WO2023116394A1

WO2023116394A1 - Film forming method, system comprising film, and application

Info

Publication number: WO2023116394A1
Application number: PCT/CN2022/136356
Authority: WO
Inventors: 朱睿; 代威; 任世龙
Original assignee: 成都齐碳科技有限公司
Priority date: 2021-12-21
Filing date: 2022-12-02
Publication date: 2023-06-29
Also published as: CN116297721A

Abstract

Provided are a film forming method, a system comprising a film, and an application. A method for forming, in a structural unit (200), a film containing an amphiphilic molecular layer comprises the following steps: forming, in a spatial region (210) of the structural unit (200), a first polar medium phase (10), a film-forming phase (20), and a third polar medium phase (30) which are sequentially distributed in a first direction, wherein the first direction is the thickness direction of the film, and the film-forming phase (20) is formed by a film forming mixture containing a second polar medium, an amphiphilic molecule, and a non-polar medium; and providing conditions so that the film-forming phase (20) forms an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and the second polar medium contained in the film-forming phase (20) is distributed to the first polar medium phase (10) and/or the third polar medium phase (30).

Description

Membrane-forming methods, systems comprising membranes, and applications

Cross References to Related Applications

This application claims priority to Chinese patent application 202111576352.8, filed on December 21, 2021, entitled "Membrane Formation Method, System Containing Membrane, and Application", the entire content of which is incorporated herein by reference.

technical field

The present application relates to the field of sequencing technology, in particular to a membrane comprising an amphiphilic molecular layer and a method for forming the same, a system comprising the membrane, a nanopore sequencing device and applications.

Background technique

Nanopore sequencing needs to embed nanopore proteins in a membrane containing an amphiphilic molecular layer, so the thickness of the membrane is required to be within a suitable range, generally nanoscale. A general method for forming a membrane containing an amphiphilic molecular layer is carried out in an array structure composed of multiple structural units, and a membrane containing an amphiphilic molecular layer is formed in the structural units. However, this method has a low film formation rate.

Contents of the invention

The application provides a membrane containing an amphiphilic molecular layer and its formation method, a nanopore sequencing device and its application, aiming to solve the problem of low film formation rate of the amphiphilic molecular layer membrane forming method in the related art.

In the first aspect, the embodiment of the present 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:

A first polar dielectric phase, a film-forming phase and a third polar dielectric phase sequentially distributed along a first direction are formed in the spatial region of the structural unit; wherein, the first direction is the thickness direction of the film, The film-forming phase is formed from a film-forming mixture comprising a second polar medium, amphiphilic molecules, and a non-polar medium;

Conditions are provided so that the film-forming phase forms an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and the second polar medium contained in the film-forming phase is distributed to the first polar medium phase and/or the The third polar medium phase.

In some embodiments, the spatial region of the structural unit comprises an opening.

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 the first direction in the spatial region of the structural unit includes:

In the spatial region of the structural unit, the first polar medium, the film-forming mixture and the third polar medium are sequentially introduced to form the film-forming medium phase between the first polar medium phase and the third polar medium phase Mutually;

Wherein, the film-forming mixture includes a second polar medium, amphiphilic molecules and a non-polar medium.

S1: In the spatial region of the structural unit, the first polar medium and the film-forming mixture A are sequentially introduced to form the first polar medium phase and the film-forming A phase sequentially distributed along the first direction; wherein, the The film-forming mixture A comprises amphiphile molecules and a non-polar medium;

S2: adding a film-forming mixture B to the film-forming phase A to form the film-forming phase;

Wherein, the film-forming mixture B comprises a second polar medium and a non-polar medium;

S3: Passing a third polar medium on the side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.

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

A volume of the film-forming A phase beyond the space region is at least partially removed.

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

Pass into the film-forming mixture B into the film-forming A phase, and let stand to form the film-forming phase; or

The film-forming mixture B is sprayed on the surface of the film-forming phase A to form the film-forming phase.

In some embodiments, the step of introducing the first polarity medium in the spatial region of the structural unit includes:

The structural unit is placed in the first polar medium to allow the first polar medium to enter the spatial region of the structural unit.

In some embodiments, the step of passing the film-forming mixture into the spatial region of the structural unit includes:

placing the structural unit in the corresponding film-forming mixture, taking it out, standing still, and allowing the corresponding film-forming mixture to enter the space region; or

The structural unit is placed in the corresponding film-forming mixture A, taken out, and left to stand, so that the corresponding film-forming mixture A enters the space region, wherein the film-forming mixture A contains amphiphilic molecules and non-polar medium; then the structural unit is placed in the corresponding film-forming mixture B, taken out, and left to stand, so that the corresponding film-forming mixture B enters the space region, wherein the film-forming mixture B contains the second polarity media and non-polar media.

In some embodiments, the step of introducing a third polarity medium into the spatial region of the structural unit includes:

placing the structural unit comprising the first polar medium phase and the film-forming phase in a third polar medium; and/or

The step of distributing the second polar medium of the film-forming phase to the first polar medium phase and/or the third polar medium phase comprises:

The structural unit that has passed through the medium of the third polarity is left to stand.

S11: Attach the film-forming mixture A to the surface outside the spatial region of the structural unit; wherein, the film-forming mixture A includes amphiphilic molecules and a non-polar medium;

S22: Passing a first polar medium into the space region of the structural unit to form a first polar medium phase;

S33: adding the film-forming mixture B to the first polar medium phase, and dissolving the amphiphilic molecules in the film-forming mixture A attached to the surface into the film-forming mixture B to form the film-forming phase; wherein, The film-forming mixture B comprises a second polar medium and a non-polar medium;

S44: Passing a third polar medium into a side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.

The second aspect of the embodiments of the present application provides a film-forming system, wherein the system includes a structural unit, and the spatial region of the structural unit contains a first polar medium phase sequentially distributed along a first direction, a film-forming phase and a third polar medium phase; wherein, the first direction is the thickness direction of the film, and the film-forming phase includes a second polar medium, amphiphilic molecules and non-polar medium to form amphiphilic molecules layer or a membrane containing an amphiphile layer;

Wherein, 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.

A third aspect of the embodiments of the present application provides a microdroplet, wherein the microdroplet 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 non-polar medium to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer;

In some embodiments, a transmembrane pore is provided at the amphiphile layer; optionally, the transmembrane pore is a transmembrane protein pore;

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

In some embodiments, the volume of the second polar medium is 5%-45% of the volume of the non-polar medium, optionally 5%-30%, further optionally 5%-15%.

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 phase of the first polar medium , and the second polar medium is soluble in the third polar medium or the phase of the third polar medium.

In some embodiments, the second polar medium is selected from methanol, ethanol, isopropanol, cyclohexanol, toluene, ethyl acetate, propyl acetate, isopropyl acetate, acetone, methyl ethyl ketone, cyclohexanone , acetonitrile, propionitrile, dimethylsulfoxide, N,N'-dimethylformamide (DMF) and one or more of N,N'-dimethylacetamide;

And/or, the non-polar medium is selected from methylphenyl silicone oil, simethicone, optionally with different end-capped simethicone, hexadecane, tetradecane, dedecane, bromodecane, One or more of bromotetradecane and squalene.

In some embodiments, when the non-polar medium is methyl phenyl silicone oil, dimethicone oil, optional dimethicone oil with different end caps, hexadecane or a mixed medium of silicone oil and hexadecane, the The second polar medium is dimethyl sulfoxide;

Optionally, the volume of the dimethyl sulfoxide is 5-20% of the volume of the non-polar medium.

In some embodiments, the first polar medium is an aqueous solution of a first buffer selected from phosphate buffer solution, carbonate buffer solution, acetate buffer solution, trimethylol One or more of aminomethane buffer solution, 3-morpholinopropanesulfonic acid buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, borate buffer solution or citrate buffer solution;

Optionally, the concentration of the first aqueous buffer solution is 5-100mM; more alternatively, the first aqueous buffer solution is 10mM phosphate buffer solution or 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution, and/or

The third polar medium is a third aqueous buffer solution, and the third aqueous buffer solution is selected from phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris buffer solution, 3 -one or more of morpholinopropanesulfonic acid buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, borate buffer solution or citrate buffer solution;

Optionally, the concentration of the third aqueous buffer solution is 5-100mM; further optionally, the third aqueous buffer solution is 10mM phosphate buffer solution or 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution;

Optionally, the third polar medium is the same or different from the first polar medium;

Optionally, the concentration of the third aqueous buffer solution is the same or different from that of the first aqueous buffer solution.

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

Optionally, the concentration of the potassium salt is 400-800mM;

Optionally, the potassium salt is potassium chloride.

In some embodiments, the osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium maintain the first polar medium phase and the third polar medium phase impermeable to each other steady state.

In some embodiments, the amphiphile is selected from the group consisting of phospholipids, fatty acids, fatty acyl groups, glycerides, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, polyketides, and One or more of amphiphilic block copolymers.

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

The amphiphilic block copolymer comprises at least two polymer segments, a hydrophilic polymer segment A and a hydrophobic polymer segment B.

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

The fourth aspect of the embodiments of the present application provides an amphiphilic molecular layer or a film containing an amphiphilic molecular layer prepared according to the method described in the first aspect of the embodiments of the present application.

The fifth aspect of the embodiment of the present application provides a nanopore sequencing device, which includes the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer prepared by the method described in the first aspect of the embodiment of the present application, the second aspect of the embodiment of the present application The film-forming system described in one aspect, the microdroplet described in the third aspect of the embodiment of the present application, or the amphiphilic molecular layer or the film containing the amphiphilic molecular layer described in the fourth aspect of the embodiment of the present application, such as a biochip, or sequencer.

The sixth aspect of the embodiments of the present application provides a method for characterizing a target analyte, including:

(a) contacting the target analyte with a transmembrane pore embedded in an amphiphilic molecular layer in any of the methods, membrane-forming systems or microdroplets described above; optionally, the The pore is a transmembrane protein pore;

(b) determining one or more electrical signals as the analyte moves relative to the pore or when the analyte is present within the pore, wherein the determination is indicative of one or more of the analyte of interest Various features to characterize the target analyte.

