcogenide. The chalcogenide may be a monochalcogenide (MX), a dichalcogenide (MX ２ , TMDC), trichalcogenides (MX ３ ), and tetrachalcogenides (MX ４ M represents a transition metal such as molybdenum (Mo), tungsten (W), niobium (Nb), or tantalum (Ta), and X represents a chalcogen atom selected from sulfur (S), selenium (Se), and tellurium (Te). The chalcogenide may be Bi ２ Te ３ , Bi ２ Se ３ , and Sb ２ Te ３ It may also be a topological insulator such as

[0023] The thin film is MoS ２ The thin film may be a monolayer (two-dimensional material) of a chalcogenide such as MoS. The thin film may have a monolayer at least in the pore region. The thin film may be, for example, MoS. ２ Such monolayers can be fabricated from bulk crystals using mechanical exfoliation (ME) with Scotch tape, chemical vapor deposition (CVD), mist CVD, molecular beam epitaxy (MBE), or other known or emerging methods.

[0024] In some embodiments, the number of atomic layers in the region of interest, whether the region of interest in the thin film is a monolayer, etc. can be measured or confirmed using Raman spectroscopy. The thickness of the thin film can be measured or confirmed using, for example, but not limited to, a transmission electron microscope (TEM), an atomic force microscope (AFM), an optical microscope, etc.

[0025] Electron Beam The electron beam may be generated by an electron microscope. The electron beam may be generated and directed toward the thin film using a transmission electron microscope (TEM), a scanning electron microscope (SEM), a scanning transmission electron microscope (STEM), or other electron gun. The electron beam may be a focused electron beam.

[0026] The electron beam may have a beam diameter smaller than the desired pore size (diameter or minimum diameter) when irradiating the thin film. The electron beam diameter may be defined by the so-called spot size of the electron microscope. Other definitions of the electron beam diameter may also be used. The electron beam diameter may be 50 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2.8 nm, 2.6 nm, 2.5 nm, 2.4 nm, 2.3 nm, 2.2 nm, 2.1 nm, 2.0 nm, 1.9 nm, 1.8 nm, 1.7 nm, 1.6 nm, 1.5 nm, 1.4 nm, 1.3 nm, 1.2 nm, 1.1 nm, 1.0 nm, 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, etc.

[0027] The acceleration voltage of the electron beam source may be 200 kV, 160 kV, 120 kV, 100 kV, 80 kV, 60 kV, 50 kV, 40 kV, 30 kV, 20 kV, 10 kV, 9 kV, 8 kV, 7 kV, 6 kV, 5 kV, 4 kV, 3 kV, 2 kV, 1 kV, etc.

[0028] The current of the electron beam is, for example but not limited to, 0.5 nA (1 nm probe).

[0029] Although this disclosure primarily describes electron beam irradiation, in some embodiments, irradiation of thin films may also use other energy beams, such as particle beams (e.g., ion beams, focused ion beams, focused atomic beams, etc.).

[0030] <Pore> As used herein, "pore" (sometimes referred to as an opening or a through-hole) generally refers to a through-hole formed in the thickness direction of a thin film. "Irradiating the edge of a pore" refers to a portion of the thin film near the pore, and does not mean irradiating a region spaced apart from the pore or irradiating the interior of the pore, but means irradiating the region so that it is geometrically or substantially in contact with or overlaps the pore.

[0031] Repeated Irradiation and Nanopore Observation Irradiation with an electron beam may remove or etch atoms, atomic clusters, molecules, or molecular clusters (hereinafter simply referred to as "atoms") from a thin film. The kinetic energy of the electrons or the heat generated by the electron irradiation ejects atoms from the thin film. This can ablate the thin film, opening pores after a sufficient irradiation time, and / or removing atoms in the thin film near the pores. However, the mechanism for removing thin film atoms need not be the one described above, and other or different explanations may exist.

[0032] In some embodiments, the method may include sequentially irradiating the thin film with an electron beam under certain conditions to form pores in the thin film or observing the shape of the thin film. In some embodiments, the observation and pore formation may be performed simultaneously. For example, the electron beam irradiation may be observed (e.g., in a scanning mode) such that the electron beam irradiation results in pore formation or a change in the shape of the pores.

