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 membrane forming apparatus and a membrane 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 modification of DNA molecules and can directly sequence single molecules continuously. Compared with the first and second generation gene sequencing technologies, the next generation nanopore-based gene sequencing technology has advantages such as low cost, long read length, easy integration and portability, and is widely used in various fields.

[0003]

[0003] Current sequencing technology based on biological nanopores is considered the most potential and disruptive of many nanopore technologies and has been successfully commercialized. However, current sequencing technology based on biological nanopores still faces many challenges, one of the biggest challenges comes from the stability of the sequencing chip. In order to maintain the stability of biological nanopore proteins, it is necessary to embed the pore proteins in a carrier membrane with a thickness of a monolayer of molecules. The carrier membrane is usually formed by the self-assembly of amphiphilic molecules, and the membrane material is usually 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 that includes 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, wherein a hollow portion of the aperture member is configured to form a film.

[0005]

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

[0006]

[0006] In some embodiments, the body includes a first body layer disposed on a surface of the base and a second body layer located on a 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]

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

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

[0010]

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

[0011]

[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 less than or equal to the thickness of the root portion of the flange in a direction perpendicular to the base.

[0014]

[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]

[0016] In some embodiments, at least a portion is located 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]

[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 containing 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 aperture member has a plurality of 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 apparatus 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 aperture 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 aperture 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]

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

[0028]

[0028] In one aspect of the present disclosure, there is provided a membrane formation method including the steps of preparing a membrane 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 material 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 material and the sealing cover plate and allowing a non-polar medium solution and a second polar medium solution to cover the opening material, wherein at least one of the non-polar medium solution and the second polar medium solution contains amphiphilic molecules that form an amphiphilic molecular membrane 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 a step of introducing the non-polar media solution into the flow path so that the non-polar media solution covers the opening material, and a step of 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 media solution and the second polar media solution to cover the aperture material includes the steps of removing the sealing cover plate, coating the body and the aperture material with the non-polar media solution and allowing the non-polar media solution to cover the aperture material, 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 media solution into the flow path so that it covers the aperture material.

[0032]

[0032] In some embodiments, the step of removing the first polar media solution between the opening material and the sealing cover plate and covering the opening material with the non-polar media solution and the second polar media solution includes a step of introducing a non-polar media solution into the flow path to achieve removal of the first polar media solution between the body and the sealing cover plate and between the opening material and the sealing cover plate with the non-polar media solution and covering the opening material with the non-polar media solution, and a step of introducing a second polar media solution into the flow path so that the second polar media 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 present disclosure.

[0035]

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

[0036] [Figure 1] 1 is a schematic, partially cutaway, three-dimensional structural diagram of a film-forming apparatus according to some embodiments of the present disclosure. [Diagram 2] 1 is a schematic longitudinal cross-sectional view of a film-forming apparatus according to some embodiments of the present disclosure. [Diagram 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. [Diagram 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 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. [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 shows cross-sectional shapes of hollow portions and containing chambers in some embodiments of a film-forming apparatus according to the present disclosure. [Figure 9] 13 shows the shape of the flange of the aperture member in some embodiments of the film forming apparatus according to the present disclosure. [Figure 10]1 shows the longitudinal cross-sectional shape of a receiving chamber in some embodiments of a film-forming apparatus according to the present disclosure. [Figure 11] 13 illustrates the arrangement of multiple recesses in a body in some embodiments of a film forming device according to the present disclosure. [Figure 12] 1 is a schematic structural diagram 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] 4 is a schematic flow chart 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 a series of steps of some embodiments of a film formation method according to the present disclosure. [Figure 16] 1A-1C 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] 1A-1C 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 a first embodiment of a film forming apparatus according to the present disclosure. [Figure 19] 1 is a scanning electron microscope phenogram of a second embodiment of the film forming apparatus according to the present disclosure. [Figure 20] 13 is a partially cutaway scanning electron microscope phenogram of a third embodiment of the film forming apparatus according to the present disclosure. [Figure 21] 13 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]11 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] 13 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. [Diagram 25] 13 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] 1 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] 1 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 schematic diagram showing a film-forming apparatus according to a related art; and FIG. 1B is a schematic diagram showing a film-forming apparatus according to an embodiment of 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 PREFERRED EMBODIMENTS

[0037]

[0061] Various exemplary embodiments of the present disclosure will be described in detail below with reference to the drawings. The description of the exemplary embodiments is merely illustrative and is in no way intended to limit the present disclosure and its application or use. The present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that the disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Please note that unless otherwise specified, the relative arrangement of components and steps described in these embodiments, composition of materials, formulas and numerical values ​​should be interpreted as merely illustrative and not limiting.

