Biological nanopore sensor as well as preparation method and application thereof

Abstract

The invention provides a biological nanopore sensor and a preparation method thereof. The method for preparing the biological nanopore sensor comprises the following steps: a) preparing hydrophilic protein into lipidosome; and b) mixing the lipidosome with the membrane layer to promote the lipidosome to be inserted into the membrane layer to obtain the biological nanopore sensor, and the hydrophilic protein has a nanopore channel. The method can solve the problem that the hydrophilic protein is difficult to prepare into the biological nanopore sensor in the prior art, and is suitable for the field of nanopore sequencing.

Description

Biological nanopore sensor, preparation method and application thereof Technical Field

[0001] The present invention relates to the field of nanopore sequencing, and in particular to a biological nanopore sensor, a preparation method thereof, and applications thereof. Background Art

[0002] Proteins are the material basis of life activities. Different protein sequences and structures determine their different functions in life activities. Changes in protein sequence and structure may alter cell signaling pathways and metabolic pathways, thereby inducing various diseases. Therefore, it is necessary to develop a simple and reliable method to detect proteins or achieve protein sequencing.

[0003] Nanopore technology, as a simple and efficient single-molecule analysis method, has broad applications in biomedicine. Analytes, under the influence of an electric field, pass through biological or solid-state pores, interacting with them and inducing a change in current. Identification is achieved by analyzing the unique electrical signatures of different analytes. Biological nanopores are mostly naturally occurring membrane proteins that can be inserted into phospholipid membranes, forming nanopores capable of detecting analytes.

[0004] However, most biological nanopore proteins in existing technologies have low expression levels in recombinant expression systems and are highly hydrophobic, resulting in low solubility. Currently, most biological pores used in the nanopore field are endomembrane proteins, such as staphylococcal alpha-hemolysin, aerolysin, Escherichia coli membrane protein (CsgG), and Mycobacterium smegmatis MspA. Surfactants are often required during the purification process to improve the solubility of these highly hydrophobic proteins and facilitate polymer assembly. Consequently, the cost of preparing biological nanopore sensors is high; these surfactants can also affect the stability of the phospholipid membrane into which the pore protein is inserted, leading to detection failure. Furthermore, it is difficult to prepare biological nanopore sensors from highly hydrophilic proteins using existing technologies.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a biological nanopore sensor, a preparation method and application thereof, so as to solve the problem in the prior art that it is difficult to prepare a biological nanopore sensor from a hydrophilic protein.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for preparing a biological nanopore sensor is provided, the method comprising: a) preparing a hydrophilic protein into a liposome; b) mixing the liposome with a membrane layer to cause the liposome to insert into the membrane layer, thereby obtaining a biological nanopore sensor, wherein the hydrophilic protein has nanopores.

[0008] Furthermore, the hydrophilic protein includes MCM protein, and a) includes preparing the MCM protein into MCM liposomes.

[0009] Furthermore, the MCM protein includes a protein composed of two or more MCM protein monomers linked by covalent or non-covalent bonding; preferably, the MCM protein monomer includes: 1) a protein consisting of the amino acid sequence of SEQ ID NO: 1; 2) a protein mutant, wherein the amino acid sequence of the protein mutant is substituted, deleted, and / or one or more amino acids are added at at least one of the following positions of the amino acid sequence of SEQ ID NO: 1: 318, 331, 346, 359, 366, 374, 404, 423, 424, 430, 448, 473, 488, and the protein mutant has the same protein binding and DNA function and has the function of forming a nanopore; 3) a porin monomer that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein in 1) or 2), and has the same protein binding and DNA function and has the function of forming a nanopore; preferably, the MCM protein monomer contains the protein of SEQ ID NO: 1. The amino acid sequence shown in NO: 2 has a core region with a greater than 90% identity.

[0010] Furthermore, the MCM protein includes a protein composed of 6 MCM protein monomers linked non-covalently.

[0011] Furthermore, a) comprises: placing an organic solution containing phospholipids in a container to form a lipid film on the container wall; adding a solution containing a hydrophilic protein to the container and shaking to hydrate the lipid film; extruding and filtering the hydrated lipid film solution to obtain liposomes; preferably, the phospholipid comprises diphytylphosphatidylcholine, more preferably 1,2-diphytanoyl-sn-glyceryl-3-phosphocholine; preferably, the filtration comprises filtering using a polycarbonate membrane, more preferably the diameter of the polycarbonate membrane is 0.1-1 μm, more preferably 0.4 μm.

[0012] Furthermore, b) includes: mixing liposomes and a membrane layer in a solution and applying a voltage to both sides of the solution to cause the liposomes to insert into the membrane layer, while monitoring the current in the solution; when the current is constant, it indicates that the liposomes are inserted into the membrane layer, and a biological nanopore sensor is obtained; preferably, b) further includes: calculating the conductivity, conductivity = current / voltage; when the conductivity is ≥2.8nS, the liposomes are inserted into the membrane layer, and a biological nanopore sensor is obtained; preferably, the voltage is greater than 0V and less than or equal to 0.5V; preferably, after obtaining the biological nanopore sensor, the voltage is reduced to 0V.

[0013] Furthermore, after obtaining the biological nanopore sensor, the method further includes washing with a buffer solution to remove residual free hydrophilic proteins.

[0014] Further, the membrane layer includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or a liposome; preferably, the liposome includes a multilayer liposome or a unilamellar liposome; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine, more preferably a phospholipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine.

[0015] In order to achieve the above object, according to a second aspect of the present invention, a biological nanopore sensor is provided. The biological nanopore sensor is prepared by the above method.

