Method for embedding nanopore protein into biomimetic membrane and application

Abstract

The invention discloses a method for embedding nanopore protein into a biomimetic membrane and application of the biomimetic membrane. The method comprises the steps that the biomimetic membrane of a multi-block copolymer is prepared, and the multi-block copolymer contains a hydrophilic segment and a hydrophobic segment; embedding the nanopore protein into the biomimetic membrane; wherein the multi-block copolymer comprises a copolymer with two or more blocks; the biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid, facing one side of the biomimetic membrane, of the transmembrane region of the nanopore protein. According to the method, the number of single holes and / or the single hole reserving rate during hole embedding of the biomimetic membrane can be increased.

Description

Method and application of embedding nanoporous protein into biomimetic membrane Technical Field

[0001] The present invention relates to the field of nanopore sequencing, and in particular to a method for embedding nanopore protein into a biomimetic membrane and its application. Background Art

[0002] In the field of nanopore sequencing, the stability of porins on the membrane is a key factor influencing core metrics such as nanopore sequencer throughput and accuracy. Currently, conventional membrane materials use phospholipids. As the primary structural component of biological cell membranes, these materials possess natural amphiphilic properties and strong affinity for transmembrane proteins, providing a matrix for membrane-protein biosensors. However, conventional phospholipid membrane systems suffer from poor stability, hindering commercialization. Unlike traditional phospholipid membrane materials, biomimetic membrane materials possess excellent chemical and mechanical stability. Biomimetic membranes, or amphiphilic block copolymers, are novel membrane-forming materials that mimic phospholipid molecules. Their membrane-forming principles are similar to those of phospholipid membranes. However, the block structure can be manipulated in multiple dimensions, such as type, length, and functionality, to produce polymers that form stable membranes. While biomimetic membranes offer superior stability to phospholipid bilayers, their compatibility with porins requires further optimization to improve pore embedding efficiency.

[0003] Patent CN104936682B discloses the use of amphiphilic block polymers to self-assemble at the interface of two polar solution droplets to form a bilayer membrane. Porin is then embedded on both sides of the bilayer membrane by applying an electric current. However, the main drawback of the membrane-forming materials used in existing pore embedding methods is the insufficient binding strength between the protein and the membrane itself. Even if pores can be embedded, the porins quickly fall off, resulting in a limited number of single pores available for sequencing.

[0004] Summary of the Invention

[0005] The main purpose of the present invention is to provide a method and application for embedding nanoporous proteins into biomimetic membranes, so as to solve the problem of low embedding efficiency of biomimetic membranes in the prior art.

[0006] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for embedding a nanoporous protein into a biomimetic membrane is provided, the method comprising: preparing a biomimetic membrane of a multi-block copolymer, the multi-block copolymer comprising a hydrophilic segment and a hydrophobic segment; embedding the nanoporous protein into the biomimetic membrane; wherein the multi-block copolymer comprises a copolymer of two or more blocks; the biomimetic membrane comprises an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanoporous protein comprises an aromatic ring structure, and the aromatic ring structure is located on an amino acid in the transmembrane region of the nanoporous protein on the side facing the biomimetic membrane.

[0007] Furthermore, both the biomimetic membrane and the nanoporous protein contain aromatic ring structures, thereby forming a π-π stacking non-covalent interaction.

[0008] Furthermore, the aromatic ring includes a pure carbon ring and a heterocycle; preferably, the pure carbon ring is a five-membered ring or a six-membered ring; preferably, the six-membered ring is a benzene ring; preferably, the heterocycle is piperazine.

[0009] Furthermore, the biomimetic membrane containing a benzyl ring structure is selected from a biomimetic membrane of a multi-block copolymer with benzylethyl as a bridge bond or a biomimetic membrane of a multi-block copolymer with benzylpropyl as a bridge bond; preferably, the biomimetic membrane is a biomimetic membrane of a multi-block copolymer with benzylethyl as a bridge bond.

[0010] Furthermore, the biomimetic membrane is a biomimetic membrane of a multi-block copolymer with benzylethyl as a bridge bond; preparing the biomimetic membrane of the multi-block copolymer with benzylethyl as a bridge bond includes: dissolving the multi-block copolymer with benzylethyl as a bridge bond in a solvent to form a polymer solution; using the polymer solution to perform a film-forming operation on the chip to obtain the biomimetic membrane.

[0011] Furthermore, the multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer; preferably a diblock copolymer or a triblock copolymer.

[0012] Furthermore, the triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33P DMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA; preferably, the solvent is selected from any one of the following: silicone oil AR20, PDMS-OH 65cSt, PMS-H03, DMS-T12, hexadecane, decane or nonane; preferably, the concentration of the triblock copolymer with benzylethyl as the bridge bond in the polymer solution is 2-20 mg / mL.

[0013] Furthermore, the nanopore protein is selected from the bacterial amyloid protein secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, aerobic bacterial cytolytic protein or OmpG; preferably, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, PC162 protein shown in SEQ ID NO: 4; preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification.

[0014] Furthermore, based on the amino acid sequence of the bacterial amyloid protein secretion channel CsgG shown in SEQ ID NO: 1, the aromatic ring structure is located on any one of the following conserved amino acid segments on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215; preferably, the aromatic ring structure is located on any one or more of the following amino acids in the conserved segment: F, Y, or W.

[0015] Furthermore, embedding the nanoporous protein into the biomimetic membrane comprises: diluting the nanoporous protein with an electrochemical buffer to obtain a poron solution; and injecting the poron solution into an electrochemical reaction cell containing the biomimetic membrane to embed the nanoporous protein into the biomimetic membrane.

[0016] Furthermore, embedding the nanoporous protein into the bionic membrane comprises: embedding the nanoporous protein into the bionic membrane by applying a voltage on both sides of the bionic membrane; preferably, the voltage is 0.18V-0.35V, and the duration of applying the voltage is 1-2h.

[0017] Furthermore, the electrochemical buffer comprises: KCI or potassium ferrocyanide; preferably, the concentration of the porin solution is 0.01-0.02 μg / mL.

[0018] According to a second aspect of the present invention, a nanopore biomimetic membrane chip is provided, which comprises: a biomimetic membrane having a multi-block copolymer formed on the chip, the multi-block copolymer containing a hydrophilic segment and a hydrophobic segment; and a nanopore protein embedded in the biomimetic membrane to form a nanopore; wherein the multi-block copolymer comprises a copolymer of two blocks or more; the biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane.

[0019] Furthermore, both the biomimetic membrane and the nanopore protein contain aromatic ring structures, thereby forming a non-covalent π-π stacking effect; preferably, the aromatic ring structure includes a pure carbon ring and a heterocycle; preferably, the pure carbon ring is a five-membered ring or a six-membered ring; preferably, the six-membered ring is a benzene ring; preferably, the heterocycle is piperazine.

