Film formation methods, membrane-containing systems and applications

Abstract

This application discloses a film-forming method, a system comprising a membrane, and its applications. Specifically, it discloses a method for forming a membrane containing an amphiphilic molecular layer within a structural unit. The method includes the following steps: forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within a spatial region of the structural unit; wherein the first direction is the thickness direction of the membrane; and the film-forming phase is formed from a film-forming mixture comprising a second polar dielectric, amphiphilic molecules, and a non-polar dielectric; providing conditions for the film-forming phase to form an amphiphilic molecular layer or a membrane containing an amphiphilic molecular layer, and distributing the second polar dielectric contained in the film-forming phase to the first polar dielectric phase and / or the third polar dielectric phase. This film-forming method not only improves the film-forming rate but also makes the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer thinner and more conducive to pore embedding and sequencing.

Description

Technical Field

[0001] This application relates to the field of sequencing technology, and in particular to membranes containing amphiphilic molecular layers and methods for their formation, systems containing membranes, nanopore sequencing devices and their applications. Background Technology

[0002] Nanopore sequencing requires the embedding of nanoporous proteins within membranes containing amphiphilic molecular layers, thus necessitating membrane thickness within a suitable range, typically at the nanometer scale. Common methods for forming membranes with amphiphilic molecular layers involve arrays of multiple structural units, with the membrane forming within each unit. However, this method results in low membrane formation rates. Summary of the Invention

[0003] This application provides a membrane containing an amphiphilic molecular layer, a method for forming the same, a nanopore sequencing device, and its application, aiming to solve the problem of low membrane formation rate in existing methods for forming membranes with amphiphilic molecular layers.

[0004] In a first aspect, this application provides a method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer in a structural unit, the method comprising the following steps:

[0005] A first polar medium phase, a film-forming phase, and a third polar medium phase are formed sequentially along a first direction within the spatial region of the structural unit; wherein, the first direction is the thickness direction of the film, and the film-forming phase is formed by a film-forming mixture comprising a second polar medium, amphiphilic molecules, and a nonpolar medium;

[0006] Conditions are provided to enable the film-forming phase to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and to allow the second polar medium contained in the film-forming phase to be distributed to the first polar medium phase and / or the third polar medium phase.

[0007] In some embodiments, preferably, the spatial region of the structural unit includes an opening.

[0008] In some embodiments, the step of forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within the spatial region of the structural unit includes:

[0009] A first polar medium, a film-forming mixture, and a third polar medium are sequentially introduced into the spatial region of the structural unit to form the film-forming phase between the first polar medium phase and the third polar medium phase;

[0010] The film-forming mixture comprises a second polar medium, amphiphilic molecules, and a nonpolar medium.

[0011] In some embodiments, the step of forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within the spatial region of the structural unit includes:

[0012] S1: A first polar medium and a film-forming mixture A are sequentially introduced into the spatial region of the structural unit to form a first polar medium phase and a film-forming mixture A phase sequentially distributed along a first direction; wherein, the film-forming mixture A contains amphiphilic molecules and a nonpolar medium;

[0013] S2: Add film-forming mixture B to the film-forming phase A to form the film-forming phase;

[0014] The film-forming mixture B comprises a second polar medium and a non-polar medium;

[0015] S3: A third polar medium is introduced into the side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.

[0016] In some embodiments, the step between S1 and S2 further includes:

[0017] At least partially remove the volume of the film-forming A phase that extends beyond the spatial region.

[0018] In some embodiments, in step S2, adding film-forming mixture B to the film-forming phase A to form the film-forming phase includes:

[0019] The film-forming mixture B is introduced into the film-forming phase A and allowed to stand to form the film-forming phase; or

[0020] Film-forming mixture B is sprayed onto the surface of the film-forming phase A to form the film-forming phase.

[0021] In some embodiments, the step of introducing a first polar medium into the spatial region of the structural unit includes:

[0022] The structural unit is placed in the first polar medium and left to stand, so that the first polar medium enters the spatial region of the structural unit.

[0023] In some embodiments, the step of introducing the film-forming mixture into the spatial region of the structural unit includes:

[0024] The structural unit is placed in the corresponding film-forming mixture, removed, and allowed to stand, allowing the corresponding film-forming mixture to enter the spatial region; or

[0025] The structural unit is placed in a corresponding film-forming mixture A, removed, and allowed to stand, allowing the corresponding film-forming mixture A to enter the spatial region, wherein the film-forming mixture A contains amphiphilic molecules and a nonpolar medium; then the structural unit is placed in a corresponding film-forming mixture B, removed, and allowed to stand, allowing the corresponding film-forming mixture B to enter the spatial region, wherein the film-forming mixture B contains a second polar medium and a nonpolar medium.

[0026] In some embodiments, the step of introducing a third polar medium into the spatial region of the structural unit includes:

[0027] The structural unit comprising a first polar medium phase and a film-forming phase is placed in a third polar medium; and / or

[0028] The step of distributing the second polar medium of the film-forming phase into the first polar medium phase and / or the third polar medium phase includes:

[0029] The structural unit, which had been introduced into a third polar medium, was left to stand still.

[0030] In some embodiments, the step of forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within the spatial region of the structural unit includes:

[0031] S11: Adhere the film-forming mixture A to the surface outside the spatial region of the structural unit; wherein the film-forming mixture A comprises amphiphilic molecules and a nonpolar medium;

[0032] S22: A first polar medium is introduced into the spatial region of the structural unit to form a first polar medium phase;

[0033] S33: A film-forming mixture B is added to the first polar medium phase, and the amphiphilic molecules in the film-forming mixture A attached to the surface are dissolved into the film-forming mixture B to form the film-forming phase; wherein the film-forming mixture B contains a second polar medium and a non-polar medium;

[0034] S44: A third polar medium is introduced into the side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.

[0035] A second aspect of the present invention provides a film-forming system, wherein the system includes a structural unit, and the spatial region of the structural unit includes a first polar medium phase, a film-forming phase, and a third polar medium phase sequentially distributed along a first direction; wherein the first direction is the thickness direction of the film, and the film-forming phase includes a second polar medium, amphiphilic molecules, and a nonpolar medium to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer;

[0036] The second polar medium of the film-forming phase can be distributed to the first polar medium phase and / or the third polar medium phase.

[0037] A third aspect of the present invention provides a microdroplet, wherein the microdroplet comprises: a first polar medium phase, a film-forming phase, and a third polar medium phase sequentially distributed along a first direction; wherein the first direction is the thickness direction of the film, and the film-forming phase comprises a second polar medium, amphiphilic molecules, and a nonpolar medium to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer;

[0038] The second polar medium of the film-forming phase can be distributed to the first polar medium phase and / or the third polar medium phase.

[0039] In some embodiments, a transmembrane pore is provided at the amphiphilic molecular layer; preferably, the transmembrane pore is a transmembrane protein pore.

[0040] The transmembrane protein pores are selected from any one or a combination of the following: hemolysin, leukocidin, Mycobacterium smegmatis pore protein A (MspA), MspB, MspC, MspD, lysenin, CsgG, outer membrane pore protein F (OmpF), outer membrane pore protein G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), and WZA.

[0041] In some embodiments, the volume of the second polar medium is 5% to 45% of the volume of the non-polar medium, preferably 5% to 30%, and more preferably 5% to 15%.

[0042] In some embodiments, the second polar medium is soluble in the non-polar medium, and the second polar medium is soluble in the first polar medium or the first polar medium phase, and the second polar medium is soluble in the third polar medium or the third polar medium phase.

[0043] In some embodiments, the second polar medium is selected from one or more of methanol, ethanol, isopropanol, cyclohexanol, toluene, ethyl acetate, propyl acetate, isopropyl acetate, acetone, butanone, cyclohexanone, acetonitrile, propionitrile, dimethyl sulfoxide, N,N'-dimethylformamide (DMF), and N,N'-dimethylacetamide.

[0044] And / or, the nonpolar medium is selected from methylphenyl silicone oil, dimethyl silicone oil, preferably one or more of dimethyl silicone oil with different end caps, hexadecane, tetradecane, decadecane, bromodecane, bromotetradecane, and squalene.

[0045] In some embodiments, when the nonpolar medium is methylphenyl silicone oil, dimethyl silicone oil, preferably dimethyl silicone oil with different end caps, hexadecane, or a mixture of silicone oil and hexadecane, the second polar medium is dimethyl sulfoxide.

[0046] Preferably, the volume of the dimethyl sulfoxide is 5-20% of the volume of the nonpolar medium.

[0047] In some embodiments, the first polar medium is an aqueous solution of a first buffer, which is selected from one or more of phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution or citrate buffer solution;

[0048] Preferably, the concentration of the first buffer aqueous solution is 5-100 mM; more preferably, the first buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution, and / or

[0049] The third polar medium is an aqueous solution of a third buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution.

[0050] Preferably, the concentration of the third buffer aqueous solution is 5-100 mM; more preferably, the third buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution.

[0051] Optionally, the third polar medium may be the same as or different from the first polar medium;

[0052] Optionally, the concentration of the third buffer aqueous solution may be the same as or different from the concentration of the first buffer aqueous solution.

[0053] In some embodiments, both the first buffer aqueous solution and the third buffer aqueous solution contain potassium salts;

[0054] Preferably, the concentration of the potassium salt is 400-800 mM;

[0055] Preferably, the potassium salt is potassium chloride.

[0056] In some embodiments, the osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium maintain a stable state in which the first polar medium phase and the third polar medium phase do not permeate each other.

[0057] In some embodiments, the amphiphilic molecule is selected from one or more of phospholipids, fatty acids, fatty acyl groups, glycerides, glycerophospholipids, sphingolipids, sterol lipids, isopentenyl lipids, glycolipids, polyketides, and amphiphilic block copolymers.

[0058] In some embodiments, the amphiphilic block copolymer comprises at least three polymer segments, wherein the hydrophilic polymeric segments A1 and A2 are connected to opposite ends of the hydrophobic polymeric segment B; or

[0059] The amphiphilic block copolymer comprises at least two polymer segments: a hydrophilic polymeric segment A and a hydrophobic polymeric segment B.

[0060] In some embodiments, the copolymer is poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), poly(2-methyloxazoline)-polyethylene-poly(2-methyloxazoline), or poly(ethylene glycol)-poly(dimethylsiloxane)-poly(ethylene glycol).

[0061] A fourth aspect of the present invention provides an amphiphilic molecular layer or a membrane containing an amphiphilic molecular layer prepared by the method according to the first aspect of the present invention.

[0062] A fifth aspect of the present invention provides a nanopore sequencing device comprising an amphiphilic molecular layer or a membrane containing an amphiphilic molecular layer prepared by the method described in the first aspect of the present invention, a membrane forming system described in the second aspect of the present invention, a microdroplet described in the third aspect of the present invention, or an amphiphilic molecular layer or a membrane containing an amphiphilic molecular layer described in the fourth aspect of the present invention, such as a biochip or a sequencer.

[0063] A sixth aspect of the present invention provides a method for characterizing a target analyte, comprising:

[0064] (a) Contacting the target analyte with a transmembrane pore, the transmembrane pore being embedded in an amphiphilic molecular layer in any of the methods, film-forming systems or microdroplets described above; preferably, the pore is a transmembrane protein pore;

[0065] (b) When the analyte moves relative to the orifice or when the analyte is present in the orifice, one or more electrical signals are measured, wherein the measurement indicates one or more characteristics of the target analyte to characterize the target analyte.

[0066] The seventh aspect of the present invention provides the use of the amphiphilic molecular layer or membrane containing an amphiphilic molecular layer prepared by the method of the first aspect of the present invention, the film forming system of the second aspect of the present invention, the microdroplets of the third aspect of the present invention, or the amphiphilic molecular layer or membrane containing an amphiphilic molecular layer of the fourth aspect of the present invention in characterizing analytes or preparing products containing characterizing analytes.

