Film forming apparatus and film forming method

Abstract

A membrane forming apparatus and a method are provided, the membrane forming apparatus including a base (10), a body (20) disposed on the base (10) and having a recess, and an aperture member (30) disposed inside a recess port at an end of the recess remote from the base (10), the hollow portion (32) of the aperture member (30) configured to form an amphiphilic molecular membrane (80), and the cross-sectional area of ​​the hollow portion (32) of the aperture member (30) is smaller than the cross-sectional area of ​​the containment chamber (21).

Description

[Technical Field]

[0001]

[0001] The present disclosure relates to the technical field of biopharmaceuticals, and in particular to a film forming apparatus and a film forming method. [Background technology]

[0002]

[0002] Nanopore-based single-molecule gene sequencing is considered a disruptive technology in the field of genetic testing. Nanopore sequencing technology has unique advantages in principle due to its simple sequencing principle, which does not require DNA molecule modification and can directly sequence single molecules continuously. Compared with first- and second-generation gene sequencing technologies, next-generation nanopore-based gene sequencing technologies have advantages such as low cost, long read length, easy integration and portability, and are widely used in various fields.

[0003]

[0003] Current sequencing technologies based on biological nanopores are considered the most potential and disruptive of many nanopore technologies and have been successfully commercialized. However, current sequencing technologies based on biological nanopores still face many challenges, one of the biggest being the stability of the sequencing chip. To maintain the stability of biological nanopore proteins, it is necessary to embed the pore proteins in a carrier membrane with a molecular monolayer thickness. The carrier membrane is typically formed by the self-assembly of amphiphilic molecules, and the membrane material is typically a phospholipid molecule or a block polymer. Summary of the Invention

[0004]

[0004] In one aspect of the present disclosure, a film forming device is provided, comprising a base, a body disposed on the base and having a recess, and an aperture member disposed inside a recess port at an end of the recess away from the base, the hollow portion of the aperture member being configured to form a film.

[0005]

[0005] In some embodiments, the recess is configured to define a storage chamber for storing a polar medium solution, the membrane is an amphiphilic molecular membrane, and the cross-sectional area of ​​the hollow portion of the opening member is smaller than the cross-sectional area of ​​the storage chamber.

[0006]

[0006] In some embodiments, the body includes a first body layer disposed on the surface of the base and a second body layer located on the side of the first body layer away from the base, the recess penetrates the second body layer and the first body layer in a direction perpendicular to the base, and the opening member is connected to a portion of the recess that penetrates the second body layer.

[0007] In some embodiments, the aperture member is integrally formed with the second body layer.

[0008] In some embodiments, the aperture member is integrally formed with the body.

[0009] In some embodiments, the aperture member includes a flange that projects radially inward relative to the recess port, the inner edge of the flange forming the hollow portion.

[0010] In some embodiments, the inner edges of the flanges lie in the same plane.

[0011] In some embodiments, the protruding end of the flange is chamfered or rounded.

[0012] In some embodiments, the cross section of the flange is T-shaped or in the shape of a convex polygon.

[0013]

[0013] In some embodiments, the thickness of the protruding end of the flange in a direction perpendicular to the base is equal to or less than the thickness of the root portion of the flange in a direction perpendicular to the base.

[0014] In some embodiments, the thickness of the flange in a direction perpendicular to the base gradually decreases from the root portion to the protruding end.

[0015] In some embodiments, the flange has at least a portion that protrudes or is recessed from a surface of the flange in a direction perpendicular to the base.

[0016] In some embodiments, at least a portion is located at least on a side of the flange away from the base.

[0017]

[0017] In some embodiments, the inner edge of the flange has a plurality of inwardly projecting projections.

[0018] In some embodiments, the cross section of the hollow portion is circular or in the shape of a convex polygon, and the cross section of the receiving chamber is circular or in the shape of a convex polygon.

[0019] In some embodiments, the longitudinal cross section of the containment chamber is rectangular, trapezoidal, curved edge trapezoidal, or drum shaped.

[0020] In some embodiments, the film-forming apparatus further includes a first electrode disposed outside the containing chamber and a second electrode disposed inside the containing chamber.

[0021]

[0021] In some embodiments, the base includes a substrate and at least one passivation layer disposed between the substrate and the body, and the second electrode is exposed from the bottom of the recess, and the second electrode is located between the substrate and the at least one passivation layer, or the second electrode is located on a side of the at least one passivation layer away from the substrate, or the second electrode is located between adjacent passivation layers when the at least one passivation layer is configured as multiple passivation layers.

[0022] In some embodiments, the body includes a plurality of recesses, and the plurality of recesses in the body are arranged in a rectangular array, a parallelogram array, a honeycomb array, or irregularly.

[0023] In some embodiments, the apertured member has multiple hollow portions.

[0024] In some embodiments, the film-forming apparatus further includes a sealing cover plate configured to be assembled with the body.

[0025]

[0025] In some embodiments, the film forming device further includes a sealing cover plate configured to be assembled with the main body, an amphiphilic molecular film formed in the hollow portion of the opening material, a first polar medium solution contained in the accommodating chamber, and a second polar medium solution contained outside the accommodating chamber and positioned above the main body, the opening material and the amphiphilic molecular film.

[0026]

[0026] In some embodiments, the amphiphilic molecular membrane is a monolayer lipid membrane, a bilayer lipid membrane, or a polymeric membrane.

[0027] In some embodiments, the membrane-forming device further comprises a membrane channel embedded in the amphiphilic molecular membrane.

[0028]

[0028] In one aspect of the present disclosure, there is provided a film formation method including the steps of preparing a film formation apparatus as described in any one of the preceding claims, assembling a sealing cover plate on the upper side of a main body to form a flow path between the sealing cover plate and the main body, introducing a first polar medium solution into the flow path so that the first polar medium solution flows from the hollow portion of the opening member into a recess in the main body and fills the recess in the main body, and removing the first polar medium solution between the opening member and the sealing cover plate, and causing a non-polar medium solution and a second polar medium solution to cover the opening member, wherein at least one of the non-polar medium solution and the second polar medium solution contains amphiphilic molecules that form an amphiphilic molecule film in the hollow portion.

[0029]

[0029] In some embodiments, the step of removing the first polar medium solution between the opening material and the sealing cover plate includes a step of introducing a gas medium into the flow path so that the gas medium removes the first polar medium solution between the body and the sealing cover plate and between the opening material and the sealing cover plate.

[0030]

[0030] In some embodiments, the step of causing the non-polar media solution and the second polar media solution to cover the opening material includes the steps of introducing the non-polar media solution into the flow path so that the non-polar media solution covers the opening material, and introducing the second polar media solution into the flow path so that the second polar media solution covers the opening material.

[0031]

[0031] In some embodiments, the step of allowing the non-polar medium solution and the second polar medium solution to cover the aperture member includes the steps of removing the sealing cover plate, coating the body and aperture member with the non-polar medium solution and allowing the non-polar medium solution to cover the aperture member, assembling the sealing cover plate on top of the body to form a flow path between the sealing cover plate and the body, and introducing the second polar medium solution into the flow path so that it covers the aperture member.

[0032]

[0032] In some embodiments, the step of removing the first polar medium solution between the opening material and the sealing cover plate and covering the opening material with the non-polar medium solution and the second polar medium solution includes the steps of introducing a non-polar medium solution into the flow path so as to remove the first polar medium solution between the body and the sealing cover plate and the first polar medium solution between the opening material and the sealing cover plate with the non-polar medium solution and cover the opening material with the non-polar medium solution, and introducing a second polar medium solution into the flow path so that the second polar medium solution covers the opening material.

[0033]

[0033] In some embodiments, the membrane formation method further comprises embedding a membrane channel in the amphiphilic molecular membrane.

[0034]

[0034] The accompanying drawings, which form a part of the description, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure.