[0020] Furthermore, the biomimetic membrane containing a benzyl ring structure is selected from a biomimetic membrane of a multi-block copolymer with a benzylethyl group as a bridge bond or a biomimetic membrane of a multi-block copolymer with a benzylpropyl group as a bridge bond.

[0021] Furthermore, the multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer, preferably a diblock copolymer or a triblock copolymer.

[0022] Preferably, the triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PD MS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA; preferably, the biomimetic membrane containing a benzene ring structure is a biomimetic membrane of a triblock copolymer with benzylethyl as a bridge bond.

[0023] Furthermore, the nanopore protein is formed by non-covalently linking 7 to 11 porin monomers; preferably, the nanopore protein is formed by non-covalently linking 9 porin monomers.

[0024] Furthermore, the nanopore protein is selected from the bacterial amyloid protein secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, aerobic bacterial cytolytic protein or OmpG; preferably, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, PC162 protein shown in SEQ ID NO: 4; preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification.

[0025] Furthermore, based on the amino acid sequence of the bacterial amyloid protein secretion channel CsgG shown in SEQ ID NO: 1, the aromatic ring structure is located on any one of the following conserved amino acid segments on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215; preferably, the aromatic ring structure is located on any one or more of the following amino acids in the conserved segment: F, Y, or W.

[0026] Furthermore, the pore diameter of the nanopore protein is 1 to 2 nm.

[0027] According to a third aspect of the present invention, a kit is provided, comprising at least one of a biomimetic membrane having a multi-block copolymer and a nanoporous protein, wherein the multi-block copolymer comprises a hydrophilic segment and a hydrophobic segment; wherein the multi-block copolymer comprises a copolymer of two or more blocks; the biomimetic membrane comprises an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanoporous protein comprises an aromatic ring structure, and the aromatic ring structure is located on an amino acid on the side of the transmembrane region of the nanoporous protein facing the biomimetic membrane.

[0028] Furthermore, both the biomimetic membrane and the nanoporous protein contain aromatic ring structures, thereby forming a π-π stacking non-covalent interaction.

[0029] Furthermore, the aromatic ring structure includes pure carbon rings and heterocycles; preferably, the pure carbon ring is a five-membered ring or a six-membered ring; preferably, the six-membered ring is a benzene ring; preferably, the heterocycle is piperazine.

[0030] Furthermore, the biomimetic membrane containing a benzyl ring structure is selected from a biomimetic membrane of a multi-block copolymer with benzylethyl as a bridge bond or a biomimetic membrane of a multi-block copolymer with benzylpropyl as a bridge bond; preferably, the biomimetic membrane is a biomimetic membrane of a multi-block copolymer with benzylethyl as a bridge bond.

[0031] Furthermore, the multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer and a hexablock copolymer, preferably a diblock copolymer or a triblock copolymer.

[0032] Furthermore, the triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA.

[0033] Furthermore, the nanopore protein is selected from the bacterial amyloid protein secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, aerobic bacterial cytolytic protein or OmpG; preferably, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, PC162 protein shown in SEQ ID NO: 4; preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification.

[0034] Furthermore, based on the amino acid sequence of the bacterial amyloid protein secretion channel CsgG shown in SEQ ID NO: 1, the aromatic ring structure is located on any one of the following conserved amino acid segments on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215; preferably, the aromatic ring structure is located on any one or more of the following amino acids in the conserved segment: F, Y, or W.

[0035] Furthermore, the kit further includes at least one of the following: a solvent, an electrochemical buffer, an assembled chip and a sequencing buffer; preferably, the solvent is selected from any one of the following: silicone oil AR20, PDMS-OH 65cSt, PMS-H03, DMS-T12, hexadecane, decane or nonane; preferably, the electrochemical buffer includes: KCI or potassium ferrocyanide.

[0036] According to a fourth aspect of the present invention, a sequencing method is provided, which comprises: using the above-mentioned nanopore biomimetic membrane chip to detect and analyze the electrical signal generated when the biomolecule to be tested passes through the nanopore of the nanopore protein, and determining the sequence of the biomolecule to be tested.

[0037] Furthermore, the biomolecule to be detected includes any one of the following modified or unmodified biomolecules: DNA, RNA or polypeptide.

[0038] According to a fifth aspect of the present invention, there is provided a method for embedding nanopore protein into a biomimetic membrane, the nanopore biomimetic membrane chip or the kit, and their use in small molecule detection, DNA sequencing, RNA sequencing or polypeptide sequencing.

[0039] The biomimetic membrane and / or nanoporous protein of the present invention contain an aromatic ring structure, wherein the aromatic ring structure in the biomimetic membrane is connected between the hydrophilic and hydrophobic segments of the multi-block copolymer. When the nanoporous protein contains an aromatic ring structure, the aromatic ring structure is located on an amino acid on the side of the transmembrane region of the nanoporous protein facing the biomimetic membrane. By having at least one of the aromatic ring structures in the biomimetic membrane increase the number of single pores and / or the single pore retention rate when the biomimetic membrane is embedded.

[0040] When the biomimetic membrane or nanoporous protein contains aromatic ring structures, this method can embed pores, increasing the number of single pores and / or the retention rate of single pores during embedding, but the resulting membrane pore stability is insufficient. However, when both the biomimetic membrane and the nanoporous protein contain aromatic ring structures, this method further enhances the binding ability between the nanoporous protein and the membrane through the π-π stacking non-covalent bonding between the aromatic rings, thereby improving membrane pore stability and achieving the goal of increasing the embedding rate of the nanoporous protein in the biomimetic membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0042] FIG1 shows a schematic diagram of the three-dimensional structure of the PC28 protein that has not been mutated in this application;

[0043] FIG2 shows a partial enlarged schematic diagram of the three-dimensional structure of the PC28 protein that has not been mutated in this application;

[0044] FIG3 shows a schematic diagram of the three-dimensional structure of the mutant protein PC160 in the present application;

[0045] FIG4 shows a partially enlarged schematic diagram of the three-dimensional structure of the mutant protein PC160 in the present application;

[0046] FIG5 shows a schematic diagram of the three-dimensional structure of the mutant protein PC161 in the present application;

[0047] FIG6 shows a partially enlarged schematic diagram of the three-dimensional structure of the mutant protein PC161 in the present application;

[0048] FIG7 shows a schematic diagram of the three-dimensional structure of the mutant protein PC162 in the present application;

[0049] FIG8 shows a partially enlarged schematic diagram of the three-dimensional structure of the mutant protein PC162 in the present application;

[0050] FIG9 shows a schematic side view of the three-dimensional structure of the porin PC28 interacting with the membrane after the pore is completed in the present application;

[0051] FIG10 shows a schematic top view of the three-dimensional structure of the interaction between the porin PC28 and the membrane after the pore is completed in the present application;