[0067] Preferably, the characterization is nanopore characterization; more preferably, the pore is a transmembrane protein pore.

[0068] In some embodiments, the target analyte is a metal ion, inorganic salt, polymer, amino acid, peptide, protein, nucleotide, polynucleotide, polysaccharide, lipid, dye, bleaching agent, drug, diagnostic reagent, explosive or environmental pollutant.

[0069] Preferably, the polynucleotide includes DNA and / or RNA and their analogues / derivatives.

[0070] According to the method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer in a structural unit according to embodiments of this application, a first polar medium phase, a film-forming phase, and a third polar medium phase are formed sequentially distributed along a first direction within the spatial region of the structural unit; wherein, the first direction is the thickness direction of the film, and the film-forming phase is formed by a film-forming mixture comprising a second polar medium, amphiphilic molecules, and a non-polar medium. The hydrophilic segment of the amphiphilic molecule is readily soluble in the second polar medium, and the oleophilic segment is readily soluble in the non-polar medium. When the second polar medium is distributed to the first polar medium phase and / or the third polar medium phase, the hydrophilic segment of the amphiphilic molecule is carried into the corresponding polar medium phase, while the oleophilic segment remains in the non-polar medium, allowing the amphiphilic molecules to complete the self-assembly process to form a thin film, thereby obtaining an amphiphilic molecular layer or a film containing an amphiphilic molecular layer. Simultaneously, the distribution process of the second polar medium effectively reduces the thickness of the amphiphilic molecular layer and the film containing the amphiphilic molecular layer, resulting in a relatively thin film. Furthermore, the method of this application also improves the film formation rate and makes the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer thinner and more conducive to pore embedding and sequencing. Attached Figure Description

[0071] The features, advantages, and technical effects of exemplary embodiments of this application will now be described with reference to the accompanying drawings.

[0072] Figure 1 This is a structural diagram of a structural unit disclosed in the prior art;

[0073] Figure 2 This is a schematic diagram of a structural unit disclosed in an embodiment of this application;

[0074] Figure 3This is a schematic diagram of another structural unit disclosed in an embodiment of this application;

[0075] Figure 4 This is a cross-sectional structural schematic diagram of a structural unit disclosed in an embodiment of this application;

[0076] Figure 5 This is a cross-sectional structural diagram of a structural unit used in conjunction with an embodiment of this application;

[0077] Figure 6 This is a schematic diagram of a structure in which a first polar dielectric phase is formed within a structural unit, as disclosed in an embodiment of this application;

[0078] Figure 7 This is a schematic diagram of a structure in which a first polar dielectric phase and a film-forming phase are formed within a structural unit, as disclosed in an embodiment of this application;

[0079] Figure 8 This is a schematic diagram of a structure in which an amphiphilic molecular layer is formed within a structural unit and a membrane containing an amphiphilic molecular layer, as disclosed in an embodiment of this application.

[0080] Figure 9 This is a schematic diagram of a structure in which the film-forming A phase is partially removed within a structural unit, as disclosed in an embodiment of this application;

[0081] Figure 10 yes Figure 9 A schematic diagram of the chip structure after removing phase A of the film-forming mixture and adding film-forming mixture B;

[0082] Figure 11 This is a schematic diagram of a chip structure and film-forming apparatus disclosed in an embodiment of this application;

[0083] Figure 12 This is a schematic diagram of a chip structure and film-forming apparatus disclosed in an embodiment of this application;

[0084] Figure 13 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 1;

[0085] Figure 14 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 2;

[0086] Figure 15 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 3;

[0087] Figure 16 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 4;

[0088] Figure 17 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 5;

[0089] Figure 18 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 6;

[0090] Figure 19 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 7;

[0091] Figure 20 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 8;

[0092] Figure 21 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 9;

[0093] Figure 22 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Comparative Example 1;

[0094] Figure 23 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Comparative Example 2;

[0095] Figure 24 This is an electrical characterization diagram of the amphiphilic molecular layer formed in Comparative Example 3;

[0096] Figure 25 This is the real-time display interface for the experimental group's polynucleotide sequencing signals;

[0097] Figure 26 This is a graph showing the sequencing stability and chip channel utilization of the experimental group;

[0098] Figure 27 This is a graph showing the sequencing stability and chip channel utilization of the control group.

[0099] The accompanying drawings are not necessarily drawn to scale.

[0100] 10. First polar medium phase; 20. Film-forming phase; 30. Third polar medium phase; 22. Film-forming phase A; 23. Film-forming mixture B;

[0101] 100. Chip structure;

[0102] 200. Structural unit; 210. Spatial region; 220. Opening; 230. Supporting component; 211. First channel; 212. Second channel;

[0103] 300, film-forming device; 310, support frame; 320, cover; 330, gasket; 340, receiving cavity; 331, cavity; 321, liquid inlet; 322, liquid outlet. Detailed Implementation

[0104] The embodiments of this application will be described in further detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application, that is, this application is not limited to the described embodiments.

[0105] In the description of this application, it should be noted that, unless otherwise stated, "multiple" means two or more. The terms "upper," "lower," "left," "right," "inner," and "outer," indicating orientation or positional relationships, are merely for the convenience of describing this application and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation; therefore, they should not be construed as limitations on this application. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. "Vertical" is not strictly vertical, but within the allowable tolerance range. "Parallel" is not strictly parallel, but within the allowable tolerance range.

[0106] In the description of this application, it should also be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can be a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this application depending on the specific circumstances.

[0107] Structural units 200 are typically disposed within chip structure 100. Chip structure 100 includes multiple structural units 200. The multiple structural units 200 are typically arranged in an array. The walls of each structural unit 200 surround and form a spatial region 210 with a certain accommodating space. This spatial region 210 has an opening 220. In some embodiments, various raw materials for the formation of amphiphilic material films, such as polar and non-polar media, mainly enter the spatial region 210 through the opening 220.

[0108] For ease of description, the end of structural unit 200 with opening 220 is defined as the top, and the end away from opening 220 is defined as the bottom. The spatial regions 210 of each structural unit 200 can be interconnected, such as the tops of adjacent structural units 200 being interconnected. For example, refer to... Figure 1 The top wall of structural unit 200 is discontinuous, consisting of spaced-apart support members 230. The gaps between the support members form a first channel 211. Adjacent structural units 200 share some support members 230, and are connected at the top through the first channel 211. Adjacent structural units 200 can also allow partial media flow through the first channel 211.

[0109] In preparing an amphiphilic molecular layer or a film containing an amphiphilic molecular layer using the aforementioned chip structure 100, the process mainly involves sequentially introducing a polar medium, a non-polar medium dispersed with amphiphilic material, and a polar medium onto the chip structure 100. The later-introduced medium displaces the previously introduced medium, thus occupying a portion of the space region 210, ultimately forming a film containing an amphiphilic molecular layer located between two polar medium phases. However, in practice, using this method results in a relatively small number of structural units 200 forming the film containing the amphiphilic molecular layer, leading to a low film formation rate. Furthermore, the structural units 200 also exhibit a relatively thick thickness of the amphiphilic molecular layer and the film containing the amphiphilic molecular layer.

[0110] After noticing that existing methods for forming amphiphilic molecular layers and films containing amphiphilic molecular layers have problems such as a low number of structural units 200 and a low film formation rate, the applicant studied methods for forming films containing amphiphilic molecular layers. It was then discovered that the self-assembly of amphiphilic materials to form biomimetic films, i.e., amphiphilic molecular layers or films containing amphiphilic molecular layers, is a relatively fragile process that is easily affected by external polar and non-polar media, leading to the rupture of films containing amphiphilic molecular layers and the problem of a low number of structural units 200.

[0111] Based on the problems discovered by the applicant, the applicant has improved the method for forming a membrane containing an amphiphilic molecular layer in the structural unit 200. The embodiments of this application are further described below.

[0112] This application provides a method for forming an amphiphilic molecular layer and a film containing the amphiphilic molecular layer in a structural unit 200, wherein the structural unit 200 includes a spatial region 210 having an opening 220, and the method includes the following steps:

[0113] Within the spatial region 210, a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are formed in sequence along a first direction, the first direction being the thickness direction of the film. The film-forming phase 20 is formed from a mixture containing a second polar medium, amphiphilic molecules, and a nonpolar medium.

[0114] The conditions are provided for the film-forming phase to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and for the second polar medium of the film-forming phase 20 to be distributed to the first polar medium phase 10 and / or the third polar medium phase 30.

[0115] According to the film-forming method of the amphiphilic molecular layer according to the embodiments of this application, a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are disposed sequentially along a first direction in the spatial region 210 of the structural unit 200. The film-forming phase 20 is formed by a mixture of a second polar medium, amphiphilic molecules, and a non-polar medium. The hydrophilic segment of the amphiphilic molecules is readily soluble in the second polar medium, while the oleophilic segment is readily soluble in the non-polar medium. When the second polar medium is distributed to the first polar medium phase 10 and / or the third polar medium phase 30, the hydrophilic segment of the amphiphilic molecules is carried into the corresponding polar medium phase, while the oleophilic segment remains in the non-polar medium, allowing the amphiphilic molecules to complete the self-assembly process and form a thin film to obtain an amphiphilic molecular layer or a film containing an amphiphilic molecular layer. At the same time, the applicant unexpectedly discovered that the distribution process of the second polar medium effectively reduces the thickness of the film-forming phase 20, making the thickness of the amphiphilic molecular layer relatively thin.

[0116] Furthermore, due to the compatibility of the second polar medium with the first and third polar medium phases, the film-forming phase 20 undergoes a rebalancing and thinning process, thus increasing the tolerable thickness range of the initial formation of the film-forming phase 20, without needing to directly reach the target thickness, such as 5-15 nm. When the film-forming method of amphiphilic molecular layers or films containing amphiphilic molecular layers is applied to a chip structure with multiple structural units 200, if there are some thickness differences in the initial formation of the film-forming phase 20 of different structural units 200, each film-forming phase 20 can be slowly thinned from different starting points to a suitable film-forming thickness, and stop at a suitable thickness range due to the support capacity of the film-forming phase 20 itself. This improves the film formation rate while also improving the uniformity of each film-forming phase 20 within the chip structure, that is, the thickness of each film-forming phase 20 is relatively uniform.

[0117] If the film-forming phase 20 does not include the second polar medium and only contains the non-polar medium, it loses its compatibility with the first and third polar media, and thus loses its ability to rebalance the film layer. Therefore, it is crucial to ensure that the film layer thickness of the multiple structural units 200 of the chip structure simultaneously reaches a narrow range of target thickness during initial formation, which is a very demanding requirement, resulting in uneven film thickness. The aforementioned film-forming method for amphiphilic molecular layers or films containing amphiphilic molecular layers promotes film formation through the compatibility of the second polar medium with the first polar medium phase 10 and the third polar medium phase, thereby improving the film formation rate and uniformity of the amphiphilic molecular layers or films containing amphiphilic molecular layers, and resulting in thinner films. Furthermore, subsequent applications have shown that these amphiphilic molecular layers or films containing amphiphilic molecular layers can increase the monopore content of nanoporous proteins.

[0118] It should be noted that the above-described method for forming amphiphilic molecular layers or films containing amphiphilic molecular layers can be applied to chip structures 100 of various types of unit structures. Regardless of whether the spatial regions 210 of adjacent structural units 200 in the chip structure 100 are connected or not, or what kind of connection method is used, the above-described method for forming amphiphilic molecular layers or films containing amphiphilic molecular layers can be used.