[0038]

[0062] The terms "first", "second", and similar terms used in this disclosure do not indicate order, quantity, or importance, but are used only to distinguish different parts. The terms "include" or "comprise" mean that the element before the word covers the elements listed after the word, without excluding the possibility of covering other elements. The terms "upper", "lower", "left", "right", and the like are used only to indicate relative positional relationships. After the absolute positions of the described objects are changed, the relative positional relationships may 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 device or the 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] 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, unless otherwise defined. In addition, for example, terms defined in 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 in this specification.

[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 under appropriate circumstances.

[0042]

[0066] Our research has found 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 the stability of the membrane. The stability of the membrane directly affects the stability of the pore protein, and the membrane formation yield directly determines the number of single nanopores. Therefore, the membrane formation device and its membrane formation method and efficiency ultimately affect the data output and data quality of the entire sequencing system.

[0043]

[0067] In some related art, the sequencing chip includes a recess structure, and the bottom of the recess is provided with a chip electrode. 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, and using the hydrophilicity of the chip electrode to repel the hydrophobic layer to the side wall of the recess, so that the chip electrode is electrically connected to the polar medium solution; draining the polar medium solution outside the recess; and sequentially introducing the amphiphilic molecule and the polar medium solution used for forming the membrane to form a carrier membrane composed of a single / double layer of amphiphilic molecules in the recess port of the recess.

[0044]

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

[0045]

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

[0046]

[0070] 2. Based on the process requirements, the chip body needs to be pretreated with a hydrophobic coating first, 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 lifetime.

[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 carrier film formed.

[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. FIG. 2-FIG. 5 are schematic longitudinal cross-sectional views of a film-forming apparatus according to some embodiments of the present disclosure. Referring to FIG. 1-FIG. 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 containment chamber 21 for containing a polar medium solution, 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, and the membrane 80 is an amphiphilic molecular membrane.

[0052]

[0076] When the membrane forming apparatus of this embodiment is used to form a carrier membrane composed of amphiphilic molecules (i.e., an amphiphilic molecular membrane), the shape and size of the final stabilized carrier membrane are determined by the shape and size of the hollow part of the aperture material, since the formation of the carrier membrane is a dynamically stable self-assembly process. With the hollow part of the aperture material having a smaller cross-sectional area, the area of ​​the formed carrier membrane can be effectively reduced, thereby improving the stability of the carrier membrane and further improving the mechanical stability of the entire chip. Furthermore, by reducing the area of ​​the carrier membrane, the membrane capacitance of the carrier membrane can be effectively reduced, thereby reducing the electrical noise of the system. With a relatively large cross-sectional area of ​​the containing chamber, the volume of the electrochemical solution in the containing chamber and the detection life of the device can be extended.

[0053]

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

[0054]

[0078] In FIG. 1, the receiving chamber 21 can receive a polar medium solution in a liquid state. The polar medium solution can flow into the receiving chamber 21 from the upper side of the 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 body 20 can be a non-conductive photoresist or other non-conductive material. The material of the body 20 can also include a semiconductive material or a conductive material, and a non-conductive passivation layer is formed on the surface of the semiconductive material or the conductive material.

[0055]

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

[0056]

[0080] 2, in some embodiments, the body 20 is constructed as a single layer structure, and the aperture member 30 is connected to the inside of a recess port of a recess in the body 20. The aperture member 30 may be formed after 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 a 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 may be configured as 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 bonding processes. The first body layer 22 and the second body layer 23 may be made of the same material or different materials. Materials that may 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 body 20 is constructed as a single layer structure and the aperture member 30 is integrally formed with the body 20 to simplify processing. The 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 remote from the substrate 10 to perform a set function.

[0061]

[0085] In FIG. 2, the aperture member 30 may include a flange 31 that projects radially inwardly 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 lie on the same plane to facilitate processing and help form a more stable carrier film, while in other embodiments, the inner edges of flange 31 may not lie on the same plane.