[0033] In some embodiments, the method may involve irradiating the thin film with an electron beam (focused or unfocused) multiple times to form pores in or shape the thin film. For example, the focus of the focused electron beam may be moved along the thickness of the thin film. When the focus is within the thin film, or just before or just after the focus, substantial processing of the thin film by the electron beam occurs. When the focus is outside the thin film, no processing of the thin film by the electron beam occurs.

[0034] In some embodiments, electron beam irradiation and observation of a field of view including the irradiated area may be repeated for a predetermined location of a predetermined thin film. The conditions for each electron beam irradiation (irradiation time, electron beam current and voltage, electron beam diameter or spot size, focusing angle, etc.) may be set so that a single electron beam irradiation alone does not open a pore or substantially change the shape of an already formed pore. For example, the time (t ｐｏｒｅ ) is calculated in advance, and the time of each electron beam irradiation (other conditions are set to be the same) (t ｉｒｒ ) is smaller than that (t ｉｒｒ <t ｐｏｒｅ ), sufficiently small (t ｉｒｒ <<t ｐｏｒｅ The time (t ｉｒｒ ) may be set. (N × t ｉｒｒ >t ｐｏｒｅ > (N-1) x t ｉｒｒ )

[0035] The number of electron beam irradiations (N) required to open a pore or change the shape of a pore may vary depending on the thickness and other conditions, even for the same thin film material. Therefore, the time required to open a pore or change the shape of a pore (t ｐｏｒｅ ) may not be predetermined. Therefore, by using sufficiently weak electron beam irradiation conditions and repeating the electron beam irradiation multiple times, damage to the thin film can be minimized and / or smaller, more regularly shaped pores can be formed.

[0036] In some embodiments, an image of the irradiated area may be taken between any two electron beam irradiations to confirm pore formation or deformation. An image of the irradiated area may be taken after each electron beam irradiation to confirm pore formation or deformation.

[0037] When using an STEM, an image of the irradiated area may be acquired in a defocused state, for example, in spot illumination mode. In some embodiments, in the spot illumination mode of the STEM, the thin film may be processed while the focused electron beam is focused on the thin film, and the image of the irradiated area may be acquired and / or observed while the focused electron beam is defocused from the thin film. This allows for efficient processing of the thin film and observation of the irradiated area. While the defocused image in spot illumination mode may not be as clear as a bright-field image, it is still of sufficient quality to determine whether the pore has opened and / or whether the pore shape has changed.

[0038] In some embodiments, the image of the illuminated area may be acquired, for example, as a bright field image or a dark field image.

[0039] As used herein, "observing a thin film" may include obtaining and analyzing an image of the thin film, and may also include measuring any property of the thin film.

[0040] <Pore Shape> In some embodiments, to determine the shape of a pore, for example, the pore may be approximated as an ellipse, and the shape of the pore may be represented using its major and minor diameters. For example, the shape of the pore may be represented using two perpendicular diameters. For example, the maximum diameter of the pore and the length of a diameter in a direction non-parallel (e.g., perpendicular) to the maximum diameter (e.g., the minimum or maximum value of the maximum diameter) may be used. The size of the pore may be determined by performing image processing on a microscope image, etc. For example, the average diameter may be calculated based on the area of ​​the pore using image processing software, or values ​​of other geometric parameters may be calculated.

[0041] The pore shaping may be repeated until the pores are within a predetermined range. For example, the ratio of the major axis to the minor axis of an ellipse approximating the pores (ellipticity) may be used. The condition for terminating the pore processing may be that the ratio of the major axis to the minor axis (ellipticity) is 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, or greater. The condition for terminating the pore processing may be that the ratio of the difference between the major axis and the minor axis of the pore and the average diameter is 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or less.

