A copolymer film, its preparation method and use

Abstract

This application provides a copolymer membrane, its preparation method, and its application. The copolymer membrane is a hydrophilic-hydrophobic or hydrophilic-hydrophobic-hydrophilic bilayer structure, wherein the hydrophobic ends are cross-linked by unsaturated bonds. This application uses thermal cross-linking to cross-link the hydrophobic ends of the bilayer structure through unsaturated bonds. This method improves membrane stability; it also immobilizes nanoporins within the membrane, increasing the bonding strength between the pores and the membrane and reducing the risk of nanoporin detachment; furthermore, it significantly reduces the need for light-transmitting materials for UV cross-linking, lowering costs; and it avoids the adverse effects of free radicals generated by chemical cross-linking on the porous proteins.

Description

Technical Field

[0001] This application relates to the field of nanopore sequencing technology, specifically to a copolymer membrane, its preparation method, and its application. Background Technology

[0002] Nanopore sequencing (also known as fourth-generation sequencing) is a new generation of sequencing technology that has emerged in recent years. Currently, sequencing lengths can reach 150kb. The most widely accepted nanopore sequencing platform on the market is the MinION nanopore sequencer from Oxford Nanopore Technologies (ONT). Its characteristics include single-molecule sequencing, long read lengths (over 150kb), fast sequencing speed, real-time monitoring of sequencing data, and portability. After more than a decade of development, solid-state nanopore technology has become increasingly mature. Currently, there are two types of nanopores used for DNA sequencing: biological nanopores (composed of protein molecules embedded in a phospholipid membrane) and solid-state nanopores (including various silicon-based materials, SiNx, carbon nanotubes, graphene, glass nanotubes, etc.). The diameter of DNA strands is very small (double-stranded DNA is approximately 2nm in diameter, and single-stranded DNA is approximately 1nm in diameter), which places stringent requirements on the size of the nanopores used.

[0003] The field of nanopore sequencing still has room for development. A technical challenge lies in the poor stability of existing biomimetic membranes after embedding, with membrane lifespans rarely exceeding one month. Currently, some methods using UV light and photoinitiators to initiate cross-linking face difficulties, as UV light struggles to penetrate the electrolyte and PC material flow channels. It is necessary to replace the electrolyte with a transparent, isotonic KCl solution and the PC material flow channels with quartz material. The time and economic costs of this process are relatively high, making it unsuitable for mass production. Summary of the Invention

[0004] To solve the above problems, this application adopts the following technical solution: The inventive point of this application is to provide a copolymer film, which is a bilayer structure of hydrophilic end-hydrophobic end or hydrophilic end-hydrophobic end-hydrophilic end, wherein the hydrophobic ends are cross-linked by unsaturated bonds.

[0005] Optionally, the crosslinking is thermal crosslinking; the conditions for thermal crosslinking are: heating temperature of 30℃~50℃; heating time of 10min~2h.

[0006] Optionally, the thermal crosslinking requires the participation of a thermal initiator; the thermal initiator includes any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, and benzoyl peroxide.

[0007] Optionally, the unsaturated bond includes any one or more of the following: carbon-carbon double bond, carbon-carbon triple bond, carbonyl group, ester group, carboxyl group, aldehyde group, amide bond, cyano group, and imine group.

[0008] Optionally, the copolymer film is located in a non-polar solvent; the non-polar solvent further includes a crosslinkable monomer; the crosslinkable monomer is crosslinked with the hydrophobic end through unsaturated bonds.

[0009] Optionally, the crosslinkable monomer is a crosslinkable monomer with acrylate at the end, preferably one or more of dicyclopentadiene acrylate, epoxidized soybean oil acrylate, stearic acid acrylate, isobornyl methacrylate, lauryl acrylate, bisphenol A epoxy acrylate, dimethacryloyloxypropyl polydimethylsiloxane, and tricyclosepiacetic diacrylate.

[0010] Another inventive point of this application is to provide a method for preparing the copolymer film as described above.

[0011] Optionally, the preparation method includes: (1) dissolving a block copolymer containing hydrophobic ends with unsaturated bonds in a nonpolar solvent and forming a bilayer structure through self-assembly; (2) crosslinking the hydrophobic ends to obtain the copolymer film.

