WO2024037325A1 - 富集方法、表征分析物的方法及其装置 - Google Patents

富集方法、表征分析物的方法及其装置 Download PDF

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WO2024037325A1
WO2024037325A1 PCT/CN2023/110339 CN2023110339W WO2024037325A1 WO 2024037325 A1 WO2024037325 A1 WO 2024037325A1 CN 2023110339 W CN2023110339 W CN 2023110339W WO 2024037325 A1 WO2024037325 A1 WO 2024037325A1
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membrane
molecule
occupying
molecules
space
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PCT/CN2023/110339
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English (en)
French (fr)
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刘少伟
苗卉
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 WO2024037325A1 publication Critical patent/WO2024037325A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

Definitions

  • the present application belongs to the technical field of bioanalytical detection, and specifically relates to enrichment methods, methods for characterizing analytes and devices thereof, and more specifically to methods of enriching anchor molecules on amphiphilic membranes, and enriching analytes on amphiphilic membranes.
  • a molecular membrane with detectors such as nanoscale pores divides the cavity into two small chambers.
  • analytes can pass through the pores. , forming a stable and detectable ion current, which can detect different types of biomolecules based on current information.
  • Existing molecular membranes are provided with anchoring molecules to bind biomolecules through the anchoring molecules, causing biomolecules to aggregate on the surface of the molecular membrane.
  • Existing anchor molecules are usually cholesterol with tethers, and cholesterol is evenly distributed on the molecular membrane, while detectors such as nanopores are only set up on the amphipathic membrane of the molecular membrane, such as phospholipid membranes or polymer membranes, resulting in partial Biomolecules far away from the nanopore are not easy to detect.
  • the concentration of biomolecules is low, the effective current data output time is short, which affects the detection effect.
  • the inventors unexpectedly discovered that after using space-occupying molecules to bind to the non-amphiphilic membrane area of the lipid membrane, and then adding anchor molecules, the anchor molecules can be enriched on the amphiphilic membrane, thereby achieving analyte Enrichment of the detector region in the amphiphilic membrane improves detection sensitivity.
  • a first aspect of the invention relates to a method of enriching anchor molecules on an amphiphilic membrane, said method comprising:
  • the occupying molecule contacts the molecular membrane on the cis side or the trans side of the molecular membrane. More preferably, the occupying molecule contacts the molecular membrane on the cis side of the molecular membrane.
  • the space-occupying molecules include a first space-occupying molecule and optionally one or more second space-occupying molecules.
  • the first space-occupying molecule is used to connect with the grease film, and the one or more space-occupying molecules At least one of the second space-occupying molecules is used to connect with the first space-occupying molecule.
  • the first space-occupying molecule contains a hydrophobic molecule, preferably a hydrophobic polypeptide or a hydrophobic polymer. More preferably, the hydrophobic polymer is selected from the group consisting of polythiophene, polyphenylene vinylene and polyphenanthrene acetylene;
  • the second space-occupying molecule includes hydrophilic macromolecules, preferably DNA, PEG, polyacrylamide, polyacrylic acid or cellulose.
  • the anchoring molecule comprises a membrane-binding portion for binding to the amphiphilic membrane and an analyte-binding portion for binding an analyte.
  • the membrane-binding moiety is selected from lipids, fatty acids, sterols, carbon nanotubes or amino acids; the analyte-binding moiety is a nucleic acid sequence.
  • the method further includes providing a biochip for attaching the lipid film and supporting the molecular film.
  • the method further comprises inserting one or more detectors into said amphiphilic membrane.
  • the detector comprises nanopores, including solid pores and/or biological pores, including transmembrane protein pores.
  • the amphiphilic membrane is a lipid bilayer membrane
  • the lipid membrane is a mixture membrane formed of any one or more of silicone oil, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol.
  • a second aspect of the invention relates to a method of enriching an analyte on an amphiphilic membrane, comprising: binding an anchor molecule, preferably a membrane-binding portion of the anchor molecule, to the amphiphilic membrane;
  • analyte binds to the anchor molecule, preferably to an analyte binding portion of the anchor molecule.
  • a third aspect of the invention relates to a method of enriching an analyte in a region of a detector, comprising: enriching the analyte on an amphiphilic membrane in which the detector is inserted; and The analyte is drawn close to the area of the detector by the anchor molecule.
