WO2023124927A1 - 嵌入接头、锚定分子、分子膜、装置、方法及应用 - Google Patents

嵌入接头、锚定分子、分子膜、装置、方法及应用 Download PDF

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WO2023124927A1
WO2023124927A1 PCT/CN2022/138280 CN2022138280W WO2023124927A1 WO 2023124927 A1 WO2023124927 A1 WO 2023124927A1 CN 2022138280 W CN2022138280 W CN 2022138280W WO 2023124927 A1 WO2023124927 A1 WO 2023124927A1
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analyte
linker
embedded
hydrophobic
segment
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PCT/CN2022/138280
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English (en)
French (fr)
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刘少伟
张子朋
朱丽梅
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北京齐碳科技有限公司
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Publication of WO2023124927A1 publication Critical patent/WO2023124927A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present application belongs to the field of gene sequencing technology, and in particular relates to a device for embedding adapters, anchor molecules, molecular membranes, and nanopores to characterize biomolecules, a method for assembling embedded adapters in amphiphilic molecular membranes, and enriching biomolecules into transmembrane nanopores. Methods for analytes, methods and applications for nanopore characterization of analytes.
  • a molecular membrane with nanoscale pores divides the cavity into two small chambers.
  • ions or other small molecular substances can pass through the small chambers.
  • the pores form a stable and detectable ionic current, and different types of biomolecules can be detected according to the current information.
  • Anchor molecules are provided in existing molecular membranes to bind biomolecules through the anchor molecules, so that the biomolecules are aggregated on the surface of the molecular membrane.
  • the existing anchor molecules are usually cholesterol with tethers, and the cholesterol is evenly distributed on the molecular membrane, while the nanopore is only set at the amphiphilic molecular membrane of the molecular membrane, such as a phospholipid membrane or a polymer membrane, causing some parts to be far away from the nanometer membrane.
  • the biomolecules in the pores are not easy to be detected, especially when the concentration of biomolecules is low, the effective current data output time is short, which affects the detection effect.
  • the embodiments of the present application provide a device for characterizing biomolecules in embedded adapters, anchor molecules, molecular membranes, and nanopores, a method for assembling embedded adapters in amphiphilic molecular membranes, and a method for enriching analytes into transmembrane nanopores .
  • the method and application of the nanopore characterization analyte can effectively anchor the analyte near the transmembrane nanopore and increase the output time of the effective current data corresponding to the analyte.
  • an embedded linker which is used to be embedded in an amphiphilic molecular membrane, including:
  • the first hydrophilic segment, the hydrophobic segment and the second hydrophilic segment connected in sequence, the hydrophobic segment is used to cross the hydrophobic region of the amphiphilic molecular membrane, so that the embedded joint can penetrate the amphiphilic molecular membrane, and the hydrophobic segment has rigidity;
  • the first hydrophilic segment and/or the second hydrophilic segment can be attached to an analyte.
  • the hydrophobic segment is rod-shaped.
  • the hydrophobic segment is a hydrophobic polypeptide.
  • the hydrophobic polypeptide has an alpha helical structure
  • the first hydrophilic segment and/or the second hydrophilic segment is a hydrophilic polypeptide, and optionally, the embedded linker is a polypeptide.
  • the hydrophobic segment is a transmembrane polypeptide
  • the hydrophobic segment can also be a multi-membrane, non-rod-shaped, rigid polypeptide, and the polypeptide can be aggregated to the amphiphile membrane.
  • the hydrophobic segment at least includes alanine residues (A), arginine residues (R), leucine residues (L), lysine residues (K), methionine residues One or more of the groups (M).
  • the hydrophobic matching difference between the hydrophobic segment and the hydrophobic region is less than or equal to 5 nm.
  • the distribution density of acidic amino acid residues near the end of the linker is smaller than the distribution density of acidic amino acid residues at the end far away from the linker;
  • the distribution density of basic amino acid residues near the end of the linker is greater than the distribution density of basic amino acid residues away from the end of the linker.
  • the hydrophobic segment is a hydrophobic copolymer
  • the first hydrophilic segment and/or the second hydrophilic segment is a hydrophilic copolymer, and optionally, the embedded linker is a tri-block polymer.
  • non-single bonds are used between at least some adjacent molecules in the hydrophobic segment; optionally, the hydrophobic copolymer is any one of polythiophene, polyphenylene vinylene, and polyphenanthrene acetylene.
  • the present application also provides an anchor molecule, which is used to anchor the analyte on the amphiphilic molecular membrane, and the anchor molecule includes the embedding linker and the connection linker as provided in the first aspect, the linker linker A moiety capable of being attached to an analyte and a moiety capable of being attached to an embedded linker are included.
  • the connecting joint is provided at one or both ends of the embedded joint, and optionally, the connecting joint is provided at one end of the embedded joint.
  • the linker includes a capture strand capable of complementary base-pairing connection with the analyte, and the capture strand is linked to the insertion linker through a click chemical reaction.
  • the present application also provides a method for assembling an embedded joint in an amphiphilic molecular membrane, comprising contacting the embedded joint provided in the first aspect with a molecular membrane, the molecular membrane including a lipid film and an amphiphilic molecular membrane, and then embedding The linker is embedded in the amphiphilic molecular membrane.
  • the present application also provides a molecular membrane for characterizing analytes through nanopores.
  • the molecular membrane includes a lipid membrane and an amphiphilic molecular membrane, and the amphiphilic molecular membrane is embedded with a transmembrane nanopore and embedded as provided in the first aspect.
  • Linker or anchor molecule as provided on the second opposite side.
  • the present application also provides a nanopore device for characterizing analytes, including:
  • Molecular membranes including lipid membranes and amphiphilic molecular membranes distributed on the biochip, the amphiphilic molecular membranes are provided with transmembrane nanopores;
  • the present application also provides a method for enriching the analyte to the transmembrane nanopore, comprising the following steps:
  • amphiphile molecular membrane is contacted with the embedding linker as provided in the first aspect, and the embedding linker is contacted with the connecting linker connecting the analyte, or the amphiphile molecular membrane is contacted with the anchor molecule as provided in the second aspect, and the The anchor molecule is contacted with the analyte;
  • the analyte approaches the transmembrane nanopore under the pull of the embedded adapter.
  • the present application also provides a method for characterizing an analyte through a nanopore, including enriching the analyte into the transmembrane nanopore according to the method provided in the sixth aspect, and making the analyte contact the transmembrane nanopore,
  • an electrical signal is measured to characterize the analyte in order to determine one or more characteristics of the analyte.
  • the present application also provides an anchor molecule as provided in the first aspect, or an anchor molecule as provided in the second aspect, or a molecular membrane as provided in the fourth aspect, or a device as provided in the fifth aspect Application in analyte characterization;
  • the analytes include biomolecules, including polynucleotides, polypeptides, polysaccharides, or lipids;
  • the polynucleotide comprises DNA or RNA; and/or, the characterizing characteristic is selected from (i) the length of the polynucleotide; (ii) the identity of the polynucleotide; (iii) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide and (v) whether the polynucleotide has been modified.
  • the embedded linker, the anchor molecule, the molecular membrane, the device for characterizing biomolecules through nanopores, the method for enriching analytes into transmembrane nanopores, the method for characterizing analytes through nanopores, and their applications are provided with the first
  • the embedded joints of the first hydrophilic segment, the hydrophobic segment and the second hydrophilic segment cannot exist stably on the lipid membrane, but the stability of the combination in the amphiphilic molecular membrane is better, so that the anchor molecules can gradually aggregate and distribute on the amphiphilic membrane.
  • the anchor molecule can be connected to the analyte, so that the analyte connected to the anchor molecule can gather near the amphiphile molecular membrane, that is, the analyte is close to the transmembrane nanopore on the amphiphile molecular membrane , increase the number of analytes through the hole, thereby increasing the output time of the effective detection current data corresponding to the analyte;
  • the hydrophobic segment in the shape of a rigid rod can avoid the first hydrophilic segment and the second hydrophilic segment in the same anchor molecule Segment attraction makes the anchor molecule bend, so as to prevent the bent embedded joint from being fixed on the lipid film through the hydrophobic segment and not moving to the amphiphile molecular membrane.
  • Fig. 1 is a schematic structural diagram of an anchor molecule in an embodiment of the present application
  • Fig. 2 is a partial structural schematic diagram of a device for characterizing an analyte through a nanopore in an embodiment of the present application
  • Fig. 3 is a schematic structural diagram of an anchor molecule in another embodiment of the present application.
  • Fig. 4 is a transmembrane prediction diagram of an embedded linker in an embodiment of the present application.
  • Figure 5 is a chromatogram of an embedded joint in an embodiment of the present application.
  • Fig. 6 is a chromatogram of an incubation product of a linker and an embedded linker in an embodiment of the present application
  • Figure 7 is a prediction map of the transmembrane region embedded in the linker in another embodiment of the present application.
  • Fig. 8 is a fluorescence luminescence diagram of an embedded linker incubated on a molecular membrane for 0.5 h in an embodiment of the present application;
  • Fig. 9 is a fluorescence luminescence diagram of an embedded linker incubated on a molecular membrane for 12 hours in an embodiment of the present application;
  • Fig. 10 is a nanopore sequencing signal diagram of a control group of the present application.
  • Fig. 11 is a nanopore sequencing via signal diagram of an experimental group of the present application.
  • Fig. 12 is the nanopore sequencing signal frequency diagram of an experimental group and a control group of the present application.