[0119] That is, the above-mentioned film formation method of amphiphilic molecular layers or films containing amphiphilic molecular layers can be applied to chip structures 100 in which adjacent structural units 200 are interconnected, such as... Figure 1 The chip structure 100 where adjacent structural units 200 are interconnected at the top, as shown, also applies to chip structures 100 where adjacent structural units 200 are interconnected at the bottom. For example... Figure 2 As shown, the bottom wall of structural unit 200 is provided with a second channel 212 connecting to a spatial region 210 of another structural unit 200. Adjacent structural units 200 can exchange some media through the second channel 212. This also applies to chip structures 100 where two adjacent structural units 200 are connected at positions other than the top and bottom.

[0120] Furthermore, the aforementioned method for forming amphiphilic molecular layers or films containing amphiphilic molecular layers can also be applied to chip structures 100 where adjacent structural units 200 are not interconnected. For example... Figure 3 As shown, the spatial regions 210 of the structural unit 200 are independent of each other and are not interconnected. Neighboring structural units 200 cannot exchange media.

[0121] Figure 4 A cross-sectional structural schematic diagram of an exemplary structural unit 200 is provided. In the spatial region 210 of the structural unit 200, due to the effect of gravity, the first polar medium phase 10, the film-forming phase 20 and the third polar medium phase 30 are usually arranged along the extension direction of the spatial region 210, that is, arranged sequentially from the bottom to the top of the spatial region 210. The thickness direction of the film-forming phase 20 is also usually the extension direction of the spatial region 210.

[0122] The first polar medium phase 10 occupies a portion of the space within spatial region 210. The third polar medium phase 30 occupies a portion of the space within spatial region 210. The film-forming phase 20 is sandwiched between the interface of the first polar medium phase 10 and the third polar medium phase 30. The film-forming phase 20 is formed from a mixture including a second polar medium, amphiphilic molecules, and a nonpolar medium. That is, based on the nonpolar medium containing dispersed amphiphilic materials used in conventional methods for preparing film-forming phase 20, a portion of the second polar medium is added. The second polar medium and the amphiphilic molecules are dispersed or distributed in the nonpolar medium. The hydrophilic portion of the amphiphilic molecules dissolves in the second polar medium, and the lipophilic portion dissolves in the nonpolar medium.

[0123] There are various methods for forming a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 sequentially distributed along a first direction in the spatial region 210. For example, the corresponding media, especially the media for forming the film-forming phase 20, can be pre-prepared, and then the corresponding media can be sequentially introduced to form the aforementioned structure; that is, the film-forming phase 20 is formed in one step. Alternatively, the first polar medium phase 10 can be formed first, followed by the film-forming phase 20 distributed through multiple steps, and then the third polar medium phase 30 can be formed. The specific method used to form the structure in which the first polar medium phase 10, the film-forming phase 20, and the third polar medium phase 30 are sequentially distributed is not limited.

[0124] The second polar medium of the film-forming phase 20 is distributed into the first polar medium phase 10 and / or the third polar medium phase 30. Upon standing, because the second polar medium has a similar polarity to the first and third polar media, it tends to dissolve more readily in both. After standing for a certain period, the second polar medium partially or completely dissolves in the first and / or third polar medium phases 10 and 30, depending on its relative position to them. For example, when the second polar medium is distributed at the interface between the film-forming phase 20 and the first polar medium phase 10, it is more likely to dissolve in the first polar medium phase 10. Conversely, when the second polar medium is distributed at the interface between the film-forming phase 20 and the third polar medium phase 30, it is more likely to dissolve in the third polar medium phase 30. Of course, in other cases, it may also dissolve in both the first and third polar medium phases 10 and 30.

[0125] During the process of the second polar medium being distributed into the first polar medium phase 10 and / or the third polar medium phase 30, the hydrophilic segment of the amphiphilic molecule is carried into the corresponding polar medium phase, while the oleophilic segment remains in the non-polar medium, allowing the amphiphilic molecules to complete the self-assembly process and thus form a thin film.

[0126] Moreover, the applicant unexpectedly discovered that during the distribution process of the second polar medium, the thickness of the film-forming phase 20 decreased, and the film containing the amphiphilic molecular layer was formed between the two interfaces. Therefore, the thickness of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer was also relatively thin, which manifested as a reasonable increase in capacitance value that could be observed by electrical detection.

[0127] In some embodiments, the step of forming a third polar dielectric phase 30, consisting of a first polar dielectric phase 10 and a film-forming phase 20 sequentially distributed along a first direction within the spatial region 210, includes:

[0128] A first polar medium, a film-forming mixture, and a third polar medium are sequentially introduced into the spatial region 210 to form a film-forming phase 20. The film-forming mixture includes a second polar medium, amphiphilic molecules, and a nonpolar medium.

[0129] When a corresponding medium is introduced into the space region 210, the chip structure 100 can be used alone, such as by immersing the chip structure 100 in the corresponding medium or by spraying or dripping the corresponding medium onto the surface of the chip structure 100 where the opening 220 is provided. Of course, the chip structure 100 can also be used in conjunction with other devices, such as... Figure 5 As shown, the surface of the chip structure 100 with the opening 220 has a receiving cavity 340 for accommodating the corresponding medium.

[0130] Taking chip structure 100 as an example, it can also be used with other devices. A film-forming mixture can be pre-prepared to disperse the second polar medium, amphiphilic molecules, and non-polar medium relatively uniformly. For example... Figure 6 As shown, a first polar medium is introduced into the spatial region 210 to form a first polar medium phase 10. Then, as... Figure 7 As shown, a film-forming mixture is introduced into the spatial region 210. The film-forming mixture displaces a portion of the first polar medium phase 10 within the spatial region 210, occupying a portion of the first polar medium phase 10 to form a film phase 20. The film phase 20 covers the first polar medium phase 10. Then, as... Figure 8 As shown, a third polar medium is then introduced, which drives away part of the film-forming phase 20 within the spatial region 210 and occupies the area of ​​the film-forming phase 20, forming a third polar medium layer. The above-mentioned method for forming amphiphilic molecular layers or films containing amphiphilic molecular layers is relatively mature. The film-forming phase 20 is formed in one step, which is relatively simple to operate and can improve the film formation efficiency to a certain extent.

[0131] In some embodiments, the step of forming a first polar dielectric phase 10, a film-forming phase 20, and a third polar dielectric phase 30 sequentially distributed along a first direction within a spatial region 210 includes:

[0132] S1: A first polar medium and a film-forming mixture A are sequentially introduced into the spatial region 210 to form a first polar medium phase 10 and a film-forming mixture A phase 22 sequentially distributed in the first direction. The film-forming mixture A contains amphiphilic molecules and a non-polar medium.

[0133] S2: Film-forming mixture B23 is added to film-forming phase A 22 to form film-forming phase 20. Film-forming mixture B23 contains a second polar medium and a non-polar medium.

[0134] S3: A third polar medium is introduced into the side of the film-forming phase 20 away from the first polar medium phase 10 to form a third polar medium phase 30.

[0135] In step S1, a film-forming mixture A can be prepared first, in which amphiphilic molecules and a nonpolar medium are dispersed relatively uniformly. A first polar medium and film-forming mixture A are then sequentially introduced into the spatial region 210, thereby forming film-forming phase A 22 on the first polar medium phase 10. Film-forming mixture A is a solution of amphiphilic molecules dissolved in a nonpolar medium. Preferably, the concentration of the amphiphilic molecules is 5-20 mg / ml, more preferably 8-12 mg / ml, and even more preferably 10 mg / ml.

[0136] In step S2, a film-forming mixture B23 can be prepared first to disperse the second polar medium and the non-polar medium relatively uniformly. In the film-forming mixture B23, the second polar medium is dispersed in the non-polar medium, and its volume percentage concentration is 5% to 30%, preferably 5% to 15%. The film-forming mixture B23 is directly introduced into or sprayed into the film-forming phase A 22 to form the film-forming phase 20. The second polar medium is added to the film-forming phase A 22 in the form of film-forming mixture B23. Since the second polar medium is dispersed in the non-polar medium, it can be dispersed relatively uniformly in the film-forming phase A 22, and the components of the formed film-forming phase 20 are dispersed relatively uniformly, thereby improving the film-forming quality of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer. Furthermore, the second polar medium is added later in the process of forming the film-forming phase 20, which reduces the volume of the second polar medium that is prematurely allocated to the first polar medium phase during the formation of the film-forming phase 20. Therefore, the actual content of the second polar medium in the film-forming phase 20 is more consistent with the designed content of the second polar medium in the film-forming phase 20, thereby improving the film-forming quality.

[0137] In step S3, a third polar medium is introduced into the side of the film-forming phase 20 away from the first polar medium phase 10, that is, the side near the opening 220.

[0138] The above-mentioned methods for forming amphiphilic molecular layers or films containing amphiphilic molecular layers can improve the film-forming quality of amphiphilic molecular layers or films containing amphiphilic molecular layers.

[0139] In some embodiments, the process between step S1 and step S2 further includes:

[0140] At least partially remove the volume of film-forming phase A 22 that extends beyond spatial region 210.

[0141] In some embodiments, adding film-forming mixture B23 to film-forming phase A 22 to form film-forming phase 20 includes:

[0142] Film-forming mixture B23 is introduced into film-forming phase A 22, and the mixture is allowed to stand to form film-forming phase 20; or

[0143] Film-forming mixture B23 is sprayed onto the surface of film-forming phase A 22 to form film-forming phase 20.

[0144] The film-forming A phase 22 typically extends beyond the spatial region 210, meaning that part of the film-forming A phase 22 will cover outside the opening 220. In other words, the surface of the chip structure 100 with structural units 200 is covered with a film-forming A phase 22 of a certain thickness. For example... Figure 9 As shown, various methods can be used, such as blowing away with airflow, wiping or scraping, to partially or completely remove the volume of the film-forming phase A 22 that exceeds the spatial region 210, so that the film-forming phase A 22 slightly exceeds the opening 220 of the structural unit 200 or is flush with the opening 220 of the structural unit 200.

[0145] like Figure 10 As shown, after partially or completely removing the volume of film-forming phase A 22 exceeding spatial region 210, film-forming mixture B23 can be introduced into film-forming phase A 22, and allowed to stand to form film-forming phase 20. After film-forming mixture B23 enters film-forming phase A 22, it is incubated for a certain period of time, such as 10–30 min. The second polar medium in film-forming mixture B23 disperses into the non-polar medium of film-forming phase A 22, and the non-polar medium in film-forming mixture B23 and the non-polar medium in film-forming phase A 22 are miscible, thereby forming film-forming phase 20.

[0146] After partially or completely removing the volume of film-forming phase A 22 exceeding spatial region 210, another method can be used: spraying film-forming mixture B23 onto the surface of film-forming phase A 22 to form film-forming phase 20. Spraying imparts kinetic energy to film-forming mixture B23, allowing it to penetrate the interior of film-forming phase A 22 and cause slight agitation, resulting in relatively uniform mixing. The second polar medium in film-forming mixture B23 can quickly disperse into the non-polar medium of film-forming phase A 22, and the non-polar medium therein also quickly becomes miscible with the non-polar medium in film-forming phase A 22. Furthermore, this method requires a smaller amount of film-forming mixture B23, saving costs.

[0147] In some embodiments, the step of introducing a first polar medium into the spatial region 210 includes:

[0148] The structural unit 200 is placed in the first polar medium and left to stand, so that the first polar medium enters the space region 210.

[0149] Typically, multiple structural units 200 are arranged in an array on a device, such as a chip structure 100. Since the structural units 200 are relatively small, placing the structural units 200 in a first polar medium in this embodiment can be understood as placing the chip structure 100 containing the structural units 200 in a first polar medium. Similarly, in other embodiments, similar operations on the structural units 200 can be understood in the same way.