  • a fourth aspect of the invention relates to a method of characterizing an analyte, comprising enriching the analyte in a region of a detector and taking one or more measurements as the analyte moves relative to the detector, wherein said one or more Multiple measurements indicate one or more characteristics of an analyte, thereby characterizing the analyte as it moves relative to the detector.
  • the method is used to characterize multiple analytes.
  • the or each analyte is selected from one or more of polynucleotides, polypeptides, polysaccharides and lipids, preferably polynucleotides including DNA and/or RNA.
  • a fifth aspect of the invention relates to a kit for characterizing an analyte, comprising:
  • the molecular membrane includes an amphiphilic membrane and a lipid membrane.
  • the amphiphilic membrane is arranged in the lipid membrane and has one or more detectors inserted therein, so that the formed molecular membrane contains a detector inserted therein.
  • An anchoring molecule which is used to bind the analyte to the amphiphilic membrane after the lipid membrane is bound by the spacer molecule, and to bring the analyte close to the detector Area.
  • the space-occupying molecules are used to bind to the cis side of the lipid film.
  • the space-occupying molecules include a first space-occupying molecule and optionally one or more second space-occupying molecules.
  • the first space-occupying molecule is used to connect with the grease film, and the one or more space-occupying molecules At least one of the second space-occupying molecules is used to connect with the first space-occupying molecule.
  • the first space-occupying molecule contains a hydrophobic molecule, preferably a hydrophobic polypeptide or a hydrophobic polymer. More preferably, the hydrophobic polymer is selected from the group consisting of polythiophene, polyphenylene vinylene and polyphenanthrene acetylene;
  • the second space-occupying molecule includes hydrophilic macromolecules, preferably DNA, PEG, polyacrylamide, polyacrylic acid or cellulose.
  • the anchoring molecule comprises a membrane-binding portion for binding to the amphiphilic membrane and an analyte-binding portion for binding an analyte.
  • the membrane-binding moiety is selected from lipids, fatty acids, sterols, carbon nanotubes or amino acids; the analyte-binding moiety is a nucleic acid sequence.
  • the kit further includes a biochip, which is used to attach the lipid film and support the molecular film.
  • the sixth aspect of the present invention relates to a molecular membrane for nanopore characterization of analytes, wherein the molecular membrane includes an amphiphilic membrane and a lipid membrane, and the amphiphilic membrane is disposed in the lipid membrane and inserted with one or more A detector is provided, the lipid membrane is bound with a space-occupying molecule, and the amphiphilic membrane is bound with an anchoring molecule.
  • the space-occupying molecule is bound to the cis side of the lipid film.
  • the space-occupying molecules include a first space-occupying molecule and optionally one or more second space-occupying molecules, the first space-occupying molecule is connected to the lipid film, and the one or more second space-occupying molecules At least one of the space-occupying molecules is connected to the first space-occupying molecule.
  • the first space-occupying molecule contains a hydrophobic molecule, preferably a hydrophobic polypeptide or a hydrophobic polymer. More preferably, the hydrophobic polymer is selected from the group consisting of polythiophene, polyphenylene vinylene and polyphenanthrene acetylene;
  • the second space-occupying molecule includes hydrophilic macromolecules, preferably DNA, PEG, polyacrylamide, polyacrylic acid or cellulose, more preferably DNA.
  • the anchoring molecule comprises a membrane-binding portion for binding to the amphiphilic membrane and an analyte-binding portion for binding an analyte.
  • the membrane-binding moiety is selected from lipids, fatty acids, sterols, carbon nanotubes or amino acids; the analyte-binding moiety is a nucleic acid sequence.
  • a seventh aspect of the invention relates to a device for nanopore characterization of analytes, the device comprising:
  • the eighth aspect of the present invention relates to the method as provided in the first to fourth aspects, the kit as provided in the fifth aspect, the molecular membrane as provided in the sixth aspect, or the device as provided in the seventh aspect in preparing a product characterizing the analyte. or applications in characterizing analytes.
  • the present invention realizes that by using space-occupying molecules to bind to the non-amphiphilic membrane area of the lipid membrane, and then adding anchor molecules to improve the enrichment of the anchor molecules on the amphiphilic membrane, thereby improving the concentration of analytes in the amphiphilic membrane. Efficient enrichment of the detector area, especially when the analyte concentration is low, such as when the analyte concentration is as low as 20 pM, can efficiently enrich the Collect low concentration analytes.