[0042] <Adjusting Pore Shape> A method for adjusting the shape of a pore once it has been formed will be described using FIG. 4 . FIG. 4 is a schematic diagram of an actual image of a thin film having pores. As shown in FIG. 4(A), an initial through-hole or pore 20 is formed in a thin film 10. However, the pore 20 does not have the desired shape (a perfect circle in the example of FIG. 4). In FIG. 4(A), as a simple example, the pore 20 is approximated by an ellipse. The size of the pore 20 is long in one direction (major axis, length 2×a0) and short in the other direction (minor axis, length 2×b0): a0>b0.

[0043] To make the shape of the pore 20 a perfect circle, a portion of the thin film is removed, mainly from the edge in the minor axis direction. The irradiation area 30 is moved to remove the thin film at each position. Focused-defocused irradiation may also be used during this removal (FIG. 4(A)). This allows each portion to be removed gently, enabling highly accurate processing. By performing this type of irradiation in sequence, it is possible to enlarge the minor axis.

[0044] As a result, as shown in Figure 4(B), after shaping, it is possible to realize a nanopore with a target shape close to a perfect circle, with a major axis of 2 x a1 and a minor axis of 2 x b1 (a1 ≒ b1).

[0045] The condition for a perfect circle may be, for example, a predetermined ratio of major axis to minor axis (ellipticity), and the processing may be completed when the ellipticity is exceeded. The condition for a perfect circle may be, for example, specified by a predetermined circularity.

[0046] <Example 1> <1 MoS ２ Preparation of monolayer crystals > Bulk MoS ２ A weak adhesive tape was attached to the single crystal material, and its surface layer was transferred to the weak adhesive tape by mechanical peeling (ME). This was then repeatedly attached to another weak adhesive tape and peeled off, causing cleavage. As a result, MoS was finally formed on the adhesive surface of the weak adhesive tape. ２ Single-layer crystals were produced. ２ The single crystal layer was transferred to a polydimethylsiloxane (PDMS) support formed on a coverslip.

[0047] The obtained MoS ２The thin film was confirmed to be a single-layer crystal by Raman spectroscopy. ２ In thin films, MoS ２ The peak in the Raman spectrum due to the interlayer vibration of atoms (A １ｇ peak) and the peak due to vibration in the plane direction (E １ ２ｇ Peak) and 403.092 cm －１ and 304.316 cm －１ This shows that this MoS ２ It was confirmed that the thin film was a single-layer crystal. ２ The fact that the thin film is a single-layer crystal was also confirmed by the R value of the channel image of an optical microscope, which has a linear relationship with the number of layers. Specifically, the R value of the optical microscope (DSX100, Olympus) was 25 to 30 (exposure time: 50 mS, ISO value: 200). ２ It was confirmed that the thin film was a single-layer crystal.

[0048] As a support material, a free-standing silicon supported on a silicon chip ３ N ４ A film was prepared, and through-holes of 500 nm were formed in it using a focused ion beam (FIB). ３ N ４ The MoS film obtained as described above was applied to the surface of the film so as to close the through-holes. ２ The thin film of single crystals was transferred and the PDMS was removed.

[0049] <2 STEM Conditions> In this example, a STEM (JEM-ARM200F, JEOL) was used. This STEM can be switched between scanning mode and Ronchigram mode. The acceleration voltage is 200 kV. The smallest size probe was used, and the aperture was set to the smallest size. This minimized damage to areas other than the target area.

[0050] Focusing on the target region increases damage near the nanopore. Furthermore, the accuracy of the nanopore's specified size, shape, and other characteristics may be reduced. Therefore, to reduce damage to the target region due to electron beam irradiation, the STEM focus may be set in advance on the target region.

[0051] For example, Si ３ N ４ The electron beam may be focused on a support material such as a membrane and then moved to the target area. ２ The electron beam may be focused at the support material in an unsupported region of a thin film, such as a single-layer crystal, and then moved to the target region of the thin film. ２ The focus is set at the position of the support material in the region where the thin film, such as a single-layer crystal, is not supported, and then the MoS ２ The electron beam may be focused at a position of a support material in an area supporting a thin film such as a single-layer crystal, and then moved to a target area of ​​the thin film. By focusing at multiple locations before irradiating the target area, the electron beam irradiation time required for focusing on the target area can be shortened.