[0012] Optionally, the nonpolar solvent also includes a crosslinkable monomer, and the hydrophobic end is crosslinked with the crosslinkable monomer to obtain the copolymer film.

[0013] Another inventive point of this application is to provide a nanopore sequencing system.

[0014] Optionally, the sequencing system includes a membrane and nanoporous proteins embedded in the membrane; the membrane is a copolymer membrane as described above or a copolymer membrane prepared according to the preparation method described above.

[0015] Optionally, the nanopore sequencing system is prepared by the following methods: (1) dissolving a block copolymer containing hydrophobic ends with unsaturated bonds in a nonpolar solvent and forming a bilayer structure through self-assembly; (2) inserting nanoporous proteins into the bilayer structure; (3) cross-linking the hydrophobic ends to obtain the sequencing system; or, (1) dissolving a block copolymer containing hydrophobic ends with unsaturated bonds in a nonpolar solvent and forming a bilayer structure through self-assembly; (2) cross-linking the hydrophobic ends to obtain a copolymer membrane; (3) inserting nanoporous proteins into the copolymer membrane.

[0016] Another inventive point of this application is to provide the application of copolymer membranes as described above or copolymer membranes prepared by any of the preparation methods described above in nanopore sequencing.

[0017] Compared with the prior art, this application has the following advantages: This application utilizes thermal crosslinking to crosslink the hydrophobic ends of a bilayer structure through unsaturated bonds. This method can improve membrane stability, fix nanoporins between membranes, increase the binding strength between pores and membranes, and reduce the risk of nanoporin detachment. In addition, it can significantly reduce the amount of light-transmitting material required for UV crosslinking, thus reducing costs. It can also avoid the adverse effects of free radicals generated by chemical crosslinking on the porins. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the microwell structure of a chip according to an embodiment of this application; Figure 2 This is another schematic diagram of the microwell structure of a chip according to an embodiment of this application; Figure 3 This is a diagram of a crosslinked copolymer film according to an embodiment of this application; Figure 4 This is a pore signal diagram of a crosslinked copolymer film according to an embodiment of this application; Figure 5 This is a diagram of a crosslinked copolymer film (at 600mV) according to an embodiment of this application; Figure 6 This is a pore signal diagram of a crosslinked copolymer film according to an embodiment of this application; Figure 7 This is a diagram of a crosslinked copolymer film (at 600mV) according to an embodiment of this application; Figure 8 This is a pore signal diagram of a crosslinked copolymer film according to an embodiment of this application; Figure 9 This is a diagram of a crosslinked copolymer film (at 600mV) according to an embodiment of this application; Figure 10 This is a pore signal diagram of a crosslinked copolymer film according to an embodiment of this application; Figure 11 This is a diagram of a crosslinked copolymer film (at 600mV) according to an embodiment of this application; Figure 12 This is a pore signal diagram of a crosslinked copolymer film according to an embodiment of this application; Figure 13 This is a diagram of a crosslinked copolymer film (at 0mV voltage) according to an embodiment of this application; Figure 14 This is a diagram of a crosslinked copolymer film (at 300mV) according to an embodiment of this application; Figure 15 This is a diagram of a crosslinked copolymer film (at 600mV) according to an embodiment of this application; Figure 16This is a diagram of a crosslinked copolymer film (at 800mV) according to an embodiment of this application; Figure 17 This is a pore signal diagram of a crosslinked copolymer film according to an embodiment of this application. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0021] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0022] Currently, a key factor limiting nanopore sequencing is the stability of the cell-like membrane that carries the pore proteins. Cell-like membranes are commonly prepared using phospholipid bilayers. Because phospholipids naturally possess hydrophobic and hydrophilic segments, their arrangement at the interface places the hydrophobic segments in the middle and the hydrophilic segments on both sides, forming a bilayer structure. However, due to the lack of strong intermolecular interactions within the phospholipid bilayer, the resulting membrane is highly unstable. Experimental manipulations or movement of the nanopore chip can easily damage the phospholipid bilayer, severely impacting sequencing progress.