[0052] FIG11 shows a schematic diagram of the embedded pore signal of the mutant protein PC160 according to Example 1 of the present application;

[0053] FIG12 shows a schematic diagram of the embedded pore signal of the mutant protein PC161 according to Example 2 of the present application;

[0054] FIG13 shows a schematic diagram of the pore signal of the mutant protein PC162 according to Example 3 of the present application. DETAILED DESCRIPTION

[0055] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0056] As mentioned in the background art, in the field of nanopore sequencing, the stability of porins on nanomembranes is a core indicator that determines sequencing. Traditional phospholipid membrane systems have poor stability. Amphiphilic block copolymers, as a new membrane-forming material for biomimetic membranes, can be regulated from multiple dimensions such as block type, length, and functionality to obtain more stable membrane-forming polymers. However, the embedding efficiency of porins in biomimetic membranes is still not high. In the prior art, porins are embedded by applying electricity on both sides of the bilayer membrane. Due to insufficient binding force between the protein and the membrane itself, porins will quickly fall off, resulting in the defect of fewer single pores for sequencing. Therefore, in the present application, the inventors introduced aromatic ring structures into biomimetic membranes and / or porins with multi-block copolymers to achieve the purpose of improving the embedding efficiency of porins in biomimetic membranes. Among them, one of the biomimetic membrane or porin with a multi-block copolymer contains an aromatic ring structure, which improves the embedding rate compared with the existing technology; when the biomimetic membrane with a multi-block copolymer and the porin both contain aromatic ring structures, since the aromatic ring structures of the two can form a π-π stacking non-covalent bond effect, therefore, while improving the embedding rate, the binding stability of the biomimetic membrane and the porin can be further improved.

[0057] In a first typical embodiment of the present application, a method for embedding a nanoporous protein into a biomimetic membrane is provided, the method comprising: preparing a biomimetic membrane of a multi-block copolymer, the multi-block copolymer comprising a hydrophilic segment and a hydrophobic segment; embedding the nanoporous protein into the biomimetic membrane; wherein the multi-block copolymer comprises a copolymer of two or more blocks; the biomimetic membrane comprises an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanoporous protein comprises an aromatic ring structure, and the aromatic ring structure is located on an amino acid on the side of the transmembrane region of the nanoporous protein facing the biomimetic membrane.

[0058] The above method can improve the embedding rate of nanoporous protein in the biomimetic membrane by introducing an aromatic ring structure into a biomimetic membrane and / or nanoporous protein having a copolymer of two or more blocks. In a preferred embodiment, both the biomimetic membrane and the nanoporous protein contain aromatic ring structures, thereby forming a non-covalent interaction of π-π stacking. The non-covalent interaction of π-π stacking can improve the binding ability between the nanoporous protein and the biomimetic membrane, thereby stably fixing the porous protein to the membrane layer, further improving the embedding rate of the nanoporous protein in the biomimetic membrane. Among them, the nanoporous protein can stably function in the biomimetic membrane, providing a high-quality single-molecule sequencing platform. Therefore, the nanopore biomimetic membrane chip obtained using this method can obtain more accurate sequencing results in sequencing applications.

[0059] Any aromatic ring that can form a non-covalent π-π stacking between the biomimetic membrane and the nanopore protein is suitable for the present application. In a preferred embodiment, the aromatic ring includes a pure carbon ring and a heterocyclic ring; preferably, the pure carbon ring is a five-membered ring or a six-membered ring; preferably, the six-membered ring is a benzene ring; preferably, the heterocyclic ring is piperazine.

[0060] The benzene ring structure contained in the above-mentioned bionic membrane is provided by the bridge bond between the hydrophilic segment and the hydrophobic segment in the bionic membrane. Any bionic membrane having a multi-block copolymer connected by a substance with a benzene ring structure is suitable for this application. In a preferred embodiment, the above-mentioned bionic membrane containing a benzene ring structure is selected from a bionic membrane of a multi-block copolymer with benzylethyl as a bridge bond or a bionic membrane of a multi-block copolymer with benzylpropyl as a bridge bond.

[0061] In a preferred embodiment, the biomimetic membrane is a biomimetic membrane of a multi-block copolymer with benzylethyl groups as bridge bonds. Methods for preparing biomimetic membranes of any multi-block copolymer are applicable to the present application. In a preferred embodiment, preparing a biomimetic membrane of a tri-block copolymer with benzylethyl groups as bridge bonds includes: dissolving the multi-block copolymer with benzylethyl groups as bridge bonds in a solvent to form a polymer solution; and using the polymer solution to form a membrane on an assembled chip to obtain the biomimetic membrane.

[0062] The number of multi-blocks on the biomimetic membrane can be sufficient as long as it can complete the embedding of pores with the pore protein. In a preferred embodiment, the multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer. Further, considering the embedding cost and the actual sequencing requirements, in a preferred embodiment, the multi-block copolymer is a diblock copolymer or a triblock copolymer.

[0063] Any chemical substance that can constitute the above-mentioned triblock copolymer is applicable to the present application. In a preferred embodiment, the triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA; any of the above-mentioned organic solvents that can dissolve the copolymer is applicable to the present invention. In a preferred embodiment, the solvent is selected from any one of the following: silicone oil AR20, PDMS-OH 65cSt, PMS-H03, DMS-T12, hexadecane, decane or nonane; in order to form a suitable film layer on the chip, in a preferred embodiment, the concentration of the triblock copolymer with benzylethyl as the bridge bond in the polymer solution is 2-20 mg / mL.

[0064] Any nanopore protein capable of forming a porin pore is suitable for use in this application. In a preferred embodiment, the amino acids on the transmembrane region of the nanopore protein facing the biomimetic membrane have an aromatic ring structure. This aromatic ring structure can be the natural structure of the porin protein or an aromatic ring structure obtained through mutation. In a preferred embodiment, the nanopore protein is selected from the bacterial amyloid protein secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, aerobic bacterial cytolytic protein, or OmpG. In a preferred embodiment, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2 (as shown in Figures 3-4), PC161 protein shown in SEQ ID NO: 3 (as shown in Figures 5-6), and PC162 protein shown in SEQ ID NO: 4 (as shown in Figures 7-8). Preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification, such as adding a purification tag HHHHHH (SEQ ID NO: 5) or WSHPQFEK (SEQ ID NO: 6) to the carboxyl terminus. The nanoporous protein with aromatic ring structure on the amino acid of the transmembrane region facing the biomimetic membrane can be non-covalently linked to the biomimetic membrane during the biomimetic membrane embedding process, thereby increasing the embedding rate of the nanoporous protein on the biomimetic membrane.