[0016] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a biological nanopore sensor is provided, which comprises: a membrane layer and a hydrophilic protein having nanopores, wherein the nanopores of the hydrophilic protein penetrate the membrane layer, and when an electric field force is applied across the membrane layer, an electric current is formed in the pores.

[0017] Furthermore, the hydrophilic protein includes MCM protein.

[0018] Furthermore, the MCM protein includes a protein formed by covalently or non-covalently linking two or more MCM protein monomers; the MCM protein monomers include: 1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a protein mutant, the amino acid sequence of the protein mutant being the same as that shown in SEQ ID NO: 1) wherein at least one of the following positions of the amino acid sequence of SEQ ID NO: 1 is substituted, deleted, and / or one or more amino acids are added: 318, 331, 346, 359, 366, 374, 404, 423, 424, 430, 448, 473, 488, and the protein mutant has the same protein binding and DNA binding function and the ability to form a nanopore; 3) a porin monomer having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the protein of 1) or 2), and having the same protein binding and DNA binding function and the ability to form a nanopore; preferably, the MCM protein comprises a protein composed of 2-10 MCM protein monomers linked by covalent or non-covalent bonding; preferably, the MCM protein comprises a protein composed of 6 MCM protein monomers linked by non-covalent bonding; preferably, the MCM protein monomer contains a core region having more than 90% identity to the amino acid sequence of SEQ ID NO: 2.

[0019] To achieve the above-mentioned object, according to a fourth aspect of the present invention, a nanopore sequencing device is provided. The nanopore sequencing device comprises a biological nanopore sensor prepared by the above-mentioned method, or the above-mentioned biological nanopore sensor.

[0020] Furthermore, the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a biological nanopore sensor, which is located in the center of the electrolytic cell and divides the electrolytic cell and the electrolyte into a positive electrode electrolyte region and a negative electrode electrolyte region; a first electrode and a second electrode, which are respectively arranged in the positive electrode electrolyte region and the negative electrode electrolyte region.

[0021] To achieve the above-mentioned objectives, according to the fifth aspect of the present invention, a sequencing method is provided, which comprises using a biological nanopore sensor prepared by the above-mentioned method, or the above-mentioned biological nanopore sensor, or the above-mentioned nanopore sequencing device, to detect and analyze the electrical signal generated when the biological molecule to be tested passes through the nanopore channel of the hydrophilic protein, and determine the sequence of the biological molecule to be tested.

[0022] Furthermore, the hydrophilic protein includes MCM protein; preferably, the biomolecule to be detected includes modified or unmodified DNA, RNA, or polypeptide; preferably, the polypeptide includes a polypeptide monomer or a polypeptide polymer; preferably, the electrical signal includes current amplitude and / or residence time.

[0023] In order to achieve the above-mentioned purpose, according to the sixth aspect of the present invention, there is provided a biological nanopore sensor prepared by the above-mentioned method, or the above-mentioned biological nanopore sensor, or the above-mentioned nanopore sequencing device, or the above-mentioned sequencing method, and its application in nucleic acid detection, protein detection or protein conformation differentiation.

[0024] As hydrophilic proteins, MCM proteins can be expressed in large quantities in expression systems such as Escherichia coli, making them easy to purify and mass-produce. Furthermore, as multimeric proteins, MCM proteins contain both pores and helicases with unwinding activity, enabling them to function as both porins and helicases in biological nanopore sensors. Biological nanopore sensors prepared using these proteins combine the functions of two separate proteins required for nanopore sequencing in existing techniques, offering excellent application prospects. However, hydrophilic proteins cannot be directly inserted into phospholipid membranes in existing techniques, making it impossible to prepare biological nanopore sensors using MCM proteins. The present invention develops a method for preparing biological nanopore sensors. After encapsulating hydrophilic proteins such as MCM proteins in liposomes, the hydrophilic proteins are successfully inserted into the membrane layer to prepare a biological nanopore sensor. This sensor can perform operations such as sequencing and identification of biological samples, similar to other existing nanopore sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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:

[0026] FIG1 shows a schematic structural diagram of a nanopore sensor embedded with MCM protein according to Example 3 of the present invention.

[0027] 2A and 2B show the purification results of the MCM protein according to Example 1 of the present invention, wherein FIG2A shows the molecular sieve Superdex 200 purification result of the MCM protein, and FIG2B shows the gel electrophoresis result of the MCM protein.

[0028] FIG3A and FIG3B show the activity detection results of MCM according to Example 2 of the present invention, wherein FIG3A shows the activity detection result of MCM at a salt concentration of 0.15 M KCl, and FIG3B shows the activity detection result of MCM at a salt concentration of 0.5 M KCl.

[0029] FIG4 shows the current changes generated when a single MCM protein according to Example 3 of the present invention is embedded in a phospholipid bilayer.

[0030] Figures 5A to 5C show the electrical properties of the nanopore sensor according to Example 3 of the present invention, wherein Figures 5A and 5B show the current trajectory of a single MCM channel when voltages of +0.02 V, -0.02 V, +0.03 V, -0.03 V, 0.04 V, -0.04 V, and +0.05 V are applied in sequence, and Figure 5C shows the conductance distribution of the nanopore sensor (N=63).

[0031] Figures 6A to 6C show the translocation and parameter characterization of the Peptide 1 peptide through the MCM protein pore at -0.02 V according to Example 4 of the present invention, wherein Figure 6A shows the translocation signal of the Peptide 1 peptide when passing through the MCM channel at -0.02 V, Figure 6B shows the analysis results of the current amplitude when the Peptide 1 peptide perforates (N=436), and Figure 6C shows the analysis results of the residence time when the Peptide 1 peptide perforates (N=436).