  • Figure 13 is a prediction map of the transmembrane region of the polypeptide in a ratio of the present application.
  • Fig. 14 is a hydrophobicity prediction diagram of polypeptides in an embodiment of the present application and a comparative example
  • Fig. 15 is a secondary structure prediction diagram of polypeptides in an embodiment of the present application and a comparative example
  • Figure 16 is a chemical structure diagram of an embedded linker in an embodiment of the present application.
  • Fig. 17 is a diagram of the chemical structure of the embedded linker in a pair of examples of the present application.
  • the embodiment of the present application provides an embedded linker, an anchor molecule, a kit, a device for characterizing biomolecules through nanopores, a method for enriching analytes into transmembrane nanopores, and a nanopore for characterizing analytes methods and applications.
  • the anchor molecules provided in the examples of the present application are firstly introduced below.
  • the anchor molecule includes an embedding linker 1 and a connection linker 2, the embedding linker 1 is used for embedding on the amphiphilic molecular membrane, and the embedding linker 1 includes a first hydrophilic section 11, a hydrophobic section connected in sequence 12 and the second hydrophilic segment 13, the hydrophobic segment 12 is used to cross the hydrophobic region of the amphiphilic molecular membrane, so that the embedded joint 1 can penetrate the amphiphilic molecular membrane, and the hydrophobic segment 12 is a rigid rod; the first hydrophilic segment 11 and/or Or the second hydrophilic section 13 is used to connect with the connecting joint 2, and the connecting joint 2 can be connected with the analyte.
  • the analyte can be a biomolecule, and the anchor molecule provided by this application can be applied to the characterization of biomolecules based on nanopore sequencing technology, and the biomolecules can specifically be one or more of polynucleotides, polypeptides, polysaccharides, and lipids.
  • Compositions of species, polynucleotides include DNA and/or RNA.
  • the molecular film includes a mixed distribution of oil film 10 and amphiphilic molecular film 20.
  • the amphiphilic molecular film 20 is formed by amphiphilic molecules.
  • Membrane structure the amphiphile molecular membrane 20 is provided with a transmembrane nanopore 30 .
  • Amphiphiles can be synthetic or naturally occurring. For example, in naturally occurring phospholipids, the phosphoric acid part of the phospholipid is hydrophilic and the fatty acid part is hydrophobic, thus exhibiting both hydrophilic and hydrophobic properties.
  • Block copolymers in which two or more monomeric subunits are aggregated together to produce a single polymer chain.
  • Block copolymers can be engineered so that one of the monomer subunits is hydrophobic while the other is hydrophilic in aqueous media.
  • the block copolymer can have amphiphilic properties and can form structures that mimic biological membranes.
  • Block copolymers can be di-block, tri-block, tetra-block or penta-block copolymers. The present application does not limit what kind of membrane structure the amphiphilic molecular membrane 20 can adopt.
  • the grease film 10 is composed of multiple layers of molecules, and its thickness is thicker, so that the grease film 10 can be stably attached to the skeleton 40, and the amphiphilic molecular film 20 can be fixed to the skeleton 40 through the grease film 10 , to facilitate subsequent biomolecular characterization.
  • the grease film 10 can specifically be any one or a mixture of silicone oil, phosphatidyl choline (Phosphatidyl choline, PC), phosphatidyl ethanolamine (phosphatidyl ethanolamine, PE), phosphatidyl glycerol (Phosphatidyl glycerol, PG) etc. .
  • the transmembrane nanopore 30 is a through hole arranged along the thickness direction of the amphiphilic molecular membrane 20 to communicate with both sides of the amphiphilic molecular membrane 20 . Driven by the electric potential, hydrated ions can flow from one side of the amphiphile membrane 20 to the other side.
  • the transmembrane nanopore 30 may specifically be a protein pore, a polynucleotide pore, or a solid state pore, and the present application does not limit the specific form of the transmembrane nanopore 30 .
  • the analyte can pass through the transmembrane nanopore 30 in turn, and when the analyte passes through the transmembrane nanopore 30, it will cause a change in the electrical signal, and the characterization information of the analyte passing through can be further obtained according to the electrical signal .
  • the size information, sequence information, identity information, modification information, etc. of the analyte can be obtained according to the change information of the electrical signal.
  • the embedded linker 1 can specifically be a polypeptide, a block polymer, etc.
  • the first hydrophilic segment of the embedded linker 1 11 and the second hydrophilic segment 13 can be attracted to the hydrophilic region in the amphiphilic molecular membrane 20, and the hydrophobic segment 12 can be attracted to the hydrophobic region in the amphiphilic molecular membrane 20, so that the hydrophobic segment 12 appears to run through the amphiphilic molecular membrane 20
  • the hydrophobic region of , and the linker 2 connected with the embedded linker 1 are located on the surface of the amphiphile molecular membrane 20 .
  • the hydrophobic segment 12 presents a rigid, preferably rigid, rod-like structure, which avoids bending of the anchor molecule caused by the attraction of the first hydrophilic segment 11 and the second hydrophilic segment 13 in the same anchor molecule. Therefore, it is avoided that the bent embedded joint 1 is fixed on the grease film 10 through the hydrophobic section 12 and does not move toward the amphiphile molecular film 20 .
  • the hydrophobic segment 12 is a polypeptide
  • different amino acid sequences can be designed so that the hydrophobic segment 12 has a certain three-dimensional structure, presenting a rigid, preferably rigid, rod-like shape, thereby erecting the hydrophobic region across the amphiphilic molecular membrane.
  • the hydrophobic segment 12 is a block polymer, it can be ensured that the hydrophobic segment 12 Has a certain rigidity.
  • connection adapter 2 can be connected with the analyte, specifically, the connection adapter 2 can include a capture strand for complementary base pairing with the analyte, and the analyte is connected with an adapter that is complementary to the base pairing of the capture strand. It is the sequence connected to the analyte designed in advance by those skilled in the art according to the type of nanopore sequencing platform, the type of nanopore sequencing channel, the annealing temperature requirement, and the identification tag.
  • the linker 2 can be connected with different analytes through this linker.
  • a fluorophore can also be attached to the capture strand for easy observation. Specifically, the fluorophore can be FAM or Cy5.
  • the capture chain can be quickly connected to the embedded linker 1 through a click chemistry reaction.
  • Click chemistry click chemistry
  • the first linking group 211 is connected to the embedding linker 1
  • the second linking group 212 is connected to the connecting linker 2
  • a click chemical reaction occurs through the first linking group 211 and the second linking group 212, Realize quick connection of embedded joint 1 and connecting joint 2.
  • the first linking group 211 and the second linking group 212 may be linked by a carbon-carbon multiple bond addition reaction, linked by a nucleophilic ring-opening reaction, or click chemical reaction such as a cycloaddition reaction.
  • one of the first linking group 211 and the second linking group 212 can be cyclooctene (TCO), dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN) or dibenzocyclooctyne (DICO); the other can be azido (N3), tetrazine (TZ), etc.
  • TCO cyclooctene
  • DBCO dibenzocyclooctyne
  • DIFO difluorinated cyclooctyne
  • BCN bicyclononyne
  • DICO dibenzocyclooctyne
  • N3 azido
  • TZ tetrazine
  • connection adapter 2 is not limited by the type of the embedded adapter 1, and those skilled in the art can choose a suitable connection adapter 2 according to actual needs, and choose the connection method between the connection adapter 2 and the analyte .
  • the embedding linker 1 and the connection linker 2 can be stored independently, or can be stored in the form of anchor molecules after linking.
  • the embedding adapter 1 and the connecting adapter 2 are stored separately.
  • the embedding adapter 1 can be added to the solvent in contact with the molecular membrane (or the embedding adapter 1 can be mixed with the amphiphile for film formation) After film formation), the embedding adapter 1 can be embedded and penetrated on the molecular membrane, and then the connecting adapter is added to the solvent in contact with the molecular membrane, so that the connecting adapter 2 can be connected with the embedding adapter 1 through a click chemical reaction.
  • Both ends of the embedded adapter 1 can be connected with first linking groups 211, that is, at least one first linking group 211 is connected with the first hydrophilic segment 11, and at least one first linking group 211 is connected with the second hydrophilic segment 13 , so that regardless of the transmembrane direction of the embedding adapter 1 in the amphiphile molecular membrane 20, the embedding adapter 1 can be connected to the connecting adapter 2 added later through the first hydrophilic segment 11 or the second hydrophilic segment 13.
  • the embedding adapter 1 and the connecting adapter 2 are connected to form an anchor molecule storage.
  • the anchor molecule can be added to the solvent in contact with the molecular membrane, so that the embedding adapter 1 can be embedded and penetrated. set on the molecular membrane. Only one end of the embedded joint 1 can be connected with the first linking group 211 , so that the connection direction of the embedded joint 1 and the connecting joint 2 is fixed.
  • the membrane 10 has fluidity, that is, the molecules in the amphiphilic molecular membrane 20 and the lipid membrane 10 are constantly moving, thus showing that the transmembrane nanopore 30 and anchor molecules can move on the amphiphilic molecular membrane 20 and the lipid membrane 10 .
  • the embedded joint 1 provided with the first hydrophilic segment 11, the hydrophobic segment 12 and the second hydrophilic segment 13 cannot exist stably on the grease film 10, but the combination stability in the amphiphilic molecular film 20 is better , so that the anchor molecules in the molecular membrane can be gradually aggregated and distributed in the amphiphilic molecular membrane 20; the linker 2 of the anchor molecule can be connected to the analyte, so that the analyte connected to the anchor molecule can be Gather near the amphiphilic molecular membrane 20 , that is, the analyte is close to the transmembrane nanopore 30 on the amphiphilic molecular membrane 20 , increasing the amount of the analyte passing through the hole, and increasing the output time of the analyte corresponding to the effective electrical signal.