[0035]

[0035] The present disclosure can be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0036] [Figure 1] 1 is a schematic, partially cutaway, three-dimensional structural view of a film-forming apparatus according to some embodiments of the present disclosure. [Figure 2] 1 is a schematic longitudinal cross-sectional view of a film-forming apparatus according to some embodiments of the present disclosure. [Figure 3] 1 is a schematic longitudinal cross-sectional view of another film-forming apparatus according to some embodiments of the present disclosure. [Figure 4] 1 is a schematic longitudinal cross-sectional view of yet another film-forming apparatus according to some embodiments of the present disclosure. [Figure 5] 1 is a schematic longitudinal cross-sectional view of yet another film-forming apparatus according to some embodiments of the present disclosure. [Figure 6A] 1 is a schematic structural diagram of a film-forming apparatus according to some embodiments of the present disclosure assembled with a sealing cover plate. [Figure 6B] 1 is a schematic diagram of a structure of a membrane-forming device including a sealing cover plate, a first polar medium solution, an amphiphilic molecular membrane with embedded membrane channels, and a second polar medium solution, according to some embodiments of the present disclosure. FIG. [Figure 7] 1 is a schematic structural diagram of an electrode arrangement in a recess of a film-forming apparatus according to some embodiments of the present disclosure. [Figure 8] 1 illustrates cross-sectional shapes of hollow portions and containing chambers in some embodiments of a film-forming apparatus according to the present disclosure. [Figure 9] 10 shows the shape of the flange of the aperture member in some embodiments of the film forming device according to the present disclosure. [Figure 10]1 shows the shape of a longitudinal cross section of a receiving chamber in some embodiments of a film-forming apparatus according to the present disclosure. [Figure 11] 10 illustrates the arrangement of multiple recesses in the body in some embodiments of the film-forming device according to the present disclosure. [Figure 12] 1A and 1B are schematic structural diagrams showing an aperture member having a plurality of hollow portions in some embodiments of a film forming apparatus according to the present disclosure. [Figure 13A] 1 is a schematic flow chart of some embodiments of a film formation method according to the present disclosure. [Figure 13B] 1 is a schematic flowchart of another embodiment of a film formation method according to the present disclosure. [Figure 14] 1A-1C are partial cross-sectional views of a film-forming apparatus in successive steps of several embodiments of a film-forming method according to the present disclosure. [Figure 15] 1A-1C are partial cross-sectional views of another film formation apparatus in successive steps of some embodiments of a film formation method according to the present disclosure. [Figure 16] 10A-10C are partial cross-sectional views of yet another film formation apparatus in a series of steps of some embodiments of a film formation method according to the present disclosure. [Figure 17] 10A-10C are partial cross-sectional views of yet another film formation apparatus in a series of steps of some embodiments of a film formation method according to the present disclosure. [Figure 18] 1 is a scanning electron microscope phenogram of the first embodiment of the film forming apparatus according to the present disclosure. [Figure 19] 10 is a scanning electron microscope phenogram of a second embodiment of the film forming apparatus according to the present disclosure. [Figure 20] 10 is a partially cutaway scanning electron microscope phenogram of a third embodiment of the film forming apparatus according to the present disclosure. [Figure 21] 10 is a partially cutaway scanning electron microscope phenogram of a fourth embodiment of the film forming apparatus according to the present disclosure. [Figure 22] 1 is a confocal fluorescence microscope phenogram of a membrane array in a first embodiment of a membrane formation method according to the present disclosure, taken from a top view and cut away. [Figure 23]10 is a confocal fluorescence microscope phenogram of a membrane array in a second embodiment of the membrane formation method according to the present disclosure. [Figure 24] 10 is a graph showing transmembrane current curves monitored when a triangular wave voltage is applied to different channels after membrane formation in an embodiment of a membrane formation apparatus according to the present disclosure. [Figure 25] 10 is a graph showing different transmembrane current curves monitored when a triangular wave voltage is applied to different channels after membrane formation in an embodiment of a membrane formation apparatus according to the present disclosure. [Figure 26] 10 is a graph showing transmembrane current curves monitored when a triangular wave voltage is applied after film formation in one embodiment of a film formation apparatus according to the present disclosure. [Figure 27] 1 is a graph showing the capacitance of multiple channels over time during a film formation process in one embodiment of a film formation apparatus according to the present disclosure. [Figure 28] 10 is a schematic diagram of a detection current signal when a single molecule is detected by an electrical method using a carrier film formed by one embodiment of the film formation apparatus according to the present disclosure. FIG. [Figure 29] 1A is a dimensional schematic diagram of a film-forming apparatus in the related art, and FIG. 1B is a dimensional schematic diagram of one embodiment of a film-forming apparatus according to the present disclosure.

[0060] It should be understood that the dimensions of various parts shown in the accompanying drawings are not drawn according to actual scale relationships. Furthermore, the same or similar reference numerals indicate the same or similar components. DETAILED DESCRIPTION OF THE INVENTION

[0037]

[0061] Various exemplary embodiments of the present disclosure will be described in detail below with reference to the drawings. The descriptions of the exemplary embodiments are merely illustrative and are not intended to limit the present disclosure and its application or uses in any way. The present disclosure may be embodied in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, the relative arrangement of components and steps, compositions of materials, formulas and numerical values ​​described in these embodiments should be interpreted as merely illustrative and not limiting.

[0038]

[0062] As used in this disclosure, similar words such as "first," "second," etc., do not indicate order, quantity, or importance, but are used only to distinguish different parts. Similar words such as "comprise" or "include" mean that the element before the word covers the elements listed after the word, without excluding the possibility of covering other elements. "Top," "bottom," "left," "right," etc. are used only to indicate relative positional relationships. After the absolute position of the described object changes, the relative positional relationships may also change accordingly.

[0039]

[0063] In this disclosure, when a particular device is described as being located between a first device and a second device, there may or may not be an intervening device between the particular device and the first or second device. When a particular device is described as being connected to other devices, the particular device may be directly connected to the other devices without an intervening device, or may have an intervening device without being directly connected to the other devices.

[0040]

[0064] Unless otherwise defined, all terms used in this disclosure (including technical or scientific terms) have the same meaning as understood by a person skilled in the art to which this disclosure belongs. It should also be understood that terms defined in, for example, a general dictionary should be interpreted to have a meaning consistent with their meaning in the context of the relevant art, and should not be interpreted in an idealized or extremely formal sense unless explicitly defined herein.

[0041]

[0065] Techniques, methods and equipment known to those skilled in the art may not be described in detail, but the techniques, methods and equipment should be considered part of the description where appropriate.

[0042]

[0066] Our research has shown that in sequencing chips, the carrier membrane is supported by a microwell structure, and the shape and opening diameter of the support structure directly affect membrane stability. Membrane stability directly affects the stability of the pore protein, and membrane formation yield directly determines the number of single nanopores. Therefore, the membrane formation device, as well as its membrane formation method and efficiency, ultimately affect the data output and data quality of the entire sequencing system.

[0043]

[0067] In some related technologies, a sequencing chip includes a recess structure, and a chip electrode is provided at the bottom of the recess. The membrane formation process of the sequencing chip includes: completely covering the entire surface of the recess with a hydrophobic layer; introducing a polar medium solution; using the hydrophilicity of the chip electrode to repel the hydrophobic layer to the sidewall of the recess so that the chip electrode is electrically connected to the polar medium solution; discharging the polar medium solution outside the recess; and sequentially introducing amphiphilic molecules and the polar medium solution used to form the membrane to form a carrier membrane composed of a monolayer / bilayer of amphiphilic molecules in the recess port of the recess.

[0044]

[0068] After research, it is found that these related technologies have the following drawbacks:

[0045]

[0069] 1. The film formation yield of the chip array is low, and the formed carrier film has low stability.

[0046]

[0070] 2. Based on the process requirements, the chip body needs to be first pre-treated with a hydrophobic coating, which makes the chip treatment process more complicated.

[0047]

[0071] 3. The large membrane formation area leads to high membrane capacitance, resulting in high electrical noise, and facilitates the implantation of multiple nanopore proteins.

[0048]

[0072] 4. The volume of electrochemical solution that can be accommodated in the recess is smaller, which leads to a shorter detection life.

[0049]

[0073] In this regard, the present disclosure provides embodiments of a film-forming apparatus and a film-forming method that can improve the stability of the formed carrier film.

[0050]

[0074] FIG. 1 is a schematic, partially cutaway, three-dimensional structural diagram of a film-forming apparatus according to some embodiments of the present disclosure. FIGS. 2 to 5 are schematic longitudinal cross-sectional views of a film-forming apparatus according to some embodiments of the present disclosure. Referring to FIGS. 1 to 5, in some embodiments, the film-forming apparatus includes a base 10, a main body 20, and an aperture member 30. The main body 20 is provided on the base 10 and has a recess. The aperture member 30 is provided inside a recess port on the side of the recess away from the base 10, and a hollow portion 32 of the aperture member 30 is configured to form a film 80.

[0051]

[0075] In some embodiments, the recess defines a storage chamber 21 for storing a polar medium solution, the cross-sectional area of ​​the hollow portion 32 of the opening member 30 is smaller than the cross-sectional area of ​​the storage chamber 21, and the membrane 80 is an amphiphilic molecular membrane.