[0032] Figures 7A to 7C show the translocation and parameter characterization of the Peptide 2 peptide through the MCM protein pore at -0.02V according to Example 4 of the present invention, wherein Figure 7A shows the translocation signal of the Peptide 2 peptide when passing through the MCM channel at -0.02V, Figure 7B shows the analysis results of the current amplitude when the Peptide 2 peptide perforates (N=270), and Figure 7C shows the analysis results of the residence time when the Peptide 2 peptide perforates (N=270).

[0033] Figures 8A to 8C show the translocation and parameter characterization of the TCEP-treated Peptide 2 peptide through the MCM channel at -0.02V according to Example 5 of the present invention, wherein Figure 8A shows the translocation signal of the TCEP-treated Peptide 2 peptide when passing through the MCM channel at -0.02V, Figure 8B shows the analysis results of the current amplitude when the TCEP-treated Peptide 2 peptide perforates (N=270), and Figure 8C shows the analysis results of the residence time when the TCEP-treated Peptide 2 peptide perforates (N=270). DETAILED DESCRIPTION

[0034] 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.

[0035] As mentioned in the background, most existing biological nanopore proteins have low expression levels in recombinant expression systems, are difficult to purify, and are challenging to prepare. Therefore, in this application, the inventors attempted to develop a method for preparing a biological nanopore sensor. After encapsulating a hydrophilic protein with liposomes, they successfully inserted the hydrophilic protein into a membrane layer to produce a biological nanopore sensor. Consequently, a series of protection schemes are proposed in this application.

[0036] In a first typical embodiment of the present application, a method for preparing a biological nanopore sensor is provided, the method comprising: a) preparing a hydrophilic protein into a liposome; b) mixing the liposome with a membrane layer to cause the liposome to insert into the membrane layer to obtain a biological nanopore sensor; the above-mentioned hydrophilic protein has nanopores.

[0037] In a preferred embodiment, the hydrophilic protein includes MCM protein, and a) includes preparing the MCM protein into MCM liposomes.

[0038] The minichromosome maintenance (MCM) protein family, encoded by the MCM gene, is ubiquitous from Archaean organisms to higher eukaryotes and represents a highly conserved protein family. MCM proteins are ring-shaped, hydrophilic hexameric complexes composed of six subunits (i.e., MCM protein monomers). They possess helicase activity and play a crucial role in the initiation and elongation of DNA replication. They contain nanoscale pores within their complexes. MCM proteins are highly expressed in recombinant Escherichia coli expression systems, and their hexameric structure makes them suitable for development as biological nanopores. However, since they are not native membrane proteins, their outer surface is relatively hydrophobic, preventing them from directly inserting into phospholipids.

[0039] Therefore, the inventors of this application have provided a novel method for inserting non-native membrane proteins into membrane layers. In this method, a hydrophilic protein, such as MCM protein, is first encapsulated with a lipophilic material, such as a phospholipid, to form MCM liposomes. The MCM liposomes are then mixed with the membrane layer, allowing the MCM liposomes, and thus the MCM protein, to penetrate the membrane layer, resulting in a bionanopore sensor with a pore connecting both sides.

[0040] In the existing technology, strand sequencing is a relatively mature nanopore sequencing method, commercialized by Oxford Nanopore Technologies (ONT) in the UK. The core principle of ONT strand sequencing is that a helicase unwinds double-stranded DNA into single-stranded DNA and guides the single-stranded DNA through a nanopore protein (helicase and porin are two independent proteins). The novel biological nanopore sensor constructed in this invention differs from ONT technology: the MCM protein has been reported in the literature to be a helicase with unwinding activity and contains a pore; in this invention, the MCM protein is used to perform both the functions of a porin and a helicase, combining the functions of two independent proteins in strand sequencing.

[0041] The biological nanopore sensor prepared by the above method can be used for DNA sequencing. In the examples of this application, enzyme activity assays demonstrate that the biological nanopore sensor constructed in the present invention has unwinding activity, while conductance and analyte permeation demonstrate the formation of pores in the sensor. These two key results demonstrate the potential of this biological nanopore sensor for DNA sequencing.

[0042] In a preferred embodiment, the MCM protein comprises a protein composed of two or more MCM protein monomers linked by covalent or non-covalent linkage; preferably, the MCM protein monomer comprises: 1) a protein consisting of the amino acid sequence of SEQ ID NO: 1; 2) a protein mutant, wherein the amino acid sequence of the protein mutant is substituted, deleted, and / or added with one or more amino acids at at least one of the following positions of the amino acid sequence of SEQ ID NO: 1: 318, 331, 346, 359, 366, 374, 404, 423, 424, 430, 448, 473, 488, and the protein mutant has the same protein binding and DNA binding function and nanopore formation function; 3) a porin monomer that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein in 1) or 2), and has the same protein binding and DNA binding function and nanopore formation function; preferably, the MCM protein monomer comprises a protein consisting of the amino acid sequence of SEQ ID NO: 1; The amino acid sequence shown in NO: 2 has a core region with a greater than 90% identity.

[0043] In a preferred embodiment, the MCM protein comprises a protein composed of 6 MCM protein monomers linked non-covalently.

[0044] In this application, a biological nanopore sensor specifically refers to a detector composed of a nanopore protein inserted into a membrane. This type of nanopore sensor incorporates a pore-bearing MCM protein embedded within the membrane. The pores of the MCM protein penetrate the membrane, connecting the two sides of the membrane. When an electric field is applied, ions on both sides of the membrane migrate through the nanopore channel, generating an electric current. A schematic diagram of a biological nanopore sensor based on MCM proteins is shown in Figure 1.

[0045] The MCM protein monomer shown in SEQ ID NO: 1 has high temperature resistance and is modified from the wild-type protein Sso MCM shown in SEQ ID NO: 3 from the thermophilic archaeon Saccharolobus solfataricus.