[0023] This application provides a copolymer film, which has a hydrophilic-hydrophobic or hydrophilic-hydrophobic-hydrophilic bilayer structure, wherein the hydrophobic ends are cross-linked by unsaturated bonds. The hydrophobic ends contain unsaturated bonds; the number of unsaturated bonds is preferably one, two, three, four, or more; the type of unsaturated bonds is preferably one, two, three, four, or more. The number and type of unsaturated bonds are specifically determined based on the groups and number of repeating units in the hydrophobic end.

[0024] The hydrophilic end includes one or more of polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyethyleneamine, polyethyleneimine, and polyacrylamide.

[0025] The hydrophobic end includes one or more of poly(1,2-butadiene), poly(1,4-butadiene), or other polymers containing one, two, or more unsaturated bonds. For example, polymers formed by the polymerization of two alkenyl groups, such as polypentadiene, polyhexadiene, and other C4-C20 polymers containing two or more alkenyl groups.

[0026] For example, when the hydrophobic end is poly(1,2-butadiene), the unsaturated bond is a carbon-carbon double bond, and there is only one such bond; the number of unsaturated bonds is determined by the number of repeating units in poly(1,2-butadiene).

[0027] Diblock copolymers can be represented by the following formula:

[0028] Where n ranges from 5 to 40; m ranges from 5 to 40; and p ranges from 5 to 40.

[0029] n can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; m can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; p can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0030] The concentration of the copolymer is 2 mg / mL to 100 mg / mL, and can be 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL or any value within this range.

[0031] The solvent for the copolymer is an alkane; preferably one or more of n-decane, n-tetradecane, and n-hexadecane.

[0032] The copolymer film is located in a non-polar solvent; the non-polar solvent also includes a crosslinkable monomer; the crosslinkable monomer is crosslinked with the hydrophobic end through unsaturated bonds.

[0033] The crosslinkable monomer is a crosslinkable monomer with acrylate at the end, preferably one or more of the following: dicyclopentadiene acrylate, epoxidized soybean oil acrylate, stearic acid acrylate, isobornyl methacrylate, lauryl acrylate, bisphenol A epoxy acrylate, dimethacryloyloxypropyl polydimethylsiloxane, and tricyclosepiacetic diacrylate.

[0034] The nonpolar solvent also contains an inert solvent for dissolving the copolymer; preferably, the crosslinkable monomer has a volume percentage of 1% to 80% in the solvent; for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or any value within this range.

[0035] The cross-linking method is thermal cross-linking.

[0036] Thermal crosslinking is the process of crosslinking the hydrophobic ends of a substance under heating conditions using a thermal initiator.

[0037] The conditions for thermal crosslinking are: The heating temperature is 30℃~50℃; it can be 30℃, 35℃, 40℃, 45℃, 50℃ or any other value within this range.

[0038] The heating time is 1 min to 2 h; it can be 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 0.5 h, 1 h, 1.5 h, 2 h or any other value within this range.

[0039] Thermal initiators include any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, and benzoyl peroxide.

[0040] The concentration of the thermal initiator is 2 mg / ml to 10 mg / ml, and can be 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL or any value within this range.

[0041] The conventional preparation method for gene sequencing chip molecular membranes is as follows: first, obtain... Figure 1 or Figure 2The chip shown has electrodes on its bottom, and a microwell structure is formed by photolithography using photoresist. Then, a first layer of electrolyte (polar solvent) is sequentially introduced into the microwell structure to wet the entire film-forming area. Next, a second layer of non-polar film-forming solution is introduced to drive away part of the first layer of electrolyte. Then, a third layer of electrolyte (polar solvent) is introduced, so that the non-polar solvent of the amphiphilic material is sandwiched between the two layers of polar solvent to form molecules.

[0042] The preparation method of the copolymer membrane in the sequencing system includes: (1) adding a polar solution to a microwell; (2) adding a non-polar film-forming solution (dissolving the copolymer containing hydrophobic ends with unsaturated bonds in a non-polar solvent) to the microwell, and forming a bilayer structure through self-assembly; (3) adding a polar solution to the microwell; and (4) crosslinking the hydrophobic ends to obtain the copolymer membrane.