[0052] Various methods can be used for each focusing, and focusing may be performed based on, for example, but not limited to, Ronchigrams, lattice images, diffraction images, diffraction contrast, etc.

[0053] MoS ２ The single-layer crystal was underfocused and the magnification was set to 20 M or more. The scanning mode was changed to Ronchigram mode. A typical image obtained at this time is shown in Figure 5(A). Figure 5(A) shows an image at 500 nm underfocus.

[0054] At the position where the pore should be formed, MoS ２ The monolayer film was focused and kept in focus until the Ronchigram was visible, a typical Ronchigram image of which is shown in Figure 5(B).

[0055] The focus was returned to underfocus to confirm the formation of pores. A typical image obtained at this time is shown in Figure 5(C). Figure 5(C) shows an image at 500 nm underfocus.

[0056] Pores can be identified when the image at the center of the Ronchigram changes or disappears. Pores can be identified in either scanning mode or Ronchigram mode.

[0057] In some embodiments, pore confirmation may be performed in scanning mode. In some embodiments, without switching modes, focus and defocus (underfocus or overfocus) may be repeated in Ronchigram mode. This saves time due to mode switching and reduces damage to the thin film. In other words, in Ronchigram mode, the focus is moved in the Z direction so as to pass through the thin film. When the focus is on the thin film, the thin film is essentially sputtered. By observing the image during defocus, it is possible to confirm whether the first pores have been formed.

[0058] <3. Pore Shaping> <Pore Shaping-1> In some embodiments, electron beam irradiation and observation may be repeated until the pores reach a predetermined size.

[0059] In some embodiments, when the first pore is formed and the pore has not yet reached a predetermined size, the pore is observed and measured, for example, in scanning mode. The pore can be enlarged by irradiating it with an electron beam for observation. FIG. 6(A) shows a TEM image immediately after the first pore was formed. Observation was then continued at a magnification of approximately 10M to 20M. FIG. 6(B) shows a TEM image after 5 minutes of irradiation, and FIG. 6(C) shows a TEM image after 7 minutes of irradiation. When the desired pore size is reached, the electron beam irradiation is stopped, observation is terminated, and pore shaping is completed. In this way, by continuously irradiating the electron beam during observation, the pore size can be accurately controlled.

[0060] <Pore Shaping-2> In some embodiments, the electron beam irradiation position may be changed while irradiation is performed, and the pores may be observed each time. Electron beam irradiation and observation may be repeated until the pores reach a predetermined size.

[0061] In some embodiments, focused-defocused irradiation may be performed while moving the in-plane position of the irradiation relative to the edge of the nanopore so as to approximate a perfect circle in the shape of the nanopore once formed.

[0062] As an example, a nanopore with a target diameter (d = 0.7 nm to 9 nm) was created. Figures 7(A) to 7(H) show STEM images of the created nanopore. The area (A) of the created nanopore was calculated by image processing. Next, the nanopore was approximated as a perfect circle, and its diameter (d ＊ ) was calculated as SQRT(4A / π). The nanopore was also approximated by an ellipse, and its ellipticity (minor axis length / major axis length) was calculated. The results are shown in Table 1.

[0063] Thus, it was demonstrated that nanopores very close to the target conditions (size and shape) could be fabricated. The above is only one experimental example, and it is clear that nanopores with shapes even closer to the target shape can be fabricated. That is, as shown in some examples in the table above, it is possible to fabricate pores with nanometer-sized diameters that have the target diameter and / or excellent ellipticity (close to perfect circle).

[0064] <Nanopore Fabrication System> Figure 8 schematically shows a nanopore fabrication system 100 according to an embodiment of the present disclosure. The nanopore fabrication system 100 includes an electron microscope main body 110 including at least an optical system and a detector 111, and a computing system 130.

[0065] The electron microscope main body 110 is generally a STEM, TEM, or TEM / STEM. The electron microscope main body 110 includes an electronic circuit unit 112, either internally as a part of the electron microscope main body 110 or externally connected to the electron microscope main body 110. The electron microscope main body 110 can irradiate a sample 120 introduced therein with an electron beam probe under desired conditions. The detector 111 can receive electrons that have passed through or been diffracted from the sample 120 and convert them into an output signal. The detector 111 is generally a bright-field (BF) detector, a dark-field (DF) detector, a high-angle annular dark-field (HAADF) detector, an EELS detector, an EDX detector, or the like.