[0150] Devices with structural units 200, such as chip structures 100, can be directly placed in and in contact with the first polar medium. After a certain period of settling, the first polar medium mainly seeps into the spatial region 210 of the structural unit 200 through the opening 220, filling the spatial region 210. The settling time is preferably such that the first polar medium fills most or all of the spatial region 210, such as 2 to 30 minutes. The entire process can be carried out in a vacuum environment. Furthermore, the first polar medium can be degassed before placing the chip to reduce the amount of air in it.

[0151] Alternatively, the chip structure 100 can be mounted on a film-forming apparatus 300, which forms a receiving cavity 340 around the surface of the chip structure 100. A first polar medium is introduced into the receiving cavity 340, and the chip structure 100 comes into contact with the first polar medium. After a certain period of settling, the first polar medium mainly seeps into the spatial region 210 of the structural unit 200 through the opening 220, filling the spatial region 210. The settling time is preferably such that the first polar medium fills most or all of the spatial region 210, such as 2 to 30 minutes. The entire process can be carried out in a vacuum environment. Furthermore, the first polar medium can be degassed before placing the chip to reduce the amount of air in it.

[0152] This application provides an exemplary film-forming apparatus 300, which includes a support frame 310, a cover 320, and a gasket 330. A chip structure 100 is disposed on the support frame 310, the cover 320 is connected to the support frame 310, and the gasket 330 is pressed onto the chip structure 100. The gasket 330 includes a cavity 331, which corresponds to an array of structural units 200 distributed on the chip structure 100. The orthographic projection of the cavity 331 onto the chip structure 100 covers the array of structural units 200. The gasket 330 is disposed between the cover 320 and the chip structure 100, thereby forming a receiving cavity 340. The cover 320 also has an inlet 321 and an outlet 322, both of which communicate with the receiving cavity 340. A first polar medium can enter the receiving cavity 340 from the inlet 321.

[0153] In some embodiments, the step of introducing film-forming mixture A or film-forming mixture B into the spatial region 210 includes:

[0154] The structural unit 200, which includes the first polar medium phase 10, is placed in the corresponding film-forming mixture within the spatial region 210. It is then removed and allowed to stand, so that the corresponding film-forming mixture replaces part of the first polar medium phase 10 within the spatial region 210.

[0155] Similarly, the structural unit 200, including the first polar medium phase 10, is placed in a corresponding film-forming mixture, which may be film-forming mixture A or film-forming mixture B.

[0156] Overall, the permeability of the first polar medium and the third polar medium entering the space region 210 is weaker than that of the non-polar medium entering the space region 210. Both film-forming mixture A and film-forming mixture B contain a certain amount of non-polar medium, therefore their permeability is greater than that of the first polar medium. Therefore, after separating the chip structure 100 of the first polar medium phase 10 from the corresponding film-forming mixture, it is allowed to stand, such as by removing it from the container containing the corresponding film-forming mixture phase, or by removing the corresponding film-forming mixture from the receiving cavity 340, to prevent the corresponding film-forming mixture from completely replacing part of the first polar medium phase 10 in the space region 210.

[0157] In some embodiments, the step of introducing a third polar medium into the spatial region 210 includes:

[0158] The structural unit 200, which includes the first polar medium phase 10 and the film-forming phase 20, is placed in the third polar medium;

[0159] The step of dispensing the second polar medium of the film-forming phase 20 into the first polar medium phase 10 and / or the third polar medium phase 30 includes:

[0160] The structural unit 200, which had been introduced with a third polar medium, was left to stand still.

[0161] The third polar medium phase 30 has relatively weak permeability. Therefore, the chip structure 100, including the first polar medium phase 10 and the film-forming phase 20, in the spatial region 210 can be directly placed in the third polar medium and brought into contact with it. Alternatively, the chip structure 100 can be clamped onto the film-forming apparatus 300, and the third polar medium can be introduced into the receiving cavity 340, bringing the chip structure 100 into contact with the third polar medium. Both methods aim to increase the total amount of the third polar medium to facilitate its permeation into the spatial structure. The third polar medium mainly permeates into the spatial region 210 of the structural unit 200 through the opening 220, replacing part of the film-forming phase 20.

[0162] In some embodiments, the step of forming a first polar dielectric phase and a film-forming phase sequentially distributed along a first direction within a spatial region includes:

[0163] S11: Solidify the film-forming mixture A onto the surface outside the spatial region of the structural unit; wherein the film-forming mixture A contains amphiphilic molecules and a nonpolar medium;

[0164] S22: A first polar medium is introduced into the space region to form a first polar medium phase;

[0165] S33: Film-forming mixture B is added to the first polar medium phase, and the amphiphilic molecules in film-forming mixture A attached to the surface are dissolved into film-forming mixture B to form a film-forming phase; film-forming mixture B contains a second polar medium and a non-polar medium;

[0166] S44: A third polar medium is introduced into the side of the film-forming phase away from the first polar medium phase to form a third polar medium phase.

[0167] In step S11, this can be understood as applying a small amount of film-forming mixture A to the surface outside the structural unit spatial region, such as the surface of the chip structure 100. This can be achieved by spraying a small amount of film-forming mixture A, allowing it to diffuse uniformly using the chip surface microstructure, and then curing it, such as by drying, to adhere it to the chip. In step S33, after curing, the amphiphilic molecules in the film-forming mixture A will dissolve back into the nonpolar medium and the second polar medium in the film-forming mixture B from the chip surface. This method uses a smaller amount of amphiphilic molecules, thus saving costs.

[0168] In some embodiments, the volume of the second polar medium is 5% to 30% of the volume of the non-polar medium, preferably 5% to 15%.

[0169] The second polar medium comprises 5% to 30% of the volume of the non-polar medium. At this ratio, the volume of the second polar medium can be dispersed relatively uniformly in the non-polar medium, and both film-forming mixture A and film-forming mixture B are relatively stable, resulting in a relatively stable film-forming phase 20. The film-forming phase 20 is less prone to stratification due to an excessively large volume of the second polar medium, and also less prone to premature distribution due to an excessively small volume of the second polar medium. This prevents the second polar medium of the film-forming phase 20 from being distributed into the first polar medium phase 10 when the third polar medium phase 30 is introduced, thus ensuring film quality.

[0170] Since the film-forming phase 20 is relatively stable, most of the second polar medium can be controlled to be distributed only when needed to the first polar medium phase 10 and / or the third polar medium phase 30. Therefore, the above-described method for forming amphiphilic molecular layers or films containing amphiphilic molecular layers is controllable, and the film-forming rate of amphiphilic molecular layers or films containing amphiphilic molecular layers is high. In some embodiments, the second polar medium is 5% to 15% of the volume of the non-polar medium. Under this condition, the above-described method for forming amphiphilic molecular layers or films containing amphiphilic molecular layers is even more controllable, and the film-forming rate of amphiphilic molecular layers or films containing amphiphilic molecular layers is further improved.

[0171] In some embodiments, the second polar medium is selected from one or more of methanol, ethanol, isopropanol, cyclohexanol, toluene, ethyl acetate, propyl acetate, isopropyl acetate, acetone, butanone, cyclohexanone, acetonitrile, propionitrile, dimethyl sulfoxide, N,N'-dimethylformamide, and N,N'-dimethylacetamide.

[0172] The nonpolar medium can be one or more of methylphenyl silicone oil, dimethyl silicone oil, dimethyl silicone oil with different end caps, hexadecane, tetradecane, decadecane, bromodecane, bromotetradecane, and squalene.

[0173] Experimental verification shows that the film-forming phase 20 can be relatively stable, and the film-forming method of the above-mentioned film containing amphiphilic molecular layers has a high film-forming rate and a thinner thickness of the amphiphilic molecular layer or film containing amphiphilic molecular layers.

[0174] In some embodiments, when the nonpolar medium is methylphenyl silicone oil, dimethyl silicone oil, dimethyl silicone oil with different end caps, hexadecane, or a mixture of silicone oil and hexadecane, the second polar medium is dimethyl sulfoxide. The volume of the dimethyl sulfoxide is 5-20% of the volume of the nonpolar medium.

[0175] When the nonpolar medium is a single medium such as methylphenyl silicone oil, dimethyl silicone oil, perfluorosilicone oil, dimethyl silicone oil with different end caps, or hexadecane, and the second polar medium is selected as dimethyl sulfoxide, which constitutes 5-15% of the volume of the nonpolar medium, the film formation rate of the above-mentioned film formation method containing an amphiphilic molecular layer is further improved, and the thickness of the amphiphilic molecular layer or the film containing an amphiphilic molecular layer is further reduced. The dimethyl silicone oil with different end caps can be dimethyl silicone oil with dihydroxyl end caps, dimethyl silicone oil with monohydroxyl end caps, dimethylphenyl silicone oil with dihydroxyl end caps, dimethylphenyl silicone oil with monohydroxyl end caps, diamino silicone oil with diamino end caps, dimethyl silicone oil with dicarboxyl end caps, dimethyl silicone oil with monocarboxyl end caps, diethylene oxide end caps, diethylene oxide end caps, dialkoxy end caps, or dialkoxy end caps.

[0176] When the nonpolar medium is a mixture such as silicone oil and hexadecane, and the second polar medium is selected as dimethyl sulfoxide, which constitutes 5-15% of the volume of the nonpolar medium, the film formation rate of the above-mentioned film formation method containing an amphiphilic molecular layer is further improved, and the thickness of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is further reduced. In the silicone oil and hexadecane mixture, the mixing ratio of silicone oil and hexadecane can be (1-4):1, such as 1:1, 7:3, 3:1, or 4:1.

[0177] In some embodiments, the first polar medium is a first buffer aqueous solution, which is selected from one of phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, and citrate buffer solution. The third polar medium is a third buffer aqueous solution, which is selected from one of phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, and citrate buffer solution. The third polar medium may be the same as or different from the first polar medium. In some embodiments, the concentration of the first buffer aqueous solution is 5-100 mM, preferably a 10 mM phosphate buffer solution, the concentration of the third buffer aqueous solution is 5-100 mM, preferably a 10 mM phosphate buffer solution, and the concentration of the third polar medium is the same as or different from the concentration of the first polar medium.

[0178] The first polar medium and the third polar medium are each independently selected from one of the above-mentioned aqueous solutions of the first and third buffers. The third polar medium may be the same as or different from the first polar medium. Both the first polar medium and the third polar medium can meet the requirements of the above-mentioned film formation method for films containing amphiphilic molecular layers.

[0179] In other embodiments, the first and third polar media can be selectively chosen based on the electrode material used in the system when the amphiphilic layer or membrane containing the amphiphilic layer is used to characterize the analyte. For example, when silver-silver chloride is used as the electrode material for constructing the electrochemical system, the first and third polar media can be selected as phosphate buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, or 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution. When gold or platinum is used as the electrode material for constructing the electrochemical system, the first and third polar media can be selected as phosphate buffer solution containing potassium ferricyanide or 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution containing potassium ferricyanide.

[0180] In some embodiments, the first and third buffer aqueous solutions each contain a potassium salt, such as KCl, at a concentration of 400-800 mM. The presence of KCl at these concentrations in the first and third buffer aqueous solutions promotes the equilibrium of the electrochemical system.

[0181] In some embodiments, the osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium can maintain a stable state in which the first polar medium and the third polar medium do not permeate each other.

[0182] The osmotic pressure of the first polar medium is equal to or close to that of the third polar medium, so that the first polar medium phase 10 and the third polar medium phase 30 do not interpenetrate through the film-forming phase 20 in the spatial region 210, thus maintaining stability. Under these conditions, the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer is relatively stable and can maintain a stable state for a longer period of time. When using the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer for testing, the testing is more convenient and the success rate of the test is improved.