The seventh aspect of the embodiment of the present application provides the amphiphilic molecular layer or the film containing the amphiphilic molecular layer prepared by the method described in the first aspect of the embodiment of the present application, and the film-forming system described in the second aspect of the embodiment of the present application , the microdroplet described in the third aspect of the embodiment of the present application or the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer described in the fourth aspect of the embodiment of the present application in characterizing the analyte or preparing a characterizing analyte product application;

Optionally, the characterization is a nanopore characterization, more optionally, the pore is a transmembrane protein pore.

In some embodiments, the target analyte is a metal ion, inorganic salt, polymer, amino acid, peptide, protein, nucleotide, polynucleotide, polysaccharide, lipid, dye, bleach, drug, diagnostic reagent, Explosive or environmental pollutants;

Optionally, said polynucleotide comprises DNA and/or RNA and analogs/derivatives thereof.

According to the method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer in a structural unit according to an embodiment of the present application, a first polar medium phase, a first polar medium phase, and a first polar medium phase sequentially distributed along a first direction are formed in the spatial region of the structural unit. A film-forming phase and a third polar medium phase; wherein, the first direction is the thickness direction of the film, and the film-forming phase is composed of a film-forming phase comprising a second polar medium, amphiphilic molecules and a non-polar medium A mixture forms. The hydrophilic segment of the amphiphile is easily soluble in the second polar medium, and the lipophilic segment is easily 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 amphiphile is carried into the corresponding polar medium phase, while the lipophilic segment remains in the non-polar medium phase. In a polar medium, amphiphile molecules complete the process of self-assembly to form a thin film to obtain an amphiphile layer or a film containing an amphiphile layer. At the same time, the thickness of the amphiphilic molecular layer and the membrane containing the amphiphilic molecular layer is effectively reduced through the distribution process of the second polar medium, so that the thickness of the amphiphilic molecular layer and the membrane containing the amphiphilic molecular layer is relatively thin. Further, using the method of the present application also improves the film formation rate, and makes the amphiphile layer or the film containing the amphiphile layer thinner and more conducive to hole insertion and sequencing.

Description of drawings

The features, advantages, and technical effects of the exemplary embodiments of the present application will be described below with reference to the accompanying drawings.

Fig. 1 is a structural schematic diagram of a structural unit disclosed in the related art;

Fig. 2 is a schematic structural diagram of a structural unit disclosed in an embodiment of the present application;

Fig. 3 is a structural schematic diagram of another structural unit disclosed in an embodiment of the present application;

Fig. 4 is a schematic cross-sectional structure diagram of a structural unit disclosed in an embodiment of the present application;

Fig. 5 is a schematic cross-sectional structural view of a structural unit disclosed in an embodiment of the present application;

Fig. 6 is a schematic structural diagram of forming a first polar dielectric phase in a structural unit disclosed in an embodiment of the present application;

Fig. 7 is a schematic structural diagram of the formation of the first polar medium phase and the film-forming phase in a structural unit disclosed in an embodiment of the present application;

Fig. 8 is a structural schematic diagram of a membrane containing an amphiphilic molecular layer and an amphiphilic molecular layer formed in a structural unit disclosed in an embodiment of the present application;

Fig. 9 is a structural schematic diagram of partially removing the film-forming phase A in a structural unit disclosed in an embodiment of the present application;

Fig. 10 is a schematic diagram of adding the film-forming mixture B after partly removing the film-forming A phase in the chip structure of Fig. 9;

Fig. 11 is a schematic structural diagram of a chip structure and a film forming device disclosed in an embodiment of the present application;

Fig. 12 is a schematic structural diagram of a chip structure and a film forming device disclosed in an embodiment of the present application;

13 is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 1;

14 is an electrical characterization diagram of the amphiphile layer formed in Example 2;

15 is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 3;

16 is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 4;

17 is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 5;

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

19 is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 7;

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

21 is an electrical characterization diagram of the amphiphilic molecular layer formed in Example 9;

22 is an electrical characterization diagram of the amphiphilic molecular layer formed in Comparative Example 1;

23 is an electrical characterization diagram of the amphiphilic molecular layer formed in Comparative Example 2;

24 is an electrical characterization diagram of the amphiphilic molecular layer formed in Comparative Example 3;

Fig. 25 is the real-time display interface of the polynucleotide sequencing signal of the experimental group;

Figure 26 is a test chart of sequencing stability and chip channel utilization of the experimental group;

Figure 27 is a test chart of sequencing stability and chip channel utilization in the control group.

In the drawings, the drawings are not necessarily drawn to scale.

10. The first polar medium phase; 20. The film-forming phase; 30. The third polar medium phase; 22. The film-forming A phase; 23. The film-forming mixture B;

100. Chip structure;

200. Structural unit; 210. Spatial area; 220. Opening; 230. Support member; 211. First channel; 212. Second channel;

300, film forming device; 310, carrier frame; 320, cover body; 330, gasket; 340, accommodation chamber; 331, cavity; 321, liquid inlet; 322, liquid outlet.

Detailed ways

The implementation manner of the present application will be further described in detail below with reference to the drawings and embodiments. The detailed description and drawings of the following embodiments are used to illustrate the principles of the application, but not to limit the scope of the application, that is, the application is not limited to the described embodiments.

In the description of this application, it should be noted that, unless otherwise specified, the meaning of "plurality" is more than two, and the terms "upper", "lower", "left", "right", "inner", " The orientation or positional relationship indicated by "outside" and so on are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a reference to this application. Application Restrictions. In addition, 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 range of error. "Parallel" is not strictly parallel, but within the allowable range of error.

In the description of this application, it should also be noted that, unless otherwise clearly specified and limited, the terms "installation", "connection", and "connection" should be interpreted in a broad sense, for example, it can be a fixed connection or a flexible connection. Disconnected connection, or integral connection, can be directly connected or indirectly connected through an intermediary. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific situations.

The structural unit 200 is usually disposed in the chip structure 100 . The chip structure 100 includes a plurality of structural units 200 . Multiple structural units 200 are usually distributed in an array. The walls of each structural unit 200 surround and form a space area 210 with a certain accommodation space. The spatial region 210 has an opening 220 . In some embodiments, various raw materials for amphiphilic material film formation, such as polar media and non-polar media, mainly enter the space region 210 through the opening 220 .

For ease of description, the end of the structural unit 200 provided with the opening 220 is defined as the top, and the end away from the opening 220 is defined as the bottom. The spatial regions 210 of the structural units 200 may communicate with each other, for example, the tops of adjacent structural units 200 communicate with each other. For example, referring to FIG. 1 , the walls at the top of the structural unit 200 are discontinuous, and are support members 230 arranged at intervals. The spaces between the supports form the first channel 211 . Adjacent structural units 200 share part of the support member 230 , and the tops of the structural units 200 communicate through the first channel 211 . Adjacent structural units 200 can partially communicate with the medium through the first channel 211 .

When the chip structure 100 of the above-mentioned unit structure prepares the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer, the polar medium, the non-polar medium dispersed with the sex medium. The medium injected later drives out the medium injected earlier to occupy part of the space region 210 , and finally forms a membrane containing an amphiphilic molecule layer between the two polar medium phases. However, in fact, using this method, the number of structural units 200 forming a film containing an amphiphilic molecular layer is relatively small, and the film formation rate is low, and in the structural unit 200 formed into a film, there are also amphiphilic molecular layers and amphiphilic molecular layers. The film thickness of the molecular layer is relatively thick.

After the applicant noticed that the film-forming method of the amphiphilic molecular layer and the film containing the amphiphilic molecular layer in the related art has the problem that the number of structural units 200 is small and the film-forming rate is low, the application of the amphiphilic molecular layer The film-forming method of the film was studied, and it was found that the self-assembly of the amphiphilic material to form a biomimetic film, that is, the amphiphilic molecular layer or the film containing the amphiphilic molecular layer, is a relatively fragile process, which is easily affected by external polarity and nonpolarity. The influence of the medium leads to the rupture of the membrane containing the amphiphilic molecular layer, and the problem that the number of structural units 200 in the membrane is small.

Based on the above problems discovered by the applicant, the applicant improved the method for forming the membrane containing the amphiphilic molecular layer in the structural unit 200, and the following further describes the embodiments of the present application.

The embodiment of the present application proposes a method for forming an amphiphilic molecular layer and a film containing the amphiphilic molecular layer in the structural unit 200. The structural unit 200 includes a space region 210 with an opening 220. The method includes the following steps:

In the space region 210, the first polar dielectric phase 10, the film-forming phase 20 and the third polar dielectric phase 30 distributed sequentially along the first direction are formed, the first direction is the thickness direction of the film, and the film-forming phase 20 consists of the second A mixture of dipolar media, amphiphilic molecules, and nonpolar media is formed.

The film-forming phase is provided with conditions to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and the second polar medium of the film-forming phase 20 is distributed to the first polar medium phase 10 and/or the third polar medium Phase 30.

According to the method for forming a film containing an amphiphilic molecular layer according to an embodiment of the present application, a first polar medium phase 10, a film-forming phase 20, and a third Polar medium phase 30. The film-forming phase 20 is formed from a mixture of a second polar medium, amphiphilic molecules and a non-polar medium. The hydrophilic segment of the amphiphile is easily soluble in the second polar medium, and the lipophilic segment is easily 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 molecule is carried into the corresponding polar medium phase, while the lipophilic segment remains In a non-polar medium, amphiphile molecules complete the process of self-assembly to form a thin film to obtain an amphiphile layer or a film containing an amphiphile layer. At the same time, the applicant unexpectedly found that the distribution process of the second polar medium effectively reduces the thickness of the film-forming phase 20, making the thickness of the amphiphile layer relatively thin.

