A cell-like membrane structure, its preparation method and use

Abstract

This application provides a cell-like membrane structure, its preparation method, and its application. The cell-like membrane structure is a block copolymer, consisting of a hydrophilic end and a hydrophobic end; the hydrophobic end contains unsaturated bonds; the hydrophobic end is cross-linked through these unsaturated bonds to form the cell-like membrane structure. This application significantly enhances the interaction forces between the bilayers by cross-linking the hydrophobic ends of the block copolymer through unsaturated bonds, thereby improving membrane stability; furthermore, the cross-linking immobilizes nanoporous proteins within the membrane, increasing the binding strength between the pores and the membrane and reducing the risk of nanoporous protein detachment.

Description

Technical Field

[0001] This application relates to the field of nanopore sequencing technology, specifically to a cell membrane-like structure, 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 lasting more than a month. Summary of the Invention

[0004] To solve the above problems, this application adopts the following technical solution: This application proposes a method for simulating phospholipid bilayer film formation using block copolymers. The block copolymers consist of hydrophilic-hydrophobic diblocks or hydrophilic-hydrophobic-hydrophilic triblocks. The film formation principle is similar to that of phospholipid bilayers, but due to the stronger intermolecular interactions, their stability is slightly better. By introducing unsaturated bonds (double bonds) into the hydrophobic segments of the block copolymer, after polymer film formation, crosslinking methods such as photocrosslinking and / or chemical crosslinking are used to firmly bind the polymer film molecules together, thereby maximizing the stability of the polymer film.

[0005] The inventive point of this application is to provide a cell membrane-like structure, which is a block copolymer divided into a hydrophilic end and a hydrophobic end; the hydrophobic end contains unsaturated bonds; the hydrophobic end is cross-linked through unsaturated bonds to form a cell membrane-like structure.

[0006] 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.

[0007] Optionally, the hydrophobic end is a polymer with carbon-carbon double bonds in the end group.

[0008] Optionally, the cell-like membrane structure is located in a nonpolar solvent; the nonpolar 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] Optionally, the crosslinking method is photocrosslinking and / or chemical crosslinking; in photocrosslinking, the light is ultraviolet light or infrared light.

[0011] Another inventive point of this application is to provide a method for preparing the cell membrane structure described above.

[0012] 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 cell membrane-like structure.

[0013] Optionally, the nonpolar solvent also includes a crosslinkable monomer, and the hydrophobic end is crosslinked with the crosslinkable monomer to obtain the cell membrane-like structure.

[0014] Another inventive point of this application is to provide a sequencing system for nanopore sequencing.

[0015] Optionally, the sequencing system includes a membrane and nanoporous proteins embedded in the membrane; the membrane is a cell-like membrane structure as described above or a cell-like membrane structure prepared according to the preparation method described above.

[0016] Optionally, the 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 membrane; (3) inserting nanoporous proteins into the membrane.

[0017] Another inventive point of this application is to provide the application of the cell membrane-like structure as described above or the cell membrane-like structure prepared by any of the preparation methods described above in nanopore sequencing.

[0018] Compared with the prior art, this application has the following advantages: This application greatly enhances the interaction force between the bilayers by crosslinking the hydrophobic ends of the block copolymer through unsaturated bonds, thereby improving the stability of the membrane. Furthermore, through crosslinking, the nanoporous proteins are fixed between the membranes, increasing the binding strength between the pores and the membrane and reducing the risk of nanoporous protein detachment. Attached Figure Description

[0019] 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 cross-linked films under different cross-linking conditions in one embodiment of this application; Figure 4 This is a diagram of a membrane after crosslinking and storage for 45 days, according to an embodiment of this application. Figure 5 This is a pore-filling signal diagram of a membrane after crosslinking and storage for 45 days, according to an embodiment of this application. Figure 6 This is a diagram of a cross-linked membrane according to an embodiment of this application; Figure 7 This is a pore signal diagram of a cross-linked membrane according to an embodiment of this application; Figure 8 This is a diagram of a cross-linked membrane according to an embodiment of this application; Figure 9 This is a pore signal diagram of a cross-linked membrane according to an embodiment of this application; Figure 10 This is a diagram of a cross-linked membrane according to an embodiment of this application; Figure 11 This is a pore signal diagram of a cross-linked membrane according to an embodiment of this application; Figure 12 This is a diagram of a cross-linked membrane according to an embodiment of this application; Figure 13 This is a pore signal diagram of a cross-linked membrane according to an embodiment of this application. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0023] 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.