  • the linker 2 is connected to the first hydrophilic segment 11 , so that the linker 2 can be effectively distributed on the outer surface of the amphiphilic molecular membrane 20 .
  • the anchor molecule is first connected to the analyte, and then the connection product is added to the pre-prepared molecular membrane. Due to the large mass and variable chemical properties of the analyte, compared with the connection The first hydrophilic segment 11 and the second hydrophilic segment 13 containing the analyte are more easily inserted into the amphiphilic molecular membrane 20, thereby ensuring that the analyte stays on one side of the molecular membrane.
  • connection joints 2 are separately arranged at both ends of the embedded joint 1, at least one connection joint 2 is connected to the first hydrophilic section 11, and at least one connection joint 2 is connected to the second hydrophilic section. 13 connection, or the first linking group 211 connected to the second linking group 212 is provided at both ends of the embedding linker 1, so that whether the first hydrophilic segment 11 or the second hydrophilic segment in the anchoring molecule or the embedding linker 1 13 Regardless of the transmembrane direction in the amphiphile molecular membrane 20, the anchor molecule or the embedded linker 1 can anchor the analyte through the first hydrophilic segment 11 or the second hydrophilic segment 13.
  • the first hydrophilic segment 11 and/or the second hydrophilic segment 13 are lower than the thickness of the amphiphilic molecular membrane 20.
  • Surface when the length of the embedded joint 1 is greater than the thickness of the amphiphile molecular membrane 20 , it shows that the first hydrophilic segment 11 and/or the second hydrophilic segment 13 are exposed on the surface of the amphiphilic molecular membrane 20 .
  • the length of the embedded linker 1 can control the length of the embedded linker 1 to adapt to the thickness of the amphiphile molecular membrane 20 by changing the number of amino acids in the polypeptide, the length of each subunit in the block polymer, etc., so that the connecting linker 2 is free on both sides.
  • the connecting linker 2 is free on both sides.
  • the free end of the connecting adapter 2 can still be connected to the analyte or the adapter of the analyte, so that the anchor molecule can be anchored to be analyzed
  • the substance is on the surface of the amphiphile molecular membrane 20.
  • the embedded linker 1 is a polypeptide
  • the hydrophobic segment 12 is a hydrophobic polypeptide.
  • Those skilled in the art can design the first hydrophilic segment 11, the hydrophobic segment 12 and the second hydrophilic segment 13 according to the hydrophilicity and hydrophobicity of amino acids, so that the first hydrophilic segment 11 and the second hydrophilic segment 13 are hydrophilic
  • the hydrophobic segment 12 exhibits hydrophobicity.
  • the hydrophobic polypeptide has an alpha-helical structure.
  • the design of polypeptides with an ⁇ -helical structure is simple and easy to implement, and the ⁇ -helical structure is stable and not easy to bend.
  • the connecting joint 2 can be connected to the N-terminal and/or C-terminal of the embedded joint 1 .
  • the embedded linker 1 can be prepared by solid-phase synthesis, liquid-phase synthesis, cell expression, in vitro expression, and the like.
  • the amino acid sequence embedded in the linker 1 can be designed so that the embedded linker 1 can exhibit the required physical and chemical properties.
  • the sequence is more likely to exhibit an ⁇ -helical structure.
  • the hydrophobic segment 12 at least includes alanine residues (A), arginine residues (R), leucine residues (L), lysine residues (K), methionine residues
  • One or more of the groups (M) to increase the possibility of forming an ⁇ -helical structure.
  • the distribution density of acidic amino acid residues near the end of the linker 2 is smaller than the distribution density of acidic amino acid residues at the end far away from the linker 2; and/or the basic amino acid residues near the end of the linker 2
  • the distribution density of residues is greater than the distribution density of basic amino acid residues at the end far away from linker 2.
  • the hydrophilic amino acid residue needs to overcome the hydrophobic Sexual energy barrier.
  • the positive charges of basic residues (such as arginine Arg (R), lysine Lys (K) and leucine His (H)) need to additionally overcome the electrostatic potential energy caused by the membrane potential of the amphiphile membrane 20 ; while acidic residues (such as aspartic acid Asp (D) and glutamic acid Glu (E)) can use this electrostatic potential energy to effectively overcome the hydrophobic energy barrier.
  • this property can be used to set the distribution density of acidic amino acid residues near the end of the linker 2 or the analyte in the hydrophobic section 12 to be smaller than the distribution density of acidic amino acid residues at the end far away from the linker 2; and/or near the linker 2 or
  • the distribution density of basic amino acid residues at one end of the analyte is greater than the distribution density of basic amino acid residues at the end far away from the linker 2.
  • the degree of matching between the thickness of the hydrophobic region and the length of the hydrophobic segment 12 is a hydrophobic mismatch.
  • the hydrophobic mismatch is defined as zero.
  • the hydrophobic segment 12 and the amphiphilic molecular membrane 20 with a larger hydrophobic matching difference are more likely to undergo conformational changes under the action of external force; on the contrary, the hydrophobic segment 12 with a smaller hydrophobic matching difference has a stronger anchoring effect in the amphiphilic molecular membrane 20.
  • the amino acid sequence of the hydrophobic segment 12 is designed to reduce the hydrophobic matching difference between the hydrophobic segment 12 and the hydrophobic region, so as to improve the affinity between the anchor molecule and the amphiphilic molecular membrane 20 .
  • the hydrophobic matching difference between the hydrophobic segment 12 and the hydrophobic region is less than or equal to 5 nm.
  • the hydrophobic segment 12 may also stably exist in the amphiphilic molecular membrane 20 by showing a protruding hydrophobic region or tilting at a certain angle. .
  • the anchor molecule sequence is as follows:
  • the anchor molecule is a tri-block polymer.
  • the first hydrophilic segment 11 and the second hydrophilic segment 13 are hydrophilic copolymers, such as polyethylene glycol (PEG), poly(2-methyl-2-oxazoline) (PMOXA), polyacrylic acid ( PAA) etc.;
  • the hydrophobic segment 12 is a hydrophobic copolymer, for example: any one of polythiophene, polyphenylene acetylene, polyphenanthrene acetylene, certainly can also be phenylene acetylene macromolecule, phenanthrene acetylene macromolecule etc.
  • the present application also provides a molecular membrane for nanopore characterization of analytes, including a lipid membrane 10 and an amphiphilic molecular membrane 20, and the amphiphilic molecular membrane 20 is embedded with a transmembrane nanopore 30 and an embedded joint 1 or anchor molecules. Since the embedding linker 1 or anchor molecule provided by any one of the above embodiments is used, it has the same technical effect and will not be repeated here.
  • the present application also provides a kit for preparing the above-mentioned anchor molecule, including the above-mentioned insertion linker 1 or the anchor molecule.
  • the kit provided in the embodiment of the present application uses the anchor molecule provided in any one of the above embodiments, so it has the same technical effect and will not be repeated here.
  • the anchor molecule When the anchor molecule is a polypeptide, the anchor molecule can be freeze-dried or stored in a buffer; when the anchor molecule is a triblock polymer, it can be directly placed in a container. Those skilled in the art can determine the appropriate anchor molecule preservation method according to the type of anchor molecule. Embedding Adapter 1 and Connecting Adapter can be stored independently and joined together when characterizing analytes.
  • the nanopore sequencing kit can also include L fragment buffer (L fragment buffer, LFB), S fragment buffer (S fragment buffer, SFB), ligation buffer (Ligation buffer, LNB), elution buffer (Elution buffer, EB), sequencing buffer (Sequencing buffer, SQB), can also include independently packaged sequencing tether (Sequencing tether, SQT), DNA control strand (DNA control strand), sequencing particles (Loading beads, LB) etc., and are not limited to this, for the convenience of users to prepare samples that can be directly used on the machine.
  • the present application also provides a nanopore device for characterizing biomolecules, including: a biochip, a molecular membrane, and the above-mentioned anchor molecule, or the above-mentioned kit, the molecule
  • the membrane includes a grease film 10 and an amphiphile molecular membrane 20 distributed on the biochip, and the amphiphile molecular membrane 20 is provided with transmembrane nanopores 30 ; the embedding joint 1 can penetrate the amphiphile molecular membrane 20 .
  • the nanopore device for characterizing biomolecules provided in the embodiments of the present application has the same technical effect due to the use of the anchor molecules provided in any of the above embodiments, and will not be repeated here.
  • the biochip is a structure that provides potential and forms a molecular film.
  • the biochip includes a skeleton 40 with a liquid storage chamber and electrodes corresponding to the liquid storage chamber.
  • the skeleton 40 is used to place reagents and analytes, and to support the molecular film.
  • Polar solvents such as buffer solutions
  • the non-polar solvent is the non-polar solvent that dissolves the amphiphilic material, specifically methylphenyl silicone oil, polydimethylsiloxane, etc., to drive the polar solvent, that is, to complete the process of "oil driving water”.
  • a polar solvent is added to drive the non-polar solvent of the amphiphilic material, that is, the process of "water driving oil” is completed.
  • Polar solvents such as buffer solutions can specifically be phosphate buffer solutions, HEPES buffer solutions containing KCl or NaCl, CAPS buffer solutions containing KCl or NaCl, and the like.