[0046]

[0047] The MCM protein monomer represented by SEQ ID NO:1 is a highly conserved protein. Proteins that share 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% or greater identity with the protein, share the same protein- and DNA-binding functions, and possess the same pore-forming structure are also MCM proteins and can be inserted into membranes to form nanopore sensors with the same functionality. Properties such as protein and aggregate stability, pore size, pore lining amino acid residues, and biomolecule passage capacity are likely identical to those of the protein represented by SEQ ID NO:1. This "same functionality" includes, but is not limited to, enabling DNA or protein biomolecules to pass through the MCM protein pore under the influence of an electric field, generating a current corresponding to each nucleotide or amino acid.

[0048] Identity as used herein refers to the "identity" between amino acid sequences or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides in an amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0049] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the sequence in 1).

[0050] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0051] Generally speaking, according to the rules of substitution, replacement, etc., amino acids with similar properties will have similar effects when substituted with each other. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0052] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0053] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0054] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0055] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0056] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0057] The MCM protein family has a highly conserved core region consisting of approximately 200 amino acid residues, as shown in SEQ ID NO: 2. This core region can significantly influence the function and three-dimensional structure of the MCM protein. Therefore, proteins with a high degree of identity (90%, 95%, 97%, 98%, 99%, or even greater than 99.5%) with this core region also have similar "same function" and three-dimensional structure (including but not limited to the pore).

[0058]

[0059] In a preferred embodiment, a) comprises: placing an organic solution containing phospholipids in a container to form a lipid film on the container wall; adding a solution containing a hydrophilic protein to the container and shaking to hydrate the lipid film; extruding and filtering the hydrated lipid film solution to obtain liposomes; preferably, the phospholipid comprises diphytylphosphatidylcholine, more preferably 1,2-diphytanoyl-sn-glyceryl-3-phosphocholine; preferably, the filtration comprises filtering using a polycarbonate membrane, more preferably the diameter of the polycarbonate membrane is 0.1-1 μm, more preferably 0.4 μm.

[0060] Hydrophilic proteins such as MCM proteins can be prepared into MCM liposomes using the above methods, or methods known in the art for preparing liposomes. Agitation includes, but is not limited to, ultrasonic agitation and vortex agitation. Maintaining the consistency of the composition of the phospholipids and porins inserted into the phospholipid membrane helps maintain system stability.

[0061] In a preferred embodiment, b) includes: mixing liposomes and a membrane layer in a solution and applying a voltage to both sides of the solution to cause the liposomes to insert into the membrane layer, while monitoring the current in the solution; when the current is constant, it indicates that the liposomes are inserted into the membrane layer, and a biological nanopore sensor is obtained; preferably, b) further includes: calculating the conductivity, conductivity = current / voltage; when the conductivity is ≥2.8nS, the liposomes are inserted into the membrane layer, and a biological nanopore sensor is obtained; preferably, the voltage is greater than 0V and less than or equal to 0.5V; preferably, after obtaining the nanopore sensor, the voltage is reduced to 0V.

[0062] After mixing the prepared liposomes with the membrane in a solution, a voltage is applied to the solution. Under the influence of the voltage, the liposomes insert into the membrane. The current in the solution is monitored in real time, and the conductivity of the membrane is calculated using the formula "conductivity = current / voltage." When the hydrophilic protein nanopores penetrate the membrane, the conductivity increases, connecting the two ends of the membrane. This increases the current and remains constant over time, indicating that a biological nanopore sensor has been obtained.

[0063] Preferably, after the conductivity increases, the applied voltage is reduced to prevent further hydrophilic proteins from continuing to insert into the membrane, thereby obtaining a bionanopore sensor with a single hydrophilic protein embedded. In this application, protein embedding or insertion refers to the situation in which proteins penetrate the membrane layer in the cell flow mosaic theory; in particular, it refers to proteins with pores that penetrate the membrane layer and connect the pores on both sides of the membrane layer, rather than proteins embedded in the membrane surface.

[0064] In a preferred embodiment, after obtaining the biological nanopore sensor, the method further comprises washing with a buffer solution to remove residual free hydrophilic proteins.

[0065] In order to prevent the hydrophilic proteins not inserted into the membrane layer from affecting subsequent operations such as sequencing, the nanopore sensor is rinsed after being obtained to remove free hydrophilic proteins.

[0066] In a preferred embodiment, the membrane layer comprises a lipid layer or an artificial polymer membrane; preferably, the lipid layer comprises amphiphilic lipids; preferably, the amphiphilic lipids comprise a phospholipid bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilayer liposome or a unilamellar liposome; preferably, the lipid layer comprises a phospholipid bilayer composed of diphytylphosphatidylcholine, more preferably a phospholipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine.

[0067] Artificial polymer membranes include, but are not limited to, polysiloxanes, polyolefins, perfluoropolyethers, perfluoroalkyl polyethers, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinyl chloride, polyalkyl acrylates, polyalkyl methacrylates, polyacrylonitrile, polypropylene, PTHF, polymethacrylates, polyacrylates, polysulfones, polyethylene ethers, poly(propylene oxide) and copolymers thereof, alkyl-substituted C1-C6 alkyl acrylates and methacrylates, acrylamide, methacrylamide, (C1-C6 alkyl) acrylamide and methacrylamide, N,N-dialkyl-acrylamide, ethoxy acrylate and methacrylate, polyethylene glycol monomethacrylate and polyethylene glycol monomethyl ether methacrylate, hydroxy-substituted (C1-C6 alkyl) acrylamide and methacrylamide, hydroxy-substituted C1-C6 alkyl vinyl ether, sodium vinyl sulfonate, sodium styrene sulfonate, 2-acrylamide-2-methyl The invention also includes one or more of ethylenically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(C1-C6 alkyl)-, mono(C1-C6 alkylamino)(C1-C6 alkyl)- and bis(C1-C6 alkylamino)(C1-C6 alkyl)-acrylates and methacrylates, allyl alcohol, 2-hydroxypropyl 3-trimethylammonium methacrylate chloride, dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide, cyclic imino ethers, vinyl ethers, cyclic ethers including epoxy derivatives, cyclic unsaturated ethers, N-substituted ethylenimines, β-lactones and β-lactams, ketene acetals, vinyl acetals or phosphoranes.