[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 receiving chamber 21 and a second electrode 52 disposed inside the receiving 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 at the bottom of the receiving chamber 21, or may be disposed on a side wall of the receiving chamber 21, or may be suspended within the receiving 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 adopt 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 a lower current, and have stability to the aqueous solution. 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, or conductors such as graphene or titanium nitride, or semiconductor materials. The materials used for the first electrode 51 and the second electrode 52 may be the same or different. The first electrode 51 and the second electrode 52 may be composite electrodes formed by stacking or mixing multiple materials. The surfaces of the first electrode 51 and the second electrode 52 may or may not be covered with materials such as conductive polymers and hydrogels.

[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] To facilitate the formation of the amphiphilic molecular film, referring to FIG. 6A, 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 removable. The sealing cover plate 60 can be assembled with the main body 20 to form a flow path located above the main body 20, and the flow path 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 path 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 body 20 to form a flow path located above the body 20, and the flow path 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 accommodation chamber 21, and the second polar medium solution M4 is contained outside the accommodation 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 to the upper and / or lower sides of 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 non-polar medium solution M3 may remain in the layer in which 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 non-polar 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 FIG. 6B, in some embodiments, the membrane channel is embedded in the amphiphilic molecular membrane. Optionally, the membrane channel is, for example, a nanopore protein. Nanopore protein refers to a protein that can form a transmembrane protein pore. The 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 other monomers).

[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 body 20. The second electrode 52 is exposed from the bottom of the recess so as to contact the aqueous solution in the receiving 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 the two passivation layers 12, 13 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 epoxy resin photoresist or polyimide film, or an insulating paint commonly used to passivate printed circuit boards, such as acrylic resin.

[0078]

[0102] (a)-(g) of FIG. 8 respectively show the cross-sectional shapes of the hollow portion and the receiving chamber in some embodiments of the film-forming apparatus according to the present disclosure. Referring to FIG. 8, the orthogonal projection of the cross-section of the hollow portion 32 on the base is located within the cross-section of the receiving chamber. The cross-section of the hollow portion 32 and the cross-section of the receiving chamber 21 may be the same or different in shape, the cross-section of the hollow portion 32 may be in the shape of a circle or a convex polygon, and the cross-section of the receiving chamber 21 may be in the shape of a circle or a convex polygon. The convex polygon may be in the shape of a triangle, a rectangle, a square, a hexagon, an octagon, etc. The hollow portion having a cross-section of a circle or a convex polygon is more likely to form a stable carrier film. The receiving chamber having a cross-section of a circle or a convex polygon 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 protruding inwardly. The non-uniform edge microstructure formed by the multiple protrusions can form a carrier film with higher stability.

[0081]

[0105] 9(a)-(i) show the shapes of the flanges of the aperture members 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 the T-shaped cross section in the direction perpendicular to the base 10 is equal to or less than the thickness of the root part of the flange 31 in the direction perpendicular to the base 10. The aperture member of 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 in shape. 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 gentle acute-angle edge 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 flange 31 is rectangular in shape. In Fig. 9(d), the cross section of flange 31 is convex pentagonal in shape, which means that the base of flange 31 is rectangular and the protruding end is triangular. In Fig. 9(g)-(h), the cross section of flange 31 is convex quadrilateral in shape, and the position of the inner edge of the protruding end may be flush with the highest or lowest point of flange 31.

[0084]

[0108] The increase in the specific surface area of ​​the flange 31 can make the non-polar medium solution adhere more easily and improve the stability of the formed carrier film. Referring to FIG. 9(i), the protruding end of the flange 31 may be chamfered or rounded. Referring to FIG. 9(e) and FIG. 9(f), 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 be a discrete or continuous raised or recessed microstructure. In FIG. 9(f), the at least a portion is located at least on the side of the flange 31 away from the base 10. The carrier film may 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)-(d) show the shape of the longitudinal cross section of the accommodation 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 accommodation chamber 21 is rectangular, trapezoidal, curved-edge trapezoidal, or drum-shaped. The sidewall of the recess defining the accommodation cavity 21 may be a vertical structure, an inclined structure, or a curved structure, or any combination of curved, inclined, and vertical structures.