  • a structure of polar solvent-non-polar solvent-polar solvent is formed in each structural unit, and the non-polar solvent of the amphiphilic material is sandwiched between two layers of polar solvent to form an amphiphilic molecular film 20 .
  • the anchor molecules are randomly embedded in the molecular membrane, and gradually gather into the amphiphilic molecular membrane 20 .
  • the electrodes are used to form a potential difference on both sides of the molecular membrane, so that the analyte can pass through the transmembrane nanopore 30 .
  • the present application also provides a method for enriching the analyte to the transmembrane nanopore, comprising the following steps:
  • the analyte approaches the transmembrane nanopore under the traction of the embedded joint.
  • the present application also provides a method for characterizing an analyte through a nanopore, including enriching the analyte to the transmembrane nanopore as described above, making the analyte contact the transmembrane nanopore,
  • an electrical signal is measured to characterize the analyte in order to determine one or more characteristics of the analyte.
  • the present application also provides an application of the above-mentioned anchor molecule, or the above-mentioned kit, or the above-mentioned nanopore device for characterizing biomolecules in characterizing biomolecules.
  • the applications provided in the embodiments of the present application have the same technical effect due to the use of the anchor molecules provided in any of the above embodiments, and will not be repeated here.
  • the analytes include biomolecules including one or more of polynucleotides, polypeptides, polysaccharides, or lipids.
  • Characteristic features may be (i) length of polynucleotide; (ii) identity of polynucleotide; (iii) sequence of polynucleotide; (iv) secondary structure of polynucleotide and (v) polynucleotide Is modified.
  • the application also provides the following verification tests to illustrate the embedded adapters, anchor molecules, kits, devices for characterizing biomolecules in nanopores, methods for enriching analytes in transmembrane nanopores, and nanopore characterization analysis provided by this application The method of material and the effect of application.
  • connection linker is connected to the analyte through the capture chain
  • the insertion linker is a polypeptide
  • the connection linker and the insertion linker are connected through "DBCO” and "N3".
  • Figure 4 is a prediction map of the polypeptide transmembrane region. From Figure 4, it can be seen that the 7th to 29th amino acids in the amino acid sequence (underlined in the amino acid sequence) are transmembrane regions, indicating that the anchor molecule can be across the amphiphile membrane.
  • the connecting adapter is incubated with the embedding adapter, and the incubated product is collected, and the incubated product is an anchor molecule.
  • Figure 5 is the chromatogram of the embedded joint
  • Figure 6 is the chromatogram of the incubation product.
  • the peak time of a single embedded joint is 24.227 minutes
  • the peak time of the incubation product is 31.353 minutes.
  • the anchor molecules formed by linking the linker with the embedded linker had a peak eluting time of 31.353 minutes, corresponding to the collection of the incubation product of 31.353 minutes, the anchor molecules could be collected.
  • FIG. 7 is a prediction map of the transmembrane region of the polypeptide, wherein amino acids from the 4th to the 26th (underlined in the amino acid sequence) are the transmembrane regions.
  • Figure 8 is a fluorescence microscope observation result of the molecular film after adding the embedded joint prepared in Example 2 for 30 minutes
  • Figure 9 is the molecular film after adding the embedded joint prepared in Example 2 for 12 hours. Fluorescence microscope observation results of the membrane, where the part framed by the dotted line corresponds to the amphiphilic molecular membrane located in the middle of the molecular membrane.
  • the anchor molecules prepared in Example 1 were connected with fluorescent groups, and mixed with the molecular membrane for incubation. It can also be observed that the anchor molecules converge to the amphiphilic molecular membrane in the middle of the molecular membrane as time increases.
  • Figure 10 is the via signal diagram of the control group
  • Figure 11 is the via hole signal diagram of the experimental group