And because of the miscibility of the second polar medium and the first polar medium phase and the third polar medium phase, there is a process of rebalancing thinning in the film-forming phase 20, thus improving the tolerance that can be tolerated when the film-forming phase 20 is initially formed. The thickness range does not need to directly reach the target thickness, such as the thickness of 5-15nm. When the film-forming method of an amphiphilic molecular layer or a film containing an amphiphilic molecular layer is applied to a chip structure with multiple structural units 200, when there are some thickness differences in the film-forming phase 20 of different structural units 200 when they are initially formed, Each film-forming phase 20 can be slowly thinned from different starting points to a suitable film-forming thickness, and stops at a suitable thickness range due to the supporting capacity of the film-forming phase 20 itself, thereby increasing the film-forming rate while improving the chip structure. The uniformity of each film-forming phase 20, that is, the thickness of each film-forming phase 20 is relatively uniform.

And if the film-forming phase 20 does not include the second polar medium, and the film-forming phase 20 only contains the non-polar medium, it will lose its compatibility with the first and the third polar medium, and the rebalance of the film-forming layer will be lost. Therefore, it is necessary to ensure that the thickness of the film-forming layer of the multiple structural units 200 of the chip structure reaches a narrow range of the target thickness at the same time when it is initially formed, which is very demanding, thus resulting in uneven film-forming thickness. The film-forming method of the above-mentioned amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer promotes the compatibility of the amphiphilic molecular layer or the amphiphilic molecular layer through the compatibility of the second polar medium with the first polar medium phase 10 and the third polar medium phase. The film formation of the film of the molecular hydrophilic layer improves the film formation rate and the uniformity of the film formation of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer, and the thickness of the amphiphilic molecular layer and the film containing the amphiphilic molecular layer is relatively thin. Moreover, it is found in the subsequent application process that the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer can increase the embedded porosity of the nanoporin.

It should be noted that the above method for forming the amphiphilic molecular layer or the film containing the amphiphilic molecular layer can be adapted to the chip structure 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 chip is connected, the above-mentioned method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer can be used.

That is, the method for forming the amphiphilic molecular layer or the film containing the amphiphilic molecular layer can be applied to the chip structure 100 in which adjacent structural units 200 are connected to each other, as shown in FIG. 1 . The chip structure 100 is also applicable to the chip structure 100 in which the bottoms of adjacent structural units 200 communicate with each other. As shown in FIG. 2 , the wall at the bottom of the structural unit 200 is provided with a second channel 212 communicating with the space area 210 of another structural unit 200 . Adjacent structural units 200 can partially communicate with the medium through the second channel 212 . Of course, it is also applicable to the chip structure 100 in which two adjacent structural units 200 are connected at positions other than the top and the bottom.

Moreover, the above film forming method of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer can also be applied to the chip structure 100 in which adjacent structural units 200 are not connected to each other. As shown in FIG. 3 , the spatial regions 210 of the structural unit 200 are independent from each other and not connected to each other. Adjacent structural units 200 cannot communicate with the medium.

FIG. 4 shows a schematic cross-sectional structure diagram of an exemplary structural unit 200 . In the space 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 space region 210, that is, from the space The regions 210 are arranged sequentially from the bottom to the top, and the thickness direction of the film-forming phase 20 is generally also the extension direction of the space region 210 .

The first polar medium phase 10 is the first polar medium that occupies part of the space in the space region 210 . The third polarity medium phase 30 is a third polarity medium occupying part of the space in the space region 210 . The film-forming phase 20 is sandwiched between the interfaces of the first polar medium phase 10 and the third polar medium phase 30 . The film-forming phase 20 is formed from a mixture comprising a second polar medium, amphiphilic molecules and a non-polar medium. That is, on the basis of the non-polar medium dispersed with amphiphilic materials used in the conventional method for preparing the film-forming phase 20, a part of the second polar medium is added. The second polar medium and the amphiphile are dispersed or distributed in the nonpolar medium. The hydrophilic segment of the amphiphilic molecule is soluble in the second polar medium and the lipophilic segment is soluble in the nonpolar medium.

There are many ways to form the first polar dielectric phase 10, the film-forming phase 20, and the third polar dielectric phase 30 distributed sequentially along the first direction in the space region 210, such as pre-configuring the corresponding media, especially forming a The medium of the film phase 20 is then fed into the corresponding medium in sequence to form the above-mentioned structure, that is, the film-forming phase 20 is formed in one step. It is also possible to form the first polar medium phase 10 first, and then distribute and form the film-forming phase 20 through multiple steps, and then form the third polar medium phase 30 . The specific method to form the structure in which the first polar dielectric phase 10 , the film-forming phase 20 and the third polar dielectric phase 30 are sequentially distributed is not limited.

The second polar medium of the film-forming phase 20 is distributed to the first polar medium phase 10 and/or the third polar medium phase 30, as standing still, due to the second polar medium and the first polar medium, the third polar medium Polar media are similar in polarity, and the second polar media tends to dissolve in both. After standing for a certain period of time, the second polar medium is partially or completely dissolved in the first polar medium phase 10 and/or the third polar medium phase 30 according to its relative position with the first polar medium phase 10 and the third polar medium phase 30. In the polar medium phase 30 . For example, when the second polar medium is distributed on 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 . However, when the second polar medium is distributed on 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 the first polar medium phase 10 and the third polar medium phase 30 .

During the distribution of the second polar medium to the first polar medium phase 10 and/or the third polar medium phase 30, the hydrophilic segment of the amphiphile molecule is carried into the corresponding polar medium phase, while the lipophilic segment Then it stays in the non-polar medium, so that the amphiphile completes the process of self-assembly to form a thin film.

Moreover, the applicant unexpectedly found that during the distribution process of the second polar medium, the thickness of the film-forming phase 20 is reduced, and the film containing the amphiphile molecular layer is formed between the two interfaces, so the amphiphile molecular layer or the amphiphile molecule-containing layer The thickness of the film is also relatively thin, which is manifested by a reasonable increase in the capacitance value that can be observed by electrical testing.

In some embodiments, the step of forming the first polar dielectric phase 10 and the third polar dielectric phase 30 of the film-forming phase 20 sequentially distributed along the first direction in the space region 210 includes:

The first polar medium, the film-forming mixture and the third polar medium are sequentially fed into the space region 210 to form the film-forming phase 20 , and the film-forming mixture includes the second polar medium, amphiphilic molecules and non-polar medium.

When the corresponding medium is passed into the space region 210 , the chip structure 100 can be used alone, such as immersing the chip structure 100 in the corresponding medium or spraying or dropping the corresponding medium on the surface of the chip structure 100 provided with the opening 220 . Of course, the chip structure 100 can also be used in conjunction with other devices, as shown in FIG. 5 , so that the surface of the chip structure 100 provided with the opening 220 has an accommodating cavity 340 capable of accommodating corresponding media.

Take the example that the chip structure 100 can also be used in conjunction with other devices. The film-forming mixture can be pre-configured to disperse the second polar medium, amphiphilic molecules and non-polar medium more uniformly. As shown in FIG. 6 , the first polar medium is passed into the space region 210 to form the first polar medium phase 10 . Then, as shown in FIG. 7 , the film-forming mixture is introduced into the space region 210, and the film-forming mixture drives away part of the first polar medium phase 10 in the space region 210 to occupy part of the first polar medium phase 10, A film-forming phase 20 is formed. The film-forming phase 20 covers the first polar medium phase 10 . Next, as shown in FIG. 8 , a third polar medium is introduced, and the third polar medium drives away part of the film-forming phase 20 in the space region 210 to occupy part of the film-forming phase 20 to form a third polar medium. layer. The above-mentioned method for forming the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer is relatively mature. The film-forming phase 20 is formed in one step, and the operation is relatively simple, which can improve the film-forming efficiency to a certain extent.

In some embodiments, the step of forming the first polar dielectric phase 10, the film-forming phase 20, and the third polar dielectric phase 30 sequentially distributed along the first direction in the space region 210 includes:

S1: In the space region 210, the first polar medium and the film-forming mixture A are sequentially introduced to form the first polar medium phase 10 and the film-forming A phase 22 sequentially distributed in the first direction, and the film-forming mixture A contains amphiphilic molecules and non-polar media;

S2: adding a film-forming mixture B23 to the film-forming A phase 22 to form a film-forming phase 20, and the film-forming mixture B23 includes a second polar medium and a non-polar medium;

S3: Passing a third polar medium on the side of the film-forming phase 20 away from the first polar medium phase 10 to form a third polar medium phase 30 .

In step S1, the film-forming mixture A can be configured first, and the amphiphilic molecules and the non-polar medium are more uniformly dispersed, and the first polar medium and the film-forming mixture A are sequentially introduced into the space region 210, and then the first polar The film-forming A phase 22 is formed on the neutral medium phase 10 . The film-forming mixture A is a solution in which amphiphilic molecules are dissolved in a non-polar medium. Optionally, the concentration of amphiphilic molecules is 5-20 mg/ml, further optionally, 8-12 mg/ml. Optionally, 10mg/ml.

In step S2, the film-forming mixture B23 can be configured first to disperse the second polar medium and the non-polar medium more 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%-30%, optionally 5%-15%. The film-forming mixture B23 is directly passed or sprayed into the film-forming phase A 22 to form the film-forming phase 20 . The second polar medium is added in the film-forming A phase 22 in the form of film-forming mixture B23. Since the second polar medium is dispersed in the non-polar medium, it can be more uniformly dispersed in the film-forming A phase 22, and the formed film The components of the membrane phase 20 are more uniformly dispersed, thereby improving the film-forming quality of the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer. And 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 distributed to the first polar medium phase when forming the film-forming phase 20, thus The actual content of the second polar medium in the film-forming phase 20 is relatively consistent with the designed content of the second polar medium in the film-forming phase 20, thereby improving the film-forming quality.

In step S3 , the third polar medium is injected into the side of the film-forming phase 20 away from the first polar medium phase 10 , that is, the side close to the opening 220 .

In the method for forming the amphiphilic molecular layer or the amphiphilic molecular layer-containing film, the film-forming quality of the amphiphilic molecular layer or the amphiphilic molecular layer-containing film can be improved.

In some embodiments, between the S1 step and the S2 step also includes:

The volume of the film-forming A phase 22 beyond the space region 210 is at least partially removed.