  • the present invention can reduce or avoid the attachment of anchor molecules on the surface of the chip support structure, thereby improving the enrichment effect.
  • the present invention can improve the detection sensitivity of target analytes such as target polynucleotides.
  • Figure 1 shows a partial structural diagram of a device for characterizing analytes in one embodiment of the present invention. Among them, 10. Grease membrane; 20. Amphiphilic membrane; 30. Transmembrane pore; 40. Chip matrix.
  • Figure 2 shows the fluorescence distribution in the molecular film of A) anchor molecule alone, B) spacer molecule alone, and C) spacer molecule added first and then anchor molecule added.
  • A) the fluorescence of individual anchor molecules is evenly distributed on the lipid film and amphiphilic membrane of the chip; in B), the fluorescence of individual placeholder molecules is mainly distributed in the lipid film area of the chip surface and rarely Distributed in the amphiphilic membrane area; in C), the fluorescence of the anchoring molecule is stronger in the amphiphilic membrane area than in other areas.
  • Figure 3 shows the fluorescence intensity ratio of the anchor molecule (amphiphilic membrane area/non-amphiphilic membrane area) in the cases of A) and C) of Figure 2.
  • the fluorescence intensity ratio is significantly increased, which is about 1.5 times that of adding only anchoring molecules.
  • Figure 4 shows the structure of a linker-binding enzyme, in which the enzyme is bound to the dotted region of the Y1-S single strand of the linker, and the linker is connected to the polynucleotide sequence to be tested.
  • Figure 5 shows the signal diagram of nanopore sequencing of ssDNA in the control group (ie, only anchoring molecules are added).
  • Figure 6 shows the signal diagram of nanopore sequencing of ssDNA in the experimental group (that is, the placeholder molecule is added first, and then the anchor molecule is added).
  • SEQ ID NO.: 1 shows the sequence of the DNA of the placeholder molecule of Example 1
  • SEQ ID NO.: 2 shows the sequence of the hydrophobic polypeptide of the placeholder molecule of Example 1
  • SEQ ID NO.: 3 shows the nucleic acid sequence of the anchor molecule of Example 1
  • SEQ ID NO.: 4 shows the sequence of the hydrophobic polypeptide of the placeholder molecule of Example 3
  • SEQ ID NO.: 5 shows the Y1-S nucleic acid sequence of the linker of Example 4.
  • SEQ ID NO.: 6 shows the Y2-S nucleic acid sequence of the linker of Example 4.
  • SEQ ID NO.: 7 shows the Y-Bottom-S nucleic acid sequence of the linker of Example 4.
  • the molecular membrane when characterizing biomolecules based on transmembrane pore sequencing technology, a molecular membrane needs to be prepared on the substrate 40 of the gene chip.
  • the molecular membrane includes a mixed distribution of a lipid membrane 10 and an amphiphilic membrane 20.
  • the amphiphilic membrane 20 is set In the lipid film 10 , the molecular film formed has a structure including the amphiphilic membrane 20 and the lipid film 10 surrounding the amphiphilic membrane 20 .
  • the amphiphilic membrane 20 is a membrane structure formed by amphiphilic molecules, and a detector, such as a transmembrane pore 30, is provided on the amphiphilic membrane 20.
  • Amphiphilic molecules can be synthetic or naturally occurring.
  • the phosphoric acid part of the phospholipid is hydrophilic and the fatty acid part is hydrophobic, thus exhibiting both hydrophilic and hydrophobic properties.
  • Another example is synthetic block copolymers, in which two or more monomer subunits are polymerized together to create a single polymer chain. Block copolymers can be engineered so that one of the monomer subunits is hydrophobic in aqueous media, while the other subunit is hydrophilic. In this case, the block copolymers can have amphiphilic properties and can form structures that mimic biological membranes.
  • Block total The polymer can be a diblock, triblock, tetrablock or pentablock copolymer.
  • the lipid bilayer is a model cell membrane and serves as an excellent platform for a range of experimental studies.
  • lipid bilayers can be used for in vitro studies of membrane proteins using single-channel recording.
  • lipid bilayers can be used as biosensors to detect the presence of a range of substances.
  • the lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes.