[0052]

[0076] When using the membrane-forming device of this embodiment to form a carrier film composed of amphiphilic molecules (i.e., an amphiphilic molecular film), the formation of the carrier film is a dynamically stable self-assembly process, so the shape and size of the final stabilized carrier film are determined by the shape and size of the hollow portion of the aperture member. A hollow portion of the aperture member with a smaller cross-sectional area can effectively reduce the area of ​​the formed carrier film, thereby improving the stability of the carrier film and further improving the mechanical stability of the entire chip. Furthermore, reducing the area of ​​the carrier film can effectively reduce the membrane capacitance of the carrier film, thereby reducing the electrical noise of the system. A holding chamber with a relatively large cross-sectional area can extend the volume of the electrochemical solution in the holding chamber and the detection life of the device.

[0053]

[0077] Embodiments of the membrane-forming device according to the present disclosure can be applied to the field of medical devices for in vitro diagnostic testing, such as gene sequencing, protein sequencing, and biomolecular testing. For example, nanopore proteins are embedded in the formed carrier membrane to test nucleic acid molecules. Embodiments of the membrane-forming device according to the present disclosure can also be applied to high-end manufacturing or semiconductor fields, such as MEMS sensors and biosensors, or scientific research fields, such as life science research and brain / neuroscience research.

[0054]

[0078] In FIG. 1 , the containing chamber 21 can contain a liquid polar medium solution. The polar medium solution can flow into the containing chamber 21 from the upper side of the main body 20 through the opening member 30. The polar medium solution can be an electrochemical solution such as a potassium chloride solution or a potassium ferricyanide / potassium ferrocyanide solution. The material of the main body 20 can be a non-conductive photoresist or other non-conductive material. The material of the main body 20 can also include a semiconductive material or a conductive material, with a non-conductive passivation layer formed on the surface of the semiconductive material or conductive material.

[0055]

[0079] The base 10 may be constructed as a single layer or a multi-layer structure. The main body 20 may be constructed as a single layer or a multi-layer structure. The aperture member 30 may be integrally formed with all or part of the main body 20, or may be formed separately from the main body 20 and connected to the main body 20. The base 10 can provide support and a forming surface for other parts of the film forming apparatus. In the case of a main body 20 including multiple recesses, the base 10 and the main body 20 can be made of materials capable of achieving electrical insulation to achieve electrical insulation between adjacent electrodes.

[0056]

[0080] 2, in some embodiments, the body 20 is configured as a single layer structure, and the aperture member 30 is connected to the inside of the recess port of the recess in the body 20. The aperture member 30 may be formed after the formation of the body. For example, the body 20 may be formed by performing a single photolithography step.

[0057]

[0081] 3 , in some embodiments, the body 20 includes a first body layer 22 and a second body layer 23. The first body layer 22 is provided on the surface of the base 10. The second body layer 23 is located on the side of the first body layer 22 away from the base 10. A recess penetrates the second body layer 23 and the first body layer 22 in a direction perpendicular to the base 10, and the opening member 30 is connected to a part of the recess that penetrates the second body layer 23.

[0058]

[0082] The first body layer 22 may be configured as a single layer structure or a multi-layer structure. The aperture member 30 may be integrally formed with the second body layer 23 to simplify the process. The first body layer 22 and the second body layer 23 may be formed by multiple photolithographic laminations or a bonding process. The first body layer 22 and the second body layer 23 may be made of the same material or different materials. Materials that can be used for all layers of the body include, but are not limited to, silicon-based materials, photoresists, etc. For example, in the laminated body 20, the first body layer 22 is made of a silicon-based material, and the second body layer 23 is made of a photosensitive epoxy resin photoresist.

[0059]

[0083] 4, in some embodiments, the main body 20 is constructed as a single layer structure, and the aperture member 30 is integrally formed with the main body 20 to simplify processing. The main body 20 and the aperture member 30 may be formed of the same material or different materials.

[0060]

[0084] Referring to FIG. 5, in some embodiments, a single or multi-layer structure 40, such as an electrode layer, may be provided on the side of the body 20 away from the substrate 10 to perform a set function.

[0061]

[0085] In Figure 2, the aperture member 30 may include a flange 31 that protrudes radially inward relative to the recess port. The inner edge of the flange 31 forms a hollow portion 32. The hollow portion 32 can be used to form an amphiphilic molecular membrane that allows for the insertion of a membrane channel. The membrane channel may be a nanopore, a naturally occurring pore, a mutant pore derived from a naturally occurring pore, or a synthetic pore. The pore may be a homo-oligomer (derived from the same monomer) or a hetero-oligomer (at least one monomer is different from the other monomers).

[0062]

[0086] 1-5, in some embodiments, the inner edges of flange 31 are coplanar to facilitate processing and help form a more stable carrier film, while in other embodiments, the inner edges of flange 31 may not be coplanar.

[0063]

[0087] 6A is a schematic structural diagram of a film-forming apparatus according to some embodiments of the present disclosure assembled with a sealing cover plate. Referring to FIG. 6A, in some embodiments, the film-forming apparatus further includes a first electrode 51 disposed outside the containing chamber 21 and a second electrode 52 disposed inside the containing chamber 21. The first electrode 51 may be implemented by a conductive layer deposited on a surface on one side of the body 20 away from the base 10. The second electrode 52 may be disposed on the bottom of the containing chamber 21, on a sidewall of the containing chamber 21, or suspended within the containing chamber 21. The second electrode 52 may be embedded between multiple body layers included in the body.

[0064]

[0088] The first electrode 51 and the second electrode 52 are electrically connected to the aqueous solutions outside and inside the containing chamber 21, respectively, so that the electrical signal across the amphiphilic molecule film can be measured by connecting a circuit to the first electrode 51 and the second electrode 52. The circuit can have the same structure as a conventional circuit for detecting an electrical signal across an amphiphilic molecule layer.

[0065]

[0089] The first electrode 51 and the second electrode 52 are electrochemical electrodes that can contact an aqueous solution and measure lower currents, and are stable in aqueous solutions. Materials that can be used for the first electrode 51 include, but are not limited to, silver / silver chloride. Materials that can be used for the second electrode 52 include, but are not limited to, silver / silver chloride, precious metals such as gold, platinum, and palladium, conductors such as graphene or titanium nitride, or semiconductor materials. The materials used for the first electrode 51 and the second electrode 52 can be the same or different. The first electrode 51 and the second electrode 52 can also be composite electrodes formed by layering or mixing multiple materials. The surfaces of the first electrode 51 and the second electrode 52 can be covered with materials such as conductive polymers and hydrogels, or they can be uncovered.

[0066]

[0090] In other embodiments, the membrane-forming device may not include electrodes, and correspondingly, other methods, such as optical signal detection, may be used to achieve applications such as DNA sequencing, protein analysis, single-cell analysis, or drug screening.

[0067]

[0091] 6A, to facilitate the formation of the amphiphilic molecular film, in some embodiments, the film-forming apparatus further includes a sealing cover plate 60. In this film-forming apparatus, the sealing cover plate 60 is detachable. The sealing cover plate 60 can be assembled with the main body 20 to form a flow channel located above the main body 20, and the flow channel may be connected to an inlet and an outlet (not shown) formed in the sealing cover plate 60. An operator can introduce a gas medium, a polar medium solution, or a non-polar medium solution into the flow channel through the inlet during the formation of the amphiphilic molecular film.

[0068]

[0092] 6B is a schematic diagram of a structure of a membrane-forming apparatus including a sealing cover plate, a first polar medium solution, an amphiphilic molecular membrane with an embedded membrane channel, and a second polar medium solution, according to some embodiments of the present disclosure. Referring to FIG. 6B, in some embodiments, the membrane-forming apparatus further includes a sealing cover plate 60, an amphiphilic molecular membrane 80, a first polar medium solution M1, and a second polar medium solution M4. The sealing cover plate 60 is configured to be assembled with the main body 20 to form a flow channel located above the main body 20, and the flow channel may be connected to an inlet and an outlet (not shown) formed in the sealing cover plate 60.

[0069]

[0093] The amphiphilic molecular film 80 is formed in the hollow portion 32 of the aperture member 30. The first polar medium solution M1 is contained in the storage chamber 21, and the second polar medium solution M4 is contained outside the storage chamber 21 and is located above the main body 20, the aperture member 30, and the amphiphilic molecular film 80.