[0183] In some embodiments, the amphiphilic molecule is a phospholipid, fatty acid, fatty acyl, glycerol, glycerophospholipid, sphingolipid, sterol lipid, isopentenol lipid, glycolipid, polyketide compound, or amphiphilic block copolymer.

[0184] The amphiphilic molecule can be formed using any of the above-mentioned substances, which can meet the requirements for film formation. The concentration of the amphiphilic molecule in the nonpolar solvent is 5-20 mg / mL, with a preferred concentration of 10 mg / mL.

[0185] In some embodiments, the amphiphilic block copolymer is a copolymer comprising at least three polymer segments, the copolymer having hydrophilic polymeric segments A1 and A2 at the ends of the molecular chain and hydrophobic polymeric segment B in the middle of the molecular chain, or the amphiphilic block copolymer is a copolymer comprising at least two polymeric segments, wherein the at least two polymeric segments include hydrophilic polymeric segment A and hydrophobic polymeric segment B.

[0186] The amphiphilic molecular layers or films containing amphiphilic molecular layers formed by the above-mentioned method of amphiphilic block copolymers are robust, stable, not easily degraded, and can withstand large potential differences applied and passed through them.

[0187] In some embodiments, the amphiphilic block copolymer is a copolymer comprising at least three polymer segments, wherein the copolymer is poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), poly(2-methyloxazoline)-polyethylene-poly(2-methyloxazoline), or poly(ethylene glycol)-poly(dimethylsiloxane)-poly(ethylene glycol).

[0188] The amphiphilic molecular layer or film containing the amphiphilic molecular layer formed by the above-mentioned method of amphiphilic block copolymer is more robust, more stable, less prone to degradation, and can withstand the applied potential difference that passes through it.

[0189] Secondly, embodiments of this application provide amphiphilic molecular layers or films containing amphiphilic molecular layers prepared by the above methods.

[0190] Thirdly, embodiments of this application provide a nanopore sequencing device, comprising an amphiphilic molecular layer or a membrane containing an amphiphilic molecular layer prepared by the above method.

[0191] Fourthly, embodiments of this application provide the application of the amphiphilic molecular layer or membrane containing the amphiphilic molecular layer prepared by the above method in characterizing analytes, the analytes including: biopolymers, the biopolymers being selected from one of polynucleotides, polypeptides, polysaccharides and lipids.

[0192] The analytes described above can be embedded in the amphiphilic molecular layer prepared by the above preparation method for testing.

[0193] In some embodiments, the biopolymer is a polynucleotide, which includes DNA and / or RNA and their analogs / derivatives.

[0194] Example

[0195] The following embodiments describe the disclosure of this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. All reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing. Similarly, the instruments used in the embodiments are commercially available.

[0196] Example 1

[0197] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0198] S110: This will have structural unit 200 ( Figure 2 The chip structure 100 is placed in a culture dish, and the first polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH7.5)) after degassing is added. The mixture is left to stand under vacuum (50mBar) for 2 minutes. The first polar medium fills the spatial region 210 of the structural unit 200 to form the first polar medium phase 10.

[0199] S120: After removing the chip structure 100 containing the first polar dielectric phase 10 from the structural unit 200, it is immersed in the film-forming mixture. The chip structure 100 is extracted at a speed of 10 mm / s, left to stand for 30 seconds, and excess film-forming mixture outside the structural unit 200 of the chip structure 100 is wiped off with absorbent paper. The first polar dielectric phase 10 and the film-forming phase 20 covering the first polar dielectric phase 10 are formed within the structural unit 200. Wherein:

[0200] Film-forming mixtures include second polar media, amphiphilic molecules, and nonpolar media;

[0201] The second polar medium is ethyl acetate, and its volume is 5% of the volume of the non-polar medium;

[0202] The amphiphilic molecule is PDMS-PEG (PDMS-PMOXA is also possible), and the concentration in a nonpolar medium is 10 mg / ml;

[0203] The non-polar medium is methylphenyl silicone oil, preferably AP100 (Sigma-Aldrich), but AR20 (Sigma-Aldrich) is also acceptable.

[0204] S130: The chip structure 100, which has a first polar medium phase 10 and a film-forming phase 20 within the structural unit 200, is loaded into the film-forming apparatus 300. A third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) is introduced into the receiving cavity 340 through the liquid inlet 321, displacing part of the film-forming phase 20. After standing, the first polar medium phase 10, the film-forming phase 20, and the third polar medium phase 30 are formed sequentially distributed in the first direction within the structural unit 200.

[0205] S140: After standing for 2 hours, the film-forming phase 20 forms the film containing the amphiphilic molecular layer, and the test begins.

[0206] Example 2

[0207] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0208] S210: Proceed as step S110 in Embodiment 1, except that the structure of structural unit 200 is as follows: Figure 1 As shown.

[0209] S220: After removing the chip structure 100 containing the first polar dielectric phase 10 from the structural unit 200, it is loaded into the film-forming apparatus 300, and a film-forming mixture is introduced into the receiving cavity 340 through the liquid inlet 321. After standing for 10 minutes, the first polar dielectric phase 10 and a film-forming phase 20 covering the first polar dielectric phase 10 are formed in the structural unit 200. Wherein:

[0210] Film-forming mixtures include second polar media, amphiphilic molecules, and nonpolar media;

[0211] The second polar medium is cyclohexanol, which accounts for 5% of the volume of the nonpolar medium;

[0212] The amphiphilic molecule is PDMS-PEG (PDMS-PMOXA is also possible), and the concentration in a nonpolar medium is 10 mg / ml;

[0213] The non-polar medium is methylphenyl silicone oil (silicone oil AP100, Sigma-Aldrich).

[0214] S230: A third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) is introduced into the receiving cavity 340 through the inlet 321, causing the film-forming mixture to be discharged from the outlet 322. After standing, a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are sequentially distributed in the first direction within the structural unit 200.

[0215] S240: After standing for 2 hours, the film-forming phase 20 forms the film containing the amphiphilic molecular layer, and the test begins.

[0216] Example 3

[0217] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0218] S310: Proceed as step S110 in Embodiment 1, except that the structure of structural unit 200 is as follows: Figure 3 As shown.

[0219] S320: After removing the chip structure 100 containing the first polar dielectric phase 10 from the structural unit 200, it is loaded into the film-forming apparatus 300, and the film-forming mixture A is introduced into the receiving cavity 340 through the liquid inlet 321. After standing for 10 minutes, the first polar dielectric phase 10 and the film-forming phase A 22 covering the first polar dielectric phase 10 are formed in the structural unit 200. Wherein:

[0220] Film-forming mixture A includes amphiphilic molecules and a nonpolar medium;

[0221] The concentration of the amphiphilic molecule PDMS-PEG (also PDMS-PMOXA) in a nonpolar medium is 10 mg / ml;

[0222] The non-polar medium is silicone oil AP100.

[0223] S330: Air is introduced into the receiving cavity 340 through the liquid inlet 321 to push away excess film-forming mixture A on the surface of the chip structure 100, causing it to be discharged from the liquid outlet 322. Then, film-forming mixture B23 is introduced into the receiving cavity 340 through the liquid inlet 321. After standing, a first polar dielectric phase 10 and a film-forming phase 20 covering the first polar dielectric phase 10 are formed within the structural unit 200. Wherein:

[0224] Film-forming mixture B contains a second polar medium and a non-polar medium;

[0225] The second polar medium is toluene, which accounts for 10% of the volume of the non-polar medium;

[0226] The non-polar medium is methylphenyl silicone oil.

[0227] S340: A third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) is introduced into the receiving cavity 340 through the inlet 321, causing the film-forming mixture B23 to be discharged from the outlet 322. After standing, a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are formed sequentially in the first direction within the structural unit 200.

[0228] S350: After standing for 20 minutes, the film-forming phase 20 forms the film containing the amphiphilic molecular layer, and the test begins.

[0229] Example 4

[0230] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0231] S410: Proceed according to step S110 in Example 1.

[0232] S420: Proceed according to step S320 in Example 3.

[0233] S430: Air is introduced into the receiving cavity 340 through the liquid inlet 321 to push away excess film-forming mixture A on the surface of the chip structure 100, allowing it to be discharged from the liquid outlet 322. The cover 320 is removed, and film-forming mixture B23 is sprayed into the cavity 331 surrounded by the gasket 330. After standing, a first polar dielectric phase 10 and a film-forming phase 20 covering the first polar dielectric phase 10 are formed within the structural unit 200. Wherein:

[0234] Film-forming mixture B contains a second polar medium and a non-polar medium;

[0235] The second polar medium is propyl acetate, which is 10% of the volume of the nonpolar medium;

[0236] The non-polar medium is silicone oil AP100.

[0237] S440: Same as step S340 in Example 3.

[0238] S450: Same as step S350 in Example 3.

[0239] Example 5

[0240] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0241] S510: Spray the film-forming mixture A at 5 μL per square centimeter onto the surface having the structural unit 200 ( Figure 2 On the outermost layer of the chip structure 100, after being placed at room temperature for 30 minutes, the microstructure on the chip surface is used to achieve uniform diffusion. The chip is then baked at 100 degrees Celsius for 15 minutes to ensure that the amphiphilic molecules are evenly distributed on the chip surface for later use.

[0242] The film-forming mixture A includes amphiphilic molecules and a nonpolar medium.

[0243] The amphiphilic molecule is PDMS-PEG (PDMS-PMOXA is also possible), and the concentration in a nonpolar medium is 10 mg / ml;

[0244] The non-polar medium is a mixture of silicone oil AR20 and C10 (decane) in a volume ratio of 1:9.

[0245] S520: The chip structure 100 with the processed structural unit 200 is added to a degassed first polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) and left to stand under vacuum (50mBar) for 2 minutes. The first polar medium fills the spatial region 210 of the structural unit 200 to form the first polar medium phase 10.

[0246] S530: After removing the chip structure 100 containing the first polar dielectric phase 10 and amphiphilic molecules from the structural unit 200, it is loaded into the film-forming apparatus 300. Then, the film-forming mixture B23 is introduced into the receiving cavity 340 through the liquid inlet 321 and allowed to stand for 30 minutes, forming the first polar dielectric phase 10 and the film-forming phase 20 covering the first polar dielectric phase 10 within the structural unit 200. Wherein:

[0247] Film-forming mixture B contains a second polar medium and a non-polar medium;

[0248] The second polar medium is ethyl acetate, which accounts for 25% of the volume of the non-polar medium;

[0249] The non-polar medium is silicone oil AR20;

[0250] During the 30-minute settling process, the amphiphilic molecules will dissolve back from the chip surface into the nonpolar and second polar media in the film-forming mixture B.

[0251] S540: A third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) is introduced into the receiving cavity 340 through the inlet 321, causing the film-forming mixture B23 to be discharged from the outlet 322. After standing, a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are sequentially distributed in the first direction within the structural unit 200.

[0252] S550: After standing for 20 minutes, the film-forming phase 20 forms the film containing the amphiphilic molecular layer, and the test begins.

[0253] Example 6

[0254] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0255] S610: Uniformly spray 4 μL of film-forming mixture A onto the surface having the structural unit 200 ( Figure 1 The chip structure 100 was placed at room temperature for 30 minutes.

[0256] The film-forming mixture A includes amphiphilic molecules and a nonpolar medium.

[0257] The amphiphilic molecule is a phospholipid (DPHPC), and the concentration of the amphiphilic molecule in a nonpolar medium is 10 mg / ml;

[0258] The nonpolar medium is a mixture of C16 (hexadecane) and C10 (decane) in a volume ratio of 1:4.