In some embodiments, the film-forming mixture B23 is added to the film-forming A phase 22 to form the film-forming phase 20 comprising:

Pass into the film-forming mixture B23 in the film-forming A phase 22, and let stand to form the film-forming phase 20; or

The film-forming mixture B23 is sprayed on the surface of the film-forming phase A 22 to form the film-forming phase 20 .

The film-forming A-phase 22 usually exceeds the space area 210, that is, part of the film-forming A-phase 22 will cover outside the opening 220, that is to say, the surface of the chip structure 100 provided with the structural unit 200 is covered with a certain thickness of the film-forming A-phase 22 . As shown in FIG. 9 , various methods can be adopted, such as blowing off the air flow, wiping or scraping, etc., to partially remove or completely remove the volume of the film-forming A phase 22 beyond the space region 210, so that the film-forming A phase 22 It slightly exceeds the opening 220 of the structural unit 200 or is flush with the opening 220 of the structural unit 200 .

As shown in FIG. 10 , after partially or completely removing the volume of the film-forming phase A 22 beyond the space region 210 , the film-forming mixture B23 can be passed into the film-forming phase A 22 and left standing to form the film-forming phase 20 . After the film-forming mixture B23 enters the film-forming A phase 22, it is incubated for a certain period of time, such as 10-30 minutes. The second polar medium in the film-forming mixture B23 is dispersed in the non-polar medium of the film-forming A phase 22, and the non-polar medium in the film-forming mixture B23 is mutually soluble with the non-polar medium in the film-forming A phase 22, thereby A film-forming phase 20 is formed.

After partially or completely removing the volume of the film-forming A phase 22 beyond the space region 210 , another method can also be adopted, spraying the film-forming mixture B23 on the surface of the film-forming A phase 22 to form the film-forming phase 20 . Spraying makes the film-forming mixture B23 have a certain kinetic energy, and the film-forming mixture B23 enters the interior of the film-forming A phase 22, causing slight disturbance, and the mixing of the two is relatively uniform. The second polar medium in the film-forming mixture B23 can be quickly dispersed into the non-polar medium of the film-forming A phase 22, and the non-polar medium therein is also miscible with the non-polar medium in the film-forming A phase 22 . In addition, the amount of film-forming mixture B23 used in this way is small, which saves costs.

In some embodiments, the step of passing the first polarity medium in the space region 210 includes:

Put the structural unit 200 in the first polar medium and let it stand still, so that the first polar medium enters the space region 210 .

Usually, a plurality of structural units 200 are arranged in an array on a device, such as the chip structure 100 . The volume of the structural unit 200 is relatively small. Therefore, in the embodiment of the present application, placing the structural unit 200 in the first polar medium can be understood as placing the chip structure 100 containing the structural unit 200 in the first polar medium. stand still. Similarly, in other embodiments, similar operations on the structural unit 200 can also be understood in this way.

The device with the structural unit 200, such as the chip structure 100, can be directly placed in the first polarity medium and be in contact with the first polarity medium. After standing for a certain period of time, the first polar medium mainly seeps into the space region 210 of the structural unit 200 from the opening 220 to fill the space region 210 . The standing time is suitable for filling most or all of the space region 210 with the first polar medium, such as 2 minutes to 30 minutes. The whole step can be carried out under vacuum environment. Moreover, the first polarity medium can be degassed before placing the chip to reduce the air in it.

Of course, the chip structure 100 can also be clamped on some film forming devices 300 , and the film forming devices 300 form a receiving cavity 340 around the surface of the chip structure 100 . The first polarity medium is passed into the accommodation cavity 340 , and the chip structure 100 is in contact with the first polarity medium. After standing for a certain period of time, the first polar medium mainly seeps into the space region 210 of the structural unit 200 from the opening 220 to fill the space region 210 . The standing time is suitable for filling most or all of the space region 210 with the first polar medium, such as 2 minutes to 30 minutes. The whole step can be carried out under vacuum environment. Moreover, the first polarity medium can be degassed before placing the chip to reduce the air in it.

The present application exemplarily provides a film forming device 300 , and the film forming device 300 includes a carrier 310 , a cover 320 and a gasket 330 . The chip structure 100 is disposed on the carrier 310 , the cover 320 is connected to the carrier 310 , and the gasket 330 is pressed on the chip structure 100 . The spacer 330 includes a cavity 331 , which is arranged corresponding to the structural units 200 distributed in an array on the chip structure 100 , and the orthographic projection of the cavity 331 on the chip structure 100 covers the structural units 200 distributed in an array. The spacer 330 is disposed between the cover body 320 and the chip structure 100 , thus, the spacer 330 , the cover body 320 and the chip structure 100 surround and form an accommodating cavity 340 . The cover body 320 is also provided with a liquid inlet 321 and a liquid outlet 322 , both of which communicate with the accommodating cavity 340 . The first polar medium can enter the accommodation chamber 340 from the liquid inlet 321 .

In some embodiments, the step of passing the film-forming mixture A or the film-forming mixture B into the space region 210 includes:

Place the structural unit 200 including the first polar medium phase 10 in the space region 210 in the corresponding film-forming mixture, take it out and let it stand, so that the corresponding film-forming mixture replaces part of the first polar medium phase 10 in the space region 210 .

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

Overall, the permeability of the first polar medium and the third polar medium into the space region 210 is weaker than that of the non-polar medium into the space region 210 . Both the film-forming mixture A and the film-forming mixture B include a certain amount of non-polar medium, so its permeability is greater than that of the first polar medium. Therefore, the chip structure 100 of the first polar medium phase 10 is separated from the corresponding film-forming mixture and left to stand, such as taking out from the container containing the corresponding film-forming mixture phase, or removing the corresponding film-forming mixture in the holding chamber 340 , so as not to completely replace part of the first polar medium phase 10 in the space region 210 by the corresponding film-forming mixture.

In some embodiments, the step of passing a third polarity medium in the space region 210 includes:

placing the structural unit 200 comprising the first polar medium phase 10 and the film-forming phase 20 in a third polar medium;

The step of distributing the second polar medium of the film-forming phase 20 to the first polar medium phase 10 and/or the third polar medium phase 30 includes:

The structural unit 200 passed through the third polarity medium was left to stand.

The permeability of the third polar medium phase 30 is relatively weak, and the chip structure 100 including the first polar medium phase 10 and the film-forming phase 20 in the space region 210 can be directly placed in the third polar medium and the third polar medium. media contact. Of course, the chip structure 100 can also be clamped on the film forming device 300 , the third polarity medium is passed into the accommodation cavity 340 , and the chip structure 100 is in contact with the third polarity medium. Both of the above two ways are to increase the total amount of the third polarity medium so as to penetrate into the space structure. The third polarity medium mainly penetrates into the space region 210 of the structural unit 200 from the opening 220 to replace part of the film-forming phase 20 .

In some embodiments, the step of forming the first polar medium phase and the third polar medium phase of the film-forming phase sequentially distributed along the first direction in the space region includes:

S11: making the film-forming mixture A solidified and attached to the surface outside the spatial region of the structural unit; wherein, the film-forming mixture A contains amphiphilic molecules and a non-polar medium;

S22: passing through the first polarity medium in the space region to form the first polarity medium phase;

S33: Add the film-forming mixture B to the first polar medium phase, and dissolve the amphiphilic molecules in the film-forming mixture A attached to the surface into the film-forming mixture B to form a film-forming phase; the film-forming mixture B contains the first Dipolar media and non-polar media;

S44: Passing a third polar medium on the side of the film-forming phase away from the first polar medium phase to form a third polar medium phase.

Among them, in S11, it can be understood that a small amount of film-forming mixture A is arranged on the surface outside the structural unit space area, such as the surface of the chip structure 100, such as spraying a small amount of film-forming mixture A, so that it can use the microstructure of the chip surface to achieve diffusion. Uniform and then cured, such as by drying, to adhere to the die. In step S33, after curing, the amphiphilic molecules in the film-forming mixture A will redissolve from the surface of the chip into the non-polar medium and the second polar medium in the film-forming mixture B. The amount of amphiphilic molecules used in the above method is less, which can save costs.

In some embodiments, the volume of the second polar medium is 5%-30%, optionally 5%-15%, of the volume of the non-polar medium.

The second polar medium is 5% to 30% of the volume of the non-polar medium. Under this ratio, the volume of the second polar medium can be more uniformly dispersed in the non-polar medium, regardless of the film-forming mixture A or the film-forming mixture B. It is relatively stable, and the formed film-forming phase 20 is relatively stable. The film-forming phase 20 is not easy to cause delamination of the film-forming phase 20 itself because the volume of the second polar medium is too large, and it is not easy to cause the second polar medium therein to be delaminated because the volume of the second polar medium is too small. Premature distribution, if it can be avoided, when the third polar medium phase 30 is introduced, the second polar medium of the film-forming phase 20 is distributed into the first polar medium phase 10 to ensure the film-forming quality.

Since the film-forming phase 20 is relatively stable, most of the second polar medium can be controlled to be distributed to the first polar medium phase 10 and/or the third polar medium phase 30 . Therefore, the process of the method for forming the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is controllable, and the film formation rate of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is high. In some of these embodiments, the second polar medium is 5% to 15% of the volume of the non-polar medium. Under this condition, the above-mentioned method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer is more feasible. Control, the film formation rate of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is further improved.

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

Non-polar media can be methylphenyl silicone oil, simethicone, simethicone with different end caps, hexadecane, tetradecane, dedecane, bromodecane, bromotetradecane, and squalane One or more of alkenes.

It has been verified by experiments that the film-forming phase 20 can be relatively stable. The film-forming method of the above-mentioned film-forming method containing the amphiphilic molecular layer has a high film-forming rate, and the thickness of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is relatively thin.

In some embodiments, when the non-polar medium is methyl phenyl silicone oil, simethicone oil, simethicone oil with different ends, hexadecane or a mixed medium of silicone oil and hexadecane, the second polarity The medium is dimethyl sulfoxide. The volume of the dimethyl sulfoxide is 5-20% of the volume of the non-polar medium.