  • Figure 12 is the number of ssDNA via holes per unit time in the control group and the experimental group. It can be seen from the through-hole signal diagram that the use of the anchor molecule provided by the present application can greatly increase the frequency of the analyte through the hole, thereby improving the detection efficiency.
  • Figure 13 is a prediction map of the polypeptide transmembrane region of Comparative Example 1, wherein the 7th to 29th amino acids in the amino acid sequence (underlined in the amino acid sequence) are transmembrane regions, indicating that the polypeptide is different from that of Example 1
  • the embedded linkers in both are the same, and both can penetrate the amphiphilic molecular membrane.
  • FIG. 14 The hydrophobicity analysis of the embedded linker in Example 1 and the polypeptide in Comparative Example 1 was performed by the hydrophobicity analysis software provided by ExPASy, and FIG. 14 was obtained.
  • Fig. 14 (a) is the hydrophobicity curve of the embedded linker of Example 1
  • Fig. 14 (b) is the hydrophobicity curve of the polypeptide of Comparative Example 1, as can be seen from the figure, in Example 1
  • Both the polypeptide embedded in the linker and the polypeptide in Comparative Example 1 are hydrophobic in the middle and hydrophilic at both ends.
  • Example 2 the embedded linker in Example 1 and the polypeptide in Comparative Example 1 were respectively connected to the fluorescent group, and then mixed with the molecular membrane for incubation for 12 hours. Observation by fluorescence microscope showed that the embedded linker in Example 1 was in The aggregation degree of the amphiphilic molecular membrane is greater than that of the polypeptide in the amphiphilic molecular membrane in Comparative Example 1, indicating that the rigid ⁇ -helical structure can improve the aggregation degree of the polypeptide in the amphiphilic molecular membrane.
  • the embedded linker was designed and synthesized, and its chemical formula is shown in Figure 16.
  • the embedded joint is a block polymer, and polyphenylene vinylene is used to form a rigid rod-like structure.
  • Connectors and insert connectors are connected via "DBCO” and "N3".
  • the fluorophore FAM is attached to the linker.
  • the embedded linker was designed and synthesized, and its chemical formula is shown in Figure 17.
  • DBCO DBCO
  • N3 are respectively set at both ends of the block polymer.
  • the difference from Example 4 is that the polysiloxane in this comparative example replaces the polyphenylene vinylene in Example 4, and the polysiloxane has a non-rigid structure.
  • Example 2 the embedded linker of Example 4 and the block polymer of Comparative Example 2 were mixed with the prepared molecular membrane and incubated respectively. It was observed by fluorescence microscopy that, as time went on, compared with Comparative Example 2, the embedded linker in Example 4 converged more towards the amphiphilic molecular membrane in the middle of the molecular membrane.

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Abstract

本申请公开了一种嵌入接头、锚定分子、分子膜、装置、方法及应用。其中，嵌入接头用于嵌设于两亲分子膜处，嵌入接头包括依次连接的第一亲水段、疏水段和第二亲水段，疏水段用于跨两亲分子膜的疏水区域，疏水段为刚性；第一亲水段和/或第二亲水段用于与待分析物连接。

Description

嵌入接头、锚定分子、分子膜、装置、方法及应用
相关申请的交叉引用
本申请要求享有于2021年12月28日提交的名称为“嵌入接头、锚定分子、分子膜、装置、方法及应用”的中国专利申请202111616038.8的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本申请属于基因测序技术领域，尤其涉及一种嵌入接头、锚定分子、分子膜、纳米孔表征生物分子的装置、在两亲分子膜中组装嵌入接头的方法、向跨膜纳米孔富集待分析物的方法、纳米孔表征分析物的方法及应用。
背景技术
现有纳米孔测序中，在充满电解液的腔内，带有纳米级小孔的分子膜将腔体分成2个小室，当电压作用于电解液时，离子或其他小分子物质可穿过小孔，形成稳定的可检测的离子电流，可根据电流信息检测不同类型的生物分子。
现有的分子膜中设置有锚定分子，以通过锚定分子结合生物分子，使得生物分子聚合在分子膜表面。现有锚定分子通常为带有系链的胆固醇，胆固醇均匀分布在分子膜上，而纳米孔仅设置在分子膜的两亲分子膜处，如磷脂膜或聚合物膜上，导致部分远离纳米孔的生物分子不易被检测到，特别是当生物分子浓度较低时，有效电流数据输出时长较短，影响检测效果。
发明内容
本申请实施例提供一种在嵌入接头、锚定分子、分子膜、纳米孔表征生物分子的装置、在两亲分子膜中组装嵌入接头的方法、向跨膜纳米孔富集待分析物的方法、纳米孔表征分析物的方法及应用，能有效将待分析物锚定在跨膜纳米孔附近，提高待分析物对应有效电流数据的输出时长。
一方面，本申请实施例提供一种嵌入接头，嵌入接头用于嵌设于两亲分子膜处，包括：
依次连接的第一亲水段、疏水段和第二亲水段，疏水段用于跨两亲分 子膜的疏水区域，以使嵌入接头能够贯穿两亲分子膜，疏水段具有刚性；
第一亲水段和/或第二亲水段能够与待分析物连接。
可选地，疏水段为棒状。
可选地，疏水段为疏水多肽。
可选地，疏水多肽为α螺旋结构；
第一亲水段和/或第二亲水段为亲水多肽，可选地，嵌入接头为多肽。
可选地，疏水段为一次跨膜多肽，
疏水段也可以是多次跨膜、非棒状、刚性的多肽，且该多肽可以向两亲分子膜聚集即可。
可选地，疏水段中至少包括丙氨酸残基(A)、精氨酸残基(R)、亮氨酸残基(L)、赖氨酸残基(K)、甲硫氨酸残基(M)中的一种或多种。
可选地，疏水段与疏水区域的疏水匹配差小于或等于5nm。
可选地，疏水段中，靠近连接接头一端的酸性氨基酸残基分布密度小于远离连接接头一端的酸性氨基酸残基分布密度；和/或
靠近连接接头一端的碱性氨基酸残基分布密度大于远离连接接头一端的碱性氨基酸残基分布密度。
可选地，疏水段为疏水性共聚物；
第一亲水段和/或第二亲水段为亲水性共聚物，可选地，嵌入接头为三嵌段聚合物。
可选地，疏水段中至少部分相邻分子之间采用非单键连接；可选地，疏水性共聚物为聚噻吩、聚苯乙炔、聚菲乙炔中的任意一种。
第二方面，本申请还提供了一种锚定分子，锚定分子用于锚定待分析物于两亲分子膜上，锚定分子包括如第一方面提供的嵌入接头和连接接头，连接接头包括能够与待分析物连接的部分和能够与嵌入接头连接的部分。
可选地，连接接头设于嵌入接头的一端或两端，可选地，连接接头设于嵌入接头的一端。
可选地，连接接头包括能够与待分析物碱基互补配对连接的捕获链，捕获链通过点击化学反应与嵌入接头连接。
第三方面，本申请还提供了一种在两亲分子膜中组装嵌入接头的方法， 包括将第一方面提供的嵌入接头与分子膜接触，分子膜包括油脂膜和两亲分子膜，随后嵌入接头嵌设于两亲分子膜处。