[0070]

[0094] The amphiphilic molecular film 80 may be formed in the hollow portion 32, but is not limited to being formed in the hollow portion 32, and may extend above and / or below the flange 31. The amphiphilic molecular film 80 may be located at the interface between the first polar medium solution M1 and the second polar medium solution M4, and a portion of the nonpolar medium solution M3 may remain in the layer where the amphiphilic molecular film 80 is located. The amphiphilic molecular film 80 may be directly attached to the flange 31, or may be maintained in the hollow portion 32 by attaching the nonpolar solution M3 to the flange 31.

[0071]

[0095] In this embodiment, the first polar medium solution M1 and the second polar medium solution M4 may be the same or different liquids, typically conductive salt solutions such as an aqueous solution of potassium chloride, or a potassium ferricyanide / potassium ferrocyanide solution, etc. The amphiphilic molecular membrane 80 may be a monolayer lipid (e.g., a phospholipid monolayer), a bilayer lipid (e.g., a phospholipid bilayer), or a polymer membrane (e.g., a diblock, triblock).

[0072]

[0096] Referring to Figure 6B, in some embodiments, a membrane channel is embedded in an amphiphilic molecular membrane. Optionally, the membrane channel is, for example, a nanopore protein. A nanopore protein refers to a protein that can form a transmembrane protein pore. A transmembrane protein pore is usually formed by multiple (e.g., 6, 7, or 8) repeating subunits. These subunits generally surround a central axis and provide a channel that allows ions to flow therethrough. The transmembrane protein pore allows ions to flow down their electrochemical gradient from one side of the membrane to the other.

[0073]

[0097] In some embodiments, the nanopore protein is selected from hemolysin, MspA, Frac, ClyA, PA63, CsgG, GspD, XcpQ, Wza, SP1, Phi29 connector, SPP1 connector, T3 connector, T4 connector, T7 connector, K ion channel protein, Na ion channel protein, and Ca ion channel protein.

[0074]

[0098] The membrane channel may be a nanopore, a naturally occurring pore, a mutant pore derived from a naturally occurring pore, or a synthetic pore. The pore may be a homo-oligomer (derived from the same monomers) or a hetero-oligomer (at least one monomer is different from the others).

[0075]

[0099] 7 is a schematic structural diagram of an electrode arrangement in a recess of a film-forming apparatus according to some embodiments of the present disclosure. Referring to FIG. 7, in some embodiments, the base 10 includes a substrate 11 and at least one passivation layer (e.g., passivation layers 12 and 13). The at least one passivation layer is disposed between the substrate 11 and the main body 20. The second electrode 52 is exposed from the bottom of the recess so as to contact the aqueous solution in the containing chamber 21.

[0076]

[0100] The second electrode 52 may be located between the substrate 11 and the at least one passivation layer, or may be located on a side of the at least one passivation layer remote from the substrate 11. If the at least one passivation layer is configured as multiple passivation layers, for example, two passivation layers 12, 13 as shown in FIG. 7, the second electrode 52 may be located between adjacent passivation layers 12, 13.

[0077]

[0101] The substrate 11 may be a silicon-based substrate, a glass substrate, a carbon-based substrate, an integrated circuit wafer, a printed circuit board, a flexible circuit board, etc. The material of the passivation layer may be a conventional silicon-based passivation material such as silicon dioxide and silicon nitride, or a thin layer of photoresist such as an epoxy resin photoresist or a polyimide film, or an insulating paint commonly used to passivate printed circuit boards, such as an acrylic resin.

[0078]

[0102] Figures 8(a) to 8(g) show the cross-sectional shapes of the hollow portion and the storage chamber, respectively, in some embodiments of the film-forming apparatus according to the present disclosure. Referring to Figure 8, the orthogonal projection of the cross-section of the hollow portion 32 on the base is located within the cross-section of the storage chamber. The cross-sections of the hollow portion 32 and the storage chamber 21 may have the same or different shapes. The cross-section of the hollow portion 32 may be circular or a convex polygon, and the cross-section of the storage chamber 21 may be circular or a convex polygon. The convex polygon may be a triangle, rectangle, square, hexagon, octagon, or other shape. A hollow portion with a circular or convex polygonal cross-section is more likely to form a stable carrier film. A storage chamber with a circular or convex polygonal cross-section can accommodate a large amount of aqueous solution.

[0079]

[0103] In (a) to (c) of Figure 8, the cross sections of the storage chambers 21 are all circular. In (d) and (e) of Figure 8, the cross sections of the storage chambers 21 are all square. In (a) and (d) of Figure 8, the cross sections of the hollow portions 32 are all circular. In (b), (c), and (e) of Figure 8, the cross sections of the hollow portions 32 are rectangular and triangular, respectively.

[0080]

[0104] To improve adhesion of hollow portion 32 to the carrier film, in some embodiments, referring to Figures 8(f) and (g), the inner edge of flange 31 has multiple protrusions that protrude inward. The non-uniform edge microstructure formed by the multiple protrusions can form a carrier film with higher stability.

[0081]

[0105] 9(a) to 9(i) show the shapes of the flanges of the apertured member in some embodiments of the film-forming apparatus according to the present disclosure. Referring to FIG. 9, in some embodiments, the cross section of the flange 31 may be T-shaped or convex polygonal. In FIG. 9(c), the thickness of the protruding end of the flange 31 having a T-shaped cross section in a direction perpendicular to the base 10 is equal to or less than the thickness of the root portion of the flange 31 in a direction perpendicular to the base 10. An apertured member with this structure is more reliable and can form a carrier film with higher stability.

[0082]

[0106] 9(a), the cross section of the flange 31 is triangular. The thickness of the flange 31 in the direction perpendicular to the base 10 gradually decreases from the root portion to the protruding end. This gently sharp edge of the flange 21 can increase the assembly stress of the carrier film and improve the stability of the carrier film.

[0083]

[0107] In Fig. 9(b), the cross section of the flange 31 is rectangular. In Fig. 9(d), the cross section of the flange 31 is convex pentagonal, which means that the base of the flange 31 is rectangular and the protruding end is triangular. In Fig. 9(g) to (h), the cross section of the flange 31 is convex quadrilateral, and the position of the inner edge of the protruding end may be flush with the highest or lowest point of the flange 31.

[0084]

[0108] Increasing the specific surface area of ​​the flange 31 can facilitate adhesion of the nonpolar medium solution and improve the stability of the formed carrier film. Referring to (i) of FIG. 9, the protruding end of the flange 31 may be chamfered or rounded. Referring to (e) and (f) of FIG. 9, in some embodiments, the flange 31 has at least a portion that protrudes or is recessed from the surface of the flange 31 in a direction perpendicular to the base 10. This portion may have a discrete or continuous raised or recessed microstructure. In (f) of FIG. 9, at least the portion is located at least on the side of the flange 31 away from the base 10. The carrier film need not be limited to the hollow portion and may be formed on the upper or lower surface of the flange 31. The localized microstructure helps to maintain the carrier film on the surface of the flange 31.

[0085]

[0109] 10(a) to 10(d) show the shapes of longitudinal cross sections of the storage chamber in some embodiments of the film-forming apparatus according to the present disclosure. Referring to FIG. 10, in some embodiments, the longitudinal cross section of the storage chamber 21 is rectangular, trapezoidal, curved-edge trapezoidal, or drum-shaped. The sidewalls of the recess defining the storage cavity 21 may have a vertical structure, an inclined structure, a curved structure, or any combination of a curved surface, an inclined surface, and a vertical structure.

[0086]

[0110] In Figures 10(a) and (d), the longitudinal cross section of the storage chamber 21 is rectangular, but differs in that Figure 10(d) realizes a larger rectangular shape, thereby forming a storage chamber with a larger volume, which is beneficial for extending the detection life of the film-forming device. In Figures 10(b) and (c), the longitudinal cross section of the storage chamber 21 is trapezoidal and curved-edge trapezoidal, respectively.

[0087]

[0111] 11(a) to 11(c) respectively show the arrangement of multiple recesses in the main body in some embodiments of the film forming apparatus according to the present disclosure. Referring to FIG. 11, in some embodiments, the main body 20 may include multiple recesses. From a top view, the multiple recesses may be arranged in the main body 20 in various forms, such as a rectangular array, a parallelogram array, a honeycomb array, or an irregular pattern. For example, in FIG. 11(a), the multiple recesses are arranged in a rectangular array, with the recesses arranged at approximately equal row spacing and column spacing. In FIG. 11(b), the multiple recesses are arranged in a honeycomb array. In FIG. 11(c), the multiple recesses are arranged in an irregular pattern.