[0068] In a second typical embodiment of the present application, a biological nanopore sensor is provided. The biological nanopore sensor is a biological nanopore sensor prepared by the above method.

[0069] In a third typical embodiment of the present application, a biological nanopore sensor is provided, which includes: a membrane layer and a hydrophilic protein with nanopores, the nanopores of the hydrophilic protein penetrate the membrane layer, and when an electric field force is applied across the membrane layer, an electric current is formed in the pores.

[0070] In a preferred embodiment, the hydrophilic protein comprises MCM protein.

[0071] In a preferred embodiment, the MCM protein comprises a protein formed by covalently or non-covalently linking two or more MCM protein monomers; the MCM protein monomers comprise: 1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a protein mutant, wherein the amino acid sequence of the protein mutant is shown in SEQ ID NO: 1) wherein at least one of the following positions of the amino acid sequence of SEQ ID NO: 1 is substituted, deleted, and / or one or more amino acids are added: 318, 331, 346, 359, 366, 374, 404, 423, 424, 430, 448, 473, 488, and the protein mutant has the same protein binding and DNA function and the ability to form a nanopore; 3) a porin monomer having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein of 1) or 2), and having the same protein binding and DNA function and the ability to form a nanopore; preferably, the MCM protein comprises a protein composed of 2-10 MCM protein monomers linked by covalent or non-covalent bonding; preferably, the MCM protein comprises a protein composed of 6 MCM protein monomers linked by non-covalent bonding; preferably, the MCM protein monomer contains a core region having more than 90% identity with the amino acid sequence of SEQ ID NO: 2.

[0072] In a fourth typical embodiment of the present application, a nanopore sequencing device is provided, which includes a biological nanopore sensor prepared by the above method, or the above biological nanopore sensor.

[0073] The aforementioned nanopore sequencing device, with the exception of the improved nanopore sensor, can maintain the same structural components as those in the prior art, or may be adjusted accordingly based on the differences in the nanopore sensor of the present application. In a preferred embodiment, the nanopore sequencing device comprises: an electrolytic cell containing an electrolyte; a nanopore sensor located in the center of the electrolytic cell, dividing the cell and electrolyte into a positive electrolyte region and a negative electrolyte region; and a first electrode and a second electrode, the first electrode and the second electrode being disposed in the positive electrolyte region and the negative electrolyte region, respectively.

[0074] In a fifth typical embodiment of the present application, a sequencing method is provided, which includes using a biological nanopore sensor prepared by the above method, or the above biological nanopore sensor, or the above nanopore sequencing device to detect and analyze the electrical signal generated when the biological molecule to be tested passes through the nanopore channel of the hydrophilic protein, and determine the sequence of the biological molecule to be tested.

[0075] In a preferred embodiment, the hydrophilic protein includes an MCM protein; preferably, the biomolecule to be detected includes modified or unmodified DNA, RNA, or polypeptide; preferably, the polypeptide includes a polypeptide monomer or a polypeptide polymer; preferably, the electrical signal includes current amplitude and / or residence time.

[0076] In a sixth typical embodiment of the present application, a biological nanopore sensor prepared by the above method, or the above biological nanopore sensor, or the above nanopore sequencing device, or the above sequencing method is provided, and its application in nucleic acid detection, protein detection or protein conformation differentiation is provided.

[0077] In the detection of biological samples, the aforementioned nanopore sensors, or methods for preparing nanopore sensors, or nanopore sequencing devices, or sequencing methods can sequence biological samples (including but not limited to DNA, RNA, or polypeptides) to obtain their specific sequences; they can also identify and differentiate the three-dimensional structures (e.g., monomers, polymers, secondary structures, tertiary structures, or quaternary structures) of the aforementioned biological samples, such as detecting and differentiating different polypeptides, and can also be used to distinguish between polypeptide monomers and polymers. The aforementioned protein detection includes protein marker detection, as well as further application to screening for related diseases or tumors.

[0078] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0079] Example 1 Cloning, expression and purification of MCM

[0080] The MCM gene was synthesized into the pet28a(+) vector with his-tag and transformed into BL21 competent cells for expansion culture. 600 When the p-value reaches 0.6-1, 500 μM IPTG is added and the temperature is adjusted to 16°C overnight to induce protein expression. The harvested cells are crushed under high pressure and centrifuged at 20,000 rpm to remove unbroken cells and relatively large membrane pieces; the supernatant is collected and the protein is purified by IMAC (Immobilized metal affinity chromatography), ion-exchange, and SEC (size-exclusion chromatography). After purification, a large amount of MCM protein with good purity can be obtained, with an average of about 10 mg of target protein purified per gram of bacteria. The molecular sieve Superdex 200 purification results of the MCM protein and the gel image results are shown in Figures 2A and 2B.

[0081] Example 2 ATPase activity detection of MCM protein

[0082] (1) Preparation of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA)

[0083] DNA1 (SEQ ID NO: 4) and DNA2 (SEQ ID NO: 5) were dissolved in TE buffer (pH = 8) to prepare stock solutions with a final concentration of 100 μM.

[0084] DNA1 (SEQ ID NO: 4): agcgtcgaaaagcagtacttaggcatt.