[0065] SEQ ID NO: 2:

[0066] SEQ ID NO: 3:

[0067] SEQ ID NO: 4:

[0068] The PC28 protein (shown in Figures 1-2) is a member of the bacterial amyloid secretion channel CsgG. As a transmembrane protein, amino acids in its transmembrane region facing the biomimetic membrane are mutated. The mutated amino acids contain aromatic ring structures, enabling non-covalent bonding with the biomimetic membrane, which also contains aromatic ring structures, thereby enhancing the binding between the biomimetic membrane and the nanopore protein. It should be noted that while the CsgG sequences vary between species, they all maintain a high degree of structural conservation. Therefore, even with different transmembrane region sequences, the methods of this application can theoretically be used to enhance the interaction with the biomimetic membrane. Transmembrane nanopore proteins such as Mycobacterium smegmatis porins, α-hemolysin, aerobic cytolytic proteins, or OmpG, while significantly different in structure and sequence from CsgG, can also be used to enhance the binding between biomimetic membranes and nanopore proteins using the improved approach of this application.

[0069] Therefore, in a preferred embodiment, based on the amino acid sequence of the bacterial amyloid protein secretion channel Csg G (PC28 protein) shown in SEQ ID NO: 1, the aromatic ring structure is located at any one of the following conserved amino acid segments on the biomimetic membrane side of the nanopore protein's transmembrane region: positions 152-158, positions 178-183, or positions 210-215. Introducing the aromatic ring structure into the amino acids in these three conserved segments can further improve the nanopore protein's embedding efficiency in the biomimetic membrane. Based on the natural amino acids in these three conserved segments, in a preferred embodiment, the aromatic ring structure is located at any one or more of the following amino acids in the conserved segments: F, Y, or W.

[0070] SEQ ID NO: 1:

[0071] In a preferred embodiment, embedding the nanoporous protein into the biomimetic membrane includes: diluting the nanoporous protein with an electrochemical buffer to obtain a poron solution; and injecting the poron solution into an electrochemical reaction cell containing the biomimetic membrane to embed the nanoporous protein into the biomimetic membrane. The resulting three-dimensional structure of the embedded pore biomimetic membrane is shown in Figures 9-10. To embed the nanoporous protein into the membrane layer, a suitable poron solution must be prepared to ensure stable and efficient embedding of the poron during the preparation of the nanoporous biomimetic membrane.

[0072] To further enhance the stability of the porins embedded in the biomimetic membrane, in a preferred embodiment, a voltage is applied across the membrane to embed the nanoporins. Any voltage parameter that can embed the nanoporins in the biomimetic membrane is applicable to the present invention. In a preferred embodiment, the voltage is 0.18V-0.35V, and the voltage is applied for 1-2 hours.

[0073] Any electrochemical buffer capable of diluting the porins is suitable for use in the present invention. In a preferred embodiment, the electrochemical buffer comprises KCl or potassium ferrocyanide. Furthermore, to embed a suitable density of nanoporins into the biomimetic membrane, in a preferred embodiment, the porin solution has a concentration of 0.01-0.02 μg / mL.

[0074] In a second typical embodiment of the present application, a nanopore biomimetic membrane chip is provided, which comprises: a biomimetic membrane having a multi-block copolymer formed on the chip, the multi-block copolymer containing a hydrophilic segment and a hydrophobic segment; and a nanopore protein embedded in the biomimetic membrane to form a nanopore; wherein the multi-block copolymer comprises a copolymer of two blocks or more; the biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane.

[0075] The structure of the nanopore biomimetic membrane chip comprises a biomimetic membrane of a multi-block copolymer and / or a porin containing an aromatic ring structure, which can increase the embedding rate of the nanoporin in the biomimetic membrane. In a preferred embodiment, both the biomimetic membrane and the nanoporin contain aromatic ring structures, thereby forming a non-covalent π-π stacking effect. The non-covalent π-π stacking effect can improve the binding ability between the nanoporin and the biomimetic membrane, thereby stably fixing the porin to the membrane layer, further increasing the embedding rate of the nanoporin in the biomimetic membrane. The nanopore biomimetic membrane chip can provide a high-quality single-molecule sequencing platform, enabling more accurate sequencing results in sequencing applications.

[0076] Any aromatic ring that can form a non-covalent π-π stacking between the biomimetic membrane and the nanopore protein is suitable for the present application. In a preferred embodiment, the aromatic ring includes a pure carbon ring and a heterocyclic ring; preferably, the pure carbon ring is a five-membered ring or a six-membered ring; preferably, the six-membered ring is a benzene ring; preferably, the heterocyclic ring is piperazine.

[0077] The aromatic ring structure contained in the above-mentioned bionic membrane is provided by the bridge bond between the hydrophilic segment and the hydrophobic segment in the bionic membrane. Any bionic membrane with a multi-block copolymer connected by a substance with an aromatic ring structure is suitable for this application. In a preferred embodiment, the above-mentioned bionic membrane containing an aromatic ring structure is selected from a bionic membrane of a multi-block copolymer with benzylethyl as a bridge bond or a bionic membrane of a multi-block copolymer with benzylpropyl as a bridge bond.

[0078] The number of multi-blocks on the biomimetic membrane can be sufficient as long as it can complete the embedding of pores with the pore protein. In a preferred embodiment, the multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer. Further, considering the embedding cost and the actual sequencing requirements, in a preferred embodiment, the multi-block copolymer is a diblock copolymer or a triblock copolymer.

[0079] Any chemical substance that can form the above-mentioned triblock copolymer is suitable for use in this application. In a preferred embodiment, the triblock copolymer is selected from any of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO, and 3PMOXA-34PDMS-3PMOXA. In a preferred embodiment, the biomimetic membrane containing a benzyl ring structure is a biomimetic membrane of a triblock copolymer with benzylethyl as a bridge bond.

[0080] The porin protein that satisfies the sequencing function on the nanopore biomimetic membrane is composed of any number of porin monomers. In a preferred embodiment, the porin protein is composed of 7-11 porin monomers linked covalently or non-covalently.

[0081] In a preferred embodiment, the porin is composed of 9 porin monomers linked non-covalently.

[0082] Porin monomers can spontaneously aggregate together through hydrogen bonds, ionic bonds, hydrophobic interactions, and other forces to form porins. Therefore, the porin monomers obtained by expression and purification exist as multimers, especially nonamers, under non-denaturing conditions, while denaturing the protein results in the existence of porin monomers.

[0083] Any nanopore protein suitable for sequencing on a nanopore biomimetic membrane chip is suitable for this application. In a preferred embodiment, the amino acids on the transmembrane region of the nanopore protein facing the biomimetic membrane have an aromatic ring structure. This aromatic ring structure can be a natural structure of the porin or an aromatic ring structure obtained through mutation. In a preferred embodiment, the nanopore protein is selected from the bacterial amyloid protein secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, aerobic bacterial cytolytic protein, or OmpG. In a preferred embodiment, the nanopore protein is selected from any of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, or PC162 protein shown in SEQ ID NO: 4. Preferably, the end of the nanopore protein sequence contains a tag sequence for protein purification. Nanopore proteins with aromatic ring structures on the amino acids on the transmembrane region facing the biomimetic membrane can form a non-covalent bond with the biomimetic membrane during the biomimetic membrane embedding process, thereby increasing the embedding rate of the nanopore protein on the biomimetic membrane.