[0086]

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

[0087]

[0111] 11(a)-(c) show the arrangement of a plurality of 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 a plurality of recesses. From a top view, the plurality of 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 shape. For example, in FIG. 11(a), the plurality of recesses are arranged in a rectangular array, and the recesses are arranged at approximately the same row intervals and approximately the same column intervals. In FIG. 11(b), the plurality of recesses are arranged in a honeycomb array. In FIG. 11(c), the plurality of recesses are arranged in an irregular shape.

[0088]

[0112] 12(a)-(c) are schematic structural diagrams showing an aperture member having multiple hollow portions in some embodiments of the membrane forming apparatus according to the present disclosure. Referring to FIG. 12, in some embodiments, an 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 form smaller carrier membranes, respectively. Depending on application requirements, membrane channels such as nanopore proteins may be embedded in the carrier membrane formed in one or more hollow portions 32 of the aperture member 30.

[0089]

[0113] In Fig. 12(a) and (c), the apertured member 30 has a greater number of hollow portions 32, and in Fig. 12(b), the apertured member 30 has a lesser number of hollow portions 32. The multiple hollow portions 32 may be disposed at equal or different intervals. The cross section of each hollow portion 32 may be in the shape of a circle or a convex polygon.

[0090]

[0114] Based on the above-mentioned embodiments of the film forming apparatus according to the present disclosure, the present disclosure further provides a corresponding film forming method. Figure 13A is a schematic flow chart 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 passage 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 non-polar medium solution M3 and the second polar medium solution M4 to cover the aperture member 30.

[0094]

[0118] In this embodiment, at least one of the non-polar 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 to the upper and / or lower sides of the flange 31. The layer in which the amphiphilic molecular film 80 is located can hold a portion of the non-polar medium solution M3. 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 non-polar 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 molecules include, but are not limited to, lipids, such as 1,2-bis(diphenylphosphine)ethane (DPPE) phospholipid molecules, which may be polymers, such as diblock and triblock. The lipids may include head groups and hydrophobic tail groups. In the formed amphiphilic molecular membrane, the lipid head groups may face the polar medium solution, and the hydrophobic tail groups may face the non-polar 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 an aqueous solution of potassium chloride, or potassium ferricyanide / potassium ferrocyanide solution, etc. The non-polar medium solution M3 may be a water-insoluble solvent, such as n-hexadecane, n-decane, n-hexane, and silicone oil.

[0097]

[0121] Figure 13B is a schematic flow chart of some embodiments of the membrane formation method according to the present disclosure. Referring to Figure 13B, compared with Figure 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 some embodiments of a film-forming method according to the present disclosure. Referring to (a) and (b) of FIG. 14 to (a) and (b) of FIG. 17, the assembly of the sealing cover plate 60 and the 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 Figures 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 of air, nitrogen, argon, etc., or a combination thereof. 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 in the hollow portion 32 of the aperture member 30.

[0101]

[0125] In other embodiments, the first polar media solution M1 between the aperture member 30 and the sealing cover plate 60 may also be removed by other means, such as by introducing a liquid that is insoluble with the first polar media solution M1, such as a non-polar media 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 material 30 in step S30 includes the steps of introducing the non-polar media solution M3 into the flow path such that the non-polar media solution M3 covers the opening material 30, and introducing the second polar media solution M4 into the flow path such that the second polar media solution M4 covers the opening material 30.

[0103]

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

[0104]

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

[0105]

[0129] Referring to FIG. 16, in some embodiments, in step S30, the step of causing the non-polar media solution M3 and the second polar media solution M4 to cover the opening material 30 includes the steps of removing the sealing cover plate 60, coating the main body 20 and the opening material 30 with the non-polar media solution M3 and causing the non-polar media solution M3 to cover the opening material 30, assembling the sealing cover plate 60 to the upper side 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 opening material 30.

[0106]

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

[0107]

[0131] The amphiphilic molecules can be dissolved in the non-polar media solution M3, or the second polar media solution M4, or both the non-polar media solution M3 and the second polar media solution M4. Referring to FIG. 16(g), a membrane 80 separating the first polar media solution M1 and the second polar media 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 thin and expand in area by 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 material 30 and the sealing cover plate 60 and covering the opening material 30 with the nonpolar media solution M3 and the second polar media solution M4 includes a step of introducing the nonpolar media solution M3 into the flow path so as to achieve the removal of 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 material 30 and the sealing cover plate 60 with the nonpolar media solution M3, and the covering of the opening material 30 with the nonpolar media solution M3, and a step of introducing the second polar media solution M4 into the flow path so that the second polar media solution M4 covers the opening material 30.