第四方面，本申请还提供了一种纳米孔表征分析物用的分子膜，分子膜包括油脂膜和两亲分子膜，两亲分子膜中嵌入跨膜纳米孔以及如第一方面提供的嵌入接头或如第二反面提供的锚定分子。
第五方面，本申请还提供了一种纳米孔表征分析物的装置，包括：
生物芯片；
分子膜，包括分布在生物芯片上的油脂膜和两亲分子膜，两亲分子膜上设置有跨膜纳米孔；和
如第一方面提供的嵌入接头，或如第二方面提供的锚定分子。
第六方面，本申请还提供了一种向跨膜纳米孔富集待分析物的方法，包括如下步骤：
提供嵌入跨膜纳米孔的两亲分子膜；
使两亲分子膜与如第一方面提供的嵌入接头接触，且使嵌入接头与连接有待分析物的连接接头接触，或使两亲分子膜与如第二方面提供的锚定分子接触，且使锚定分子与待分析物接触；
待分析物在嵌入接头的牵引下靠近跨膜纳米孔。
第七方面，本申请还提供了一种纳米孔表征分析物的方法，包括如第六方面提供的方法向跨膜纳米孔富集待分析物，使待分析物接触跨膜纳米孔，
当待分析物相对于跨膜纳米孔运动时或当跨膜纳米孔内存在待分析物时，测定电信号，以测定待分析物的一种或多种特征表征待分析物。
第八方面，本申请还提供了一种如第一方面提供的锚定分子、或如第二方面提供的锚定分子、或如第四方面提供的分子膜、或如第五方面提供的装置在分析物表征上的应用；
可选地，分析物包括生物分子，生物分子包括多核苷酸、多肽、多糖、或脂质；
更可选地，多核苷酸包括DNA或RNA；和/或，表征的特征选自(i)多核苷酸的长度；(ii)多核苷酸的一致性；(iii)多核苷酸的序列；(iv) 多核苷酸的二级结构和(v)多核苷酸是否经修饰。
本申请实施例的嵌入接头、锚定分子、分子膜、纳米孔表征生物分子的装置、向跨膜纳米孔富集待分析物的方法、纳米孔表征分析物的方法及应用中，设置有第一亲水段、疏水段和第二亲水段的嵌入接头在油脂膜上无法稳定存在，而在两亲分子膜中结合的稳定性较好，从而使得锚定分子能逐渐聚集分布在两亲分子膜中；通过锚定分子可以与待分析物连接，从而使得与锚定分子连接的待分析物可聚集在两亲分子膜附近，即待分析物靠近两亲分子膜上的跨膜纳米孔，提高过孔的待分析物数量，从而提高与待分析物对应的有效检测电流数据的输出时长；呈刚性棒状的疏水段可避免同一锚定分子中的第一亲水段和第二亲水段吸引，使得锚定分子弯曲，从而避免弯曲后的嵌入接头通过疏水段固定于油脂膜上，而不向两亲分子膜移动。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图作简单的介绍，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本申请一个实施例中锚定分子的结构示意图；
图2是本申请一个实施例中纳米孔表征分析物的装置的部分结构示意图；
图3是本申请另一个实施例中锚定分子的结构示意图；
图4是本申请一实施例中嵌入接头的跨膜预测图；
图5是本申请一实施例中嵌入接头的色谱图；
图6是本申请一实施例中连接接头与嵌入接头的孵育产物的色谱图；
图7是本申请另一实施例中嵌入接头的跨膜区域预测图；
图8是本申请一实施例中嵌入接头在分子膜上孵育0.5h的荧光发光图；
图9是本申请一实施例中嵌入接头在分子膜上孵育12h的荧光发光图；
图10是本申请一对照组的纳米孔测序过孔信号图；
图11是本申请一实验组的纳米孔测序过孔信号图；
图12是本申请一实验组和对照组的纳米孔测序过孔信号频率图；
图13是本申请一对比例中多肽的跨膜区域预测图；
图14是本申请一实施例和对比例中多肽的疏水性预测图；
图15是本申请一实施例和对比例中多肽的二级结构预测图；
图16是本申请一实施例中嵌入接头的化学结构图；
图17是本申请一对比例中嵌入接头的化学结构图。
附图标记说明：
1、嵌入接头；11、第一亲水段；12、疏水段；13、第二亲水段；2、连接接头；211、第一连接基团；212、第二连接基团；10、油脂膜；20、两亲分子膜；30、跨膜纳米孔；40、骨架。
具体实施方式
下面将详细描述本申请的各个方面的特征和示例性实施例，为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及具体实施例，对本申请进行进一步详细描述。应理解，此处所描述的具体实施例仅意在解释本申请，而不是限定本申请。对于本领域技术人员来说，本申请可以在不需要这些具体细节中的一些细节的情况下实施。下面对实施例的描述仅仅是为了通过示出本申请的示例来提供对本申请更好的理解。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括……”限定的要素，并不排除在包括要素的过程、方法、物品或者设备中还存在另外的相同要素。
为了解决相关技术问题，本申请实施例提供了一种嵌入接头、锚定分子、试剂盒、纳米孔表征生物分子的装置、向跨膜纳米孔富集待分析物的方法、纳米孔表征分析物的方法及应用。下面首先对本申请实施例所提供 的锚定分子进行介绍。
请参阅图1和图2，锚定分子包括嵌入接头1和连接接头2，嵌入接头1用于嵌设于两亲分子膜上，嵌入接头1包括依次连接的第一亲水段11、疏水段12和第二亲水段13，疏水段12用于跨两亲分子膜的疏水区域，以使嵌入接头1能够贯穿两亲分子膜，疏水段12为刚性棒状；第一亲水段11和/或第二亲水段13用于与连接接头2相连接，连接接头2能够与待分析物连接。
待分析物可以为生物分子，本申请提供的锚定分子可以应用于基于纳米孔测序技术的生物分子表征中，生物分子具体可以是多核苷酸、多肽、多糖和脂质中的一种或多种的组合物，多核苷酸包括DNA和/或RNA。
基于纳米孔测序技术进行生物分子表征时，需要在基因芯片的骨架40上制备分子膜，分子膜包括混合分布的油脂膜10和两亲分子膜20，两亲分子膜20为两亲分子形成的膜结构，两亲分子膜20上设置有跨膜纳米孔30。两亲分子可以是合成的或天然存在的。例如，天然存在的磷脂，磷脂中磷酸部分亲水，脂肪酸部分疏水，从而同时表现出亲水性和疏水性。还比如人工合成的嵌段共聚物，其中，两个或更多个的单体亚单元聚合在一起产生单一聚合物链。可以对嵌段共聚物进行改造，使得其中一个单体亚单元在水性介质中时是疏水的，而另一个亚单元是亲水的。在这种情况下，嵌段共聚物可以具有两亲性质并且可以形成模拟生物膜的结构。嵌段共聚物可以是二嵌段、三嵌段、四嵌段或五嵌段等共聚物。本申请中不限定两亲分子膜20具体可以采用何种形式的膜结构。与两亲分子膜20相比，油脂膜10由多层分子构成，其厚度较厚，使得油脂膜10可以稳定的附着在骨架40上，两亲分子膜20可以通过油脂膜10与骨架40固定，方便后续生物分子表征的进行。油脂膜10具体可以是硅油、磷脂酰胆碱(Phosphatidyl choline，PC)、磷脂酰乙醇氨(phosphatidyl ethanolamine，PE)、磷脂酰甘油(Phosphatidyl glycerol，PG)等中的任一种或多种的混合物。跨膜纳米孔30为沿两亲分子膜20厚度方向设置的通孔，以连通两亲分子膜20两侧。在电势的驱动下，水合离子可以从两亲分子膜20的一侧流到另一侧。跨膜纳米孔30具体可以是蛋白质孔、多核苷酸孔或固态孔等， 本申请不限定跨膜纳米孔30的具体形成形式。在电势差的作用下，待分析物可以依次从跨膜纳米孔30通过，待分析物从跨膜纳米孔30通过时，会引起电信号变化，可以进一步根据电信号得到通过待分析物的表征信息。例如根据电信号变化信息得到待分析物的尺寸信息、序列信息、同一性信息、修饰信息等。
嵌入接头1具体可以是多肽、嵌段聚合物等，锚定分子与上述分子膜接触时，由于两亲分子膜20和锚定分子的嵌入接头1结构类似，嵌入接头1的第一亲水段11和第二亲水段13可以与两亲分子膜20中的亲水区域吸引，疏水段12可以与两亲分子膜20中的疏水区域吸引，使得疏水段12表现为贯穿两亲分子膜20的疏水区域，与嵌入接头1连接的连接接头2位于两亲分子膜20的表面。
疏水段12呈现刚性优选刚性棒状结构，避免同一锚定分子中的第一亲水段11和第二亲水段13吸引造成的锚定分子弯曲。从而避免弯曲后的嵌入接头1通过疏水段12固定于油脂膜10上，而不向两亲分子膜20移动。当疏水段12为多肽时，可以通过设计不同的氨基酸序列，使得疏水段12具有一定的三维结构，呈现刚性优选刚性棒状，从而直立的跨两亲分子膜的疏水区域。当疏水段12为嵌段聚合物时，可以通过苯乙炔类高分子、菲乙炔类高分子等由双键、三键、苯环或其他杂原子环等组成的聚合物，以保证疏水段12具有一定的刚性。
连接接头2能够与待分析物连接，具体地，连接接头2可以包括用于与待分析物碱基互补配对的捕获链，待分析物上连接有与捕获链碱基互补配对的接头，该接头为本领域技术人员预先根据纳米孔测序平台类型、纳米孔测序通道类型、退火温度需求、识别标签等设计的与待分析物连接的序列。连接接头2通过该接头可以与不同的待分析物连接。还可以在捕获链上连接荧光基团，以方便观察。荧光基团具体可以是FAM或Cy5。捕获链具体可以通过点击化学反应实现与嵌入接头1快速连接，点击化学(click chemistry)为通过小单元的拼接，来快速地完成不同分子的化学合成。在本实施例中，嵌入接头1上连接有第一连接基团211，连接接头2连接有第二连接基团212，通过第一连接基团211和第二连接基团212发 生点击化学反应，实现嵌入接头1和连接接头2快速连接。第一连接基团211和第二连接基团212可以是通过碳碳多键加成反应连接、通过亲核开环化反应连接、通过环加成反应等点击化学反应。例如：第一连接基团211和第二连接基团212中一个可以是环辛烯(TCO)、二苯并环辛炔(DBCO)、二氟化环辛炔(DIFO)、二环壬炔(BCN)或二苯并环辛炔(DICO)中的任意一种；另一个可以是叠氮基(N3)、四嗪基(TZ)等。本领域技术人员可以理解的是，本实施例不限定第一连接基团211和第二连接基团212具体为哪种可发生点击化学反应的基团。本领域技术人员可以理解的是，连接接头2的选择不受嵌入接头1的类型限制，本领域技术可以根据实际需要自行选择合适的连接接头2，并选择连接接头2与待分析物的连接方式。
本领域技术人员可以理解的是，嵌入接头1和连接接头2可以独立存储，也可以连接后以锚定分子的形式存储。在一实施例中，嵌入接头1和连接接头2分别存储，进行分析物表征时，可以先将嵌入接头1加入与分子膜接触的溶剂中(或将嵌入接头1与成膜用两亲分子混合后成膜)，使得嵌入接头1可以嵌入并贯穿设置在分子膜上，再将连接接头加入与分子膜接触的溶剂中，使得连接接头2可以与嵌入接头1通过点击化学反应连接。嵌入接头1的两端均可以连接第一连接基团211，即至少一个第一连接基团211与第一亲水段11连接，至少一个第一连接基团211与第二亲水段13连接，使得无论嵌入接头1在两亲分子膜20中跨膜的方向如何，嵌入接头1均可以通过第一亲水段11或第二亲水段13与后加入的连接接头2连接。