[0084] PC28 protein is a member of the bacterial amyloid secretion channel CsgG. As a transmembrane protein, amino acids in its transmembrane domain facing the biomimetic membrane are mutated. The mutated amino acids contain aromatic ring structures, which can form non-covalent bonds with the biomimetic membrane, thereby enhancing the binding between the biomimetic membrane and the nanopore protein. In a preferred embodiment, based on the amino acid sequence of the bacterial amyloid secretion channel CsgG shown in SEQ ID NO:1, the aromatic ring structure is located at any one of the following conserved amino acid segments on the transmembrane domain facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215. Introducing aromatic ring structures into amino acids within these three conserved segments can further enhance the embedding efficiency of the nanopore protein on the biomimetic membrane. Based on the naturally occurring amino acids within these three conserved segments, in a preferred embodiment, the aromatic ring structure is located at any one or more of the following amino acids: F, Y, or W.

[0085] Porins of any diameter that can provide sequencing functionality on nanopore biomimetic membranes are applicable to this application. In a preferred embodiment, the pore diameter of the porin is 1 to 2 nm. Within a certain range, the smaller the pore diameter of the porin, the higher its accuracy when used for sequencing. If the pore diameter of the porin is too large, more than one molecule may pass through the pore at a time, making it difficult to meet the requirements of single-molecule sequencing. When the biomolecule to be tested passes through an overly large pore, the current signal generated may be missed or erroneous, resulting in low sequencing accuracy. In single-molecule sequencing, accurate sequencing results are obtained by sequencing the same molecule multiple times. Therefore, the higher the sequencing accuracy, the shorter the number of sequencing times and time required. Using porins with high sequencing accuracy for sequencing can greatly reduce sequencing time and reduce costs. This advantage is particularly evident in high-throughput sequencing.

[0086] In a third exemplary embodiment of the present application, a kit is provided, comprising at least one of a biomimetic membrane and a nanoporous protein comprising a multi-block copolymer; the multi-block copolymer comprising a hydrophilic segment and a hydrophobic segment; wherein the multi-block copolymer comprises a copolymer of two or more blocks; the biomimetic membrane comprising an aromatic ring structure, wherein the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanoporous protein comprising an aromatic ring structure, wherein the aromatic ring structure is located on an amino acid on the side of the transmembrane region of the nanoporous protein facing the biomimetic membrane. The contents of the kit can be used to conveniently and quickly perform single-molecule sequencing during the sequencing process to obtain accurate sequencing results.

[0087] Introducing an aromatic ring structure into the biomimetic membrane and / or nanoporous protein of the multi-block copolymer contained in the above-mentioned test kit can improve the accuracy of single molecule sequencing using this test kit. In a preferred embodiment, the biomimetic membrane and the nanoporous protein both contain aromatic ring structures, thereby forming a non-covalent interaction of π-π stacking. Through the non-covalent interaction of π-π stacking, the binding ability between the nanoporous protein and the biomimetic membrane can be improved, and then the porin and the membrane layer can be stably fixed, further improving the embedding rate of the nanoporous protein in the biomimetic membrane. Among them, the nanoporous protein can stably perform its function in the biomimetic membrane, providing a high-quality single molecule sequencing platform. Therefore, the nanopore biomimetic membrane chip obtained using this method can obtain more accurate sequencing results in sequencing applications.

[0088] Any aromatic ring that can form a non-covalent π-π stacking between the biomimetic membrane and the nanopore protein is suitable for the present application. In a preferred embodiment, the aromatic ring includes a pure carbon ring and a heterocyclic ring; preferably, the pure carbon ring is a five-membered ring or a six-membered ring; preferably, the six-membered ring is a benzene ring; preferably, the heterocyclic ring is piperazine.

[0089] The benzene ring structure contained in the above-mentioned biomimetic membrane is provided by the bridge bond connecting the hydrophilic segment and the hydrophobic segment in the biomimetic membrane. Any biomimetic membrane having a multi-block copolymer connected by substances having a benzene ring structure is suitable for this application. In a preferred embodiment, the above-mentioned biomimetic membrane containing a benzene ring structure is selected from a biomimetic membrane of a multi-block copolymer with a benzylethyl group as a bridge bond or a biomimetic membrane of a multi-block copolymer with a benzylpropyl group as a bridge bond. In a preferred embodiment, the biomimetic membrane is a biomimetic membrane of a multi-block copolymer with a benzylethyl group as a bridge bond.

[0090] The number of multi-blocks on the biomimetic membrane can be sufficient as long as it can complete the embedding of pores with the pore protein. In a preferred embodiment, the multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer. Further, considering the embedding cost and the actual sequencing requirements, in a preferred embodiment, the multi-block copolymer is a diblock copolymer or a triblock copolymer.

[0091] Any kit contents capable of performing nanopore protein membrane sequencing are applicable to the present application. In a preferred embodiment, the triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA.

[0092] Any nanoporous protein capable of porin embedding is applicable to the present application. In a preferred embodiment, the amino acids on the transmembrane region of the nanoporous protein facing the biomimetic membrane have an aromatic ring structure. This aromatic ring structure can be the natural structure of the porin or an aromatic ring structure obtained through mutation. In a preferred embodiment, the nanoporous protein is selected from the bacterial amyloid protein secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, aerobic bacterial cytolytic protein, or OmpG. In a preferred embodiment, the nanoporous protein is selected from any of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, and PC162 protein shown in SEQ ID NO: 4. Preferably, the sequence end of the nanoporous protein contains a tag sequence for protein purification. Nanoporous proteins with aromatic ring structures on the amino acids on the transmembrane region facing the biomimetic membrane can form a non-covalent bond with the biomimetic membrane during the biomimetic membrane embedding process, thereby increasing the embedding rate of the nanoporous protein on the biomimetic membrane.

[0093] PC28 protein is a member of the bacterial amyloid secretion channel CsgG. As a transmembrane protein, the aromatic ring structure within its transmembrane domain can form a non-covalent bond with the embedded nanopore protein, further enhancing the binding affinity between the biomimetic membrane and the nanopore protein. In a preferred embodiment, based on the amino acid sequence of the bacterial amyloid secretion channel CsgG shown in SEQ ID NO:1, the aromatic ring structure is located at any one of the following conserved amino acid segments on the transmembrane side of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215. Introducing the aromatic ring structure within the amino acids within these three conserved segments can further enhance the embedding efficiency of the nanopore protein within the biomimetic membrane. Based on the naturally occurring amino acids within these three conserved segments, in a preferred embodiment, the aromatic ring structure is located at any one or more of the following amino acids: F, Y, or W.