[0066] The electronic circuit unit 112 includes, for example, a converter, a modulator, an amplifier, an arithmetic unit, etc. (not shown) that process the signal output from the detector 111. The electronic circuit unit 112 is incorporated into many electron microscopes. The electronic circuit unit 112 can output information such as the obtained image data and imaging condition data to the outside.

[0067] The calculation system 130 includes a central processing unit (CPU), a memory, etc. The calculation system 130 processes image information of the sample 120 (bright-field image or dark-field image, diffraction image, Ronchigram image, etc.) output from the electronic circuit unit 112, and can determine whether changes have occurred in the thin film due to electron beam irradiation, whether pores have been formed, the shape of the pores, etc.

[0068] Based on these determination results, the calculation system 130 transmits an instruction for the next processing irradiation to the electron microscope main body 110. The calculation system 130 may also transmit irradiation conditions (processing irradiation conditions) for the electron beam probe. The irradiation conditions include, but are not limited to, electron beam conditions, movement of the irradiation position (movement of the sample 120 relative to the optical axis of the electron microscope main body 110), setting of the focal position (focus irradiation, defocus irradiation, etc.), and a focal position movement profile (conditions such as the number of focus irradiations to be performed before the next observation, time, etc.). A driver that actually drives the electron microscope main body 110 based on these irradiation conditions may be incorporated into the calculation system 130 or the electron microscope main body 110, or may be externally connected to them (not shown).

[0069] When the electronic circuit unit 112 receives information indicating that the predetermined processing irradiation has been completed and information on the observed image, the calculation system 130 processes the image information, makes a judgment regarding the pore, and transmits an instruction for the next processing irradiation. This process is continued until it is judged that the predetermined pore has been formed.

[0070] When the pore processing is completed, the user is notified of an instruction to remove the sample 120 from the electron microscope main body 110, or an instruction to remove the sample 120 to the electron microscope main body 110 is issued.

[0071] The electron microscope main body 110 may include a sample holder (not shown) for holding a plurality of samples 120. This allows, for example, a large number of nanopores to be efficiently manufactured by drawing a vacuum once.

[0072] <Example Applications> The nanopores provided in the present disclosure can be used to measure a variety of target substances. In some embodiments, a target substance may be passed through the nanopore, and a change in an electrical signal may be measured.

[0073] When a thin film with a nanopore is immersed in a conductive fluid and a voltage potential is applied across it, an ionic current passing through the nanopore can be observed. This ionic current is determined by the effective cross-sectional area of ​​the target substance. When a target substance passes through the nanopore, the target substance partially blocks the nanopore, changing the magnitude of the current passing through the nanopore. Because each target substance, in whole or in part, has a unique shape and size, the ionic current changes depending on the respective passage site. By reading this change, the target substance can be determined.

[0074] In some embodiments, the target substance may be a biomolecule. In some embodiments, the target substance may be a non-biomolecule (organic or inorganic substance). In some embodiments, the target substance may be a molecule containing the biomolecule to be measured, or may be a complex of biomolecules.

[0075] The target substance may be a nucleic acid. The nucleic acid may be deoxyribonucleic acid (DNA) or may contain DNA. The nucleic acid may be ribonucleic acid (RNA) or may contain ribonucleic acid (RNA). The nucleotide sequence can be determined by passing the nucleic acid (DNA, RNA) through a nanopore and measuring the change in the electrical signal that occurs during the passage.

[0076] The biomolecule may be, include, or be selected from proteins, viruses, bacteria, cells (red blood cells, white blood cells, immune cells, etc.), and the like.