[0088]

[0112] 12(a) to 12(c) are schematic structural diagrams showing an aperture member having multiple hollow portions in some embodiments of a membrane-forming device according to the present disclosure. Referring to FIG. 12, in some embodiments, the aperture member 30 has multiple hollow portions 32. This aperture member 30 does not include a flange 31, but multiple through-holes are formed in the solid aperture member as multiple hollow portions 32. The multiple hollow portions 32 may each form a smaller carrier membrane. Depending on application requirements, a membrane channel such as a nanopore protein may be embedded in the carrier membrane formed in one or more hollow portions 32 of the aperture member 30.

[0089]

[0113] 12(a) and (c), the apertured member 30 has a greater number of hollow portions 32, while in FIG. 12(b), the apertured member 30 has a lesser number of hollow portions 32. The hollow portions 32 may be arranged at equal or different intervals. The cross section of each hollow portion 32 may be circular or convex polygonal.

[0090]

[0114] Based on the above-described embodiments of the film forming apparatus according to the present disclosure, the present disclosure further provides corresponding film forming methods. Figure 13A is a schematic flowchart of some embodiments of the film forming method according to the present disclosure. Referring to Figure 13A, in some embodiments, the film forming method includes steps S10 to S30.

[0091]

[0115] In step S10, any of the embodiments of the film-forming apparatus described above is provided, and a sealing cover plate 60 is assembled on the top of the body 20 to form a flow path between the sealing cover plate 60 and the body 20.

[0092]

[0116] In step S20, the first polar medium solution M1 is introduced into the flow path, so that the first polar medium solution M1 flows from the hollow portion 32 of the opening member 30 into the recess of the main body 20 and fills the recess of the main body 20.

[0093]

[0117] In step S30, the first polar medium solution M1 between the aperture member 30 and the sealing cover plate 60 is removed, allowing the aperture member 30 to be covered with the non-polar medium solution M3 and the second polar medium solution M4.

[0094]

[0118] In this embodiment, at least one of the nonpolar medium solution M3 and the second polar medium solution M4 contains amphiphilic molecules that form an amphiphilic molecular film 80 in the hollow portion 32. The amphiphilic molecular film 80 may be formed in the hollow portion 32, but is not limited to being formed in the hollow portion 32, and may extend above and / or below the flange 31. The layer on which the amphiphilic molecular film 80 is located can retain a portion of the nonpolar medium solution M3. The amphiphilic molecular film 80 may be attached directly to the flange 31, or may be maintained in the hollow portion 32 by attaching the nonpolar solution M3 to the flange 31.

[0095]

[0119] The amphiphilic molecular membrane may be a monolayer / bilayer molecular membrane composed of amphiphilic molecules.The amphiphilic molecule may include, but is not limited to, lipids, such as 1,2-bis(diphenylphosphine)ethane (DPPE) phospholipid molecules, which may be polymers, such as diblock and triblock.The lipid may comprise a head group and a hydrophobic tail group.In the formed amphiphilic molecular membrane, the head group of the lipid may face the polar medium solution, and the hydrophobic tail group may face the nonpolar medium solution.

[0096]

[0120] The first polar medium solution M1 and the second polar medium solution M4 may be the same or different liquids, typically conductive salt solutions such as aqueous potassium chloride solutions or potassium ferricyanide / potassium ferrocyanide solutions, etc. The non-polar medium solution M3 may be a water-insoluble solvent such as n-hexadecane, n-decane, n-hexane, or silicone oil.

[0097]

[0121] 13B is a schematic flowchart of some embodiments of a membrane formation method according to the present disclosure. Referring to FIG. 13B, compared to FIG. 13A, in some embodiments, the membrane formation method further includes step S40. In step S40, the membrane channel 90 is embedded in the amphiphilic molecule membrane 80. Step S40 may be performed after the formation of a stable amphiphilic molecule membrane.

[0098]

[0122] 14 to 17 are partial cross-sectional views of a film-forming apparatus in a series of steps of several embodiments of a film-forming method according to the present disclosure. Referring to FIGS. 14(a) and 14(b) to 17(a) and 17(b), assembly of the sealing cover plate 60 and introduction of the first polar medium solution M1 are performed by steps S10 and S20, respectively. In the process of introducing the first polar medium solution M1, the first polar medium solution M1 flows from the hollow portion 32 of the opening member 30 into the recess of the main body 20 and fills the recess of the main body 20.

[0099]

[0123] Referring to (c) and (d) of Figures 14 to (c) and (d) of Figure 16, in some embodiments, in step S30, the step of removing the first polar medium solution M1 between the opening member 30 and the sealing cover plate 60 includes the step of introducing a gas medium M2 into the flow path so that the gas medium M2 removes the first polar medium solution M1 between the main body 20 and the sealing cover plate 60 and between the opening member 30 and the sealing cover plate 60.

[0100]

[0124] The gas medium M2 may be any one or a combination of air, nitrogen, argon, etc. The introduced gas medium M2 can displace the first polar medium solution M1 above the body 20 and the aperture member 30, thereby forming an interface between the first polar medium solution M1 and the gas medium M2 within the hollow portion 32 of the aperture member 30.

[0101]

[0125] In other embodiments, the first polar medium solution M1 between the opening member 30 and the sealing cover plate 60 may also be removed by other means, such as by introducing a liquid that is insoluble in the first polar medium solution M1, such as a non-polar medium solution.

[0102]

[0126] 14 and 15, in some embodiments, the step of causing the non-polar media solution M3 and the second polar media solution M4 to cover the opening member 30 in step S30 includes the steps of introducing the non-polar media solution M3 into the flow path so that the non-polar media solution M3 covers the opening member 30, and introducing the second polar media solution M4 into the flow path so that the second polar media solution M4 covers the opening member 30.

[0103]

[0127] In step S30, as shown in (e) of Figure 14, the second polar medium solution M4 may be introduced into the flow path immediately after the nonpolar medium solution M3 is introduced into the flow path, or as shown in (e) and (f) of Figure 15, the nonpolar medium solution M3 may be introduced into the flow path, and after waiting for a certain period of time, the second polar medium solution M4 may be introduced into the flow path.

[0104]

[0128] The amphiphilic molecules can be dissolved in the nonpolar medium solution M3, the second polar medium solution M4, or both the nonpolar medium solution M3 and the second polar medium solution M4. Referring to Figures 14(f) and 15(g), after the nonpolar medium solution M3 or the second polar medium solution M4 containing the amphiphilic molecules is introduced, a membrane 80 separating the first polar medium solution M1 and the second polar medium solution M4 is formed in the hollow portion of the aperture member 30. The membrane 80 contains amphiphilic molecules that can self-assemble, and gradually thins and expands in area through self-assembly until it reaches stability.

[0105]

[0129] Referring to FIG. 16, in some embodiments, in step S30, the step of covering the aperture member 30 with the non-polar media solution M3 and the second polar media solution M4 includes the steps of removing the sealing cover plate 60, coating the main body 20 and the aperture member 30 with the non-polar media solution M3 and covering the aperture member 30 with the non-polar media solution M3, assembling the sealing cover plate 60 to the top of the main body 20 to form a flow path between the sealing cover plate 60 and the main body 20, and introducing the second polar media solution M4 into the flow path so that the second polar media solution M4 covers the aperture member 30.

[0106]

[0130] In FIG. 16(e), the sealing cover plate 60 is removed, thereby exposing the main body 20 and the aperture member 30. A layer of non-polar medium solution M3 is then applied as uniformly as possible to the surfaces of the main body 20 and the aperture member 30. The non-polar medium solution M3 may be applied by dripping, atomizing, spraying, or other methods. In FIG. 16(f), the sealing cover plate 60 is reassembled on top of the main body 20, and then a second polar medium solution M4 is introduced into the formed flow path. The second polar medium solution M4 can expel the non-polar medium solution M3 outside the aperture member 30.

[0107]

[0131] The amphiphilic molecules can be dissolved in the nonpolar medium solution M3, the second polar medium solution M4, or both the nonpolar medium solution M3 and the second polar medium solution M4. Referring to (g) of Figure 16, a membrane 80 separating the first polar medium solution M1 and the second polar medium solution M4 is formed in the hollow portion of the aperture member 30. The membrane 80 contains amphiphilic molecules that can self-assemble, and gradually thins and expands in area through self-assembly until it reaches stability.