  • the grease film 10 is composed of multiple layers of molecules and has a thicker thickness, so that the grease film 10 can be stably attached to the matrix 40.
  • the amphiphilic membrane 20 can be fixed to the matrix 40 through the grease film 10, which is convenient. Subsequent biomolecular characterization was performed.
  • the grease film 10 may specifically be any one or a mixture of silicone oil, phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), etc. .
  • the transmembrane pore 30 is a through hole provided along the thickness direction of the amphiphilic membrane 20 to connect both sides of the amphiphilic membrane 20 .
  • the transmembrane pore 30 may specifically be a protein pore, a polynucleotide pore, a solid pore, etc.
  • the present application does not limit the specific formation form of the transmembrane pore 30 .
  • the analyte can pass through the transmembrane pore 30 in sequence.
  • the characterization information of the passing analyte can be further obtained based on the electrical signal. For example, the size information, sequence information, identity information, modification information, etc. of the analyte can be obtained based on the electrical signal change information.
  • the molecular membrane of the present invention is a molecular membrane used to characterize analytes in nanopores, wherein the molecular membrane includes an amphiphilic membrane and a lipid membrane, and the amphiphilic membrane is disposed in the lipid membrane and inserted with one or more In the detector, the lipid membrane is bound with space-occupying molecules, and the amphiphilic membrane is bound with anchoring molecules.
  • the “cis-side” refers to the entry side of the analyte through which the analyte or modified analyte enters the opening of the nanopore;
  • the “trans-side” refers to the outflow side of the analyte through which the analyte or modified analyte passes.
  • Side outflow nanopore opening For example, the cis side is on the top of the chip and the trans side is on the bottom of the chip. In some cases, molecules pass through the pore from the cis-side to the trans-side of the membrane or pore.
  • Space-occupying molecules preferentially bind to the cis side of the lipid film.
  • the placeholder molecule of the present invention is used to characterize biomolecules based on transmembrane pore sequencing technology. Before the biomolecules are combined with the amphiphilic membrane around the transmembrane pore, they are first bound to the non-amphiphilic membrane area (that is, there is no arrangement on the lipid membrane). area of the amphiphilic membrane). This substance occupies the non-amphiphilic membrane area on the lipid membrane so that other substances cannot bind to this area, so it is called an occupying molecule.
  • the space-occupying molecule comprises a first space-occupying molecule and optionally one or more second space-occupying molecules.
  • the space-occupying molecule may be composed of a first space-occupying molecule and optionally one or more second space-occupying molecules.
  • the first space-occupying molecule is used to connect to the lipid film, preferably the non-amphiphilic membrane region on the lipid film, and at least one of the one or more second space-occupying molecules is used to connect to the first space-occupying molecule.
  • the first space-occupying molecule contains or is a hydrophobic molecule, preferably a hydrophobic polypeptide or a hydrophobic polymer.
  • the hydrophobic polymer is selected from polythiophene, polyphenylene vinylene and polyphenanthrene acetylene; the second space-occupying molecule contains or It is a hydrophilic macromolecule, preferably DNA, PEG, polyacrylamide, polyacrylic acid or cellulose.
  • the spacer molecule is a hydrophobic polypeptide.
  • the spacer molecule is a structure in which a hydrophobic polypeptide is attached to DNA.
  • a first of the spacer molecules is linked to a second spacer molecule through a click chemical reaction.
  • Click chemistry is a method of quickly completing the chemical synthesis of different molecules through the splicing of small units.
  • the first spacer molecule is connected with a first linking group
  • the second spacer molecule is connected with a second linking group
  • click chemistry occurs through the first linking group and the second linking group. reaction to achieve rapid connection between the first space-occupying molecule and the second space-occupying molecule.
  • the first linking group and the second linking group may be connected through a carbon-carbon multi-bond addition reaction, a nucleophilic ring-opening reaction, or a click chemical reaction such as a cycloaddition reaction.
  • one of the first linking group and the second linking group can be cyclooctene (TCO), dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), or bicyclononyne (BCN). ) or any one of dibenzocyclooctyne (DICO); the other can be an azide group (N3), a tetrazinyl group (TZ), etc.