[0094] To further enhance operational convenience, in a preferred embodiment, the kit further includes at least one of the following: a solvent, an electrochemical buffer, an assembled chip, and a sequencing buffer. In a preferred embodiment, the solvent is selected from any of the following: silicone oil AR20, PDMS-OH 65cSt, PMS-HO3, DMS-T12, hexadecane, decane, or nonane; and the electrochemical buffer includes KCl or potassium ferricyanide.

[0095] In a fourth typical embodiment of the present application, a sequencing method is provided, which comprises: using the above-mentioned nanopore biomimetic membrane chip to detect and analyze the electrical signal generated when the biomolecule to be tested passes through the nanopore of the nanopore protein, and determining the sequence of the biomolecule to be tested.

[0096] In a preferred embodiment, the biomolecule to be detected includes any of the following modified or unmodified biomolecules: DNA, RNA, or polypeptide, wherein the modified biomolecule includes: methylated or phosphorylated DNA, methylated RNA, phosphorylated or acetylated protein, etc.

[0097] When a voltage is applied across the pore, ions in the solution pass through the central gating region of the porin, generating an electric current. As the target nucleic acid sequence (including DNA or RNA) moves through the pore, the electrical signal passing through the pore is measured. Because different nucleotide types have different sizes, they block the current to varying degrees. Consequently, different nucleotide compositions produce different current signals when passing through the pore. By analyzing this current signal, the sequence of the target nucleic acid can be determined.

[0098] In a fifth typical embodiment of the present application, a method for embedding the above-mentioned nanopore protein into a biomimetic membrane, the above-mentioned nanopore biomimetic membrane chip or the above-mentioned kit are provided, and their use in small molecule detection, DNA sequencing, RNA sequencing or polypeptide sequencing.

[0099] The above-mentioned small molecules include but are not limited to small molecule compounds such as nucleotides, amino acids, polysaccharides or vitamins.

[0100] Nanopore sequencing offers a major advantage over traditional sequencing: it avoids errors that can negatively impact accuracy, enabling extremely long read lengths. This can compensate for gaps that inevitably occur when assembling short fragments in traditional sequencing, allowing for the identification of long deletions, duplications, inversions, and translocations within chromosomes. This allows for full-length coverage of transcriptomes, typically several kilobases in length, providing a novel solution for scientific research on genome assembly, structural variation, and alternative splicing.

[0101] Since nanopore sequencing does not require PCR amplification, the original base modification information on the nucleic acid molecule to be tested can be retained, and the type, site and abundance of the modified base can be directly sequenced at one time. Therefore, the pore protein of the present application can also detect several nucleic acid molecules with DNA / RNA modified bases: including 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine (m7G), pseudouracil (pseudouridine, Ψ), etc. By performing specific model training and algorithm development on various modified bases, nanopore sequencing can complete the identification and positioning of more modified bases, thereby constructing a more complete genome / transcriptome modification map.

[0102] Furthermore, from a clinical perspective, nanopore sequencing's long read length, high portability, fast sequencing speed, and real-time readout make it highly suitable for critical epidemic monitoring and rapid pathogen detection (e.g., in large-scale epidemics such as Zika, Ebola, dengue, and the novel coronavirus), providing a highly timely response. In addition to viruses, nanopore sequencing can also be used for the rapid detection of other pathogens, such as bacteria and fungi.

[0103] Based on the common composition of proteins and nucleic acid molecules, nanopore sequencing platforms also have enormous application potential in the field of protein sequencing. For example, based on the research currently underway, by using protein unfolding enzymes as rate-control tools, characteristic protein signals have been successfully observed, and preliminary identification of protein types and modification states has been achieved, validating the feasibility of pore protein sequencing. In future developments, by further optimizing the rate-control system and developing suitable pore proteins and signal analysis algorithms, fingerprinting and even sequence identification of proteins at the single-molecule level may be achieved.

[0104] In addition to its applications in sequencing, the nanopore platform can also serve as a foundational detection platform, combined with sensing techniques to enable metabolomics analysis of various small and large molecules. By integrating genomics, proteomics, and metabolomics, the nanopore platform could ultimately develop into a universal measurement platform that meets the needs of comprehensive omics analysis, providing a powerful research tool for a deeper understanding of the laws of life and the mechanisms of disease.

[0105] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0106] The protein with the amino acid sequence shown in SEQ ID NO: 1 in the present application is the wild-type PC28 protein; the PC160 protein in the examples (amino acid sequence shown in SEQ ID NO: 2) is a mutant protein of the wild-type PC28 protein after the N208Y mutation; the PC161 protein (amino acid sequence shown in SEQ ID NO: 3) is a mutant protein of the wild-type PC28 protein after the L181F mutation; and the PC162 protein (amino acid sequence shown in SEQ ID NO: 4) is a mutant protein of the wild-type PC28 protein after the L154F mutation.

[0107] In the triblock copolymer (3PMOXA-34PDMS-3PMOXA) involved in the examples of the present application, PMOXA is poly(2-methyloxazoline) and PDMS is polydimethylsiloxane.

[0108] Example 1 Mutant PC160 protein embedded in biomimetic membrane 1

[0109] A triblock copolymer with benzylethyl as a bridge bond (3PMOXA-34PDMS-3PMOXA, Polymer source, Product No. P43506) was dissolved in silicone oil AR20 to obtain a 15 mg / mL membrane solution 1;

[0110] Use electrochemical buffer to dilute the 1 μg / mL mutant PC160 protein stock solution to obtain a diluted 0.01 μg / mL PC160 protein solution. The specific components of the electrochemical buffer are: 1mM KCl + 10 mM potassium ferricyanide.

[0111] The chip was assembled with reference to the method disclosed in patent WO2023122883A1. The membrane solution of the above step was injected into the electrochemical reaction cell and allowed to stand for 24 hours to complete the membrane formation operation. Then, the diluted 0.01 μg / mL PC160 protein solution was injected into the electrochemical reaction cell, and a voltage of 0.18 V was applied to both sides of the polymer membrane, with a maximum voltage not exceeding 0.35 V, to allow the protein to be embedded in the polymer membrane bilayer, completing the protein embedding operation and obtaining the embedding signal shown in Figure 11.

[0112] Example 2: Mutant PC161 protein embedded in biomimetic membrane 1

[0113] Referring to the steps of Example 1, PC161 protein was used instead of PC160 protein, and PC161 protein was embedded in the biomimetic membrane 1 to obtain the embedded pore signal shown in FIG12 .

[0114] Example 3: Mutant PC162 protein embedded in biomimetic membrane 1

[0115] Referring to the steps of Example 1, PC162 protein was used instead of PC160 protein, and PC162 protein was embedded in the biomimetic membrane 1 to obtain the embedded pore signal shown in FIG13 .