[0109]

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

[0110]

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

[0111]

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

[0112]

[0136] FIG. 18 is a scanning electron microscope phenogram of the first embodiment of the 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 part of the aperture member of the film-forming apparatus is about 80 μm. A plurality of sets of circumferentially arranged micro-groove structures are provided on the flange of the aperture member, and each set of micro-groove structures includes a plurality of radially arranged micro-groove structures. This micro-groove structure is beneficial for forming an interface between the gas medium or non-polar medium solution and the first polar medium solution and maintaining the carrier film. The structure is formed by spin-coating SU-8 photoresist (final thickness 95 μm) on a silicon substrate, and then carrying out processes such as pre-baking, exposure, post-baking, developing, and hard-baking.

[0113]

[0137] FIG. 19 is a scanning electron microscope phenogram of the second embodiment of the 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 part of the aperture member of the film-forming apparatus is less than 50 μm, and is finally measured to be about 47 μm. A plurality of sets of circumferentially arranged micro-groove structures are provided on the flange of the aperture member, and each set of micro-groove structures includes a plurality of radially arranged micro-groove structures. This micro-groove structure is beneficial for forming an interface between the gas medium or non-polar medium solution and the first polar medium solution and maintaining the carrier film. The structure is formed by spin-coating SU-8 photoresist (final thickness 95 μm) on a silicon substrate, and then carrying out processes such as pre-baking, exposure, post-baking, developing, and film curing.

[0114]

[0138] FIG. 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 about 80 μm. The shape of the hollow portion of the aperture member is approximately circular, and the flange is smooth without microstructure. The sidewall of the containing chamber in the aperture member is a smooth curved surface.

[0115]

[0139] FIG. 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 aperture member of the film-forming apparatus is about 75 μm. The shape of the hollow portion of the aperture member is approximately circular with an irregular pattern, and its inner edge has a plurality of about 3 μm raised microstructures protruding inward. The raised microstructures can increase the stability of the carrier film. The sidewall of the containing chamber in the aperture member is a smooth curved surface.

[0116]

[0140] FIG. 22 is a confocal fluorescence microscope phenogram of a membrane array in a first embodiment of a membrane-forming method according to the present disclosure, cut away from a top view. Referring to FIG. 14, in the first embodiment of the membrane-forming method, an aqueous solution of potassium chloride (500 mM) and Hepes (5 mM) as a first polar medium solution is introduced into a flow channel of a membrane-forming device including a plurality of recesses ((b) of FIG. 11), and the aqueous solution fills the receiving chamber. Then, 10 mL of air is forced through the flow channel of the membrane-forming device to completely drain the aqueous solution directly above the 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) in which amphiphilic molecules (1,2-bis(diphenylphosphine)ethane (DPPE) phospholipid molecules) (50 mg / mL) were dissolved, and 1 mL of an aqueous solution of potassium chloride (500 mM) and Hepes (5 mM) as a second polar medium solution were sequentially pushed into the cavity.

[0117]

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

[0118]

[0142] The object depicted in FIG. 22 is an array of carrier membranes formed on an array of multiple recesses with an aperture diameter of about 80 μm. The fluorescence confocal microscope (FCFM) model used in the experiment is Nikon C2+. The first polar media solution in the receiving chamber in each recess contains 0.02% by weight of Rhodamine B fluorescent dye. The excitation peak wavelength and emission peak wavelength of Rhodamine B are 546 nm and 568 nm, respectively. The second polar media solution above the hollow part of the aperture material does not contain Rhodamine B. 2 μM of BODIPY FL fluorescent dye is mixed into the non-polar media solution. The excitation peak and emission peak of BODIPY FL are 503 nm and 512 nm, respectively. Since BODIPY FL is readily soluble in the non-polar media solution, the area containing the non-polar media solution in the array device appears green.

[0119]

[0143] In the microscopic phenogram shown in FIG. 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 has no fluorescence and shows a black background. The non-polar medium solution containing phospholipids appears light green in the phenogram (indicated by arrow G). As can be seen from the fluorescence confocal cross section shown in FIG. 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 FIG. 22, the boundary of the carrier membrane is a relatively regular circular shape.