在另一实施中，嵌入接头1和连接接头2连接后形成锚定分子存储，进行分析物表征时，可以先将锚定分子加入与分子膜接触的溶剂中，使得嵌入接头1可以嵌入并贯穿设置在分子膜上。嵌入接头1可以仅一端与第一连接基团211连接，使得嵌入接头1与连接接头2连接的方向固定。
在表征生物分子过程中，在跨膜纳米孔30附近游离的生物分子越多，生物分子经过跨膜纳米孔30的效率越高，从而表现出检测效率越高，而两亲分子膜20和油脂膜10具有流动性，即两亲分子膜20和油脂膜10中 的分子是在不断运动的，从而表现出跨膜纳米孔30和锚定分子可以在两亲分子膜20和油脂膜10上移动。在本申请中设置有第一亲水段11、疏水段12和第二亲水段13的嵌入接头1在油脂膜10上无法稳定存在，而在两亲分子膜20中结合的稳定性较好，从而使得锚定分子在分子膜中，能逐渐聚集分布在两亲分子膜20中；通过锚定分子的连接接头2可以与待分析物连接，从而使得与锚定分子连接的待分析物可聚集在两亲分子膜20附近，即待分析物靠近两亲分子膜20上的跨膜纳米孔30，提高过孔的待分析物数量，提高待分析物对应有效电信号的输出时长。
在一实施例中，连接接头2与第一亲水段11连接，使得连接接头2可有效的分布在两亲分子膜20的外表面。在对待分析物进行检测时，先将锚定分子与待分析物连接，再将连接产物加入已预先制备的分子膜中，由于待分析物质量较大、化学性质多变，使得相较于连接有待分析物的第一亲水段11，第二亲水段13更容易插入两亲分子膜20中，从而保证待分析物留在分子膜的一侧。
请结合参阅图3，在另一实施例中，连接接头2分设于嵌入接头1的两端，至少一个连接接头2与第一亲水段11连接，至少一个连接接头2与第二亲水段13连接，或者嵌入接头1两端均设有与第二连接基团212连接的第一连接基团211，使得无论锚定分子或嵌入接头1中第一亲水段11和第二亲水段13在两亲分子膜20中跨膜的方向如何，锚定分子或嵌入接头1均可以通过第一亲水段11或第二亲水段13锚定待分析物。
本领域技术人员可以理解的是，当嵌入接头1的长度小于两亲分子膜20的厚度时，表现出第一亲水段11和/或第二亲水段13低于两亲分子膜20的表面；当嵌入接头1的长度大于两亲分子膜20的厚度时，表现出第一亲水段11和/或第二亲水段13外露在两亲分子膜20的表面。本领域技术人员可以通过改变多肽中氨基酸的个数、嵌段聚合物中各亚单元长度等，控制嵌入接头1的长度与两亲分子膜20的厚度适配，从而使连接接头2游离在两亲分子膜20上。当然无论嵌入接头1的两端凸出或凹陷于两亲分子膜20的表面，连接接头2游离的一端仍然可以与待分析物或待分析物的接头连接，从而锚定分子可以锚定待分析物在两亲分子膜20的表面。
在一实施例中，嵌入接头1为多肽，疏水段12为疏水多肽。本领域技术人员可以根据氨基酸的亲水性和疏水性设计第一亲水段11、疏水段12和第二亲水段13，使得第一亲水段11和第二亲水段13表现亲水性，疏水段12表现疏水性。可选地，疏水多肽为α螺旋结构。呈α螺旋结构的多肽设计简单易实现，同时α螺旋结构结构稳定、不易弯折。连接接头2可以与嵌入接头1的N端和/或C端连接。嵌入接头1可以通过固相合成、液相合成、细胞表达、体外表达等方式制备得到。
本领域技术人员可以理解的是，不同氨基酸分子具有不同理化性质，从而可以通过设计嵌入接头1中的氨基酸序列，以使嵌入接头1表现出所需的理化性质。当氨基酸序列中丙氨酸残基、精氨酸残基、亮氨酸残基、赖氨酸残基、甲硫氨酸残基的含量较多时，该序列表现出α螺旋结构的可能性更大，所以疏水段12中至少包括丙氨酸残基(A)、精氨酸残基(R)、亮氨酸残基(L)、赖氨酸残基(K)、甲硫氨酸残基(M)中的一种或多种，以提高形成α螺旋结构的可能性。
在另一实施例中，疏水段12中，靠近连接接头2一端的酸性氨基酸残基分布密度小于远离连接接头2一端的酸性氨基酸残基分布密度；和/或靠近连接接头2一端的碱性氨基酸残基分布密度大于远离连接接头2一端的碱性氨基酸残基分布密度。
在进行待测分析物检测时，需要从分子膜的一侧加入锚定分子或嵌入接头1时，当锚定分子或嵌入接头1进入两亲分子膜20时，亲水氨基酸残基需要克服疏水性能量势垒。碱性残基(如精氨酸Arg(R)、赖氨酸Lys(K)和亮氨酸His(H))的正电荷需要额外地克服两亲分子膜20的膜电位所造成的静电势能；而酸性残基(如天冬氨酸Asp(D)和谷氨酸Glu(E))则可以利用该静电势能来有效地克服疏水性能量势垒。因此，碱性残基倾向于被滞留在锚定分子或嵌入接头1加入侧，而酸性残基则更容易地被推送到另一侧。因此可以利用该性质，设置疏水段12中，靠近连接接头2或待分析物一端的酸性氨基酸残基分布密度小于远离连接接头2一端的酸性氨基酸残基分布密度；和/或靠近连接接头2或待分析物一端的碱性氨基酸残基分布密度大于远离连接接头2一端的碱性氨基酸残基分 布密度。以使嵌入接头1中远离连接接头2或待分析物的一端或用于连接连接接头2或待分析物的相对端可以优先插入两亲分子膜20中，使得锚定分子在两亲分子膜20中的方向可控。
两亲分子膜20的厚度与嵌入接头1的长度是否适配，可以影响两者亲和性。疏水区域的厚度与疏水段12的长度之间的匹配程度为疏水匹配差(hydrophobic mismatch)，当疏水区域的厚度与疏水段12的长度一致，疏水匹配差被定义为零。疏水匹配差越大的疏水段12和两亲分子膜20，越容易在外力作用下发生构象变化；相反，疏水匹配差越小的疏水段12，在两亲分子膜20中的锚定作用越明显，所以设计疏水段12的氨基酸序列，减小疏水段12与疏水区域的疏水匹配差，以提高锚定分子与两亲分子膜20的亲和性。可选地，疏水段12与疏水区域的疏水匹配差为小于或等于5nm。当然在疏水段12和两亲分子膜20的疏水匹配差在一定范围内时，疏水段12也可能通过表现为凸出疏水区域、或倾斜一定角度等方式，稳定存在于两亲分子膜20中。
例如一实施例中，锚定分子序列如下所示：
Figure PCTCN2022138280-appb-000001
其中，“GGTCGGTGCTGGACTTTTTTTTTTTTTTTTTTTTT”为捕获链，用于与待分析物的接头碱基互补配对连接；“WERNLPSVSGLLKIIGFSTSVTALGFVLYKYKLLPRS”为嵌入接头；“DBCO-N3”为第一连接基团和第二连接基团，以使得捕获链与嵌入接头连接。
请再次参阅图1，在另一实施例中，锚定分子为三嵌段聚合物。第一亲水段11和第二亲水段13为亲水性共聚物，例如：聚乙二醇(PEG)、聚(2-甲基-2-噁唑啉)(PMOXA)、聚丙烯酸(PAA)等；疏水段12为疏水性共聚物，例如：聚噻吩、聚苯乙炔、聚菲乙炔中的任意一种，当然还可以是苯乙炔类高分子、菲乙炔类高分子等由双键、三键、苯环或其他杂原子环等组成的聚合物，以保证疏水段12具有一定的刚性，避免同一锚定分子中的第一亲水段11和第二亲水段13吸引造成的锚定分子弯曲。从而避 免弯曲后的三嵌段聚合物通过疏水段12存在与油脂膜10上，而不向的两亲分子膜20移动。本领域技术人员可以根据不同基团的亲水性和疏水性，选择三嵌段聚合物中需要聚合的基团，再通过活性聚合、正离子聚合转化法、气相本体法或液相本体法等合成得到所需的三嵌段聚合物。
另一方面，本申请还提供了一种纳米孔表征分析物用的分子膜，包括油脂膜10和两亲分子膜20，两亲分子膜20中嵌入跨膜纳米孔30以及的嵌入接头1或的锚定分子。由于采用了以上任意一实施例提供的嵌入接头1或锚定分子，因而具有相同的技术效果，这里不再赘述。
本申请还提供了一种用于制备如上述锚定分子的试剂盒，包括如上述的嵌入接头1或锚定分子。本申请实施例提供的试剂盒，由于采用了以上任意一实施例提供的锚定分子，因而具有相同的技术效果，这里不再赘述。
当锚定分子为多肽，锚定分子可以采用冻干或存储于缓冲剂中；当锚定分子为三嵌段聚合物，可以直接放置于容器中。本领域技术人员可以根据锚定分子的类型，决定合适的锚定分子保存方式。嵌入接头1和连接接头可以分别独立保存，表征分析物时再连接在一起。
具体地，该纳米孔测序试剂盒还可以包括L片段缓冲液(L fragment buffer，LFB)、S片段缓冲液(S fragment buffer，SFB)、连接缓冲液(Ligation buffer，LNB)、洗脱缓冲液(Elution buffer，EB)、测序缓冲液(Sequencing buffer，SQB)，还可以包括独立包装的测序系绳(Sequencing tether，SQT)、DNA控制链(DNA control strand)、测序颗粒(Loading beads，LB)等中的一样或多样，且不限于此，以方便用户制备可直接上机的样品。
再一方面，请参阅图1和图2，本申请还提供了一种纳米孔表征生物分子的装置，包括：生物芯片、分子膜和如上述的锚定分子，或如上述的试剂盒，分子膜包括分布在生物芯片上的油脂膜10和两亲分子膜20，两亲分子膜20上设置有跨膜纳米孔30；嵌入接头1能够贯穿两亲分子膜20。本申请实施例提供的纳米孔表征生物分子的装置，由于采用了以上任意一实施例提供的锚定分子，因而具有相同的技术效果，这里不再赘述。
生物芯片为提供电势、形成分子膜的结构，生物芯片上包括具有储液 腔的骨架40和对应储液腔设置的电极，骨架40用于放置试剂、待分析物，并用于支撑分子膜。可以在生物芯片上通入极性溶剂，如缓冲溶液，具体可为磷酸盐缓冲溶液、含有KCl或NaCl的HEPES缓冲溶液、含有KCl或NaCl的CAPS缓冲溶液等，再通入两亲性材料的非极性溶剂即溶解两亲性材料的非极性溶剂，具体可为甲基苯基硅油、聚二甲基硅氧烷等，对极性溶剂进行驱赶，即完成“油赶水”过程。在此基础上，再通入极性溶剂对两亲性材料的非极性溶剂进行驱赶，即完成“水赶油”过程。极性溶剂如缓冲溶液，具体可为磷酸盐缓冲溶液、含有KCl或NaCl的HEPES缓冲溶液、含有KCl或NaCl的CAPS缓冲溶液等。此时，每个结构单元中形成极性溶剂-非极性溶剂-极性溶剂的结构，两亲性材料的非极性溶剂夹在两层极性溶剂之间形成两亲分子膜20。加入锚定分子后，锚定分子随机嵌入分子膜中，并逐渐汇集到两亲分子膜20中。电极用于在分子膜的两侧形成电势差，使得待分析物可以穿过跨膜纳米孔30。
又一方面，本申请还提供了一种向跨膜纳米孔富集待分析物的方法，包括如下步骤：
S1、提供嵌入跨膜纳米孔的两亲分子膜；
S2、使两亲分子膜与如上述的嵌入接头接触，且使嵌入接头与连接有待分析物的连接接头接触；或使两亲分子膜与如上述的锚定分子接触，且使锚定分子与待分析物接触；
S3、待分析物在嵌入接头的牵引下靠近跨膜纳米孔。
又一方面，本申请还提供了一种纳米孔表征分析物的方法，包括如前述的方法向跨膜纳米孔富集待分析物，使待分析物接触跨膜纳米孔，
当待分析物相对于孔运动时或当孔内存在分析物时，测定电信号，以测定待分析物的一种或多种特征表征待分析物。
又一方面，本申请还提供了一种如上述的锚定分子、或如上述的试剂盒、或如上述的纳米孔表征生物分子的装置在表征生物分子上的应用。本申请实施例提供的应用，由于采用了以上任意一实施例提供的锚定分子，因而具有相同的技术效果，这里不再赘述。