When the non-polar medium is a single medium such as methylphenyl silicone oil, dimethyl silicone oil, perfluorosilicone oil, simethicone oil with different end caps, hexadecane, and the second polar medium is a corresponding non-polar medium When the volume is 5-15% of dimethyl sulfoxide, the film formation rate of the film-forming method of the above-mentioned amphiphilic molecular layer is further improved, and the thickness of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is also further reduced. . Dimethicone with different endcaps can be dihydroxyl-terminated simethicone, monohydroxyl-terminated simethicone, dihydroxyl-terminated methylphenyl silicone, monohydroxyl-terminated methylphenyl silicone, diamino Dimethicone blocked, monoamino blocked dimethicone, dicarboxy terminated dimethicone, monocarboxy terminated dimethicone, dioxirane terminated dimethicone, monooxirane terminated dimethicone Alkane-terminated simethicone oil, bis-alkoxy-terminated simethicone oil, and mono-alkoxy-terminated simethicone oil.

When the non-polar medium is a mixed medium such as a mixed medium of silicone oil and hexadecane, and the second polar medium correspondingly selects dimethyl sulfoxide with 5-15% of the volume of the non-polar medium, the above-mentioned amphiphile-containing layer The film forming rate of the film forming method of the film is further improved, and the thickness of the amphiphilic molecular layer or the film containing the amphiphilic molecular layer is further reduced. In the mixed medium of silicone oil and hexadecane, the mixing ratio of silicone oil and hexadecane can be (1-4):1, such as 1:1, 7:3, 3:1 or 4:1.

In some embodiments, the first polar medium is an aqueous buffer solution selected from phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris buffer solution , 3-morpholinepropanesulfonic acid buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, borate buffer solution and citrate buffer solution, the third polar medium is the third buffer Aqueous solution, the third buffer aqueous solution is selected from phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris buffer solution, 3-morpholine propanesulfonic acid buffer solution, 4-hydroxyethyl One of piperazineethanesulfonic acid buffer solution, borate buffer solution and citrate buffer solution, the third polar medium is the same as or different from the first polar medium. In some embodiments, the concentration of the first aqueous buffer solution is 5-100 mM, optionally, the first aqueous buffer solution is 10 mM phosphate buffer solution, and the concentration of the third aqueous buffer solution is 5-100 mM, optionally , the third aqueous buffer solution is 10 mM phosphate buffer solution, and the concentration of the third polarity medium is the same as or different from that of the first polarity medium.

The first polar medium and the third polar medium are independently selected from one of the first aqueous buffer solution and the third aqueous buffer solution, and the third polar medium is the same as or different from the first polar medium . Both the above-mentioned first polar medium and the third polar medium can meet the requirements of the film-forming method of the above-mentioned amphiphilic molecular layer-containing film.

In some other embodiments, the first polar medium and the third polar medium can be selectively selected according to the electrode material used in the application of the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer in characterizing the analyte. . For example, when silver-silver chloride is used as an electrode material to construct an electrochemical system, the first polarity medium and the third polarity medium can be selected as phosphate buffer solution, tris (Tris) buffer solution or 4 - Hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution. When gold or platinum is used as the electrode material to construct the electrochemical system, the first polarity medium and the third polarity medium can be selected as phosphate buffer solution containing potassium ferricyanide or 4-hydroxyethyl alcohol containing potassium ferricyanide Piperazineethanesulfonic acid (HEPES) buffer solution.

In some embodiments, the first aqueous buffer solution and the third aqueous buffer solution respectively include a potassium salt, such as potassium chloride (KCl), and the concentration of KCl is 400-800 mM. The first buffer aqueous solution and the third buffer aqueous solution have the above-mentioned concentration of KCl, which can promote the balance of the electrochemical system.

In some embodiments, the osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium can keep the first polar medium and the third polar medium in a stable state that does not penetrate each other.

The osmotic pressure of the first polar medium is equal to or close to the osmotic pressure of the third polar medium, so that the first polar medium phase 10 and the third polar medium phase 30 in the space region 210 do not penetrate each other through the film-forming phase 20 , keep it stable. Under this condition, the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer is relatively stable and can maintain a stable state for a long time. When the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer is used for testing, the test is more stable For convenience, and to improve the success rate of the test.

In some embodiments, the amphiphilic molecule is a phospholipid, fatty acid, fatty acyl, glyceride, glycerophospholipid, sphingolipid, sterol lipid, prenol lipid, glycolipid, polyketide, or amphiphilic block copolymers.

Any one of the above-mentioned substances can be used for the amphiphile to meet the film-forming requirements of the amphiphile. The concentration of the amphiphile in the non-polar solvent is 5-20 mg/mL, and the optional concentration is 10 mg/mL.

In some embodiments, the amphiphilic block copolymer is a copolymer comprising at least three polymer segments, and the copolymer has hydrophilic polymer segments A1 and A2 at the end of the molecular chain and a polymer segment at the middle of the molecular chain. A hydrophobic polymeric segment B, or an amphiphilic block copolymer is a copolymer comprising at least two polymeric segments, wherein the at least two polymeric segments include a hydrophilic polymeric segment A and a hydrophobic polymeric segment B.

The amphiphilic molecular layer or the film containing the amphiphilic molecular layer formed by the method of the above-mentioned amphiphilic block copolymer is strong, stable, not easy to degrade, and can withstand a large potential difference applied and passed through itself.

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

The method of adopting the above-mentioned amphiphilic block copolymer to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer is stronger, more stable, less prone to degradation, and can withstand the potential difference applied and passed through itself, which can be further improved. improve.

In the second aspect, the embodiment of the present application provides the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer prepared by the above method.

In a third aspect, the embodiment of the present application provides a nanopore sequencing device, comprising the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer prepared by the above method.

In the fourth aspect, the embodiment of the present application provides the application of the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer prepared by the above method in characterizing the analyte, the analyte includes: biopolymer, the biopolymer is selected from polynucleotide, One of polypeptides, polysaccharides and lipids.

The above-mentioned analyte can be embedded in the amphiphile molecular layer prepared by the above-mentioned preparation method for testing.

In some embodiments, the biopolymer is a polynucleotide, and the polynucleotide includes deoxyribonucleic acid (English DeoxyriboNucleic Acid, abbreviated as DNA) and/or ribonucleic acid (abbreviated as RNA, ie Ribonucleic Acid) and analogs/derivatives thereof thing.

Example

The following examples describe the content disclosed in the present application more specifically, and these examples are for illustrative purposes only, because various modifications and changes within the scope of the disclosed content of the application will be obvious to those skilled in the art. All reagents used in the examples are commercially available or synthesized according to conventional methods, and can be used directly without further treatment, and the instruments used in the examples are all commercially available.

Example 1

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

S110: Place the chip structure 100 having the structural unit 200 (Fig. 2) in a petri dish, add the degassed first polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)), vacuum (50 mBar) for 2 minutes, the first polar medium is filled into the space region 210 of the structural unit 200 to form the first polar medium phase 10 .

S120: After taking out the chip structure 100 with the first polar dielectric phase 10 in the structural unit 200, immerse it in the film-forming mixture. The chip structure 100 was pulled out at a speed of 10 mm/s, and left to stand for 30 seconds, and the excess film-forming mixture outside the structural units 200 of the chip structure 100 was wiped off with oil-absorbing paper. 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 . in:

The film-forming mixture includes a second polar medium, amphiphilic molecules and a non-polar medium;

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

The amphiphile is PDMS-PEG (or PDMS-PMOXA), and the concentration in the non-polar medium is 10 mg/ml;

The non-polar medium is methyl phenyl silicone oil, specifically AP100 (Sigma-Aldrich) or AR20 (Sigma-Aldrich).

S130: Load the chip structure 100 having the first polar medium phase 10 and the film-forming phase 20 in the structural unit 200 into the film-forming device 300, and pass the third polar medium ( Phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) and drive away part of the film-forming phase 20. After standing still, the first polar medium phase 10 , the film-forming phase 20 and the third polar medium phase 30 are sequentially distributed in the first direction in the structural unit 200 .

S140: after standing for 2 hours, the film-forming phase 20 forms a film to form the film containing the amphiphile layer, and the test starts.

Example 2

S210: Proceed as in step S110 in Embodiment 1, the difference is that the structure of the structural unit 200 is as shown in Figure 1 .

S220: After the chip structure 100 with the first polar medium phase 10 in the structural unit 200 is taken out, it is loaded into the film forming device 300 , and the film forming mixture is introduced into the containing chamber 340 from the liquid inlet 321 . After standing still for 10 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 . in:

The second polar medium is cyclohexanol, which is 5% of the volume of the non-polar medium;

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

S230: Pass the third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) into the holding chamber 340 from the liquid inlet 321, so that the film-forming mixture is discharged from the liquid outlet 322. After standing still, the first polar medium phase 10 , the film-forming phase 20 and the third polar medium phase 30 are sequentially distributed in the first direction in the structural unit 200 .

S240: After standing still for 2 hours, the film-forming phase 20 forms a film to form the film containing the amphiphile layer, and the test is started.

Example 3

S310: Perform as step S110 in Embodiment 1, except that the structure of the structural unit 200 is shown in FIG. 3 .

S320: After the chip structure 100 with the first polar medium phase 10 in the structural unit 200 is taken out, it is loaded into the film forming device 300 , and the film forming mixture A is introduced into the containing chamber 340 from the liquid inlet 321 . After standing still for 10 minutes, the first polar medium phase 10 and the film-forming A phase 22 covering the first polar medium phase 10 are formed in the structural unit 200 . in:

Film-forming mixture A includes amphiphile molecules and non-polar medium;

Amphiphile PDMS-PEG (also PDMS-PMOXA), the concentration in the non-polar medium is 10mg/ml;

The non-polar medium is silicone oil AP100.

S330: Air is introduced into the accommodation cavity 340 from the liquid inlet 321 to push away the excess film-forming mixture A on the surface of the chip structure 100, so that it is discharged from the liquid outlet 322. Then, the film-forming mixture B23 is introduced into the chamber 340 from the liquid inlet 321 , and 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 after standing. in:

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

The second polar medium is toluene, which is 10% of the volume of the non-polar medium;

The non-polar medium is methylphenyl silicone oil.