[0259] S620: The chip structure 100 with the processed structural unit 200 is added to a degassed first polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) and left to stand under vacuum (50mBar) for 2 minutes. The first polar medium fills the spatial region 210 of the structural unit 200 to form the first polar medium phase 10.

[0260] S630: After removing the chip structure 100 containing the first polar medium phase 10 and the film-forming mixture A on the surface from the structural unit 200, it is loaded into the film-forming device 300. Then, the film-forming mixture B23 is introduced into the receiving cavity 340 through the liquid inlet 321. After standing for 30 minutes, the first polar medium phase 10 and the film-forming phase 20 covering the first polar medium phase 10 are formed in the structural unit 200.

[0261] Wherein: film-forming mixture B contains amphiphilic molecules, a second polar medium, and a nonpolar medium;

[0262] The second polar medium is DMF, which accounts for 43% of the volume of the non-polar medium;

[0263] The nonpolar medium is C16 (hexadecane).

[0264] S640: A third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) is introduced into the receiving cavity 340 through the inlet 321, causing the film-forming mixture B23 to be discharged from the outlet 322. After standing, a first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are sequentially distributed in the first direction within the structural unit 200.

[0265] S650: After standing for 20 minutes, the film-forming phase 20 forms the film containing the amphiphilic molecular layer, and the test begins.

[0266] Example 7

[0267] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0268] S710: Proceed according to step S110 in Example 1, except that: the first polar medium is a buffer solution containing 600mM KCl and 10mM 3-morpholinopropanesulfonic acid, and the system is allowed to stand under vacuum (50mBar) for 5 minutes.

[0269] S720: The second polar medium is 10% of the volume of the nonpolar medium. The second polar medium is methanol, the amphiphilic molecule is glycerophospholipid, and the nonpolar medium is squalene. The concentration of the amphiphilic molecule in the nonpolar medium volume is 10 mg / mL. The rest is the same as S120 in Example 1.

[0270] S730: The third polar medium is a 10 mM 3-morpholinopropanesulfonic acid buffer solution containing 600 mM KCl. The rest is the same as S130 in Example 1.

[0271] S740: The settling time is 2 hours. The rest is the same as S140 in Example 1.

[0272] Example 8

[0273] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0274] S810: The first polar medium is a buffer solution containing 600 mM KCl and 10 mM acetate. The system is allowed to stand under vacuum (50 mBar) for 5 minutes. The rest is the same as S110 in Example 1.

[0275] S820: The second polar medium is 15% of the volume of the nonpolar medium. The second polar medium is ethyl acetate, the amphiphilic molecule is dimethyl silicone oil with dihydroxyl-terminated ends, and the nonpolar medium is tetradecane bromo, with the concentration of the amphiphilic molecule in the nonpolar medium volume being 20 mg / mL. The rest is the same as S120 in Example 1.

[0276] S830: The third polar medium is a 10 mM citrate buffer solution containing 600 mM KCl. The rest is the same as S130 in Example 1.

[0277] S840: The settling time is 2 hours. The rest is the same as S140 in Example 1.

[0278] Example 9

[0279] A membrane containing an amphiphilic molecular layer and a method for forming the same, comprising the following steps:

[0280] S910: The first polar medium is a carbonate buffer solution containing 600 mM KCl and 10 mM. The system is allowed to stand under vacuum (50 mBar) for 5 minutes. The rest is the same as S110 in Example 1.

[0281] S920: The second polar medium is 10% of the volume of the nonpolar medium. The second polar medium is N,N'-dimethylformamide, the amphiphilic molecule is dihydroxyl-terminated dimethyl silicone oil, and the nonpolar medium is a mixture of silicone oil and hexadecane in a volume ratio of 3:1. The concentration of the amphiphilic molecule in the nonpolar medium is 10 mg / mL. The rest is the same as S120 in Example 1.

[0282] S930: The third polar medium is a citrate buffer solution containing 600 mM KCl. The rest is the same as S130 in Example 1.

[0283] S940: The settling time is 2 hours. The rest is the same as S140 in Example 1.

[0284] Comparative Example 1

[0285] A membrane containing an amphiphilic molecular layer was prepared according to the method of Example 1, the main difference being that the film-forming mixture did not contain a second polar medium, as detailed below:

[0286] S'110: The chip structure 100 having the structural unit 200 is then filled with a degassed first polar medium. Figure 2 As shown. The first polar medium is a phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5). The system is allowed to stand under vacuum (50mBar) for 2 minutes, and the first polar medium fills the spatial region 210 of the structural unit 200 to form the first polar medium phase 10. (Same as step S110 in Example 1)

[0287] S'120: After removing the chip structure 100 containing the first polar dielectric phase 10 from the structural unit 200, it is loaded into the film-forming apparatus 300. Then, the film-forming mixture B23 is introduced into the receiving cavity 340 through the liquid inlet 321. After standing for 30 minutes, the first polar dielectric phase 10 and the film-forming phase 20 covering the first polar dielectric phase 10 are formed in the structural unit 200. The film-forming mixture B contains amphiphilic molecules and non-polar dielectrics. The amphiphilic molecules and non-polar dielectrics are the same as in step S120 of Example 1.

[0288] S'130: A third polar medium is introduced into the receiving cavity 340 through the inlet 321, causing the film-forming mixture B23 to be discharged from the outlet 322. A first polar medium phase 10, a film-forming phase 20, and a third polar medium phase 30 are sequentially distributed in the first direction within the structural unit 200.

[0289] S'140: After standing for 20 minutes, the film-forming phase 20 forms the film containing the amphiphilic molecular layer.

[0290] Comparative Example 2

[0291] S'210: Same as step S'110 of Comparative Example 1.

[0292] S'220: Film-forming mixture B contains amphiphilic molecules and a nonpolar medium. The concentration of the amphiphilic molecules in the nonpolar medium is 10 mg / ml. The amphiphilic molecules are PDMS-PEG or PDMS-PMOXA, and the nonpolar medium is a mixture of AR20 and C10 in a volume ratio of 1:4. The rest is the same as step S'120 of Comparative Example 1.

[0293] S'230: Same as step S'130 of Comparative Example 1.

[0294] S'240: Same as step S'140 of Comparative Example 1.

[0295] Comparative Example 3

[0296] S'310: Same as step S'110 of Comparative Example 1.

[0297] S'320: Film-forming mixture B contains amphiphilic molecules and a nonpolar medium. The concentration of the amphiphilic molecules in the nonpolar medium is 10 mg / ml. The amphiphilic molecules are DPHPC, and the nonpolar medium is a mixture of C16 and C10 molecules in a volume ratio of 1:4. The rest is the same as step S'120 of Comparative Example 1.

[0298] S'330: Same as step S'130 in Comparative Example 1.

[0299] S'340: Same as step S'140 of Comparative Example 1.

[0300] Characterization Examples Figure 2The chip structure of structural unit 200 shown, the films containing amphiphilic molecular layers in Examples 1-9, and the films containing amphiphilic molecular layers in Comparative Examples 1-3 were tested. Chip structure 100 includes multiple first electrodes, each corresponding to a structural unit 200. Each first electrode is located at the end of structural unit 200 away from the opening, communicating with the spatial region 210 surrounded by structural unit 200, and contacting the first polar dielectric phase 10 of the film containing the amphiphilic molecular layer. Simultaneously, the film-forming apparatus 300 may include a second electrode communicating with the receiving cavity 340. The second electrode contacts the third polar dielectric phase 30 at the end away from the first polar dielectric phase. The first electrode and the second electrode are connected to the testing device to test the film containing the amphiphilic molecular layer. Each structural unit 200 is actually a film capacitor, and the electrical characterization will differ depending on the thickness of the film containing the amphiphilic molecular layer. Each rectangular block in the figure represents a film capacitor, corresponding to one structural unit 200. The value in the rectangular block represents the capacitance value of that film capacitor. Furthermore, the depth of the color displayed by each unit of the instrument's electrical characterization is positively correlated with the value of the membrane capacitance; that is, the deeper the color, the greater the membrane capacitance.

[0301] The capacitance value can characterize the state of different structural units 200, such as whether a film has formed, and the thickness and state of the formed film. Specifically:

[0302] Values ​​less than 20 pF represent the instrument's background capacitance or the capacitance value in the initial state before film formation, and are displayed in light gray.

[0303] 20.1–30 pF is the film capacitance value that is unfavorable for subsequent conventional pore embedding of this type of amphiphilic molecular layer film. It is shown as medium gray and indicates that the film thickness is too large.

[0304] 30.1–65 pf is the film capacitance value suitable for subsequent conventional pore embedding of this type of amphiphilic molecular layer film, shown as dark gray, indicating that the film thickness is appropriate;

[0305] 65.1–100 pF is the film capacitance value that is unfavorable for subsequent conventional pore embedding of this type of amphiphilic molecular layer film, and is shown in black, indicating that the film thickness is too small;

[0306] A value greater than 100.1 pf indicates a broken membrane or that the amphiphilic molecular layer exhibits a membrane lacking pore-forming ability, which is displayed as a deep black color.

[0307] The test results are shown in the figure. Specifically:

[0308] First, the test was performed. Figure 2 The electrical characterization of the chip structure of the structural unit 200 shown shows that the capacitance value of each structural unit is less than 20pF, and is mostly concentrated between 12pF and 13pF (results not shown).

[0309] Figure 13The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 1. Figure 13 The data shows that the capacitance values ​​corresponding to 6 structural units are greater than 100.1 pF, the capacitance values ​​corresponding to 4 structural units are less than or close to 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 97.40%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 1 reached 97.40%.

[0310] Figure 14 This is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 2. Figure 14 The data shows that the capacitance values ​​corresponding to two structural units are greater than 100.1 pF, the capacitance values ​​corresponding to five structural units are less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 98.18%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 2 reached 98.18%.

[0311] Figure 15 This is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 3. Figure 15 The data shows that the capacitance values ​​corresponding to 3 structural units are greater than 100.1 pF, the capacitance value corresponding to 1 structural unit is between 65.1 and 100 pF, the capacitance values ​​corresponding to 18 structural units are less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 94.27%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 3 reached 94.27%.

[0312] Figure 16 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 4. Figure 16 The data shows that the capacitance values ​​corresponding to 5 structural units are greater than 100.1 pF, the capacitance values ​​corresponding to 14 structural units are less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 95.05%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 4 reached 95.05%.

[0313] Figure 17 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 5. Figure 17 The data shows that the capacitance value corresponding to one structural unit is greater than 100.1 pF, the capacitance value corresponding to three structural units is between 65.1 and 100 pF, the capacitance value corresponding to seven structural units is less than 20 pF, and the capacitance value corresponding to the remaining structural units is between 30.1 and 65 pF, accounting for 97.40%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 5 reached 97.14%.

[0314] Figure 18 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 6. Figure 18 The data shows that the capacitance values ​​corresponding to 3 structural units are greater than 100.1 pF, the capacitance values ​​corresponding to 5 structural units are between 65.1 and 100 pF, the capacitance values ​​corresponding to 3 structural units are between 20.1 and 30 pF, the capacitance values ​​corresponding to 5 structural units are less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 95.83%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 6 reached 95.83%.

[0315] Figure 19 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 7. Figure 19 The data shows that the capacitance values ​​corresponding to three structural units are greater than 100.1 pF, the capacitance value corresponding to one structural unit is between 65.1 and 100 pF, the capacitance value corresponding to one structural unit is between 20.1 and 30 pF, the capacitance value corresponding to 13 structural units is less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 95.31%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 7 reached 95.31%.

[0316] Figure 20 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 8. Figure 20 The data shows that the capacitance values ​​corresponding to 9 structural units are greater than 100.1 pF, the capacitance values ​​corresponding to 2 structural units are between 65.1 and 100 pF, the capacitance value corresponding to 1 structural unit is less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 96.61%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 8 reached 96.88%.