[0077] The present disclosure also includes the following embodiments: A001 A method for processing a thin film, comprising: (a) providing a thin film; (b) irradiating the thin film with an electron beam; (c) observing the thin film to determine whether pores satisfying predetermined conditions have been formed; and (d) if it is determined in (c) that the pores satisfying the predetermined conditions have not been formed, repeating (b) and (c). A002 A method for processing a thin film, comprising: (a0) providing a thin film; (b1) irradiating the thin film with a first electron beam to form a pore in the thin film; (b2) irradiating the edge of the pore with a second electron beam to change the shape of the pore; (c2) observing the shape of the pore; and (d2) if the observed shape of the pore does not satisfy the predetermined conditions, repeating (b2) and (c2). A003 A method for processing a thin film, comprising: (A0) providing a thin film; (B1) irradiating the thin film with a first electron beam (to form pores in the thin film); (C1) observing the thin film to confirm whether pores satisfying predetermined conditions have been formed; (D1) if it is determined in (C1) that the pores satisfying the predetermined conditions have not been formed, repeating (B1) and (C1); (B2) irradiating the edge of the pore in the thin film with a second electron beam; (C2) observing the shape of the pore; and (D2) if the shape of the observed pore does not satisfy the predetermined conditions, repeating (B2) and (C2). A011 The method of any one of embodiments A001 to A003, wherein the thin film has a thickness of 10 nm or less. A012 The method of any one of embodiments A001 to A011, wherein the thin film is a crystalline material, and the crystalline material is a monolayer. A015 The method of any one of embodiments A001 to A012, wherein the thin film comprises a monolayer of a transition metal dichalcogenide or graphene. A016 The method of any one of embodiments A001 to A015, wherein the thin film is a crystalline MoS ２A021 The method of any one of embodiments A001 to A016, wherein a diameter of the electron beam, the first electron beam, and / or the second electron beam is 2 nm or less. A022 The method of embodiment A021, wherein a diameter of the electron beam, the first electron beam, and / or the second electron beam is 1 nm or less. A031 The method of any one of embodiments A001 to A022, wherein the electron beam, the first electron beam, and / or the second electron beam is a focused electron beam. A032 The method of embodiment A031, wherein irradiating the first electron beam and / or the second electron beam comprises repeatedly moving the focal point of the focused electron beam through the thin film. A033 The method of embodiment A032, wherein (d), (d2), and / or (D2) comprise acquiring an image when the focal point of the focused electron beam is outside the thin film. A041 The method of any one of embodiments A001 to A033, wherein (d), (d2) and / or (D2) comprise acquiring an image of the electron beam irradiation area or a field of view including the pore. A042 The method of embodiment A041, wherein observing the shape of the pore or observing the thin film comprises measuring the size of the pore from the acquired pore image. A051 The method of any one of embodiments A001 to A042, wherein (b), (c), (B1), (C1), (B2) and / or (C2) are performed using a scanning transmission electron microscope (STEM). A052 The method of A051, wherein the irradiation of the electron beam, first electron beam and / or second electron beam is performed in spot mode of the STEM. B001 A program for causing a computer to execute the method of any one of A001 to A052. C001 A storage medium on which the program described in B001 is recorded.D001 A system comprising: a pore processing unit, comprising: an electron gun and a lens system capable of spot irradiating an introduced sample; the lens system being a focusing lens and comprising an objective lens that can repeatedly move the focus of the spot irradiation between in-focus and defocus conditions relative to the sample; a bright-field detector that can acquire an image at the time of defocusing; and a computer that receives an output including the defocused image from the bright-field detector, analyzes the image, determines the presence or absence of a pore in the sample, evaluates the shape of the pore, and determines based on the result whether to perform the next spot irradiation or terminate processing, and if the next spot irradiation is to be performed, determines the position of the next spot irradiation and instructs the lens system to perform the spot irradiation at that position. D002 The system according to D001, comprising an electron microscope (scanning transmission electron microscope). E001 A thin film having a nanopore, wherein the maximum width of the nanopore is 10 nm or less, and the approximate ellipticity of the nanopore is 80% or more. E101: A nanopore device comprising the thin film having the nanopore. E111: A nucleic acid sequencer for detecting nucleic acid molecules or nucleotides passing through the nanopore.