[0024] This application provides a cell membrane-like structure, which is a block copolymer divided into a hydrophilic end and a hydrophobic end. The hydrophobic end contains unsaturated bonds. The hydrophobic end is cross-linked through unsaturated bonds to form the cell membrane-like structure. The hydrophobic end contains 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.

[0025] The hydrophilic end of the block copolymer includes one or more of polyethylene glycol, ethylene oxide, polyvinyl alcohol, polyethyleneamine, polyethyleneimine, and polyacrylamine; 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 and polyhexadiene, are 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 cell membrane-like structure is located in a nonpolar solvent; the nonpolar solvent also includes crosslinkable monomers; the crosslinkable monomers are crosslinked with hydrophobic ends through unsaturated bonds.

[0033] 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.

[0034] The cross-linking methods are photocross-linking and / or chemical cross-linking.

[0035] The light intensity is 10~100W; it can be 10W, 20W, 30W, 40W, 50W, 60W, 70W, 80W, 90W, 100W or any other value within this range.

[0036] The illumination time is 1 to 10 minutes; it can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes or any other value within this range.

[0037] Photoinitiated crosslinking also requires a photoinitiator; photoinitiators include any one or more of benzoin dimethyl ether, trimethylbenzoylphenyl phosphate ethyl ester, 1-hydroxycyclohexylphenyl ketone, 2,4,6-trimethylbenzoylphenylphosphonate ethyl ester, and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide.

[0038] In photocrosslinking, the light is ultraviolet light, infrared light, or visible light.

[0039] The ultraviolet crosslinking uses a light source with a wavelength of 254 nm, and the monomers used are photoinitiators at the corresponding wavelength, including 184 (1-hydroxycyclohexylphenyl ketone) and 1173 (2-hydroxy-2-methyl-1-phenyl-1-propanone).

[0040] Place the membrane at a distance of 3-5 cm under a UV light source and irradiate for 3-60 minutes to perform UV crosslinking.

[0041] Visible light uses a light source with a wavelength of 400nm~800nm ​​(preferably 400nm), and the initiator used is a photocrosslinking agent with the corresponding initiation wavelength, including tpo-l (ethyl 2,4,6-trimethylbenzoylphenylphosphonate), 819 (phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide), etc.

[0042] Chemical crosslinking is a double bond polymerization reaction initiated by a chemical initiator after the addition of a chemical initiator.

[0043] The chemical crosslinking time is 1 min to 50 min, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min or any other value within this range.

[0044] Chemical initiators include organic peroxides and inorganic peroxides.

[0045] Inorganic peroxides include persulfates, specifically potassium persulfate, sodium persulfate, and ammonium persulfate.

[0046] Organic peroxides include one or more of the following: ketones, phenyl esters, phenyl ketones, azo compounds, and phenylphosphides.

[0047] Phenyl esters include one or more of the following: tert-butyl peroxybenzoate (TBPB), tert-butyl peroxy(2-ethylhexanoate) (TBPO), and 2-ethylhexyl 4-dimethylaminobenzoate.

[0048] Ketones include cyclohexanone peroxide (CHP) and / or methyl ethyl ketone peroxide (MEKPO).

[0049] Phenyl ketones include one or more of the following: 1-hydroxycyclohexylphenyl ketone, methyl benzoylformate, 2,2-methoxy-phenylacetophenone, benzoyl peroxide (BPO), and Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetone).

[0050] Azo compounds include: 2,2-azobis(2-methylphenylimidazolium) dihydrochloride.

[0051] Phenylphosphorus compounds include one or more of the following: (2,4,6-trimethylbenzoyl)diphenylphosphine oxide, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, and phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite.

[0052] Preferably, the chemical initiator includes two or more types; by combining the initiators, the cross-linking is more complete, resulting in a better estimate of the polymer film effect.

[0053] When two chemical initiators are selected, the molar ratio between them is 2:1 to 20:1; for example, it can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 or any value within this range.

[0054] When two or more chemical initiators are selected, they can be added in proportion; preferably, the two initiators should conform to the above-mentioned proportion relationship.

[0055] The concentration of the initiator is 5 mg / mL to 200 mg / mL; for example, it can be 5 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, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, or any other value within this range.

[0056] The conventional preparation method for gene sequencing chip molecular membranes is as follows: first, obtain... Figure 1 or Figure 2 The 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.