[0120]

[0144] FIG. 23 is a confocal fluorescence microscope phenogram of a membrane array in a second embodiment of a membrane formation method according to the present disclosure. Referring to FIG. 16, in the 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 flow channel of a membrane formation device including a plurality of recesses ((b) of FIG. 11), and the aqueous solution fills the receiving chamber. The aqueous solution also contains 0.02% by mass of Rhodamine B. Then, 10 mL of air is forced through the flow channel of the membrane formation device to completely drain the aqueous solution directly above the 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 non-polar medium solution (n-hexadecane) containing 50 mg / mL of amphiphilic molecules (1,2-bis(diphenylphosphine)ethane (DPPE) phospholipid molecules) is then dropped directly onto the main body and left for 10 minutes. At 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 flow channel of the membrane-forming device. The device is then left for 24 hours without disturbance, during which time the membrane self-assembles to form a stable phospholipid bilayer.

[0122]

[0146] After the carrier membrane is stabilized, 5 mL of a solution of 500 mM potassium chloride and 5 mM Hepes without Rhodamine is pushed through the liquid inlet for flushing. Referring to Figure 23, the circular fluorescent spot appearing in the microscope phenogram represents the formation of a stable carrier membrane. If the membrane formation fails, the diffusion of the fluorescent solvent cannot be blocked at the bottom of the recess after the membrane ruptures. The fluorescent dye in the accommodation chamber of the recess diffuses quickly into the non-fluorescent second polar solution outside the accommodation chamber, and finally appears gray-black in the fluorescence microscope phenogram. In Figure 23, there is strong fluorescence at the position corresponding to each recess, which indicates that the membrane formation yield has reached 100%.

[0123]

[0147] 24 and 25 are graphs showing the transmembrane current curves monitored when triangular 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 with a hollow part of about 47 μm in diameter shown in FIG. 19, after membrane is formed by the membrane formation method disclosed herein, the carrier membrane is stabilized for 24 hours, and then a triangular 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 FIG. 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 about 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 FIG. 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 about 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] FIG. 26 is a graph showing a transmembrane current curve monitored when a triangular wave voltage is applied after membrane formation in one embodiment of a membrane formation apparatus according to the present disclosure. Based on a membrane formation apparatus having a hollow part with a diameter of about 90 μm, after a 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. Referring to FIG. 26, when driven by a triangular wave voltage, the transmembrane current appears as a quasi-square wave with a peak value of about 200-220 pA. The membrane capacitance is then calculated to be 100 pF, which is within a reasonable capacitance range (10-150 pF) for normal membrane formation.

[0126]

[0150] In another embodiment, a membrane formation apparatus having a hollow part with a diameter of about 80 μm shown in FIG. 18 is used, and capacitance statistics are performed after a membrane is formed by the membrane formation method of the present disclosure. Immediately after the membrane is formed, there is still a relatively large amount of non-polar medium solution in the membrane, so the membrane thickness is large or the membrane area is small, and therefore the membrane capacitance of most channels is measured to be 0-20 pF. After 24 hours, the capacitance is monitored again. The membrane capacitance of most channels increases to about 30-40 pF and tends to stabilize. It can be seen that the membrane capacitance values ​​are all within a reasonable capacitance range (10-150 pF) of normal membrane formation. After monitoring, it is found that only one channel out of 128 channels has a membrane capacitance above 300 pF, because it was determined that the channel was short-circuited due to membrane rupture, but the remaining 127 channels all have intact carrier membranes, and therefore the membrane formation yield is 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 the film formation apparatus according to the present disclosure. A film formation apparatus with a hollow portion with a diameter of about 110 μm is used. The film formation apparatus uses silver / silver chloride electrodes. After a film is formed using the film formation method described herein, the membrane capacitance curves of multiple independent channels in the film formation apparatus are obtained by long-term measurement. The data recording mode of the membrane capacitance records the capacitance of all channels every 10 minutes. With reference to FIG. 27, the x-axis represents time in the smallest unit of time (h) and the y-axis represents capacitance in picofarads (pF). FIG. 27 records the capacitance data of multiple independent channels for 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 0-20 pF. After that, the carrier film spontaneously thins due to the van der Waals forces between molecules, resulting in the area of ​​the carrier film continuing to increase and the capacitance gradually increasing.