[0043] The polar solvent is an aqueous solution containing KCl, K3Fe(CN)6, and K4Fe(CN)6.

[0044] The preferred aqueous solution is 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6.

[0045] The non-polar film-forming solution also includes an inert solvent; this inert solvent is used to dissolve the copolymer film and the non-polar crosslinkable solvent. The inert solvent is preferably an alkane; preferably one or more of n-decane, n-tetradecane, and n-hexadecane, or a commercially available blended oil; such as N14 mineral oil (source: Paragon standard oil, model: ALK-N14), white oil, etc.

[0046] A nanopore sequencing system includes a membrane and nanopore proteins embedded in the membrane; said membrane is a copolymer membrane as described above or a copolymer membrane prepared according to the preparation method described above.

[0047] The preparation method of the nanopore sequencing system further includes: embedding a pore protein between step (3) and step (4), or embedding a pore protein after step (4).

[0048] Method I: Embed the porin between steps (3) and (4). That is, embed the porin first, and then perform self-crosslinking of the solvent.

[0049] Method II: Porin embedding after step (5). That is, pore embedding is performed after solvent self-crosslinking.

[0050] In this application, any suitable porin may be used; including but not limited to porins derived from Mycobacterium smegmatis A, Mycobacterium smegmatis B, Mycobacterium smegmatis C, Mycobacterium smegmatis D, hemolysin, cytolysin, interleukin, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, WZA, or Neisseria autotransporter lipoprotein, etc.; for example, it may be a mutant of MspA, CsgG, FraC, ClyA, especially MspA or CsgG proteins; more specifically: CsgG nanopores (specifically CsgG-Y51A / F56Q / R97W in WO2017 / 149318A1) or MspA nanopores (MspA protein sequence is SEQ ID NO:31, according to Michael Faller et al., “The Structure of a Mycobacterial Outer-Membrane Channel”, Science). The preparation was carried out as described in 303,1189(2004); DOI:10.1126 / science.1094114.

[0051] Test method for membrane breakdown voltage: Initial voltage 140mV, voltage increased by 10mV every 3s; due to the fluidity of the copolymer membrane, when the voltage across the copolymer membrane reaches the critical value, the copolymer membrane will rupture. This phenomenon can be observed under a microscope.

[0052] Example 1

[0053] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution (the raw materials of the copolymer film structure are added to the solvent to form a non-polar film-forming solution) is added to the microwell, and a bilayer structure is formed through self-assembly; The nonpolar film-forming solution comprises: a block copolymer with 10 mg / ml of hydrophobic end poly(1,2-butadiene) (degree of polymerization 10) and hydrophilic end poly(ethylene oxide) (degree of polymerization 10); a thermal initiator of 5 mg / ml azobisisobutyronitrile (AIBN); and a solvent of N14 mineral oil. (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) After the membrane is formed, the membrane protein (CsgG membrane protein) is introduced and a DC voltage is applied to make it self-assemble on the copolymer membrane. The current is detected. When the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0054] (5) Heat the membrane to 50 degrees Celsius and maintain for 10 minutes.

[0055] pass Figure 3 It is known that the cross-linked copolymer membrane has the advantages of clear boundaries and poor diffusion resistance, with a breakdown voltage of 600mV. The activity of the porin is unaffected, sequencing is still possible, and the sequencing signal is normal. Figure 4 ).

[0056] Example 2

[0057] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution (the raw materials of the copolymer film structure are added to the solvent to form a non-polar film-forming solution) is added to the microwell, and a bilayer structure is formed through self-assembly; The nonpolar film-forming solution comprises: a block copolymer with 10 mg / ml of hydrophobic end poly(1,2-butadiene) (degree of polymerization 10) and hydrophilic end polyethylene glycol (degree of polymerization 10); a thermal initiator of 5 mg / ml azobisisobutyronitrile (AIBN); and a solvent of N14 mineral oil. (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) After the membrane is formed, the membrane protein (CsgG membrane protein) is introduced and a DC voltage is applied to make it self-assemble on the copolymer membrane. The current is detected. When the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0058] (5) Heat the membrane to 40 degrees Celsius and maintain for 60 minutes.