[0317] Figure 21 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Example 9. Figure 21 The data shows that the capacitance values ​​corresponding to three structural units are greater than 100.1 pF, the capacitance values ​​corresponding to one structural unit are between 65.1 and 100 pF, the capacitance values ​​corresponding to two structural units are between 20.1 and 30 pF, the capacitance values ​​corresponding to ten structural units are less than 20 pF, and the capacitance values ​​corresponding to the remaining structural units are all between 30.1 and 65 pF, accounting for 96.09%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Example 9 reached 95.83%.

[0318] Figure 22This is an electrical characterization diagram of the film containing the amphiphilic molecular layer formed in Comparative Example 1. Figure 22 The data shows that the capacitance value corresponding to one structural unit is greater than 100.1 pF, the capacitance value corresponding to three structural units is between 65.1 and 100 pF, the capacitance value corresponding to 126 structural units is less than 20 pF, and the capacitance value corresponding to 254 structural units is between 30.1 and 65 pF, accounting for 66.15%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Comparative Example 1 is only 66.15%.

[0319] Figure 23 This is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Comparative Example 2. Figure 23 The data shows that 21 structural units correspond to capacitance values ​​greater than 100.1 pF, 53 structural units correspond to capacitance values ​​between 65.1 and 100 pF, 16 structural units correspond to capacitance values ​​less than 20 pF, and 294 structural units correspond to capacitance values ​​between 30.1 and 65 pF, accounting for 76.56%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Comparative Example 2 is only 76.56%.

[0320] Figure 24 The image shows the electrical characterization of the membrane containing the amphiphilic molecular layer formed in Comparative Example 3. Figure 24 The data shows that the capacitance values ​​corresponding to 12 structural units are greater than 100.1 pF, the capacitance values ​​corresponding to 62 structural units are between 65.1 and 100 pF, the capacitance values ​​corresponding to 17 structural units are less than 20 pF, and the capacitance values ​​corresponding to 293 structural units are all between 30.1 and 65 pF, accounting for 76.3%. In other words, the film formation rate of the film containing the amphiphilic molecular layer in Comparative Example 3 is only 76.3%.

[0321] Application Examples:

[0322] Experimental group: Take the chip structure containing the membrane with amphiphilic molecular layer formed in Example 1, and introduce a third polar medium containing nanoporous protein (Mycobacterium smegmatis porin A, abbreviated as MspA, SEQ ID NO:1, concentration between 10ng / ml and 100ng / ml) into the receiving cavity 340 through the liquid inlet 321. Incubate statically for 1 hour, and then introduce 5 times the fluid volume of the third polar medium into the receiving cavity 340 through the liquid inlet 321 to replace the solution containing nanoporous proteins, thus completing the pore embedding process.

[0323] Control group: The experimental group was treated the same way, except that no second polar medium was added to complete the pore embedding process.

[0324] The chips from both the experimental and control groups were connected to the electrical system (QNome-9604 nanopore sequencer). After embedding, a single nanopore was considered usable if the opening current was 60-70 pA under a constant voltage of 80 mV. The QNome-9604 nanopore sequencer's automatic pore-screening program (under a constant voltage of 80 mV, a single nanopore was considered usable if the opening current was 60-70 pA and the noise was less than 1.5 pA) screened out the following:

[0325] The experimental group contained 274 single pores, with a single pore embedding rate of 71.35%, accounting for 73.25% of the film formation rate (97.4%).

[0326] The control group had 171 single pores, with a single pore embedding rate of only 44.53%, which accounted for 67.31% of the film formation rate (66.15%).

[0327] Figure 25 This is a real-time display interface for the polynucleotide sequencing signals of the experimental group, used to determine the signal.

[0328] Figure 26 This is a graph showing the sequencing stability and chip channel utilization of the experimental group. The chip channel utilization is approximately 80% (light gray area).

[0329] Figure 27 The graph shows the sequencing stability and chip channel utilization of the control group. The chip channel utilization is approximately 50% (light gray area).

[0330] Comparing the results of the experimental group and the control group, it can be seen that the experimental group, with the addition of a second polar medium, has a higher single-pore embedding rate of nanoporous proteins in amphiphilic molecular layers or membranes containing amphiphilic molecular layers, which is more conducive to embedding, sequencing stability and chip channel utilization.

[0331] Although this application has been described with reference to preferred embodiments, various modifications can be made thereto and components can be replaced with equivalents without departing from the scope of this application. In particular, the technical features mentioned in the various embodiments can be combined in any manner, provided there is no structural conflict. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer in a structural unit, characterized in that, The method includes the following steps: A first polar medium phase, a film-forming phase, and a third polar medium phase are formed sequentially along a first direction within the spatial region of the structural unit; wherein, the first direction is the thickness direction of the film, and the film-forming phase is formed by a film-forming mixture comprising a second polar medium, amphiphilic molecules, and a nonpolar medium; Conditions are provided to enable the film-forming phase to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and to allow the second polar medium contained in the film-forming phase to be distributed to the first polar medium phase and / or the third polar medium phase.

2. The method according to claim 1, characterized in that, The spatial region of the structural unit includes openings.

3. The method according to claim 1, characterized in that, The step of forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within the spatial region of the structural unit includes: A first polar medium, a film-forming mixture, and a third polar medium are sequentially introduced into the spatial region of the structural unit to form the film-forming phase between the first polar medium phase and the third polar medium phase; The film-forming mixture comprises a second polar medium, amphiphilic molecules, and a nonpolar medium.

4. The method according to claim 1, characterized in that, The step of forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within the spatial region of the structural unit includes: S1: A first polar medium and a film-forming mixture A are sequentially introduced into the spatial region of the structural unit to form a first polar medium phase and a film-forming mixture A phase sequentially distributed along a first direction; wherein, the film-forming mixture A contains amphiphilic molecules and a nonpolar medium; S2: Add film-forming mixture B to the film-forming phase A to form the film-forming phase; The film-forming mixture B comprises a second polar medium and a non-polar medium; S3: A third polar medium is introduced into the side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.

5. The method according to claim 4, characterized in that, The step between S1 and S2 also includes: At least partially remove the volume of the film-forming A phase that extends beyond the spatial region.

6. The method according to claim 4, characterized in that, In step S2, adding film-forming mixture B to the film-forming phase A to form the film-forming phase includes: The film-forming mixture B is introduced into the film-forming phase A and allowed to stand to form the film-forming phase; or Film-forming mixture B is sprayed onto the surface of the film-forming phase A to form the film-forming phase.

7. The method according to any one of claims 3 to 6, characterized in that, The step of introducing a first polar medium into the spatial region of the structural unit includes: The structural unit is placed in the first polar medium and left to stand, so that the first polar medium enters the spatial region of the structural unit.

8. The method according to any one of claims 3 to 6, characterized in that, The step of introducing the film-forming mixture into the spatial region of the structural unit includes: The structural unit is placed in the corresponding film-forming mixture, removed, and allowed to stand, allowing the corresponding film-forming mixture to enter the spatial region; or The structural unit is placed in a corresponding film-forming mixture A, removed, and allowed to stand, allowing the corresponding film-forming mixture A to enter the spatial region, wherein the film-forming mixture A contains amphiphilic molecules and a nonpolar medium; then the structural unit is placed in a corresponding film-forming mixture B, removed, and allowed to stand, allowing the corresponding film-forming mixture B to enter the spatial region, wherein the film-forming mixture B contains a second polar medium and a nonpolar medium.

9. The method according to any one of claims 3 to 6, characterized in that, The step of introducing a third polar medium into the spatial region of the structural unit includes: The structural unit comprising a first polar medium phase and a film-forming phase is placed in a third polar medium; and / or The step of distributing the second polar medium of the film-forming phase into the first polar medium phase and / or the third polar medium phase includes: The structural unit, which had been introduced into a third polar medium, was left to stand still.

10. The method according to claim 1, characterized in that, The step of forming a first polar dielectric phase, a film-forming phase, and a third polar dielectric phase sequentially distributed along a first direction within the spatial region of the structural unit includes: S11: Adhere the film-forming mixture A to the surface outside the spatial region of the structural unit; wherein the film-forming mixture A comprises amphiphilic molecules and a nonpolar medium; S22: A first polar medium is introduced into the spatial region of the structural unit to form a first polar medium phase; S33: A film-forming mixture B is added to the first polar medium phase, and the amphiphilic molecules in the film-forming mixture A attached to the surface are dissolved into the film-forming mixture B to form the film-forming phase; wherein the film-forming mixture B contains a second polar medium and a non-polar medium; S44: A third polar medium is introduced into the side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.

11. The method according to any one of claims 1-6, characterized in that, The volume of the second polar medium is 5% to 45% of the volume of the non-polar medium.

12. The method according to any one of claims 1-6, characterized in that, The volume of the second polar medium is 5% to 30% of the volume of the non-polar medium.

13. The method according to any one of claims 1-6, characterized in that, The volume of the second polar medium is 5% to 15% of the volume of the non-polar medium.

14. The method according to any one of claims 1-6, characterized in that, The second polar medium is soluble in the non-polar medium, and the second polar medium is soluble in the first polar medium or the first polar medium phase, and the second polar medium is soluble in the third polar medium or the third polar medium phase.

15. The method according to any one of claims 1-6, characterized in that, The second polar medium is selected from one or more of methanol, ethanol, isopropanol, cyclohexanol, toluene, ethyl acetate, propyl acetate, isopropyl acetate, acetone, butanone, cyclohexanone, acetonitrile, propionitrile, dimethyl sulfoxide, N,N'-dimethylformamide (DMF), and N,N'-dimethylacetamide. The nonpolar medium is selected from one or more of methylphenyl silicone oil, dimethyl silicone oil, hexadecane, tetradecane, decadecane, bromodecane, bromotetradecane, and squalene.

16. The method according to claim 15, characterized in that, The dimethyl silicone oil is a dimethyl silicone oil with different end caps. When the nonpolar medium is methylphenyl silicone oil, dimethyl silicone oil with different end caps, hexadecane, or a mixture of silicone oil and hexadecane, the second polar medium is dimethyl sulfoxide.

17. The method according to claim 16, characterized in that, The volume of the dimethyl sulfoxide is 5-15% of the volume of the nonpolar medium.

18. The method according to any one of claims 1-6, characterized in that, The first polar medium is an aqueous solution of a first buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholinopropanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution; and / or The third polar medium is an aqueous solution of a third buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution. The third polar medium may be the same as or different from the first polar medium; The concentration of the third buffer solution may be the same as or different from the concentration of the first buffer solution.

19. The method according to claim 18, characterized in that, The concentration of the first buffer aqueous solution is 5-100 mM.

20. The method according to claim 18, characterized in that, The first buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

21. The method according to claim 18, characterized in that, The concentration of the third buffer solution is 5-100 mM.

22. The method according to claim 18, characterized in that, The third buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

23. The method according to claim 18, characterized in that, Both the first and third buffer aqueous solutions contain potassium salts.

24. The method according to claim 23, characterized in that, The concentration of the potassium salt is 400-800 mM.

25. The method according to claim 23, characterized in that, The potassium salt is potassium chloride.

26. The method according to any one of claims 1-6, characterized in that, The osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium maintain a stable state in which the first polar medium phase and the third polar medium phase do not permeate each other.

27. The method according to any one of claims 1-6, characterized in that, The amphiphilic molecule is selected from one or more of phospholipids, fatty acids, fatty acyl groups, glycerides, glycerophospholipids, sphingolipids, sterol lipids, isopentenyl lipids, glycolipids, polyketides, and amphiphilic block copolymers.