可选地，分析物包括生物分子，生物分子包括多核苷酸、多肽、多糖、 或脂质中的一种或多种。表征的特征可以是(i)多核苷酸的长度；(ii)多核苷酸的一致性；(iii)多核苷酸的序列；(iv)多核苷酸的二级结构和(v)多核苷酸是否经修饰。
本申请还提供了以下验证试验，以说明本申请提供的嵌入接头、锚定分子、试剂盒、纳米孔表征生物分子的装置、向跨膜纳米孔富集待分析物的方法、纳米孔表征分析物的方法及应用的效果。
实施例1：一种锚定分子的制备
设计并合成连接接头，其核苷酸序列如下：
Figure PCTCN2022138280-appb-000002
设计并合成嵌入接头，其氨基酸序列如下：
Figure PCTCN2022138280-appb-000003
本实施例中，连接接头通过捕获链与待分析物连接，嵌入接头为多肽，连接接头和嵌入接头通过“DBCO”和“N3”连接。请参阅图4，为该多肽跨膜区域预测图，从图4可以看出氨基酸序列中第7位到第29位氨基酸(氨基酸序列中以下划线区别)为跨膜区域，说明该锚定分子可以贯穿两亲分子膜。
将该连接接头与嵌入接头进行孵育，收集孵育后的产物，该孵育产物为锚定分子。请一并参阅图5和图6，图5为嵌入接头的色谱图，图6为孵育产物的色谱图，单一嵌入接头的出峰时间为24.227min，孵育产物的出峰时间为31.353min，说明连接接头与嵌入接头连接形成的锚定分子出峰时间为31.353min，对应收集31.353min的孵育产物即可实现收集锚定分子。
实施例2：一种嵌入接头的制备及检测
设计并合成嵌入接头，其氨基酸序列如下：
Figure PCTCN2022138280-appb-000004
请参阅图7，为该多肽跨膜区域预测图，其中，第4位到第26位氨基酸(氨基酸序列中以下划线区别)为跨膜区域。
将荧光基团FAM或Cy5连接至嵌入接头，在齐碳科技有限公司的基因测序仪QNome-9604的生物芯片的储液腔中加入缓冲溶液(500mM KCl， 20mM HEPES，pH 8.0)，再加入40μl硅油和磷脂酰胆碱(PC，10mg/mL)的混合油，最后再加入前述缓冲溶液，制备得到分子膜，其结构如图2所示；将连接有荧光基团的嵌入接头与分子膜混合，使嵌入接头在分子膜加入侧所在溶液中的浓度为0.5μM，室温孵育1小时。请参阅图8和图9，其中，图8为在加入实施例2制备的嵌入接头30分钟后，分子膜的荧光显微镜观察结果图，图9是加入实施例2制备的嵌入接头12h后，分子膜的荧光显微镜观察结果，其中虚线框选的部分对应位于分子膜中间的两亲分子膜。对比图8和图9可见，图9虚线所示部分明度提高，证明随着时间的增加，嵌入接头向分子膜中间的两亲分子膜汇聚，从而可以进一步将待分析物富集在两亲分子膜所在区域。
同样将实施例1制备的锚定分子连接荧光基团，与分子膜混合进行孵育，也可以观察到随着时间增加，锚定分子向分子膜中间的两亲分子膜汇聚现象。
实施例3：使用锚定分子对分析物进行纳米孔测序
(1)设置实验组和对照组，实验组和对照组中，均采用相同的生物芯片，并按实施例2的方法制备得到分子膜；
(2)在实验组的分子膜上，加入实施例1制备的锚定分子，使嵌入接头在分子膜加入侧所在溶液中的浓度为0.5μM，室温孵育60分钟；在对照组的分子膜上，未加入任何锚定分子，室温孵育60分钟；
(3)在实验组和对照组中均加入相同浓度的样品，使用齐碳科技有限公司的基因测序仪QNome-9604对样品中的ssDNA进行测序，获得过孔信号图。
请参阅图10、图11和图12，其中图10为对照组过孔信号图，图11为实验组过孔信号图，图12为对照组和实验组的单位时间内ssDNA过孔数量图。由过孔信号图可以看出，采用本申请提供的锚定分子能大幅提高待分析物过孔频率，从而提高检测效率。
对比例1：制备非刚性疏水多肽
按以下氨基酸序列合成多肽：
Figure PCTCN2022138280-appb-000005
其中，“WERNLP”与实施例1中的第一亲水段序列相同，“KYKLLPRS”与实施例1中的第二亲水段序列相同，对比例1中的氨基酸序列仅中间部分与实施例1中的疏水段不相同。
请参阅图13，为对比例1的多肽跨膜区域预测图，其中，氨基酸序列中第7位到第29位氨基酸(氨基酸序列中以下划线区别)为跨膜区域，说明该多肽与实施例1中的嵌入接头相同，均可以贯穿两亲分子膜。
通过ExPASy提供的疏水性分析软件对实施例1中的嵌入接头和对比例1中的多肽进行疏水性分析，得到图14。请参阅图14，图14(a)为实施例1的嵌入接头疏水性曲线图，图14(b)为对比例1的多肽疏水性曲线图，从图中可以看出，实施例1中的嵌入接头和对比例1中的多肽均呈现中间部分疏水、两端亲水。
通过蛋白质二级结构预测工具JPred对实施例1中的嵌入接头和对比例1中的多肽进行二级结构分析，得到图15。请参阅图15，图15(a)为实施例1的二级结构预测，图15(b)为对比例1的多肽的二级结构预测，其中，加粗显示的部分表示α螺旋结构，即实施例1中的嵌入接头的疏水段为α螺旋结构，对比例2中的多肽不具有α螺旋结构。
参照实施例2，将实施例1中的嵌入接头和对比例1中的多肽分别与荧光基团连接，再与分子膜混合进行孵育12h，通过荧光显微镜观察得到，实施例1中的嵌入接头在两亲分子膜的聚集程度大于对比例1中的多肽在两亲分子膜的聚集程度，说明具有刚性的α螺旋结构能提高多肽在两亲分子膜的聚集程度。
实施例4：刚性嵌段聚合物的嵌入接头的制备
设计并合成嵌入接头，其化学式如图16。
本实施例中，嵌入接头为嵌段聚合物，通过聚苯乙炔形成刚性棒状结构。连接接头和嵌入接头通过“DBCO”和“N3”连接。荧光基团FAM连接至连接接头。
对比例2：非刚性嵌段聚合物的制备
设计并合成嵌入接头，其化学式如图17。
本实施例中，嵌段聚合物的两端分别设置“DBCO”和“N3”。与实 施例4不同之处在于，本对比例中的聚硅氧烷替代实施例4中的聚苯乙炔，聚硅氧烷为非刚性结构。
参照实施例2，将实施例4的嵌入接头和对比例2的嵌段聚合物分别与已制备完成的分子膜混合孵育。通过荧光显微镜观察到，随着时间增加，相较于对比例2，实施例4中的嵌入接头更多的向分子膜中间的两亲分子膜汇聚。
另外，本文中术语“和/或”，仅仅是一种描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。另外，本文中字符“/”，一般表示前后关联对象是一种“或”的关系。应理解，在本申请实施例中，“与A相应的B”表示B与A相关联，根据A可以确定B。但还应理解，根据A确定B并不意味着仅仅根据A确定B，还可以根据A和/或其它信息确定B。
以上，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以权利要求的保护范围为准。

Claims (17)

  1. 一种嵌入接头，所述嵌入接头用于嵌设于两亲分子膜处，所述嵌入接头包括：依次连接的第一亲水段、疏水段和第二亲水段，
    所述疏水段用于跨所述两亲分子膜的疏水区域，以使所述嵌入接头能够贯穿所述两亲分子膜，所述疏水段具有刚性；
    所述第一亲水段和/或所述第二亲水段能够与待分析物连接。
  2. 根据权利要求1所述的嵌入接头，其中，所述疏水段为棒状。
  3. 根据权利要求1或2所述的嵌入接头，其中，所述疏水段为疏水多肽。
  4. 根据权利要求3所述的嵌入接头，其中，所述疏水多肽为α螺旋结构；
    所述第一亲水段和/或所述第二亲水段为亲水多肽。
  5. 根据权利要求3所述的嵌入接头，其中，所述疏水段中至少包括丙氨酸残基(A)、精氨酸残基(R)、亮氨酸残基(L)、赖氨酸残基(K)、甲硫氨酸残基(M)中的一种或多种。
  6. 根据权利要求3所述的嵌入接头，其中，所述疏水段与所述疏水区域的疏水匹配差小于或等于5nm。
  7. 根据权利要求3所述的嵌入接头，其中，所述疏水段中，靠近所述待分析物一端的酸性氨基酸残基分布密度小于远离所述待分析物一端的酸性氨基酸残基分布密度；和/或
    靠近所述待分析物一端的碱性氨基酸残基分布密度大于远离所述待分析物一端的碱性氨基酸残基分布密度。
  8. 根据权利要求1或2所述的嵌入接头，其中，所述疏水段为疏水性共聚物；
    所述第一亲水段和/或所述第二亲水段为亲水性共聚物。
  9. 根据权利要求8所述的嵌入接头，其中，所述疏水段中至少部分相邻分子之间采用非单键连接；和/或，所述疏水性共聚物为聚噻吩、聚苯乙炔、聚菲乙炔中的任意一种。
  10. 一种锚定分子，包括连接接头和如权利要求1至9中任一项所述 的嵌入接头，所述连接接头包括能够与所述待分析物连接的部分和能够与所述嵌入接头连接的部分。
  11. 根据权利要求10所述的锚定分子，其中，所述连接接头设于所述嵌入接头的一端或两端；
    和/或，所述连接接头包括能够与待分析物通过碱基互补配对连接的捕获链，所述捕获链通过点击化学反应与所述嵌入接头连接。
  12. 一种在两亲分子膜中组装嵌入接头的方法，包括将权利要求1至9中任一项所述的嵌入接头与分子膜接触，所述分子膜包括油脂膜和两亲分子膜，随后所述嵌入接头嵌设于所述两亲分子膜处。
  13. 一种纳米孔表征分析物用的分子膜，所述分子膜包括油脂膜和两亲分子膜，所述两亲分子膜中嵌入跨膜纳米孔以及如权利要求1至9中任一项所述的嵌入接头或如权利要求10至11中任一项所述的锚定分子。
  14. 一种纳米孔表征分析物的装置，包括：
    生物芯片；
    分子膜，包括分布在所述生物芯片上的油脂膜和两亲分子膜，所述两亲分子膜上设置有跨膜纳米孔；和
    如权利要求1至9中任一项所述的嵌入接头，或如权利要求10至11中任一项所述的锚定分子。
  15. 一种向跨膜纳米孔富集待分析物的方法，包括如下步骤：
    提供分子膜，所述分子膜包括油脂膜和嵌入跨膜纳米孔的两亲分子膜；
    使所述两亲分子膜与如权利要求1至9中任一项所述的嵌入接头接触，且使所述嵌入接头与连接有待分析物的连接接头接触，或使所述两亲分子膜与如权利要求10至11中任一项所述的锚定分子接触，且使所述锚定分子与待分析物接触；
    待分析物在所述嵌入接头的牵引下靠近所述跨膜纳米孔。
  16. 一种纳米孔表征分析物的方法，包括如权利要求15所述方法向跨膜纳米孔富集待分析物，使待分析物接触所述跨膜纳米孔，
    当待分析物相对于所述跨膜纳米孔运动时或当所述跨膜纳米孔内存在待分析物时，测定电信号，以测定待分析物的一种或多种特征表征待分析 物。
  17. 一种如权利要求1至9中任一项所述的嵌入接头、或如权利要求10至11中任一项所述的锚定分子、或如权利要求13所述的分子膜、或如权利要求14所述的装置在分析物表征上的应用。
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