[0057] The method for preparing this type of cell membrane structure in the sequencing system includes: (1) adding a polar solution to a microwell; (2) adding a non-polar film-forming solution (dissolving a block 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) cross-linking the hydrophobic ends to obtain the cell membrane structure.

[0058] The polar solvent is an aqueous solution containing KCl, K3Fe(CN)6, and K4Fe(CN)6.

[0059] The preferred aqueous solution is 200 mM KCl, 150 mM K3Fe(CN)6, and 100 mM K4Fe(CN)6.

[0060] 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.

[0061] A nanopore sequencing system includes a membrane and nanopore proteins embedded in the membrane; the membrane is a cell-like membrane structure as described above or a cell-like membrane structure prepared according to the preparation method described above.

[0062] The preparation method of the sequencing system further includes: embedding porin between step (3) and step (4), or embedding porin after step (4).

[0063] Method I: Embed the porin between steps (3) and (4). That is, embed the porin first, and then perform self-crosslinking of the solvent.

[0064] Method II: Porin embedding after step (5). That is, pore embedding is performed after solvent self-crosslinking.

[0065] 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.

[0066] Example 1

[0067] (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 for the film structure are added to a crosslinkable solvent to form a non-polar film-forming solution) is added to a 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 20) and hydrophilic end poly(ethylene oxide) (degree of polymerization 15); the solvent is 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) Add chemical initiators of K2S2O8 and Na2S2O5 in a molar ratio of 2:1 (molar concentrations of 20mM:10mM, 30mM:15mM, 80mM:40mM, and 100mM:50mM, respectively), and react for a certain time (20min) to crosslink the hydrophobic double bond region of the copolymer.

[0068] For cross-linking effects and storage period, see Figure 3 and Figure 4 .

[0069] Figure 3 The cross-linked membrane, Figure 4 The membrane remained unchanged after 45 days of storage following chemical cross-linking (20mM:10mM), while the uncross-linked membrane diffused or ruptured, indicating a significant improvement in membrane stability. The membranes stored for 45 days were then subjected to pore embedding. The embedding steps were: embedding nanoporous proteins (CsgG membrane proteins); applying a DC voltage to induce self-assembly on the copolymer membrane; and monitoring the current. When the current value was approximately 300pA, it was considered that only one nanoporous protein had been inserted. The embedding current was as follows: Figure 5 As shown, the activity of the pore protein is unaffected, it can still be sequenced, and the sequencing signal is normal.

[0070] Example 2

[0071] (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 for the film structure are added to a crosslinkable solvent to form a non-polar film-forming solution) is added to a microwell, and a bilayer structure is formed through self-assembly. The nonpolar film-forming solution includes: a block copolymer with 10 mg / ml of hydrophobic end poly(1,4-butadiene) (degree of polymerization 20) and hydrophilic end poly(ethylene oxide) (degree of polymerization 15); the photoinitiator is 1-hydroxycyclohexylphenyl ketone (184) at a concentration of 5 mg / ml; the solvent is 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.

[0072] (5) After embedding, UV crosslinking was performed using 254nm near-ultraviolet light (80W). Before irradiation, the upper buffer solution was replaced with 1M KCl. The embedded membrane was placed under the UV light source at a position of about 3cm for 10min to perform UV crosslinking. The crosslinking effect is shown in [the figure]. Figure 6 and Figure 7 .

[0073] The membrane after UV cross-linking has clear boundaries. Figure 6 Furthermore, it is not easily diffused; after ultraviolet irradiation, the activity of porin is not affected, it can still be sequenced, and the sequencing signal is normal. Figure 7 ).

[0074] Example 3

[0075] (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 for the film structure are added to a crosslinkable solvent to form a non-polar film-forming solution) is added to a 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 20) and hydrophilic end poly(ethylene oxide) (degree of polymerization 15); a photoinitiator of 1-hydroxycyclohexylphenyl ketone (184) at a concentration of 5 mg / ml; and a crosslinking monomer of laurate acrylate at a volume ratio of 5% of the solvent, wherein the solvent is 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.

[0076] (5) After embedding, UV crosslinking was performed using 254nm near-ultraviolet light (80W). Before irradiation, the upper buffer solution was replaced with 1M KCl. The embedded membrane was placed under the UV light source at a position of about 3cm for 10min to perform UV crosslinking. The crosslinking effect is shown in [the figure]. Figure 8 It can still generate normal sequencing signals ( Figure 9 ).