S340: Pass the third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) into the holding chamber 340 from the liquid inlet 321, so that the film-forming mixture B23 is discharged from the liquid outlet 322. After standing still, the first polar medium phase 10 , the film-forming phase 20 and the third polar medium phase 30 are sequentially distributed in the first direction in the structural unit 200 .

S350: After standing still for 20 minutes, the film-forming phase 20 forms a film to form the film containing the amphiphile layer, and the test starts.

Example 4

S410: Perform according to step S110 in Embodiment 1.

S420: Perform according to step S320 in Embodiment 3.

S430 : Air is introduced into the accommodation chamber 340 from the liquid inlet 321 to push away excess film-forming mixture A on the surface of the chip structure 100 , so that it is discharged from the liquid outlet 322 . Remove the cover 320, spray the film-forming mixture B23 in the cavity 331 surrounded by the gasket 330, and form the first polar medium phase 10 and the film-forming film covering the first polar medium phase 10 in the structural unit 200 after standing. Phase 20. in:

The second polar medium is propyl acetate, which is 10% of the volume of the non-polar medium;

The non-polar medium is silicone oil AP100.

S440: Same as step S340 in Embodiment 3.

S450: Same as step S350 in Embodiment 3.

Example 5

S510: Spray 5 ul of the film-forming mixture A on the outermost layer of the chip structure 100 having the structural unit 200 ( FIG. 2 ) at a rate of 5 ul per square centimeter. After standing at room temperature for 30 minutes, use the chip surface microstructure to achieve uniform diffusion, and heat the stage 100 Bake at high temperature for 15 minutes, so that the amphiphile molecules are evenly distributed on the surface of the chip for later use.

Wherein, the film-forming mixture A includes amphiphilic molecules and non-polar media,

The amphiphile is PDMS-PEG (or PDMS-PMOXA), and the concentration in the non-polar medium is 10mg/ml;

The non-polar medium is a mixture of silicone oil AR20 and C10 (decane), the volume ratio is 1:9.

S520: adding the processed chip structure 100 having the structural unit 200 to the degassed first polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)), under vacuum (50mBar) After standing still for 2 minutes, the first polar medium is filled into the space region 210 of the structural unit 200 to form the first polar medium phase 10 .

S530: After taking out the chip structure 100 with the first polar medium phase 10 in the structural unit 200 and the amphiphilic molecule on the surface, load it into the film forming device 300, and then pass it into the forming chamber 340 from the liquid inlet 321. The film mixture B23 was left to stand for 30 minutes to form the first polar medium phase 10 and the film-forming phase 20 covering the first polar medium phase 10 in the structural unit 200 . in:

The second polar medium is ethyl acetate, which is 25% of the volume of the non-polar medium;

The non-polar medium is silicone oil AR20;

During 30 minutes of standing, the amphiphile redissolves from the surface of the chip into the non-polar medium and the second polar medium in film-forming mixture B.

S540: Pass the third polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) into the holding chamber 340 from the liquid inlet 321, so that the film-forming mixture B23 is discharged from the liquid outlet 322. After standing still, the first polar medium phase 10 , the film-forming phase 20 and the third polar medium phase 30 are sequentially distributed in the first direction in the structural unit 200 .

S550: After standing still for 20 minutes, the film-forming phase 20 forms a film to form the film containing the amphiphile layer, and the test is started.

Example 6

S610: Evenly spray 4 μL of the film-forming mixture A onto the chip structure 100 having the structural unit 200 ( FIG. 1 ), and leave it at room temperature for 30 minutes.

The amphipathic molecule is phospholipid (DPHPC), and the concentration of the amphiphilic molecule in the non-polar medium is 10mg/ml;

The non-polar medium is a mixture of C16 (hexadecane) and C10 (decane) with a volume ratio of 1:4.

S620: adding the processed chip structure 100 having the structural unit 200 to the degassed first polar medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)), under vacuum (50mBar) After standing still for 2 minutes, the first polar medium is filled into the space region 210 of the structural unit 200 to form the first polar medium phase 10 .

S630: After taking out the chip structure 100 with the first polar medium phase 10 in the structural unit 200 and the film-forming mixture A on the surface, load it into the film-forming device 300 , and then pass it into the holding chamber 340 from the liquid inlet 321 After the film-forming mixture B23 is left to stand 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 .

Wherein: the film-forming mixture B comprises amphiphilic molecules, a second polar medium and a non-polar medium;

The second polar medium is DMF, which is 43% of the volume of the non-polar medium;

The non-polar medium is C16 (hexadecane).

S640: Pass the third polarity medium (phosphate buffer solution (600mM KCl, 10mM potassium phosphate, pH 7.5)) into the holding chamber 340 from the liquid inlet 321, so that the film-forming mixture B23 is discharged from the liquid outlet 322. After standing still, the first polar medium phase 10 , the film-forming phase 20 and the third polar medium phase 30 are sequentially distributed in the first direction in the structural unit 200 .

S650: After standing still for 20 minutes, the film-forming phase 20 forms a film to form the film containing the amphiphile layer, and the test is started.

Example 7

S710: Carry out according to step S110 in Example 1, except that the first polar medium is 3-morpholine propanesulfonic acid buffer solution containing 600 mM KCl and 10 mM, and the system is left standing under vacuum (50 mBar) for 5 minutes.

S720: the second polar medium is 10% of the volume of the non-polar medium. The second polar medium is methanol, the amphiphile is glycerophospholipid, the non-polar medium is squalene, and the volume concentration of the amphiphile in the non-polar medium is 10 mg/mL. The rest are the same as S120 of Embodiment 1.

S730: The third polar medium is 10mM 3-morpholine propanesulfonic acid buffer solution containing 600mM KCl. The rest are the same as S130 of Embodiment 1.

S740: The standing time is 2 hours. All the other are the same as S140 of embodiment 1.

Example 8

S810: The first polar medium is acetate buffer solution containing 600mM KCl and 10mM. The system was left to stand under vacuum (50 mBar) for 5 minutes. The rest are the same as S110 of Embodiment 1.

S820: the second polar medium is 15% of the volume of the non-polar medium. The second polar medium is ethyl acetate, the amphiphile is dihydroxy-terminated simethicone, the non-polar medium is tetradecane bromide, and the volume concentration of the amphiphile in the non-polar medium is 20 mg/mL. The rest are the same as S120 of Embodiment 1.

S830: The third polar medium is 10mM citrate buffer solution containing 600mM KCl. The rest are the same as S130 of Embodiment 1.

S840: The standing time is 2 hours. All the other are the same as S140 of embodiment 1.

Example 9

S910: The first polar medium is carbonate buffer solution containing 600mM KCl and 10mM. The system was left to stand under vacuum (50 mBar) for 5 minutes. The rest are the same as S110 of Embodiment 1.

S920: the second polar medium is 10% of the volume of the non-polar medium. The second polar medium is N,N'-dimethylformamide, the amphiphile is dihydroxy-terminated dimethyl silicone oil, the non-polar medium is a mixed medium of silicone oil and hexadecane, and the volume ratio of the two is 3 :1, the concentration of the amphiphile molecule in the non-polar medium volume is 10mg/mL. The rest are the same as S120 of Embodiment 1.

S930: The third polar medium is a citrate buffer solution containing 600mM KCl. The rest are the same as S130 of Embodiment 1.

S940: The standing time is 2 hours. All the other are the same as S140 of embodiment 1.

Comparative example 1

According to the method of Example 1, the film containing the amphiphilic molecular layer is prepared, the main difference is that the film-forming mixture does not contain the second polar medium, as follows:

S'110: Add the chip structure 100 having the structural unit 200 into the degassed first polarity medium. The structure of the structural unit 200 is shown in FIG. 2 . The first polar medium is a phosphate buffered saline solution (600mM KCl, 10mM potassium phosphate, pH 7.5). The system was left to stand under vacuum (50 mBar) for 2 minutes, and the first polar medium was filled into the space region 210 of the structural unit 200 to form the first polar medium phase 10 . (with embodiment 1 step S110)

S'120: After taking out the chip structure 100 with the first polar medium phase 10 in the structural unit 200, load it into the film forming device 300, and then pass the film forming mixture B23 into the holding chamber 340 from the liquid inlet 321, After standing still 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 . Film-forming mixture B contains amphiphilic molecules and a non-polar medium. The amphiphile and non-polar medium are the same as step S120 of Example 1.

S'130: Pass the third polar medium into the accommodation chamber 340 from the liquid inlet 321, and discharge the film-forming mixture B23 from the liquid outlet 322. The first polar medium phase 10 , the film-forming phase 20 and the third polar medium phase 30 are sequentially distributed in the first direction in the structural unit 200 .

S'140: After standing for 20 minutes, the film-forming phase 20 forms a film to form the film containing the amphiphile layer.

Comparative example 2

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

S'220: Film-forming mixture B contains amphiphilic molecules and a non-polar medium. The concentration of the amphiphile in the non-polar medium is 10 mg/ml. The amphiphile is PDMS-PEG or PDMS-PMOXA, and the non-polar medium is a mixture of AR20 and C10 with a volume ratio of 1:4. All the other are the same as step S'120 of comparative example 1.

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

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

Comparative example 3

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

S'320: Film-forming mixture B contains amphiphilic molecules and a non-polar medium. The concentration of the amphiphile in the non-polar medium is 10 mg/ml. The amphiphile is DPHPC, and the non-polar medium is a mixture of C16 and C10 with a volume ratio of 1:4. All the other are the same as step S'120 of comparative example 1.

S'330: Same as step S'130 of Comparative Example 1.