[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, ε0 represents the vacuum permittivity, and ε r where C represents the dielectric constant of the membrane molecules, S represents the effective area of ​​the membrane, and d represents the thickness of the membrane. In general, when amphiphilic molecules self-assemble to form a stable monolayer or bilayer membrane, the membrane thickness d and the dielectric constant of the membrane molecules remain constant, and the vacuum dielectric constant ε0 is constant, so that 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, and conversely, the smaller the effective area of ​​the membrane, the smaller the capacitance. The capacitance of most channels hops rapidly and stabilizes to about 110 pF from the 1st hour to the 10th hour after membrane formation. This capacitance value is within a reasonable capacitance value range (10-150 pF) for successful membrane formation. Only a few channels have not yet achieved a 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-mentioned membrane-forming apparatus and membrane-forming method can be used for detecting a single molecule by, for example, 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 the stimulation of the voltage, 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 a nucleic acid molecule to be tested is added to the flow channel inlet of the membrane-forming apparatus, and then a voltage of 180 mV is applied to the electrode of the membrane-forming apparatus. The single nucleic acid molecule passes through the nanopore protein under the action of the electric field force, forming a continuous ionic current blocking 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 (b) are schematic diagrams showing dimensions of a membrane-forming device in the related art and one embodiment of the membrane-forming device according to the present disclosure. In conventional electrochemical detection, consumable electrodes such as silver / silver chloride electrodes are usually used. 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 the solution in the receiving chamber of the membrane-forming device.

[0131]

[0155] However, in another electrochemical detection system, the detection electrode may be a non-consumable electrode (such as gold or platinum and other inert metal electrodes), and the solution in the accommodation chamber of the film-forming device needs to be replaced with a consumable electron pair solution (such as 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 usually designed to be very small, and the volume of the solution in the accommodation chamber is very small (usually nanoliter scale). Therefore, it is necessary to focus on the effect of the internal volume of the recess on the detection lifetime. With reference to (a) and (b) of FIG. 29, calculations and comparisons are made between the total volume of the solution in the accommodation chamber of the related art shown in (a) of FIG. 29 and the total volume of the solution in the accommodation chamber of the embodiment of the film-forming device according to the present disclosure shown in (b) of FIG. 29. In the case of a non-consumable electrode and a consumable electron pair solution, it can be seen that the accommodation chamber of the embodiment of the film-forming device according to the present disclosure has great advantages.

[0132]

[0156] 29(a) and (b), the accommodation chamber of the related art and the accommodation 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 accommodation 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 accommodation chamber has a diameter d1, a height h1, a height of the opening member is h2, and a diameter of the hollow portion of the opening 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 d0 = 100μm, d1 = 200μm, h0 = 100μm, h1 = 100μm, then V0 = 7.85×10 5 μm 3 and V1 = 3.14×10 6 μm 3 It 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 various experiments described above are technically interoperable and interrelated. 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, the single-channel capacitance measurement results are presented using the film penetration current curves for the hollow parts with diameters of about 47μm and 90μm shown in FIGS. 24 - 26. The capacitance change diagram for 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] Above, various embodiments of the present disclosure have been described in detail. Some details well known in the art are not described in order to avoid obscuring the concept of the present disclosure. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein 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 merely for illustrative purposes 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 base (10); A body (20) disposed on the base (10) and having a recess; an aperture member (30) disposed inside a recess port at an end of the recess remote from the base (10), the aperture member (30) being configured such that a hollow portion (32) of the aperture member (30) forms a membrane (80); A film forming apparatus comprising:

2. 2. The membrane-forming apparatus 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 a cross-sectional area of ​​the hollow portion (32) of the opening member (30) is smaller than a cross-sectional area of ​​the storage chamber (21).

3. The body (20) a first body layer (22) disposed on a surface of the base (10); a second body layer (23) located on a side of the first body layer (22) away from the base portion (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 penetrating the second body layer (23). The film forming apparatus according to claim 1 or 2.

4. The film forming apparatus according to any one of claims 1 to 3, wherein the opening member (30) is formed integrally with the second main body layer (23).

5. The film forming apparatus according to any one of claims 1 to 4, wherein the opening member (30) is integrally formed with the main body (20).