[0108]

[0132] Referring to FIG. 17, in some embodiments, in step S30, the step of removing the first polar media solution M1 between the opening member 30 and the sealing cover plate 60 and covering the opening member 30 with the non-polar media solution M3 and the second polar media solution M4 includes the steps of: introducing the non-polar media solution M3 into the flow path so as to remove the first polar media solution M1 between the main body 20 and the sealing cover plate 60 and the first polar media solution M1 between the opening member 30 and the sealing cover plate 60 with the non-polar media solution M3, and to cover the opening member 30 with the non-polar media solution M3; and introducing the second polar media solution M4 into the flow path so as to cover the opening member 30.

[0109]

[0133] In FIG. 17(c), a nonpolar medium solution M3 is introduced into the flow channel. Under the action of shear force, an interface between the first polar medium solution M1 and the nonpolar medium solution M3 is formed in the hollow portion of the aperture member 30, as shown in FIG. 17(d). In FIG. 17(e), a second polar medium solution M4 is introduced into the flow channel. Amphiphilic molecules can be dissolved in the nonpolar medium solution M3, the second polar medium solution M4, or both the nonpolar medium solution M3 and the second polar medium solution M4. Referring to FIG. 17, after the nonpolar medium solution M3 or the second polar medium solution M4 containing amphiphilic molecules is introduced, a membrane 80 separating the first polar medium solution M1 and the second polar medium solution M4 is formed in the hollow portion of the aperture member 30. The membrane 80 contains amphiphilic molecules that can self-assemble, and gradually thins and expands in area through self-assembly until it reaches stability.

[0110]

[0134] Compared with the related art, the various embodiments of the membrane formation method described above in the present disclosure do not require hydrophobic pretreatment of the base, body, opening material, etc. of the membrane formation apparatus, reducing the difficulty of preparing the membrane formation apparatus. Based on the opening material of the membrane formation apparatus, the shear uniformity of the interface between the gas medium or nonpolar medium solution and the first polar medium solution during the membrane formation process is optimized, so that the amphiphilic molecular membrane can be completed in one go, which simplifies the membrane formation process and improves the membrane formation yield and membrane stability.

[0111]

[0135] The effects of the film forming apparatus and film forming method according to the embodiments will be described below from several aspects based on experimental data.

[0112]

[0136] FIG. 18 is a scanning electron microscope phenogram of a first embodiment of a film-forming apparatus according to the present disclosure. Referring to FIG. 18, in the first embodiment of the film-forming apparatus, the diameter of the hollow portion of the aperture member of the film-forming apparatus is approximately 80 μm. A plurality of sets of circumferentially arranged microgroove structures are provided on the flange of the aperture member, and each set of microgroove structures includes a plurality of radially arranged microgroove structures. These microgroove structures are beneficial for forming an interface between the gas medium or nonpolar medium solution and the first polar medium solution and for maintaining the carrier film. The structure is formed by spin-coating SU-8 photoresist (final thickness 95 μm) on a silicon substrate, followed by processes such as pre-baking, exposure, post-baking, development, and hard-baking.

[0113]

[0137] FIG. 19 is a scanning electron microscope phenogram of a second embodiment of a film-forming apparatus according to the present disclosure. Referring to FIG. 19, in the second embodiment of the film-forming apparatus, the diameter of the hollow portion of the aperture member of the film-forming apparatus is less than 50 μm, ultimately measuring approximately 47 μm. A plurality of sets of circumferentially arranged microgroove structures are provided on the flange of the aperture member, each set including a plurality of radially arranged microgroove structures. These microgroove structures are beneficial for forming an interface between the gas medium or nonpolar medium solution and the first polar medium solution and for maintaining the carrier film. The structure is formed by spin-coating SU-8 photoresist (final thickness 95 μm) onto a silicon substrate, followed by processes such as pre-baking, exposure, post-baking, development, and film curing.

[0114]

[0138] 20 is a partially cutaway scanning electron microscope phenogram of a third embodiment of a film-forming apparatus according to the present disclosure. Referring to FIG. 20, in the third embodiment of the film-forming apparatus, the diameter of the hollow portion of the aperture member of the film-forming apparatus is approximately 80 μm. The shape of the hollow portion of the aperture member is approximately circular, and the flange is smooth without microstructures. The sidewall of the storage chamber within the aperture member is a smoothly curved surface.

[0115]

[0139] 21 is a partially cutaway scanning electron microscope phenogram of a fourth embodiment of a film-forming apparatus according to the present disclosure. Referring to FIG. 21, in the fourth embodiment of the film-forming apparatus, the diameter of the hollow portion of the apertured member of the film-forming apparatus is approximately 75 μm. The shape of the hollow portion of the apertured member is roughly circular with an irregular pattern, and its inner edge has a plurality of approximately 3 μm raised microstructures protruding inward. These raised microstructures can enhance the stability of the carrier film. The sidewall of the containing chamber within the apertured member is a smoothly curved surface.

[0116]

[0140] Figure 22 is a confocal fluorescence microscope phenogram of a membrane array in a first embodiment of the membrane formation method according to the present disclosure, cut away from a top view. Referring to Figure 14, in the first embodiment of the membrane formation method, an aqueous solution of potassium chloride (500 mM) and Hepes (5 mM) as a first polar medium solution is introduced into a channel of a membrane formation device containing multiple recesses (Figure 11(b)), and the aqueous solution fills the holding chamber. Next, 10 mL of air is forced through the channel of the membrane formation device to completely expel the aqueous solution directly above the main body and the opening member while retaining the aqueous solution in the recesses. Due to the effect of fluid shear force, an interface between the aqueous solution and air is formed in the hollow portion of the opening member. Next, 5 μL of a nonpolar medium solution (n-hexadecane) containing an amphiphilic molecule (1,2-bis(diphenylphosphine)ethane (DPPE) phospholipid molecule) (50 mg / mL) dissolved therein and 1 mL of an aqueous solution of potassium chloride (500 mM) and Hepes (5 mM) as a second polar medium solution are sequentially inserted.

[0117]

[0141] Due to the effect of surface tension of the liquid, the nonpolar medium solution and the amphiphilic molecules dissolved in it naturally lie flat in the hollow portion of the opening material, completely covering the first polar medium solution located in the hollow portion, thus forming a water / oil / water interfacial layer in the hollow portion. The phospholipid molecules spontaneously self-assemble at the interface between the aqueous solution and the oil (n-hexadecane). Finally, under intermolecular forces, some regions of phospholipid molecules self-assemble to form a bilayer phospholipid membrane. Over time, the membrane area becomes larger and more stable.

[0118]

[0142] The object shown in Figure 22 is a carrier membrane array formed on an array of multiple recesses with an aperture diameter of approximately 80 μm. The Nikon C2+ fluorescence confocal microscope (FCFM) used in the experiment was used. The first polar medium solution in the receiving chamber of each recess contained 0.02% by weight of Rhodamine B fluorescent dye. The excitation and emission peak wavelengths of Rhodamine B are 546 nm and 568 nm, respectively. The second polar medium solution above the hollow portion of the aperture member did not contain Rhodamine B. 2 μM of BODIPY FL fluorescent dye was mixed into the nonpolar medium solution. The excitation and emission peaks of BODIPY FL are 503 nm and 512 nm, respectively. Because BODIPY FL is readily soluble in nonpolar medium solutions, the areas of the array device containing the nonpolar medium solution appear green.

[0119]

[0143] In the microscopic phenogram shown in Figure 22, Rhodamine B is dissolved in the aqueous solution below the membrane and appears red in the phenogram (indicated by arrow R). The aqueous solution above the membrane does not contain Rhodamine B and therefore does not fluoresce, showing a black background. The nonpolar medium solution containing phospholipids appears pale green in the phenogram (indicated by arrow G). As can be seen from the fluorescence confocal cross section shown in Figure 22, at the membrane interface, the first polar medium solution in the receiving chamber of the recess exhibits a slightly convex shape, indicating that the actual shape of the carrier membrane is a slightly convex curved surface. As can be seen from the top view shown in Figure 22, the boundary of the carrier membrane is a relatively regular circle.

[0120]

[0144] Figure 23 is a confocal fluorescence microscope phenogram of a membrane array in a second embodiment of the membrane formation method according to the present disclosure. Referring to Figure 16, in this second embodiment of the membrane formation method, an aqueous solution of potassium chloride (500 mM) and Hepes (5 mM) as a first polar medium solution is introduced into a channel of a membrane formation device containing multiple recesses (Figure 11(b)), and the aqueous solution fills the holding chamber. The aqueous solution also contains 0.02% by weight of rhodamine B. Next, 10 mL of air is forced through the channel of the membrane formation device to completely expel the aqueous solution directly above the main body and the opening member while retaining the aqueous solution in the recesses. Due to the effect of fluid shear force, an interface between the aqueous solution and air is formed in the hollow portion of the opening member.