28. The method according to claim 27, characterized in that, The amphiphilic block copolymer comprises at least three polymer segments, wherein hydrophilic polymeric segments A1 and A2 are connected to opposite ends of hydrophobic polymeric segment B; or The amphiphilic block copolymer comprises at least two polymer segments: a hydrophilic polymeric segment A and a hydrophobic polymeric segment B.

29. The method according to claim 28, characterized in that, The copolymer is poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), poly(2-methyloxazoline)-polyethylene-poly(2-methyloxazoline), or poly(ethylene glycol)-poly(dimethylsiloxane)-poly(ethylene glycol).

30. The amphiphilic molecular layer or membrane containing an amphiphilic molecular layer prepared by the method of any one of claims 1-29.

31. A film-forming system, characterized in that, The system includes a structural unit, and within the spatial region of the structural unit are a first polar medium phase, a film-forming phase, and a third polar medium phase distributed sequentially along a first direction; wherein, the first direction is the thickness direction of the film, and the film-forming phase includes a second polar medium, amphiphilic molecules, and a nonpolar medium to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer. The second polar medium of the film-forming phase can be distributed to the first polar medium phase and / or the third polar medium phase.

32. The film-forming system according to claim 31, characterized in that, Transmembrane pores are embedded in the amphiphilic molecular layer.

33. The film-forming system according to claim 32, characterized in that, The transmembrane pores are transmembrane protein pores.

34. The film-forming system according to any one of claims 31-33, characterized in that, The volume of the second polar medium is 5% to 45% of the volume of the non-polar medium.

35. The film-forming system according to claim 34, characterized in that, The volume of the second polar medium is 5% to 30% of the volume of the non-polar medium.

36. The film-forming system according to claim 34, characterized in that, The volume of the second polar medium is 5% to 15% of the volume of the non-polar medium.

37. The film-forming system according to any one of claims 31-33, characterized in that, The second polar medium is soluble in the non-polar medium, and the second polar medium is soluble in the first polar medium or the first polar medium phase, and the second polar medium is soluble in the third polar medium or the third polar medium phase.

38. The film-forming system according to any one of claims 31-33, characterized in that, The second polar medium is selected from one or more of methanol, ethanol, isopropanol, cyclohexanol, toluene, ethyl acetate, propyl acetate, isopropyl acetate, acetone, butanone, cyclohexanone, acetonitrile, propionitrile, dimethyl sulfoxide, N,N'-dimethylformamide (DMF), and N,N'-dimethylacetamide. The nonpolar medium is selected from one or more of methylphenyl silicone oil, dimethyl silicone oil, hexadecane, tetradecane, decadecane, bromodecane, bromotetradecane, and squalene.

39. The film-forming system according to claim 38, characterized in that, The dimethyl silicone oil is a dimethyl silicone oil with different end caps. When the nonpolar medium is methylphenyl silicone oil, dimethyl silicone oil with different end caps, hexadecane, or a mixture of silicone oil and hexadecane, the second polar medium is dimethyl sulfoxide.

40. The film-forming system according to claim 39, characterized in that, The volume of the dimethyl sulfoxide is 5-15% of the volume of the nonpolar medium.

41. The film-forming system according to any one of claims 31-33, characterized in that, The first polar medium is an aqueous solution of a first buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholinopropanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution; and / or The third polar medium is an aqueous solution of a third buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution. The third polar medium may be the same as or different from the first polar medium; The concentration of the third buffer solution may be the same as or different from the concentration of the first buffer solution.

42. The film-forming system according to claim 41, characterized in that, The concentration of the first buffer aqueous solution is 5-100 mM.

43. The film-forming system according to claim 41, characterized in that, The first buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

44. The film-forming system according to claim 41, characterized in that, The concentration of the third buffer solution is 5-100 mM.

45. The film-forming system according to claim 41, characterized in that, The third buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

46. ​​The film-forming system according to claim 41, characterized in that, Both the first and third buffer aqueous solutions contain potassium salts.

47. The film-forming system according to claim 46, characterized in that, The concentration of the potassium salt is 400-800 mM.

48. The film-forming system according to claim 47, characterized in that, The potassium salt is potassium chloride.

49. The film-forming system according to any one of claims 31-33, characterized in that, The osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium maintain a stable state in which the first polar medium phase and the third polar medium phase do not permeate each other.

50. The film-forming system according to any one of claims 31-33, characterized in that, The amphiphilic molecule is selected from one or more of phospholipids, fatty acids, fatty acyl groups, glycerides, glycerophospholipids, sphingolipids, sterol lipids, isopentenyl lipids, glycolipids, polyketides, and amphiphilic block copolymers.

51. The film-forming system according to claim 50, characterized in that, The amphiphilic block copolymer comprises at least three polymer segments, wherein hydrophilic polymeric segments A1 and A2 are connected to opposite ends of hydrophobic polymeric segment B; or The amphiphilic block copolymer comprises at least two polymer segments: a hydrophilic polymeric segment A and a hydrophobic polymeric segment B.

52. The film-forming system according to claim 51, characterized in that, The copolymer is poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), poly(2-methyloxazoline)-polyethylene-poly(2-methyloxazoline), or poly(ethylene glycol)-poly(dimethylsiloxane)-poly(ethylene glycol).

53. A microdroplet, characterized in that, The microdroplet comprises: a first polar medium phase, a film-forming phase, and a third polar medium phase distributed sequentially along a first direction; wherein, the first direction is the thickness direction of the film, and the film-forming phase comprises a second polar medium, amphiphilic molecules, and a nonpolar medium to form a film containing an amphiphilic molecular layer; The second polar medium of the film-forming phase can be distributed to the first polar medium phase and / or the third polar medium phase to form a film containing an amphiphilic molecular layer.

54. The microdroplet according to claim 53, characterized in that, Transmembrane pores are embedded in the amphiphilic molecular layer.

55. The microdroplet according to claim 54, characterized in that, The transmembrane pores are transmembrane protein pores.

56. The microdroplet according to any one of claims 53-55, characterized in that, The volume of the second polar medium is 5% to 45% of the volume of the non-polar medium.

57. The microdroplet according to claim 56, characterized in that, The volume of the second polar medium is 5% to 30% of the volume of the non-polar medium.

58. The microdroplet according to claim 56, characterized in that, The volume of the second polar medium is 5% to 15% of the volume of the non-polar medium.

59. The microdroplet according to any one of claims 53-55, characterized in that, The second polar medium is soluble in the non-polar medium, and the second polar medium is soluble in the first polar medium or the first polar medium phase, and the second polar medium is soluble in the third polar medium or the third polar medium phase.

60. The microdroplet according to any one of claims 53-55, characterized in that, The second polar medium is selected from one or more of methanol, ethanol, isopropanol, cyclohexanol, toluene, ethyl acetate, propyl acetate, isopropyl acetate, acetone, butanone, cyclohexanone, acetonitrile, propionitrile, dimethyl sulfoxide, N,N'-dimethylformamide (DMF), and N,N'-dimethylacetamide. The nonpolar medium is selected from one or more of methylphenyl silicone oil, dimethyl silicone oil, hexadecane, tetradecane, decadecane, bromodecane, bromotetradecane, and squalene.

61. The microdroplet according to claim 60, characterized in that, The dimethyl silicone oil is a dimethyl silicone oil with different end caps. When the nonpolar medium is methylphenyl silicone oil, dimethyl silicone oil with different end caps, hexadecane, or a mixture of silicone oil and hexadecane, the second polar medium is dimethyl sulfoxide.

62. The microdroplet according to claim 61, characterized in that, The volume of the dimethyl sulfoxide is 5-15% of the volume of the nonpolar medium.

63. The microdroplet according to any one of claims 53-55, characterized in that, The first polar medium is an aqueous solution of a first buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution. and / or The third polar medium is an aqueous solution of a third buffer, which is selected from one or more of the following: phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, borate buffer solution, or citrate buffer solution. The third polar medium may be the same as or different from the first polar medium; The concentration of the third buffer solution may be the same as or different from the concentration of the first buffer solution.

64. The microdroplet according to claim 63, characterized in that, The concentration of the first buffer aqueous solution is 5-100 mM.

65. The microdroplet according to claim 63, characterized in that, The first buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

66. The microdroplet according to claim 63, characterized in that, The concentration of the third buffer solution is 5-100 mM.

67. The microdroplet according to claim 63, characterized in that, The third buffer aqueous solution is a 10 mM phosphate buffer solution or a 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

68. The microdroplet according to claim 63, characterized in that, Both the first and third buffer aqueous solutions contain potassium salts.

69. The microdroplet according to claim 68, characterized in that, The concentration of the potassium salt is 400-800 mM.

70. The microdroplet according to claim 68, characterized in that, The potassium salt is potassium chloride.

71. The microdroplet according to any one of claims 53-55, characterized in that, The osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium maintain a stable state in which the first polar medium phase and the third polar medium phase do not permeate each other.

72. The microdroplet according to any one of claims 53-55, characterized in that, The amphiphilic molecule is selected from one or more of phospholipids, fatty acids, fatty acyl groups, glycerides, glycerophospholipids, sphingolipids, sterol lipids, isopentenyl lipids, glycolipids, polyketides, and amphiphilic block copolymers.

73. The microdroplet according to claim 72, characterized in that, The amphiphilic block copolymer comprises at least three polymer segments, wherein hydrophilic polymeric segments A1 and A2 are connected to opposite ends of hydrophobic polymeric segment B; or The amphiphilic block copolymer comprises at least two polymer segments: a hydrophilic polymeric segment A and a hydrophobic polymeric segment B.

74. The microdroplet according to claim 73, characterized in that, The copolymer is poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), poly(2-methyloxazoline)-polyethylene-poly(2-methyloxazoline), or poly(ethylene glycol)-poly(dimethylsiloxane)-poly(ethylene glycol).

75. A nanopore sequencing device, characterized in that, Includes the amphiphilic molecular layer or membrane containing the amphiphilic molecular layer prepared by the method of any one of claims 1-29, the film-forming system of any one of claims 31-52, or the microdroplets of any one of claims 53-74.

76. A method for characterizing a target analyte, characterized in that, include: (a) Contacting the target analyte with a transmembrane pore, the transmembrane pore being embedded in the amphiphilic molecular layer of the method of any one of claims 1-29, the film-forming system of any one of claims 31-52, or the microdroplet of any one of claims 53-74; (b) When the target analyte moves relative to the orifice or when the target analyte is present in the orifice, one or more electrical signals are measured, wherein the measurement indicates one or more characteristics of the target analyte to characterize the target analyte.

77. The method according to claim 76, characterized in that, The pores are transmembrane protein pores.

78. The method according to claim 76 or 77, characterized in that, The target analytes are metal ions, inorganic salts, polymers, amino acids, peptides, proteins, nucleotides, polynucleotides, polysaccharides, lipids, dyes, bleaching agents, drugs, diagnostic reagents, explosives, or environmental pollutants.

79. The method according to claim 78, characterized in that, The polynucleotides include DNA and / or RNA and their analogs / derivatives.

80. The use of the method of any one of claims 1-29, or the amphiphilic molecular layer or membrane containing the amphiphilic molecular layer prepared therefrom, the system of any one of claims 31-52, or the microdroplets of any one of claims 53-74 in characterizing target analytes or preparing products characterizing target analytes.

81. The application according to claim 80, characterized in that, The target analytes are metal ions, inorganic salts, polymers, amino acids, peptides, proteins, nucleotides, polynucleotides, polysaccharides, lipids, dyes, bleaching agents, drugs, diagnostic reagents, explosives, or environmental pollutants.

82. The application according to claim 81, characterized in that, The polynucleotides include DNA and / or RNA and their analogs / derivatives.

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