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

Characterization Examples The chip structure of the structural unit 200 shown in FIG. 2 , the above-mentioned amphiphilic molecular layer-containing membranes of Examples 1-9, and the amphiphilic molecular layer-containing membranes of Comparative Examples 1-3 were tested. The chip structure 100 includes a plurality of first electrodes, each first electrode is arranged correspondingly to each structural unit 200, each first electrode is arranged at an end of the structural unit 200 away from the opening, communicates with the space region 210 surrounded by the structural unit 200, and communicates with the structural unit 200. The first polar medium phase 10 of the membrane comprising the amphiphilic molecular layer is in contact. Meanwhile, the film forming apparatus 300 may include a second electrode communicated with the receiving chamber 340 . The second electrode is in contact with the third polar medium phase 30 away from the end of the first polar medium phase. The first electrode and the second electrode are connected to the test device, and the membrane containing the amphiphile molecular layer is tested. Each structural unit 200 is actually a membrane capacitor, and the thickness of the membrane containing the amphiphilic molecular layer is different, and the electrical characteristics will be different. Each rectangular block in the figure represents a film capacitor, that is, corresponds to a structural unit 200 . The numerical value in the rectangular box represents the capacitance value of the film capacitor. Moreover, the display color depth of the electrical representation of each unit in the instrument is positively correlated with the value of the membrane capacitance, that is, the darker the color, the greater the membrane capacitance.

The value of the capacitance value can represent the states of different structural units 200 , such as whether to form a film, and the thickness and state of the film formed. Specifically:

Less than 20pf is the background capacitance value of the instrument or the capacitance value of the initial state without film formation, which is displayed in light gray;

20.1-30pf is the film capacitance value that is not conducive to the subsequent conventional hole embedding of this kind of amphiphile layer film, and it is displayed in medium gray, indicating that the thickness of the film is too large;

30.1～65pf is suitable for the film capacitance value of this kind of amphiphile layer film for subsequent conventional hole embedding, it is displayed in dark gray, and the thickness of the film is suitable;

65.1～100pf is the film capacitance value that is not conducive to the subsequent conventional hole embedding of this kind of amphiphile layer film, it is displayed in black, and the thickness of the film is too small;

More than 100.1pf means membrane rupture or the amphiphilic molecular layer does not have the ability to insert holes, which is displayed in dark black.

The test results are shown in the figure, specifically:

Firstly, the electrical characterization of the chip structure of the structural unit 200 shown in FIG. 2 was tested, wherein the capacitance values of each structural unit were all less than 20pf, and mostly concentrated between 12pf-13pf (results not shown).

FIG. 13 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 1. FIG. Figure 13 shows that the capacitance values corresponding to 6 structural units are greater than 100.1pf, the capacitance values corresponding to 4 structural units are less than or close to 20pf, and the capacitance values corresponding to the remaining structural units are all between 30.1 and 65pf, accounting for 97.40%. , That is to say, the film formation rate of the film containing amphiphile layer in Example 1 reached 97.40%.

FIG. 14 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 2. FIG. Figure 14 shows that the capacitance values corresponding to 2 structural units are greater than 100.1pf, the capacitance values corresponding to 5 structural units are less than 20pf, and the capacitance values corresponding to the remaining structural units are all between 30.1 and 65pf, accounting for 98.18%. That is to say, the film forming rate of the film forming method of the amphiphilic molecular layer in Example 2 reached 98.18%.

FIG. 15 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 3. FIG. Figure 15 shows that the capacitance values corresponding to 3 structural units are greater than 100.1pf, the capacitance values corresponding to 1 structural unit are between 65.1 and 100pf, the capacitance values corresponding to 18 structural units are less than 20pf, and the capacitance values corresponding to the remaining structural units are average. It is between 30.1-65 pf, and its proportion is 94.27%, that is to say, the film formation rate of the film containing amphiphile layer in Example 3 reaches 94.27%.

FIG. 16 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 4. FIG. Figure 16 shows that the capacitance values corresponding to 5 structural units are greater than 100.1pf, the capacitance values corresponding to 14 structural units are less than 20pf, and the capacitance values corresponding to the remaining structural units are all between 30.1 and 65pf, accounting for 95.05%. That is to say, the film forming rate of the film forming method of the amphiphilic molecular layer in Example 4 reached 95.05%.

FIG. 17 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 5. FIG. Figure 17 shows that the capacitance value corresponding to one structural unit is greater than 100.1pf, the capacitance value corresponding to three structural units is between 65.1 and 100pf, the capacitance value corresponding to seven structural units is less than 20pf, and the capacitance values corresponding to the remaining structural units are all It is between 30.1-65pf, and its proportion is 97.40%. That is to say, the film formation rate of the film containing amphiphile layer in Example 5 reaches 97.14%.

FIG. 18 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 6. FIG. Figure 18 shows that the capacitance corresponding to 3 structural units is greater than 100.1pf, the capacitance corresponding to 5 structural units is between 65.1 and 100pf, the capacitance corresponding to 3 structural units is between 20.1 and 30pf, and the capacitance corresponding to 5 structural units is between 20.1 and 30pf. The corresponding capacitance value is less than 20pf, and the capacitance values corresponding to the other structural units are all between 30.1 and 65pf, accounting for 95.83%. It reached 95.83%.

FIG. 19 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 7. FIG. Figure 19 shows that the capacitance corresponding to three structural units is greater than 100.1pf, the capacitance corresponding to one structural unit is between 65.1 and 100pf, the capacitance corresponding to one structural unit is between 20.1 and 30pf, and the capacitance corresponding to one structural unit is between 20.1 and 30pf. The corresponding capacitance value is less than 20pf, and the capacitance values corresponding to the other structural units are all between 30.1 and 65pf, accounting for 95.31%. It reached 95.31%.

FIG. 20 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 8. FIG. Figure 20 shows that the capacitance values corresponding to 9 structural units are greater than 100.1pf, the capacitance values corresponding to 2 structural units are between 65.1 and 100pf, the capacitance value corresponding to 1 structural unit is less than 20pf, and the capacitance values corresponding to the other structural units are all It is between 30.1 to 65 pf, and its proportion is 96.61%, that is to say, the film formation rate of the film containing amphiphile layer in Example 8 reaches 96.88%.

FIG. 21 is an electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Example 9. FIG. Figure 21 shows that the capacitance value corresponding to 3 structural units is greater than 100.1pf, the capacitance value corresponding to 1 structural unit is between 65.1 and 100pf, the capacitance value corresponding to 2 structural units is between 20.1 and 30pf, and the capacitance value corresponding to 10 structural units The corresponding capacitance value is less than 20pf, and the capacitance values corresponding to the other structural units are all between 30.1 and 65pf, accounting for 96.09%. It reached 95.83%.

Figure 22 is the electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Comparative Example 1. Figure 22 shows that the capacitance corresponding to one structural unit is greater than 100.1pf, and the capacitance corresponding to three structural units is between 65.1 and 100pf. The capacitance value corresponding to each structural unit is less than 20pf, and the capacitance values corresponding to 254 structural units are all between 30.1 and 65pf, accounting for 66.15%. The film forming rate of the method is only 66.15%.

Figure 23 is the electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Comparative Example 2. Figure 23 shows that the capacitance value corresponding to 21 structural units is greater than 100.1pf, and the capacitance value corresponding to 53 structural units is between 65.1 and 100pf. The capacitance value corresponding to each structural unit is less than 20pf, and the capacitance values corresponding to 294 structural units are all between 30.1 and 65pf, accounting for 76.56%. The film forming rate of the method is only 76.56%.

Figure 24 is the electrical characterization diagram of the membrane containing the amphiphilic molecular layer formed in Comparative Example 3. Figure 24 shows that the capacitance value corresponding to 12 structural units is greater than 100.1pf, and the capacitance value corresponding to 62 structural units is between 65.1 and 100pf. 17 The capacitance value corresponding to each structural unit is less than 20pf, and the capacitance values corresponding to 293 structural units are all between 30.1 and 65pf, accounting for 76.3%. The film forming rate of the method is only 76.3%.

Application example:

Experimental group: Take the chip structure of Example 1 to form a membrane containing an amphiphilic molecular layer, pass into the third polar medium containing nanoporin (Mycobacterium smegmatis porin A, nanoporin Mycobacterium smegmatis porin A, Abbreviated as MspA, SEQ ID NO: 1, the concentration is between 10ng/ml-100ng/ml), static incubation for 1h, and a third polarity medium of 5 times the volume of the fluid is introduced into the holding chamber 340 from the liquid inlet 321, The solution containing nanopores is replaced to complete the pore embedding process.

Control group: carried out according to the method of the experimental group, the difference is: no second polarity medium is added, and the hole embedding process is completed.

The chips of the experimental group and the control group were connected to the electrical system (nanopore gene sequencer QNome-9604). After embedding the holes, under a constant voltage of 80mV, the opening current was 60pA-70pA. A single nanopore can be used if it is correct. Through the nanopore gene sequencer QNome-9604, the automatic sieve judgment program (at a constant voltage of 80mV, at the same time satisfying the opening current of 60pA-70pA and the noise less than 1.5pA is correct, a single nanopore can be used) to screen out:

The experimental group has 274 single holes, the embedded single hole rate reaches 71.35%, and the embedded single hole rate accounts for 73.25% of the film formation rate (97.4%).

There were 171 single holes in the control group, the embedded single hole rate was only 44.53%, and the embedded single hole rate accounted for 67.31% of the film forming rate (66.15%).

Fig. 25 is a real-time display interface of the polynucleotide sequencing signal of the experimental group, which is used for judging the signal.

Fig. 26 is a test chart of sequencing stability and chip channel utilization of the experimental group, and the chip channel utilization is about 80% (light gray area).

Fig. 27 is a test chart of sequencing stability and chip channel utilization in the control group, and the chip channel utilization is about 50% (light gray area).

Comparing the results of the experimental group and the control group, it can be seen that the second polar medium is added to the experimental group, and the amphiphilic molecular layer or the nanoporin of the membrane containing the amphiphilic molecular layer has a higher single-porosity insertion rate, which is more conducive to pore insertion and sequencing stability. performance and chip channel utilization.