[0121]

[0145] The sealing cover plate is then removed, and 30 μL of a nonpolar medium solution (n-hexadecane) containing 50 mg / mL of amphiphilic molecules (1,2-bis(diphenylphosphine)ethane (DPPE) phospholipid molecules) is dropped directly onto the main body and allowed to stand for 10 minutes. During this time, the phospholipid molecules self-assemble at the water-oil interface by molecular forces to form a stable phospholipid monolayer. The sealing cover plate is then assembled, and 1 mL of a second polar solution without rhodamine B is pushed through the membrane-forming device's channel. The device is then allowed to stand for 24 hours without disturbance, during which time the membrane self-assembles to form a stable phospholipid bilayer.

[0122]

[0146] After the carrier membrane stabilizes, 5 mL of a solution of 500 mM potassium chloride and 5 mM Hepes without rhodamine is pumped through the liquid inlet for flushing. Referring to Figure 23, circular fluorescent spots appearing in the microscopic phenogram indicate the formation of a stable carrier membrane. If membrane formation fails, the membrane ruptures and the diffusion of the fluorescent solvent at the bottom of the recess fails to be prevented. The fluorescent dye in the recess's containing chamber rapidly diffuses into the non-fluorescent second polar solution outside the containing chamber, eventually appearing gray-black in the fluorescence microscopic phenogram. Figure 23 shows strong fluorescence at the locations corresponding to each recess, indicating that the membrane formation yield reached 100%.

[0123]

[0147] 24 and 25 are graphs showing transmembrane current curves monitored when a triangular wave voltage is applied to different channels after membrane formation in an embodiment of the membrane formation device according to the present disclosure. Based on the membrane formation device having a hollow portion with a diameter of about 47 μm shown in FIG. 19, after the membrane is formed by the membrane formation method disclosed herein, the carrier membrane is stabilized for 24 hours, and then a triangular wave voltage with an amplitude of 50 mV is applied to the electrodes on both sides of the carrier membrane, and the current is monitored in real time.

[0124]

[0148] Referring to Figure 24, when an independent channel is driven by a triangular voltage, the transmembrane current appears as a quasi-square wave with a peak value of approximately 80-90 pA. The membrane capacitance is then calculated to be 40 pF, which is within a reasonable capacitance range for normal membrane formation (10-150 pF). Referring to Figure 25, when another independent channel is driven by a triangular voltage, the transmembrane current appears as a quasi-square wave with a peak value of approximately 60-70 pA. The membrane capacitance is then calculated to be 30.7 pF, which is within a reasonable capacitance range for normal membrane formation (10-150 pF).

[0125]

[0149] Figure 26 is a graph showing the transmembrane current curve monitored when a triangular wave voltage was applied after membrane formation in one embodiment of the membrane formation apparatus disclosed herein. Based on a membrane formation apparatus with a hollow portion approximately 90 μm in diameter, after membrane formation by the membrane formation method disclosed herein, the carrier membrane was stabilized for 24 hours. A triangular wave voltage with an amplitude of 50 mV was then applied to electrodes on both sides of the carrier membrane, and the current was monitored in real time. Referring to Figure 26, when driven by the triangular wave voltage, the transmembrane current appears as a quasi-square wave with a peak value of approximately 200-220 pA. The membrane capacitance was then calculated to be 100 pF, which is within a reasonable capacitance range (10-150 pF) for successful membrane formation.

[0126]

[0150] In another embodiment, a membrane formation device with a hollow section approximately 80 μm in diameter, as shown in Figure 18, was used. After membrane formation using the membrane formation method of the present disclosure, capacitance statistics were performed. Immediately after membrane formation, a relatively large amount of nonpolar medium solution still existed within the membrane, resulting in a large membrane thickness or small membrane area. Therefore, the membrane capacitance of most channels was measured to be 0-20 pF. After 24 hours, the capacitance was monitored again. The membrane capacitance of most channels increased to approximately 30-40 pF and tended to stabilize. All membrane capacitance values ​​were found to be within a reasonable capacitance range for normal membrane formation (10-150 pF). After monitoring, only one of the 128 channels had a membrane capacitance greater than 300 pF, which was determined to be a short circuit due to membrane rupture. However, the remaining 127 channels all had intact carrier membranes. Therefore, the membrane formation yield was calculated to be 127 / 128 = 99.2%. Therefore, not only is the film formation yield very high, but the stability of the carrier film is also very good.

[0127]

[0151] FIG. 27 is a graph showing the capacitance of multiple channels over time during the film formation process in one embodiment of a film formation apparatus according to the present disclosure. A film formation apparatus with a hollow portion approximately 110 μm in diameter was used. The film formation apparatus uses silver / silver chloride electrodes. After a film is formed using the film formation method described herein, a curve of the membrane capacitance of multiple independent channels in the film formation apparatus is obtained by long-term measurement. The membrane capacitance data recording mode records the capacitance of all channels every 10 minutes. Referring to FIG. 27, the x-axis represents time in the smallest unit of hours (h), and the y-axis represents capacitance in picofarads (pF). FIG. 27 records capacitance data of multiple independent channels over a total of 15 hours after film formation. As can be seen from the data, during the initial stage of film formation, the capacitance of most channels is between 0 and 20 pF. After that, the carrier film spontaneously thins due to intermolecular van der Waals forces, resulting in a continued increase in the area of ​​the carrier film and a gradual increase in capacitance.

[0128]

[0152] The effective area of ​​the membrane is calculated using the membrane capacitance calculation formula for a parallel plate capacitor: C = (ε0 * ε r *S) / d, where C represents the membrane capacitance, ε represents the vacuum permittivity, and ε r where σ represents the dielectric constant of the membrane molecules, S represents the effective area of ​​the membrane, and d represents the thickness of the membrane. Generally, when amphiphilic molecules self-assemble to form stable monolayer or bilayer membranes, the membrane thickness d and the dielectric constant of the membrane molecules remain constant, and the vacuum dielectric constant ε0 is constant, so the membrane capacitance C has a direct linear dependence on the effective area S of the membrane. The larger the effective area of ​​the membrane, the larger the capacitance; conversely, the smaller the effective area of ​​the membrane, the smaller the capacitance. The capacitance of most channels rapidly hops and stabilizes at approximately 110 pF between 1 and 10 hours after membrane formation. This capacitance value is within a reasonable capacitance range (10–150 pF) for successful membrane formation. Only a few channels have yet to achieve stable capacitance.

[0129]

[0153] FIG. 28 is a schematic diagram of a detection current signal when a single molecule is detected by an electrical method using a carrier membrane formed by one embodiment of the membrane-forming apparatus according to the present disclosure. The carrier membrane obtained by the above-described membrane-forming apparatus and membrane-forming method can be used, for example, for single-molecule detection by an electrical method. After a stable carrier membrane is formed, 200 μL of a solution containing a nanopore protein (MspA) is introduced into the flow channel. The nanopore protein is randomly distributed near the carrier membrane due to the flow of the solution and the thermal motion of the molecules. Then, under voltage stimulation, the single nanopore protein successfully passes through the molecular barrier in the carrier membrane and is successfully embedded in the carrier membrane. After the nanopore protein is successfully embedded, a solution containing the nucleic acid molecule to be tested is added to the flow channel inlet of the membrane-forming apparatus, and a voltage of 180 mV is then applied to the electrodes of the membrane-forming apparatus. The single nucleic acid molecule passes through the nanopore protein under the action of the electric field force, generating a continuous ionic current blockage signal. Referring to FIG. 28, when a nucleic acid molecule passes through a nanopore, it generates a current fluctuation signal of about 50-150 pA.

[0130]

[0154] 29(a) and 29(b) are schematic dimensional diagrams of a film-forming device in the related art and one embodiment of a film-forming device according to the present disclosure. Conventional electrochemical detection typically uses a consumable electrode, such as a silver / silver chloride electrode. In this case, it is usually necessary to focus on the service life of the electrode itself, instead of the volume of electrolyte and the consumption of solution in the accommodation chamber of the film-forming device.