[0078] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the invention. It is therefore intended that the present invention cover all such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for processing a thin film, comprising the steps of: (a) providing a thin film; (b) irradiating the thin film with a focused electron beam, the irradiating with the focused electron beam comprising repeatedly moving a focal point of the focused electron beam through the thin film; (c) observing the thin film to determine whether pores that meet predetermined conditions have been formed; and (d) repeating (b) and (c) if it is determined in (c) that the pores satisfying the predetermined conditions have not been formed; A method for providing the above.

2. 2. The method of claim 1 , The thin film is a crystalline material having a thickness of 10 nm or less. method.

3. 2. The method of claim 1 , The thin film comprises a monolayer of a transition metal dichalcogenide or graphene; method.

4. 2. The method of claim 1 , The diameter of the focused electron beam is 2 nm or less. method.

5. (delete)

6. (delete)

7. 2. The method of claim 1 , (d) includes acquiring an image when the focal point of the focused electron beam is outside the thin film; method.

8. 2. The method of claim 1 , (d) comprises acquiring an image of an area of ​​illumination of the focused electron beam or a field of view including the pore; method.

9. 9. The method of claim 8, Observing the thin film comprises measuring a size of the pores from the acquired pore images. method.

10. 2. The method of claim 1 , (b) and / or (c) are carried out using a scanning transmission electron microscope (STEM); method.

11. 11. The method of claim 10, The focused electron beam is irradiated in a spot mode of the STEM. method.

12. A program for causing a computer to execute the method according to any one of claims 1 to 11.

13. A pore processing unit, comprising: An electron gun and lens system capable of spot irradiating the introduced sample; The lens system is a focusing lens system, having an objective lens capable of repeatedly moving the focus of the spot illumination between in-focus and out-of-focus conditions with respect to the sample; A brightfield detector capable of acquiring the image during defocus; and A computer comprising: receiving an output from the brightfield detector including the defocused image; Analyzing the image; Determining the presence or absence of pores in the sample; Evaluating the shape of the pore and determining whether to perform the next spot irradiation or to end the process based on the result of the evaluation; and When performing the next spot irradiation, a position of the next spot irradiation is determined, and an instruction is issued to the lens system to perform the spot irradiation at the position. computer; A system comprising:

14. The system of claim 13 comprising a scanning transmission electron microscope.

15. A thin film having nanopores, The maximum width of the nanopore is 10 nm or less; The approximate ellipticity of the nanopore is 80% or more. Thin film.

16. A nanopore device comprising the thin film having a nanopore fabricated by the method according to any one of claims 1 to 14.

17. A nucleic acid sequencer that detects a nucleic acid molecule or nucleotide passing through a nanopore fabricated by the method according to any one of claims 1 to 8.

18. A method for processing a thin film, comprising the steps of: (a0) providing a thin film; (b1) irradiating the thin film with a first focused electron beam to form pores in the thin film, wherein irradiating with the first focused electron beam comprises repeatedly moving a focal point of the first focused electron beam through the thin film; (b2) irradiating an edge of the pore with a second focused electron beam to alter the shape of the pore, wherein irradiating with the second focused electron beam comprises repeatedly moving a focal point of the second focused electron beam through the thin film; (c2) observing the shape of the pore; and (d2) repeating (b2) and (c2) if the observed pore shape does not satisfy a predetermined condition; A method for providing the above.

19. A method for processing a thin film, comprising the steps of: (A0) providing a thin film; (B1) irradiating the thin film with a first focused electron beam to form pores in the thin film, the irradiating with the first focused electron beam comprising repeatedly moving a focal point of the first focused electron beam through the thin film; (C1) observing the thin film to confirm whether pores that satisfy predetermined conditions have been formed; (D1) repeating (B1) and (C1) if it is determined in (C1) that the pores satisfying the predetermined conditions are not formed; (B2) irradiating an edge of the pore of the membrane with a second focused electron beam, the irradiating with the second focused electron beam comprising repeatedly moving a focal point of the second focused electron beam through the membrane; (C2) observing the shape of the pore; and (D2) repeating (B2) and (C2) if the observed pore shape does not satisfy a predetermined condition; A method for providing the above.

20. 8. The method of claim 7, obtaining the image includes obtaining a Ronchigram. method.