[0077] The crosslinking results after incorporating the monomer show that the monomer adds crosslinking to the double bonds in the hydrophobic region of the membrane and accelerates the entire crosslinking process, which is a reliable method to maintain the stability of the membrane.

[0078] Example 4

[0079] (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 for the film structure are added to a crosslinkable solvent to form a non-polar film-forming solution) is added to a 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 20) and hydrophilic end poly(ethylene oxide) (degree of polymerization 15); a photoinitiator of 1-hydroxycyclohexylphenyl ketone (184) at a concentration of 5 mg / ml; a crosslinking monomer of laurate acrylate at a volume ratio of 5%; and N14 mineral oil as the solvent. (3) Add an aqueous solution of 200mM KCl, 150mM K3Fe(CN)6 and 100mM K4Fe(CN)6 to the micro well; (4) Introduce membrane protein (CSGG membrane protein), apply DC voltage to make it self-assemble on copolymer membrane, detect current, when the current value is about 300pA, it can be considered that only one nanoporous protein is inserted.

[0080] (5) Perform UV crosslinking. The UV light used was 254nm near-UV light (80W). Before irradiation, the upper buffer was replaced with 30mM K2S2O8. The membrane after pore embedding was placed under the UV light source at a position of about 3cm and irradiated for 3min to perform co-crosslinking. The crosslinking effect is shown in the figure. Figure 10 It can still generate normal sequencing signals ( Figure 11 ).

[0081] The results of combined chemical and UV crosslinking showed that a vigorous crosslinking reaction occurred. By adding a chemical crosslinking agent, the degree of crosslinking can be controlled, thereby improving membrane stability.

[0082] Example 5

[0083] (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 for the film structure are added to a crosslinkable solvent to form a non-polar film-forming solution) is added to a 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 20) and hydrophilic end poly(ethylene oxide) (degree of polymerization 15); the photoinitiator is ethyl 2,4,6-trimethylbenzoylphenylphosphonate (photoinitiator TPO-L) at a concentration of 5 mg / ml; and the solvent is 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 film formation, visible light crosslinking (wavelength 400nm, 80W) was performed. Before irradiation, the upper buffer solution was replaced with 1M KCl. The membrane with embedded pores was placed at a position of about 3cm under a visible light source and irradiated for 10min to perform visible light crosslinking. The crosslinking effect is shown in the figure. Figure 12 The cross-linked membrane can generate normal sequencing signals. Figure 12 ).

[0084] Visible light-induced crosslinking can greatly reduce the cumbersome experimental procedures, such as the high cost and complicated operation caused by the special requirements of ultraviolet crosslinking channels (the need to use quartz channels to transmit ultraviolet light), and minimize the impact on porins, making it a potentially advantageous crosslinking solution.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 cell membrane-like structure, characterized in that, The cell membrane-like structure is a bilayer structure formed by block copolymer, consisting of a hydrophilic end and a hydrophobic end; the hydrophobic end contains unsaturated bonds; the hydrophobic end is cross-linked through unsaturated bonds to form a cell membrane-like structure.

2. The cell membrane-like structure 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.

3. The cell membrane-like structure according to claim 1, characterized in that, The hydrophobic end is a polymer with carbon-carbon double bonds in the end group.

4. The cell membrane-like structure according to claim 1, characterized in that, The cell-like membrane structure is located in a nonpolar solvent; the nonpolar solvent also includes crosslinkable monomers; the crosslinkable monomers are crosslinked with hydrophobic ends through unsaturated bonds.

5. The cell membrane-like structure 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.

6. The cell membrane-like structure according to claim 1, characterized in that, The cross-linking method is photocross-linking and / or chemical cross-linking; in photocross-linking, the light is ultraviolet light, visible light or infrared light.

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

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 cell membrane-like structure.

9. A sequencing system for nanopore sequencing, comprising a membrane and nanoporous proteins embedded in the membrane; wherein the membrane is a cell-like membrane structure as described in any one of claims 1 to 6 or a cell-like membrane structure prepared by the preparation method according to claim 7 or 8.

10. The sequencing system according to claim 9, characterized in that, The sequencing system is prepared by the following method: (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 membrane; (3) inserting nanoporous proteins into the membrane.

11. The application of the cell-like membrane structure as described in any one of claims 1 to 6 or the cell-like membrane structure prepared by the preparation method as described in any one of claims 7 to 8 in nanopore sequencing.

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