[0131]

[0155] However, in other electrochemical detection systems, the detection electrode may be a non-consumable electrode (such as a gold or platinum electrode or other inert metal electrode), and the solution in the storage chamber of the membrane-forming device must be correspondingly replaced with a consumable electron pair solution (such as a potassium ferricyanide / potassium ferrocyanide solution). In this case, limited by the need to increase the array density and the number of detection channels, the size of the recess is typically designed to be very small, and the volume of the solution in the storage chamber is very small (usually on the nanoliter scale). Therefore, it is necessary to focus on the effect of the internal volume of the recess on the detection lifetime. Referring to Figures 29(a) and 29(b), a calculation and comparison is performed between the total volume of the solution in the storage chamber of the related art shown in Figure 29(a) and the total volume of the solution in the storage chamber of the embodiment of the membrane-forming device according to the present disclosure shown in Figure 29(b). It can be seen that in the case of a non-consumable electrode and a consumable electron pair solution, the storage chamber of the embodiment of the membrane-forming device according to the present disclosure has significant advantages.

[0132]

[0156] 29(a) and 29(b), the storage chamber of the related art and the storage chamber of the embodiment of the film-forming apparatus according to the present disclosure are both assumed to be cylindrical. The corresponding cylinder of the storage chamber of the related art has a diameter d0 and a height h0. In the embodiment of the film-forming apparatus according to the present disclosure, the corresponding cylinder of the storage chamber has a diameter d1 and a height h1, the height of the aperture member is h2, and the diameter of the hollow portion of the aperture member is d2.

[0133]

[0157] According to the volume formula, the internal solution volume of the related art containment chamber is V0 = (π * d02 It can be calculated as *h0) / 4, and the internal solution volume of the accommodation chamber of the embodiment of the film forming apparatus according to the present disclosure is V1≒(π*d1 2 It is calculated as *h1) / 4. Under the condition (d2<d0) that effectively reduces the membrane area, when d1 = 2*d0 and h1 = h0, V1≒(π*d1 2 *h1) / 4 = 4*((π*d0 2 *h0) / 4)=4*V0, that is, the internal solution volume of the accommodation chamber of the embodiment of the film forming apparatus according to the present disclosure is about 4 times that of the internal solution volume of the accommodation chamber of the related art.

[0134]

[0158] Taking typical values of d0 = 100μm, d1 = 200μm, h0 = 100μm, and h1 = 100μm, V0 = 7.85×10 5 μm 3 and V1 = 3.14×10 6 μmI 3 t can be seen that the embodiment of the film forming apparatus according to the present disclosure has great advantages compared with the related art. The storage capacity of the solution is more than 4 times that of the related art, thereby ensuring a longer detection life.

[0135]

[0159] The data presented in the above various experiments are technically interoperable and related to each other. For the formed carrier film, the measured capacitance of the carrier film is within a reasonable range (10~150 pF), which means normal film formation. Therefore, using the film penetration current curves in the cases of the hollow parts with diameters of about 47μm and 90μm shown in FIGS. 24 to 26, the single-channel capacitance measurement results are presented. The capacitance change diagram in the case of the hollow part with a diameter of about 110μm presents the dynamic change process of the capacitance from the start of film formation until a finally stable film is formed, as shown in FIG. 27. The film formation yield obtained from the multi-channel statistics of the film forming apparatus with a hollow part having a diameter of about 80μm is also provided above.

[0136]

[0160] The above has described in detail various embodiments of the present disclosure. In order to avoid obscuring the concept of the present disclosure, some details well known in the art are not described. Those skilled in the art can fully understand how to implement the technical solutions disclosed in this specification according to the above description.

[0137]

[0161] Although some specific embodiments of the present disclosure have been described in detail through examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Those skilled in the art should understand that the above embodiments can be modified or some technical features can be replaced with equivalents without departing from the scope and spirit of the present disclosure. The scope of the present disclosure is defined by the appended claims.

Claims

1. A step of preparing a film forming apparatus, the film forming apparatus comprising: a base (10); a body (20) disposed on the base (10) and having a recess; an apertured member (30) disposed inside a recess port at an end of the recess remote from the base (10), the apertured member (30) being configured such that a hollow portion (32) of the apertured member (30) forms a membrane (80); and Assembling a sealing cover plate (60) on the upper side of the body (20) to form a flow passage between the sealing cover plate (60) and the body (20); introducing a first polar medium solution (M1) into the flow path, so that the first polar medium solution (M1) flows from the hollow portion (32) of the opening member (30) into the recess of the main body (20) and fills the recess of the main body (20); removing the first polar medium solution (M1) between the aperture member (30) and the sealing cover plate (60), and allowing the non-polar medium solution (M3) and the second polar medium solution (M4) to cover the aperture member (30); Including, At least one of the non-polar medium solution (M3) and the second polar medium solution (M4) contains amphiphilic molecules that form an amphiphilic molecule film (80) in the hollow portion (32); The step of removing the first polar medium solution (M1) between the opening member (30) and the sealing cover plate (60) comprises: introducing a gas medium (M2) into the flow path so that the gas medium (M2) removes the first polar medium solution (M1) between the body (20) and the sealing cover plate (60) and between the opening member (30) and the sealing cover plate (60); A film forming method comprising:

2. 2. The membrane forming method of claim 1, wherein the recess is configured to define a storage chamber (21) for storing a polar medium solution, the membrane (80) is an amphiphilic molecular membrane, and the cross-sectional area of ​​the hollow portion (32) of the opening member (30) is smaller than the cross-sectional area of ​​the storage chamber (21).

3. The body (20) a first body layer (22) disposed on the surface of the base (10); a second body layer (23) located on the side of the first body layer (22) away from the base (10); Equipped with The recess penetrates the second body layer (23) and the first body layer (22) in a direction perpendicular to the base (10), and the opening member (30) is connected to a part of the recess that penetrates the second body layer (23). The film forming method according to claim 1 .

4. The opening member (30) a flange (31) projecting radially inwardly relative to said recessed port; Equipped with The inner edge of the flange (31) forms the hollow portion (32). The film forming method according to claim 1 .

5. 5. The method of claim 4, wherein the inner edges of the flanges (31) are located on the same plane.

6. 5. The method for forming a film according to claim 4, wherein the protruding end of the flange (31) is chamfered or rounded.

7. 5. The method for forming a film according to claim 4, wherein the cross section of the flange (31) is T-shaped or convex polygonal.

8. 5. A film forming method as described in claim 4, wherein the thickness of the protruding end of the flange (31) in a direction perpendicular to the base (10) is less than or equal to the thickness of the root portion of the flange (31) in a direction perpendicular to the base (10).

9. 5. The method for forming a film according to claim 4, wherein the thickness of the flange (31) in a direction perpendicular to the base (10) gradually decreases from the root portion of the flange (31) to the protruding end of the flange (31).

10. The film forming method according to claim 4, wherein the flange (31) has at least a portion that protrudes or is recessed from a surface of the flange (31) in a direction perpendicular to the base (10).

11. 11. The method of claim 10, wherein the at least one portion is located at least on a side of the flange (31) remote from the base (10).

12. The method of claim 4, wherein the inner edge of the flange (31) has a plurality of inwardly projecting protrusions.

13. The film forming apparatus, a first electrode (51) disposed outside the containing chamber (21); a second electrode (52) disposed inside the receiving chamber (21); Furthermore, The base (10) A substrate (11), At least one passivation layer (12, 13) disposed between the substrate (11) and the body (20); Equipped with The second electrode (52) is exposed from the bottom of the recess, the second electrode (52) is located between the substrate (11) and the at least one passivation layer (12, 13), or the second electrode (52) is located on a side of the at least one passivation layer (12, 13) remote from the substrate (11), or the second electrode (52) is located between adjacent passivation layers (12, 13) when the at least one passivation layer (12, 13) is configured as a plurality of passivation layers (12, 13). The film forming method according to claim 2 .

14. The film forming method according to claim 1, wherein the apertured member (30) has a plurality of hollow portions (32).

15. The step of covering the opening member (30) with the non-polar medium solution (M3) and the second polar medium solution (M4) introducing the non-polar medium solution (M3) into the flow path so that the non-polar medium solution (M3) covers the opening member (30); introducing the second polar medium solution (M4) into the flow path so that the second polar medium solution (M4) covers the opening member (30); The film forming method according to claim 1 , comprising:

16. The step of covering the opening member (30) with the non-polar medium solution (M3) and the second polar medium solution (M4) removing the sealing cover plate (60); coating the non-polar medium solution (M3) on the body (20) and the aperture member (30) so that the non-polar medium solution (M3) covers the aperture member (30); assembling the sealing cover plate (60) on top of the body (20) to form a flow passage between the sealing cover plate (60) and the body (20); introducing the second polar medium solution (M4) into the flow path so that the second polar medium solution (M4) covers the opening member (30); The film forming method according to claim 1 , comprising:

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