[0085] DNA2 (SEQ ID NO: 5): aatgcctaagtactgcttttcgacgcttttttttttttttttttttt.

[0086] 100 μM DNA2 was diluted to 10 μM with TE buffer (pH = 8) to prepare ssDNA. DNA1 and DNA2 were annealed to form dsDNA with 20 T residues overhanging the 3' end. The annealing process was incubation at 95°C for 5 minutes, cooling to 25°C at a rate of 0.1°C / s, and incubation for another 30 minutes. The annealing recipe is shown in Table 1.

[0087] Table 1 dsDNA annealing formula

[0088] Solution volume: 100 μM DNA 15 μL, 100 μM DNA 25 μL, TE buffer (pH = 8) 40 μL

[0089] (2) Prepare reaction buffer (2×)

[0090] Reaction buffer 1 consisted of 20 mM HEPES (pH 8.0), 4 mM ATP, 4 mM MgCl2, and 0.3 M KCl;

[0091] Reaction buffer 2 consisted of 20 mM HEPES (pH 8.0), 4 mM ATP, 4 mM MgCl2, and 1.0 M KCl.

[0092] (3) Dilute MCM protein

[0093] The MCM protein obtained in the example was diluted to 10 μM with 1×PBS.

[0094] (4) ATP hydrolysis reaction

[0095] Add the corresponding reagents according to the reaction system in Table 2, mix thoroughly, incubate at 30°C for 30 min, inactivate at 80°C for 5 min, and maintain at 4°C. ①②③ are experimental groups, and ④⑤⑥ are corresponding control groups.

[0096] Table 2 ATP hydrolysis reaction system

[0097] No. Reaction buffer (2×) DNAMCMH2O①10μL1μL (dsDNA)1μL8μL②10μL1μL (ssDNA)1μL8μL③10μL——1μL9μL④10μL1μL (dsDNA)——9μL⑤10μL1μL (ssDNA)——9μL⑥10μL————10μL

[0098] (5) Detection of the remaining ATP in the reaction

[0099] The reaction product obtained in step (4) was diluted 250 times with H2O to prepare the test sample; 0.1μM, 0.3μM, 1μM, 3μM, and 10μM ATP solutions were prepared as standard products. The residual ATP concentration of the reaction was determined using an ATP detection kit (Biyuntian, S0026B). 50μL of the ATP detection working solution in the detection kit and 10μL of the test sample or standard were mixed in a black microplate, reacted for 30 minutes, and the chemiluminescence value was detected by a microplate reader. Each sample was measured three times. Since the fluorescence intensity is proportional to the concentration of ATP, the concentration of ATP in the solution can be obtained by measuring the fluorescence intensity of the solution after the reaction. The results are shown in Figures 3A and 3B. As can be seen from Figures 3A and 3B, at salt concentrations of 0.15M and 0.5M, the presence of MCM protein will lead to a decrease in fluorescence intensity, indicating that MCM protein has the activity of hydrolyzing ATP under 0.15M and 0.5M KCl conditions. ①, ②, ③, ④, ⑤, and ⑥ are MCM+dsDNA group, MCM+ssDNA group, MCM group, dsDNA group, ssDNA group, and Control group (negative control group), respectively.

[0100] Example 3 Insertion of MCM proteoliposomes into phospholipid bilayer membranes

[0101] (1) Preparation of MCM liposomes

[0102] A 100 μL chloroform solution of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids) at a concentration of 10 mg / mL was placed in a round-bottom flask. The chloroform was removed using a rotary evaporator, forming a lipid film on the wall of the round-bottom flask. Then, 300 μL of a hydration buffer solution (3 M KCl, 5 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 250 mM Sucrose, pH 7.6) containing MCM protein (concentration: 1 mg / mL) was added and vortexed until the film was completely hydrated. The proteoliposomes were homogenized using an extruder (Avanti Polar Lipids). The prepared MCM liposomes were filtered 30 times through a 0.4 μm polycarbonate membrane and the resulting MCM liposome solution was stored at -20°C until further use.

[0103] (2) Inserting MCM proteoliposomes into the phospholipid bilayer membrane

[0104] The experiment was conducted using a phospholipid bilayer lipid membrane formed by DPhPC from Avanti Polar Lipids. 50 μL of the MCM liposome solution obtained in step (1) was mixed evenly with 250 μL of buffer (0.47 M KCl, 25 mM HEPES, 1 mM EDTA, pH = 8.1) and added to the flow cell containing the phospholipid bilayer through the injection port. A voltage of 0.1 V was applied to promote the insertion of the MCM liposome into the phospholipid membrane. Once inserted into the membrane, the current increased to about 360 pA. The original current graph is shown in Figure 4. As shown in Figure 4, the current was approximately 0 pA when not embedded. A voltage of 0.1 V was applied, and the current was approximately 360 pA after embedding into the phospholipid layer. After the MCM protein was successfully embedded in the phospholipid membrane, the voltage was changed to 0.02 V, at which time the current was approximately 90 pA.

[0105] A schematic diagram of the MCM protein nanopore biomembrane is shown in Figure 1. The MCM protein is inserted into the phospholipid bilayer membrane using liposomes. The analyte passes through the pore of the MCM protein pore (MCM channel), causing a change in the electrical signal. The analyte is analyzed by analyzing the changed electrical signal.