[0059] Crosslinked copolymer film ( Figure 5 The breakdown voltage of the cross-linked membrane is 650mV, which is much greater than the 300mV of the uncross-linked membrane; the membrane has clear boundaries and is not easily diffused; the sequencing current signal of the membrane protein is normal after cross-linking. Figure 6 ).

[0060] Example 3

[0061] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution (the raw materials of the copolymer film structure are added to the solvent to form a non-polar film-forming solution) is added to the microwell, and a bilayer structure is formed through self-assembly; The nonpolar film-forming solution comprises: a block copolymer with 10 mg / ml of hydrophobic end poly(1,4-butadiene) (degree of polymerization 10) and hydrophilic end polyethylene glycol (degree of polymerization 10); a thermal initiator of 5 mg / ml azobisisobutyronitrile (AIBN); and a solvent of N14 mineral oil. (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) After the membrane is formed, the membrane protein (CSGG membrane protein) is introduced and a DC voltage is applied to make it self-assemble on the copolymer membrane. The current is detected. When the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0062] (5) Heat the membrane to 32 degrees Celsius and maintain for 120 minutes.

[0063] Crosslinked copolymer film ( Figure 7 The breakdown voltage is 550mV; the membrane has clear boundaries and is not easily diffused; the sequencing current signal of the membrane proteins is normal after cross-linking. Figure 8 ).

[0064] Example 4

[0065] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution (the raw materials of the copolymer film structure are added to the solvent to form a non-polar film-forming solution) is added to the microwell, and a bilayer structure is formed through self-assembly; The nonpolar film-forming solution comprises: a block copolymer with 10 mg / ml of hydrophobic end poly(1,2-butadiene) (degree of polymerization 10) and hydrophilic end poly(ethylene oxide) (degree of polymerization 10); a thermal initiator of 5 mg / ml azobisisobutyronitrile (AIBN); and a solvent of 50% by volume white oil and 50% by volume crosslinkable monomer (50% by volume tricyclic sebacate diacrylate). (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) After the membrane is formed, the membrane protein (CsgG membrane protein) is introduced and a DC voltage is applied to make it self-assemble on the copolymer membrane. The current is detected. When the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0066] (5) Heat the membrane to 50 degrees Celsius and maintain for 10 minutes.

[0067] Crosslinked copolymer film ( Figure 9The breakdown voltage of the cross-linked membrane is 1200mV, much higher than the 300mV of the uncross-linked membrane; the membrane has clear boundaries and is not easily diffused; the sequencing current signal of the membrane protein is normal after cross-linking. Figure 10 ).

[0068] Example 5

[0069] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution (the raw materials of the copolymer film structure are added to the solvent to form a non-polar film-forming solution) is added to the microwell, and a bilayer structure is formed through self-assembly; The nonpolar film-forming solution comprises: a block copolymer with 10 mg / ml of hydrophobic end poly(1,2-butadiene) (degree of polymerization 10) and hydrophilic end poly(ethylene oxide) (degree of polymerization 10); a thermal initiator of 5 mg / ml azobisisobutyronitrile (AIBN); and a solvent of 50% by volume white oil and 50% by volume crosslinkable monomer (50% by volume isobornyl methacrylate). (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) After the membrane is formed, the membrane protein (CsgG membrane protein) is introduced and a DC voltage is applied to make it self-assemble on the copolymer membrane. The current is detected. When the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0070] (5) Heat the membrane to 50 degrees Celsius and maintain for 10 minutes.

[0071] Crosslinked copolymer film ( Figure 11 The breakdown voltage of the cross-linked membrane is 1200mV, much higher than the 300mV of the uncross-linked membrane; the membrane has clear boundaries and is not easily diffused; the sequencing current signal of the membrane protein is normal after cross-linking. Figure 12 ).