While the application has been described with reference to alternative embodiments, various modifications may be made thereto and equivalents may be substituted for parts thereof without departing from the scope of the application, in particular, as long as no structural Conflicts, the various technical features mentioned in each embodiment can be combined in any way. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

A method for forming an amphiphilic molecular layer or a film containing an amphiphilic molecular layer in a structural unit, wherein the method comprises the following steps:

A first polar dielectric phase, a film-forming phase and a third polar dielectric phase sequentially distributed along a first direction are formed in the spatial region of the structural unit; wherein, the first direction is the thickness direction of the film, The film-forming phase is formed from a film-forming mixture comprising a second polar medium, amphiphilic molecules, and a non-polar medium;

Conditions are provided so that the film-forming phase forms an amphiphilic molecular layer or a film containing an amphiphilic molecular layer, and the second polar medium contained in the film-forming phase is distributed to the first polar medium phase and/or the The third polar medium phase.
The method of claim 1, wherein the spatial region of the structural unit comprises an opening.
The method according to claim 1, wherein the step of forming the first polar dielectric phase, the film-forming phase and the third polar dielectric phase sequentially distributed along the first direction in the spatial region of the structural unit comprises:

In the spatial region of the structural unit, the first polar medium, the film-forming mixture and the third polar medium are sequentially introduced to form the film-forming medium phase between the first polar medium phase and the third polar medium phase Mutually;

Wherein, the film-forming mixture includes a second polar medium, amphiphilic molecules and a non-polar medium.
The method according to claim 1, wherein the step of forming the first polar dielectric phase, the film-forming phase and the third polar dielectric phase sequentially distributed along the first direction in the spatial region of the structural unit comprises:

S1: In the spatial region of the structural unit, the first polar medium and the film-forming mixture A are sequentially introduced to form the first polar medium phase and the film-forming A phase sequentially distributed along the first direction; wherein, the The film-forming mixture A comprises amphiphile molecules and a non-polar medium;

S2: adding a film-forming mixture B to the film-forming phase A to form the film-forming phase;

Wherein, the film-forming mixture B comprises a second polar medium and a non-polar medium;

S3: Passing a third polar medium on the side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.
The method according to claim 4, wherein, between the S1 step and the S2 step, further comprising:

A volume of the film-forming A phase beyond the space region is at least partially removed.
The method according to claim 4 or 5, wherein, in the S2 step, adding the film-forming mixture B to the film-forming A phase, forming the film-forming phase comprises:

Pass into the film-forming mixture B into the film-forming A phase, and let stand to form the film-forming phase; or

The film-forming mixture B is sprayed on the surface of the film-forming phase A to form the film-forming phase.
The method according to any one of claims 3 to 6, wherein the step of introducing a first polar medium into the spatial region of the structural unit comprises:

The structural unit is placed in the first polar medium to allow the first polar medium to enter the spatial region of the structural unit.
The method according to any one of claims 3 to 6, wherein the step of introducing the film-forming mixture into the space region of the structural unit comprises:

placing the structural unit in the corresponding film-forming mixture, taking it out, standing still, and allowing the corresponding film-forming mixture to enter the space region; or

The structural unit is placed in the corresponding film-forming mixture A, taken out, and left to stand, so that the corresponding film-forming mixture A enters the space region, wherein the film-forming mixture A contains amphiphilic molecules and non-polar medium; then the structural unit is placed in the corresponding film-forming mixture B, taken out, and left to stand, so that the corresponding film-forming mixture B enters the space region, wherein the film-forming mixture B contains the second polarity media and non-polar media.
The method according to any one of claims 3 to 6, wherein the step of introducing a third polarity medium into the spatial region of the structural unit comprises:

placing the structural unit comprising the first polar medium phase and the film-forming phase in a third polar medium; and/or

The step of distributing the second polar medium of the film-forming phase to the first polar medium phase and/or the third polar medium phase comprises:

The structural unit that has passed through the medium of the third polarity is left to stand.
The method according to claim 1, wherein the step of forming the first polar dielectric phase, the film-forming phase and the third polar dielectric phase sequentially distributed along the first direction in the spatial region of the structural unit comprises:

S11: Attach the film-forming mixture A to the surface outside the spatial region of the structural unit; wherein, the film-forming mixture A includes amphiphilic molecules and a non-polar medium;

S22: Passing a first polar medium into the space region of the structural unit to form a first polar medium phase;

S33: adding the film-forming mixture B to the first polar medium phase, and dissolving the amphiphilic molecules in the film-forming mixture A attached to the surface into the film-forming mixture B to form the film-forming phase; wherein, The film-forming mixture B comprises a second polar medium and a non-polar medium;

S44: Passing a third polar medium into a side of the film-forming phase away from the first polar medium phase to form the third polar medium phase.
A film-forming system, wherein the system includes a structural unit, and the spatial region of the structural unit contains a first polar medium phase, a film-forming phase and a third polar medium phase that are 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 non-polar medium to form an amphiphilic molecular layer or a film containing an amphiphilic molecular layer;

Wherein, 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.
A microdroplet, wherein the microdroplet 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 , the film-forming phase comprises a second polar medium, amphiphilic molecules and a non-polar medium, to form a film containing an amphiphilic molecular layer;

Wherein, 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.
The film-forming system according to claim 11 or the microdroplet according to claim 12, wherein a transmembrane pore is embedded in the amphiphilic molecular layer; optionally, the transmembrane pore is a transmembrane protein pore.
The method of any one of claims 1-10, the film-forming system of claim 11, or the droplet of claim 12, wherein the second polar medium volume is the non-polar 5%-45% of the volume of the medium, optionally 5%-30%, further optionally 5%-15%.
The method, film-forming system or droplet according to any one of claims 1-14, wherein the second polar medium is soluble in the non-polar medium, and the second polar medium It is soluble in the first polar medium or phase of the first polar medium, and the second polar medium is soluble in the third polar medium or phase of the third polar medium.
The method, film-forming system or droplet according to any one of claims 1-15, wherein the second polar medium is selected from the group consisting 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'-dimethylacetate One or more of amides;

The non-polar medium is selected from methyl phenyl silicone oil, simethicone, optional simethicone with different end caps, hexadecane, tetradecane, dedecane, bromodecane, bromotetradecane One or more of alkanes and squalene.
The method according to claim 16, the film-forming system or the droplet, wherein, when the non-polar medium is methylphenyl silicone oil, simethicone oil, dimethicone oil, hexadecane or silicone oil with different end caps can be selected When mixed with hexadecane, the second polar medium is dimethyl sulfoxide;

Optionally, the volume of the dimethyl sulfoxide is 5-15% of the volume of the non-polar medium.
The method, film-forming system or microdroplet according to any one of claims 1-17, wherein the first polar medium is a first aqueous buffer solution selected from phosphate buffered saline solution solution, carbonate buffer solution, acetate buffer solution, tris buffer solution, 3-morpholinepropanesulfonic acid buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, borate buffer One or more of solution or citrate buffer solution;

Optionally, the concentration of the first aqueous buffer solution is 5-100mM; further optionally, the first aqueous buffer solution is 10mM phosphate buffer solution or 10mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, and/or

The third polar medium is a third aqueous buffer solution, and the third aqueous buffer solution is selected from phosphate buffer solution, carbonate buffer solution, acetate buffer solution, tris buffer solution, 3 -one or more of morpholinopropanesulfonic acid buffer solution, 4-hydroxyethylpiperazineethanesulfonic acid buffer solution, borate buffer solution or citrate buffer solution;

Optionally, the concentration of the third aqueous buffer solution is 5-100mM; further optionally, the third aqueous buffer solution is 10mM phosphate buffer solution or 10mM 4-hydroxyethylpiperazineethanesulfonic acid buffer solution;

Optionally, the third polar medium is the same or different from the first polar medium;

Optionally, the concentration of the third aqueous buffer solution is the same or different from that of the first aqueous buffer solution.
The method, film-forming system or microdroplets according to claim 18, wherein both the first aqueous buffer solution and the third aqueous buffer solution contain potassium salts;

Optionally, the concentration of the potassium salt is 400-800mM;

Optionally, the potassium salt is potassium chloride.
The method, film-forming system or droplet according to any one of claims 1-19, wherein the osmotic pressure of the first polar medium and the osmotic pressure of the third polar medium make the first The polar medium phase and the third polar medium phase maintain a stable state of non-interpenetration.
The method, film-forming system or droplet according to any one of claims 1-20, wherein the amphiphilic molecules are selected from the group consisting of phospholipids, fatty acids, fatty acyl groups, glycerides, glycerophospholipids, sphingolipids, sterols One or more of lipids, prenol lipids, glycolipids, polyketides and amphiphilic block copolymers.
The method, film-forming system or droplet of claim 21 , wherein the amphiphilic block copolymer comprises at least three polymer segments, wherein the hydrophilic polymeric segments A1 and A2 are linked to The opposite end of the hydrophobic polymeric segment B; or

The amphiphilic block copolymer comprises at least two polymer segments, a hydrophilic polymer segment A and a hydrophobic polymer segment B.
The method, film-forming system or droplet of claim 22, wherein the copolymer is poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyl oxazoline), poly(2-methyloxazoline)-polyethylene-poly(2-methyloxazoline) or poly(ethylene glycol)-poly(dimethylsiloxane)-poly(ethylene diol).
The amphiphile molecular layer or the membrane containing the amphiphile molecular layer prepared by the method described in any one of the foregoing.
A nanopore sequencing device, which includes the amphiphilic molecular layer or the membrane containing the amphiphilic molecular layer prepared by any of the methods described above, and the film-forming system or microdroplets described in any of the foregoing.
A method of characterizing an analyte of interest, comprising:

(a) contacting the target analyte with a transmembrane pore embedded in an amphiphilic molecular layer in any of the methods, membrane-forming systems or microdroplets described above; optionally, the The pore is a transmembrane protein pore;

(b) determining one or more electrical signals as the analyte moves relative to the pore or when the analyte is present within the pore, wherein the determination is indicative of one or more of the analyte of interest Various features to characterize the target analyte.
Application of the method described in any one of the foregoing, or the amphiphilic molecular layer or membrane containing the amphiphilic molecular layer prepared therefrom, the system or microdroplets described in any one of the foregoing in characterizing an analyte or preparing a product characterizing an analyte.
The method or application according to claim 26 or 27, wherein the target analyte is a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a protein, a nucleotide, a polynucleotide, a polysaccharide, a lipid, a dye , bleach, drugs, diagnostic reagents, explosive or environmental pollutants;

Optionally, said polynucleotide comprises DNA and/or RNA and analogs/derivatives thereof.