[0106] (3) Electrical characterization

[0107] After completing the insertion, the embedded hole buffer (0.47M KCl, 25mM HEPES, 1mM EDTA, pH = 8.1) was used to push away the MCM protein remaining in the flow cell. The electrical properties of the nanopore biofilm were evaluated by applying different voltages (+0.02V, -0.02V, +0.03V, -0.03V, 0.04V, -0.04V, +0.05V) and recording the corresponding current values ​​(Figures 5A and 5B). At a voltage of 0.02V, the MCM channel was stable, and the current of a single MCM channel was about 60pA. Multiple voltage and current data after a single embedded hole were collected, and the conductivity value of MCM in the sequencing buffer was calculated by the formula (conductivity = current / voltage) to be approximately 2.8nS (as shown in Figure 5C).

[0108] Example 4: Using MCM to differentiate different polypeptides

[0109] The polypeptide used in this example was synthesized and purified by GenScript, and the sequence is:

[0110] Peptide 1 peptide: RRRRRRRRRR (SEQ ID NO: 6).

[0111] Peptide 2 peptide: CYGRKKRRQRRR (SEQ ID NO: 7).

[0112] To explore the potential biomedical applications of MCM channels, pore translocation signals from two peptides of different lengths and sequences were collected. The peptides were added to 300 μL of buffer solution (final concentration: 1-10 μg / mL), mixed thoroughly, and then added to the pre-welled flow cell via the inlet. A voltage of -0.02 V was applied to observe peptide translocation signals, and data were collected at a frequency of 5 kHz. Data were processed using Clampfit 10.7 software and MOSAIC. The raw data are shown in Figures 6A and 7A. Peptide 1 and Peptide 2 are positively charged, with Peptide 1 existing as a monomer. Peptide 2, however, forms a dimer due to the presence of a terminal cysteine. The pore opening current was denoted as I0, and the current during pore translocation was denoted as I. Pore translocation events were analyzed using MOSAIC. Compared to the current signal from Peptide 2, the amplitude of the pore translocation from Peptide 1 was smaller and the residence time was shorter (Figures 6B, 6C, 7B, and 7C). The two peptides can be distinguished by amplitude and residence time, which demonstrates the potential of MCM as a biological nanopore in distinguishing different peptides.

[0113] Example 5: Using MCM to distinguish between monomeric and polymeric forms of polypeptides

[0114] The polypeptide used in this example was synthesized and purified by GenScript, and the sequence is:

[0115] Peptide 2 peptide: CYGRKKRRQRRR (SEQ ID NO: 7).

[0116] To distinguish between different forms of polypeptides, this embodiment uses Peptide 2. Peptide 2 has a cysteine ​​residue at its terminal end. Cysteine ​​residues can form disulfide bonds between two polypeptides, thereby forming dimers. Tris(2-carboxyethyl)phosphine (TCEP) is a thiol reducing agent with the property of reducing disulfide bonds. After TCEP treatment, the disulfide bonds of the polypeptide dimers are reduced, and the polypeptides exist as monomers.

[0117] Peptides were added to a buffer solution containing 1 mM tris(2-carboxyethyl)phosphine (TCEP) (final concentration: 1-10 μg / mL) and mixed thoroughly. After incubation at room temperature for 30 minutes, the peptides were added to the flow cell via the inlet. A voltage of -0.02 V was applied to observe peptide translocation signals, and data were collected at a frequency of 5 kHz. Data were processed using Clampfit 10.7 software and MOSAIC. The raw data and processed results are shown in Figure 7. Under the influence of the electric potential, peptides passed through the MCM channel, generating distinct electrical signals. Peptide 2, which had not been treated with TCEP, was more charged and exhibited more translocation events than the reduced peptide (Figures 7A and 8A). Furthermore, the TCEP-treated Peptide 2, which was in monomeric form, exhibited lower amplitudes and shorter residence times (Figures 8B and 8C). Different peptides passed through the nanopore, generating distinct detection signals, enabling differentiation of peptides based on these signal differences. This example demonstrates the potential of the MCM as a biological nanopore for distinguishing different peptide forms.

[0118] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: Using the technical solutions of the present invention, a nanopore sensor constructed using MCM proteins and a membrane layer has pores extending through both sides of the membrane layer. When an electric field is applied across the membrane layer, charges can migrate directionally within the pores, thereby generating an electric current. During use, a biological sample to be tested can pass through the MCM protein channel under the action of an electric potential, generating different electrical signals, enabling operations such as sequencing and identification of the biological sample. MCM proteins can be expressed in large quantities in expression systems such as Escherichia coli. Since they are not native membrane proteins and cannot autonomously insert into the membrane layer of the expression system, they are easy to purify and mass-produce, significantly reducing production costs. In addition, as multimeric MCM proteins contain both pores and helicases with unwinding activity, they can simultaneously perform the functions of a porin and a helicase in a biological nanopore sensor. A biological nanopore sensor prepared using this protein can combine the functions of two independent proteins required for conventional mid-chain sequencing methods, and has excellent application prospects.

[0119] 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 preparing a biological nanopore sensor, characterized in that: The method comprises: a) preparing the hydrophilic protein into liposomes; b) mixing the liposomes with a membrane layer to enable the liposomes to insert into the membrane layer, thereby obtaining the biological nanopore sensor; Wherein, the hydrophilic protein has nanopores.

2. The method according to claim 1, characterized in that The hydrophilic protein includes MCM protein, and the step a) includes preparing the MCM protein into MCM liposomes.

3. The method according to claim 2, characterized in that The MCM protein includes a protein formed by covalently or non-covalently linking two or more MCM protein monomers; Preferably, the MCM protein monomer comprises: 1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a protein mutant, wherein the amino acid sequence of the protein mutant is substituted, deleted, and / or one or more amino acids are added at at least one of the following positions of the amino acid sequence of SEQ ID NO: 1: 318, 331, 346, 359, 366, 374, 404, 423, 424, 430, 448, 473, 488, and the protein mutant has the same protein and DNA binding function and the function of forming a nanopore; 3) a porin monomer that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the protein described in 1) or 2), has the same protein-binding and DNA-binding functions, and has the function of forming a nanopore; Preferably, the MCM protein monomer contains a core region having more than 90% identity with the amino acid sequence shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that The MCM protein includes a protein formed by non-covalently linking 6 MCM protein monomers.