[0072] Example 6

[0073] (1) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (2) A non-polar film-forming solution (the raw materials of the copolymer film structure are added to the solvent to form a non-polar film-forming solution) is added to the microwell, and a bilayer structure is formed through self-assembly; The nonpolar film-forming solution comprises: a block copolymer with 10 mg / ml of hydrophobic end poly(1,4-butadiene) (degree of polymerization 10) and hydrophilic end poly(ethylene oxide) (degree of polymerization 10); a thermal initiator of 5 mg / ml azobisisobutyronitrile (AIBN); and a solvent of 40% by volume white oil and 60% by volume crosslinkable monomers (40% by volume isobornyl methacrylate and 20% by volume tricyclic sebacate diacrylate). (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) After the membrane is formed, the membrane protein (CsgG membrane protein) is introduced and a DC voltage is applied to make it self-assemble on the copolymer membrane. The current is detected. When the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0074] (5) Heat the membrane to 50 degrees Celsius and maintain for 10 minutes.

[0075] The breakdown voltage of the cross-linked copolymer film is 1200mV, which is much greater than the 300mV of the uncross-linked film. Figure 13 , Figure 14 , Figure 15 and Figure 16 The images show the state of the copolymer membrane under different voltages. It can be seen that the membrane edge is clearly defined and remains stable until it ruptures at 1200mV (i.e., the breakdown voltage). The sequencing current signal of the membrane proteins is normal after cross-linking. Figure 17 ).

[0076] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the system described above can be found in the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0077] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working process and related descriptions of the storage device and processing device described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0078] The term "comprising" or any other similar term is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus / device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent in such process, method, article, or apparatus / device.

[0079] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A copolymer film, characterized in that, The copolymer film has a hydrophilic-hydrophobic or hydrophilic-hydrophobic-hydrophilic bilayer structure, wherein the hydrophobic ends are cross-linked by unsaturated bonds.

2. The copolymer film according to claim 1, characterized in that, The crosslinking is thermal crosslinking; the conditions for thermal crosslinking are: heating temperature of 30℃~50℃; heating time of 10min~2h.

3. The copolymer film according to claim 1, characterized in that, The thermal crosslinking requires the participation of a thermal initiator; the thermal initiator includes any one or more of azobisisobutyronitrile, cumene hydroperoxide, di-tert-butyl peroxide, and benzoyl peroxide.

4. The copolymer film according to claim 1, characterized in that, Unsaturated bonds include any one or more of the following: carbon-carbon double bonds, carbon-carbon triple bonds, carbonyl groups, ester groups, carboxyl groups, aldehyde groups, amide groups, cyano groups, and imine groups.

5. The copolymer film according to claim 1, characterized in that, The copolymer film is located in a non-polar solvent; the non-polar solvent also includes a crosslinkable monomer; the crosslinkable monomer is crosslinked with the hydrophobic end through unsaturated bonds.

6. The copolymer film according to claim 4, characterized in that, The crosslinkable monomer is a crosslinkable monomer with acrylate at the end, preferably one or more of the following: dicyclopentadiene acrylate, epoxidized soybean oil acrylate, stearic acid acrylate, isobornyl methacrylate, lauryl acrylate, bisphenol A epoxy acrylate, dimethacryloyloxypropyl polydimethylsiloxane, and tricyclosepiacetic diacrylate.

7. A method for preparing a copolymer film according to any one of claims 1 to 6, characterized in that, (1) Dissolve the copolymer film containing hydrophobic ends with unsaturated bonds in a nonpolar solvent and form a bilayer structure through self-assembly; (2) Crosslink the hydrophobic ends to obtain the copolymer film.

8. The preparation method according to claim 7, characterized in that, The nonpolar solvent also includes a crosslinkable monomer, and the hydrophobic end crosslinks with the crosslinkable monomer to obtain the copolymer film.

9. A nanopore sequencing system, comprising a membrane and nanoporous proteins embedded in the membrane; said membrane being a copolymer membrane as described in any one of claims 1 to 6 or a copolymer membrane prepared by the preparation method according to claim 7 or 8.

10. The application of the copolymer membrane according to any one of claims 1 to 6, the copolymer membrane prepared by any one of claims 7 to 8, or the nanopore sequencing system according to claim 9 in nanopore sequencing.

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