6. The opening member (30) A flange (31) projecting radially inwardly relative to said recess port. Equipped with The inner edge of the flange (31) forms the hollow portion (32). The film forming apparatus according to any one of claims 1 to 5.

7. The film forming apparatus according to any one of claims 1 to 6, wherein the inner edges of the flanges (31) are located on the same plane.

8. The film-forming apparatus according to any one of claims 1 to 7, wherein the protruding end of the flange (31) is chamfered or rounded.

9. The film forming apparatus according to any one of claims 1 to 8, wherein a cross section of the flange (31) is T-shaped or a convex polygonal shape.

10. A film forming apparatus as described in any one of claims 1 to 9, 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).

11. The film forming apparatus according to any one of claims 1 to 10, wherein a thickness of the flange (31) in a direction perpendicular to the base (10) gradually decreases from the root portion to the protruding end.

12. The film forming apparatus according to any one of claims 1 to 11, 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).

13. The film forming apparatus according to any one of claims 1 to 12, wherein the at least a portion is located at least on a side of the flange (31) away from the base (10).

14. The film forming apparatus according to any one of claims 1 to 13, wherein the inner edge of the flange (31) has a plurality of protrusions protruding inwardly.

15. A film forming apparatus as described in any one of claims 1 to 14, wherein the cross section of the hollow portion (32) is circular or convex polygonal in shape, and the cross section of the storage chamber (21) is circular or convex polygonal in shape.

16. The film-forming apparatus according to any one of claims 1 to 15, wherein the longitudinal cross section of the receiving chamber (21) is rectangular, trapezoidal, curved-edge trapezoidal or drum-shaped.

17. A first electrode (51) disposed outside the containing chamber (21); a second electrode (52) disposed inside the receiving chamber (21); The film forming apparatus according to any one of claims 1 to 16, further comprising:

18. 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 apparatus according to any one of claims 1 to 17.

19. The film forming apparatus according to any one of claims 1 to 18, wherein the main body (20) includes a plurality of recesses, and the plurality of recesses in the main body (20) are arranged in a rectangular array, a parallelogram array, a honeycomb array or irregularly.

20. The film forming apparatus according to any one of claims 1 to 19, wherein the opening member (30) has a plurality of hollow portions (32).

21. A sealing cover plate (60) configured to be assembled with said body (20). The film forming apparatus according to any one of claims 1 to 20, further comprising:

22. a sealing cover plate (60) configured to be assembled with said body (20); an amphiphilic molecular film (80) formed in the hollow portion (32) of the opening member (30); A first polar medium solution (M1) contained in the containing chamber (21); a second polar medium solution (M4) that is accommodated outside the accommodation chamber (21) and located above the main body (20), the opening member (30) and the amphiphilic molecular membrane (80); The film forming apparatus according to any one of claims 1 to 21, further comprising:

23. The membrane-forming apparatus according to any one of claims 1 to 22, wherein the amphiphilic molecular membrane (80) is a monolayer lipid membrane, a bilayer lipid membrane, or a polymer membrane.

24. A membrane channel (90) embedded in the amphiphilic molecular membrane (80). The film forming apparatus according to any one of claims 1 to 23, further comprising:

25. A method for forming a film, comprising the steps of: A film forming apparatus according to any one of claims 1 to 24, comprising the steps of assembling a sealing cover plate (60) on an upper side of the body (20) to form a flow path 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 body (20) and fills the recess of the 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 membrane (80) in the hollow portion (32).

26. 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). The method for forming a film according to claim 25 , comprising:

27. 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 method for forming a film according to claim 25 or 26, comprising:

28. The step of covering the opening member (30) with the non-polar medium solution (M3) and the second polar medium solution (M4) removing said sealing cover plate (60); coating the body (20) and the aperture member (30) with the non-polar medium solution (M3) 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 any one of claims 25 to 27, comprising:

29. 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); introducing the non-polar medium solution (M3) into the flow path so as to remove the first polar medium solution (M1) between the body (20) and the sealing cover plate (60) and the first polar medium solution (M1) between the opening member (30) and the sealing cover plate (60) by the non-polar medium solution (M3) and achieve covering the opening member (30) with the non-polar medium solution (M3); 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 any one of claims 25 to 28, comprising:

30. Embedding a membrane channel (90) in said amphiphilic molecular membrane (80). The film forming method according to any one of claims 25 to 29, further comprising:

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