[0116] Comparative Example 1 Wild-type PC28 protein embedded in biomimetic membrane 1

[0117] Referring to the steps of Example 1, PC28 protein was used instead of PC160 protein, and PC28 protein was embedded in the biomimetic membrane 1.

[0118] Comparative Example 2: Mutant PC160 protein embedded in biomimetic membrane 2

[0119] Referring to the steps of Example 1, a triblock copolymer (3PMOXA-34PDMS-3PMOXA) with propoxy as the bridge bond was used instead of a triblock copolymer (3PMOXA-34PDMS-3PMOXA) with benzylethyl as the bridge bond to prepare membrane solution 2, and PC160 protein was embedded in the biomimetic membrane 2.

[0120] Comparative Example 3 Wild-type PC28 protein embedded in biomimetic membrane 2

[0121] Referring to the steps of Example 1, a triblock copolymer (3PMOXA-34PDMS-3PMOXA) with propoxy as the bridge bond was used instead of a triblock copolymer (3PMOXA-34PDMS-3PMOXA) with benzylethyl as the bridge bond to prepare membrane solution 2, and PC28 protein was used instead of PC160 protein to embed PC28 protein into biomimetic membrane 2.

[0122] Number of single holes tested

[0123] After the embedding is completed, a sequencing buffer (500mM KCl, 25mM potassium phosphate, 1.5mM MgCl2, 1.5mM ATP, pH 8.0) is injected into the electrochemical reaction cell. The embedded holes of Examples 1-3 and Comparative Examples 1-3 are screened respectively (because the holes embedded initially are unstable, the initial embedded holes are rechecked and the single holes retained are screened), and the number of single holes is recorded. The number of initial embedded holes refers to the holes that can be embedded at the beginning, and the number of single holes refers to the holes that are still determined to exist after the initial embedded holes are rechecked. The number of single holes after screening is compared with the number of initial embedded holes to obtain the ratio of the number of single holes remaining, that is, the single hole retention rate = (number of single holes / number of initial embedded holes) × 100%.

[0124] Table 1

[0125] Figures 11, 12, and 13 show that the mutant proteins PC160, 161, and 162 all have normal pore signals. Comparing Figures 11 and 13, it can be seen that the pore signal noise of the PC162 protein in Example 3 is greater. Furthermore, the number of initial pores and the number of single pores in Table 1 indicate that this protein is relatively more difficult to pore, indicating that the position of the amino acid mutation has a significant impact on pores.

[0126] As can be seen from Table 1, the number of single pores and the single pore retention rate of the PC160 protein are significantly better than those of the PC161 protein and the PC162 protein. Comparing Example 1 with Comparative Example 1, it can be found that the mutant protein (PC160) has a higher pore retention rate than the unmutated protein (PC28). Comparing Example 1 with Comparative Example 2, it is found that compared with the biomimetic membrane without benzene ring polymer, the mutant protein PC160 has a higher number of single pores and a higher initial number of pores when embedded in the biomimetic membrane containing benzene ring polymer, and a higher single pore retention rate.

[0127] In summary, the π-π interaction formed by the structure containing benzene rings after protein mutation and the structure with benzene rings on the polymer bridge bond effectively improves the stability of membrane pores, thereby maximizing the number of single pores.

[0128] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: As shown in Comparative Examples 1-2 compared with Comparative Example 3, it can be seen that the introduction of benzene ring structures into biomimetic membranes or porins having multi-block copolymers can achieve the purpose of increasing the porosity of the biomimetic membrane. Furthermore, as shown in Comparative Example 3 compared with Comparative Example 3, it can be seen that the introduction of benzene ring structures into both the biomimetic membrane and the porin having triblock copolymers can improve the binding capacity between the porin and the membrane through the π-π stacking non-covalent bond effect, thereby improving the porosity of the biomimetic membrane (i.e., the single pore retention rate) and the stability of the biomimetic membrane.

[0129] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for embedding nanoporous proteins into a biomimetic membrane, characterized in that: The method comprises: preparing a biomimetic membrane of a multi-block copolymer, wherein the multi-block copolymer contains a hydrophilic segment and a hydrophobic segment; embedding nanopore protein into the biomimetic membrane; Wherein, the multi-block copolymer comprises a copolymer of two blocks or more; The biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or The nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane.

2. The method according to claim 1, characterized in that The bionic membrane and the nanopore protein both contain the aromatic ring structure, thereby forming a π-π stacking non-covalent effect.

3. The method according to claim 1, characterized in that The aromatic rings include pure carbon rings and heterocyclic rings; Preferably, the pure carbon ring is a five-membered ring or a six-membered ring; Preferably, the six-membered ring is a benzene ring; Preferably, the heterocycle is piperazine.

4. The method according to claim 3, characterized in that The bionic membrane containing the benzene ring structure is selected from a bionic membrane of a multi-block copolymer with benzylethyl as a bridge bond or a bionic membrane of a multi-block copolymer with benzylpropyl as a bridge bond.

5. The method according to claim 4, characterized in that The bionic membrane is a bionic membrane of a multi-block copolymer with benzylethyl as a bridge bond; Preferably, the preparation of a biomimetic membrane of a multi-block copolymer with benzylethyl as a bridge bond comprises: dissolving the multi-block copolymer with benzylethyl as a bridge bond in a solvent to form a polymer solution; The polymer solution is used to perform a film-forming operation on a chip to obtain the bionic membrane.

6. The method according to claim 5, characterized in that The multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer; preferably a diblock copolymer or a triblock copolymer.

7. The method according to claim 6, characterized in that The triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA; Preferably, the solvent is selected from any one of the following: silicone oil AR20, PDMS-OH 65cSt, PMS-H03, D MS-T12, hexadecane, decane or nonane; Preferably, the concentration of the triblock copolymer with benzylethyl as a bridge bond in the polymer solution is 2-20 mg / mL.

8. The method according to claim 1, characterized in that The nanopore protein is selected from the bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, Aerobacterium cytolytic protein or OmpG; Preferably, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, PC162 protein shown in SEQ ID NO: 4; Preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification.

9. The method according to claim 8, characterized in that Based on the amino acid sequence of bacterial amyloid secretion channel CsgG shown in SEQ ID NO: 1, the aromatic ring structure is located on the amino acids of any of the following conserved segments on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215; Preferably, the aromatic ring structure is located on any one or more of the following amino acids in the conserved segment: F, Y or W.

10. The method according to any one of claims 1 to 9, characterized in that Embedding the nanoporous protein into the biomimetic membrane comprises: diluting the nanoporin with an electrochemical buffer to obtain a poron solution; The porin solution is injected into an electrochemical reaction cell containing the biomimetic membrane, so that the nanoporin is embedded in the biomimetic membrane.