5. The method according to claim 1, wherein The a) mentioned above includes: placing an organic solution containing phospholipids in a container to form a lipid film on the container wall; adding a solution containing the hydrophilic protein into the container and shaking the container to hydrate the lipid film; Extruding and filtering the solution after the lipid membrane is hydrated to obtain the liposome; Preferably, the phospholipid comprises diphytylphosphatidylcholine, more preferably 1,2-diphytanoyl-sn-glycero-3-phosphocholine; Preferably, the filtering comprises filtering using a polycarbonate membrane, more preferably the diameter of the polycarbonate membrane is 0.1-1 μm, more preferably 0.4 μm.

6. The method according to claim 1, wherein b) includes: mixing the liposomes with the membrane layer in a solution and applying a voltage to both sides of the solution to cause the liposomes to insert into the membrane layer, while monitoring the current in the solution; When the current is constant, it indicates that the liposome is inserted into the membrane layer, thereby obtaining the biological nanopore sensor; Preferably, the step b) further comprises: calculating conductivity, wherein the conductivity = the current / the voltage; when the conductivity is ≥ 2.8 nS, inserting the liposome into the membrane layer to obtain the biological nanopore sensor; Preferably, the voltage is greater than 0V and less than or equal to 0.5V; Preferably, after obtaining the biological nanopore sensor, the voltage is reduced to 0V.

7. The method according to claim 6, characterized in that After obtaining the biological nanopore sensor, the method further comprises washing with a buffer solution to remove residual free hydrophilic proteins.

8. The method according to claim 1, characterized in that The membrane layer includes a lipid layer or an artificial polymer membrane; Preferably, the lipid layer comprises amphiphilic lipids; Preferably, the amphiphilic lipid comprises a phospholipid bilayer; Preferably, the lipid layer comprises a planar membrane layer or a liposome; Preferably, the liposomes comprise multilamellar liposomes or unilamellar liposomes; Preferably, the lipid layer comprises a phospholipid bilayer composed of diphytylphosphatidylcholine, more preferably a phospholipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine.

9. A biological nanopore sensor, characterized in that The biological nanopore sensor is a biological nanopore sensor prepared by the method according to any one of claims 1 to 8.

10. A biological nanopore sensor, characterized in that: The biological nanopore sensor comprises: a membrane layer and a hydrophilic protein having nanopores, wherein the nanopores of the hydrophilic protein penetrate the membrane layer. When an electric field is applied across the membrane, an electric current is formed in the pores.

11. The biological nanopore sensor according to claim 10, characterized in that The hydrophilic protein includes MCM protein.

12. The biological nanopore sensor according to claim 11, characterized in that The MCM protein includes a protein formed by covalently or non-covalently linking two or more MCM protein monomers; The MCM protein monomer includes: 1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a protein mutant, wherein the amino acid sequence of the protein mutant is substituted, deleted, and / or one or more amino acids are added at at least one of the following positions of the amino acid sequence of SEQ ID NO: 1: 318, 331, 346, 359, 366, 374, 404, 423, 424, 430, 448, 473, 488, and the protein mutant has the same protein and DNA binding function and the ability to form a nanopore function; 3) a porin monomer that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the protein described in 1) or 2), and has the same protein-binding and DNA-binding functions, and has the ability to form a nanopore; Preferably, the MCM protein comprises a protein composed of 2-10 MCM protein monomers linked covalently or non-covalently; Preferably, the MCM protein comprises a protein formed by non-covalently linking 6 MCM protein monomers; Preferably, the MCM protein monomer contains a core region having more than 90% identity with the amino acid sequence shown in SEQ ID NO:

2.

13. A nanopore sequencing device, characterized in that The nanopore sequencing device comprises a biological nanopore sensor prepared by the method according to any one of claims 1 to 8, or the biological nanopore sensor according to claim 9, or the biological nanopore sensor according to any one of claims 10 to 12.

14. The nanopore sequencing device according to claim 13, wherein: The nanopore sequencing device comprises: an electrolytic cell containing an electrolyte; a biological nanopore sensor, the biological nanopore sensor being located in the center of the electrolytic cell and dividing the electrolytic cell and the electrolyte into a positive electrode electrolyte region and a negative electrode electrolyte region; A first electrode and a second electrode, wherein the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte region and the negative electrode electrolyte region.

15. A sequencing method, characterized in that: The sequencing method comprises using a biological nanopore sensor prepared by the method of any one of claims 1 to 8, or the biological nanopore sensor of claim 9, or the biological nanopore sensor of any one of claims 10 to 12, or the nanopore sequencing device of claim 13 or 14 to detect and analyze the electrical signal generated when the biological molecule to be tested passes through the nanopore of the hydrophilic protein, and determine the sequence of the biological molecule to be tested.

16. The sequencing method according to claim 15, characterized in that The hydrophilic protein includes MCM protein; Preferably, the biomolecule to be detected includes modified or unmodified DNA, RNA, or polypeptide; Preferably, the polypeptide comprises a polypeptide monomer or a polypeptide polymer; Preferably, the electrical signal includes current amplitude and / or dwell time.

17. Use of a biological nanopore sensor prepared by the method of any one of claims 1 to 8, or a biological nanopore sensor according to claim 9, or a biological nanopore sensor according to any one of claims 10 to 12, or a nanopore sequencing device according to claim 13 or 14, or a sequencing method according to claim 15 or 16 in nucleic acid detection, protein detection, or protein conformation differentiation.

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