11. The method according to claim 10, characterized in that Embedding the nanopore protein into the biomimetic membrane comprises: By applying voltage on both sides of the biomimetic membrane, the nanopore protein is embedded in the biomimetic membrane; Preferably, the voltage is 0.18V-0.35V, and the duration of applying the voltage is 1 to 2 hours.

12. The method according to claim 10, characterized in that The electrochemical buffer comprises: KCI or potassium ferrocyanide, Preferably, the porin solution has a concentration of 0.01-0.02 μg / mL.

13. A nanopore bionic membrane chip, characterized in that: The nanopore bionic membrane chip comprises: A biomimetic membrane having a multi-block copolymer formed on a chip, wherein the multi-block copolymer contains a hydrophilic segment and a hydrophobic segment; and a nanoporin embedded in the biomimetic membrane to form a nanopore; Wherein, the multi-block copolymer comprises a copolymer of two blocks or more; The biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected to the hydrophilic between the hydrophobic section and the hydrophobic section; and / or The nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane.

14. The nanopore biomimetic membrane chip according to claim 13, characterized in that: The biomimetic membrane and the nanopore protein both contain aromatic ring structures, thereby forming a π-π stacking non-covalent effect; Preferably, the aromatic ring structure includes pure carbocyclic rings and heterocyclic rings; Preferably, the pure carbon ring is a five-membered ring or a six-membered ring; Preferably, the six-membered ring is a benzene ring; Preferably, the heterocycle is piperazine.

15. The nanopore biomimetic membrane chip according to claim 13, characterized in that: The bionic membrane containing a benzene ring structure is selected from a bionic membrane of a multi-block copolymer with benzylethyl as a bridge bond or a bionic membrane of a multi-block copolymer with benzylpropyl as a bridge bond.

16. The nanopore biomimetic membrane chip according to claim 15, characterized in that: The multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer or a hexablock copolymer, preferably a diblock copolymer or a triblock copolymer.

17. The nanopore biomimetic membrane chip according to claim 16, characterized in that: The triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA; Preferably, the bionic membrane containing a benzene ring structure is a bionic membrane of a triblock copolymer with benzylethyl as a bridge bond.

18. The nanopore biomimetic membrane chip according to claim 13, characterized in that: The nanoporin is composed of 7 to 11 porin monomers connected non-covalently; Preferably, the nanopore protein is composed of 9 porin monomers connected non-covalently.

19. The nanopore biomimetic membrane chip according to claim 13, characterized in that: The nanopore protein is selected from the bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, Aerobacterium cytolytic protein or OmpG; Preferably, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, PC162 protein shown in SEQ ID NO: 4; Preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification.

20. The nanopore biomimetic membrane chip according to claim 19, characterized in that: Based on the amino acid sequence of bacterial amyloid secretion channel CsgG shown in SEQ ID NO: 1, the aromatic ring structure is located on the amino acids of any of the following conserved segments on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215; Preferably, the aromatic ring structure is located on any one or more of the following amino acids in the conserved segment: F, Y or W.

21. The nanopore biomimetic membrane chip according to claim 19, characterized in that: The pore diameter of the nanopore protein is 1-2 nm.

22. A kit, characterized in that: The kit includes at least one of a biomimetic membrane and a nanoporous protein having a multi-block copolymer, wherein the multi-block copolymer contains a hydrophilic segment and a hydrophobic segment; Wherein, the multi-block copolymer comprises a copolymer of two blocks or more; The biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or The nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane.

23. The kit according to claim 22, characterized in that The bionic membrane and the nanopore protein both contain the aromatic ring structure, thereby forming a π-π stacking non-covalent effect.

24. The kit according to claim 22, characterized in that The aromatic ring structure includes pure carbon rings and heterocyclic rings; Preferably, the pure carbon ring is a five-membered ring or a six-membered ring; Preferably, the six-membered ring is a benzene ring; Preferably, the heterocycle is piperazine.

25. The kit according to claim 24, characterized in that The biomimetic membrane containing a benzene ring structure is selected from a biomimetic membrane of a multi-block copolymer with a benzylethyl group as a bridge bond or a biomimetic membrane of a multi-block copolymer with a benzylpropyl group as a bridge bond; Preferably, the biomimetic membrane is a biomimetic membrane of a multi-block copolymer using benzylethyl as a bridge bond.

26. The kit according to claim 22, characterized in that The multi-block copolymer is a diblock copolymer, a triblock copolymer, a tetrablock copolymer, a pentablock copolymer and a hexablock copolymer, preferably a diblock copolymer or a triblock copolymer.

27. The kit according to claim 26, characterized in that The triblock copolymer is selected from any one of the following: 8PEtOXZ-42PDMS-8PMOXZ, 6PMOXA-33PDMS-6PMOXA, PEO-PDMS-PEO or 3PMOXA-34PDMS-3PMOXA.

28. The kit according to claim 22, characterized in that The nanopore protein is selected from the bacterial amyloid secretion channel CsgG, Mycobacterium smegmatis porin, α-hemolysin, Aerobacterium cytolytic protein or OmpG; Preferably, the nanopore protein is selected from any one of the following: PC160 protein shown in SEQ ID NO: 2, PC161 protein shown in SEQ ID NO: 3, PC162 protein shown in SEQ ID NO: 4; preferably, the sequence end of the nanopore protein contains a tag sequence for protein purification.

29. The kit according to claim 28, characterized in that Based on the amino acid sequence of bacterial amyloid secretion channel CsgG shown in SEQ ID NO: 1, the aromatic ring structure is located on the amino acids of any of the following conserved segments on the side of the transmembrane region of the nanopore protein facing the biomimetic membrane: positions 152-158, positions 178-183, or positions 210-215; Preferably, the aromatic ring structure is located on any one or more of the following amino acids in the conserved segment: F, Y or W.

30. The kit according to claim 22, characterized in that The kit further comprises at least one of the following: a solvent, an electrochemical buffer, an assembled chip and a sequencing buffer; Preferably, the solvent is selected from any one of the following: silicone oil AR20, PDMS-OH 65cSt, PMS-H03, DMS-T12, hexadecane, decane or nonane; Preferably, the electrochemical buffer comprises: KCI or potassium ferrocyanide.

31. A sequencing method, characterized in that: The sequencing method comprises: The nanopore biomimetic membrane chip according to any one of claims 13 to 21 is used to detect and analyze the electrical signal generated when the biological molecule to be detected passes through the nanopore of the nanopore protein, so as to determine the sequence of the biological molecule to be detected.

32. The sequencing method according to claim 31, characterized in that The biomolecule to be detected includes any one of the following modified or unmodified biomolecules: DNA, RNA or polypeptide.

33. Use of the method for embedding nanopore protein into biomimetic membrane according to any one of claims 1 to 12, the nanopore biomimetic membrane chip according to any one of claims 13 to 21, or the kit according to any one of claims 22 to 30 in small molecule detection, DNA sequencing, RNA sequencing or peptide sequencing.

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