  • TCO cyclooctene
  • DBCO dibenzocyclooctyne
  • DIFO difluorinated cyclooctyne
  • BCN bicyclononyne
  • N3 azide group
  • TZ tetrazinyl group
  • the anchoring molecule of the present invention is a substance that binds to the amphiphilic membrane after the occupying molecules occupy the non-amphiphilic membrane area when characterizing biomolecules based on transmembrane pore sequencing technology.
  • the anchor molecule is anchored on the amphiphilic membrane. After the analyte binds to the anchor molecule, the analyte is drawn close to the detector area in the amphiphilic membrane under the pull of the anchor molecule.
  • the anchor molecule comprises or consists of a membrane-binding moiety and an analyte-binding moiety.
  • the membrane-binding portion is used to bind to the amphiphilic membrane
  • the analyte-binding portion is used to bind the analyte.
  • Anchor molecules are molecules that connect the analyte to the amphiphilic membrane.
  • the membrane binding moiety is selected from lipids, fatty acids, sterols, carbon nanotubes or amino acids; the analyte binding moiety is preferably a nucleic acid sequence.
  • the nucleic acid sequence can be connected to an analyte, such as a polynucleotide, through complementary base pairing.
  • the anchor molecule is a cholesterol-linked nucleic acid structure.
  • the membrane-binding moiety in the anchor molecule is linked to the analyte-binding moiety via click chemistry.
  • Click chemistry is as described above.
  • the membrane binding moiety has a first linker attached to it The analyte-binding part is connected with a second linking group, and a click chemical reaction occurs between the first linking group and the second linking group to achieve rapid connection between the membrane-binding part and the analyte-binding part.
  • the analyte can be any substance. Suitable analytes include, but are not limited to, metal ions, inorganic salts, polymers such as polymeric acids or bases, dyes, bleaches, pharmaceuticals, diagnostics, recreational drugs, explosives, and environmental pollutants.
  • the analyte may be an analyte secreted by a cell.
  • the analyte may be an analyte that is present within a cell such that the analyte must be extracted from the cell before practicing the invention.
  • the analyte is selected from one or more of polynucleotides, polypeptides, polysaccharides and lipids.
  • the analyte is preferably a polynucleotide such as a nucleic acid, including deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA).
  • Polynucleotides can be single-stranded or double-stranded. Polynucleotides can be circular.
  • the polynucleotide can be an aptamer, a probe that hybridizes to the microRNA, or the microRNA itself. Polynucleotides can be of any length.
  • a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotide pairs in length.
  • a polynucleotide may be 1,000 or more nucleotide pairs, 5,000 or more nucleotide pairs in length, or 100,000 or more nucleotide pairs in length.
  • Analytes can be present in any suitable sample.
  • the invention is typically performed on samples known to contain or suspected of containing the analyte.
  • the invention may be practiced on samples containing one or more analytes of unknown species.
  • the present invention can be performed on a sample to identify the species of one or more analytes known or expected to be present in the sample.
  • a detector can be any structure that provides a readable signal in response to the presence, absence, or signature of an analyte.
  • a detector can be any structure that provides a readable signal in response to the presence, absence, or signature of an analyte.
  • Suitable detectors are known in the art and comprise nanopores, including solid state pores and/or biological pores. They include, but are not limited to, transmembrane pores, tunnel electrodes, classical electrodes, nanotubes, FETs (field effect transistors) and photodetectors such as atomic force microscopy (AFM) and scanning tunneling microscopy (STM).
  • the detector detects the analyte electrically.
  • Electrical measurements can be made using standard single-channel recording equipment, such as Stoddart et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50) ):17961-72 and international application WO-2000/28312.
  • electrical measurements can be made using a multi-channel system, for example as described in International Application WO-2009/077734 and International Application WO-2011/067559.
  • the detector does not detect the analyte fluorescently.
  • Transmembrane pores are structures that allow an applied potential to drive the flow of hydrated ions from one side of the membrane to the other.
  • the transmembrane pore is preferably a transmembrane protein pore.
  • a transmembrane protein pore is a polypeptide or collection of polypeptides that allows the flow of hydrated ions (eg, analytes) from one side of a membrane to the other.
  • transmembrane protein pores can form pores that allow an applied potential to drive the flow of hydrated ions from one side of the membrane to the other.
  • Transmembrane protein pores preferably allow the flow of analytes (eg, nucleotides) from one side of the membrane (eg, lipid bilayer) to the other.
  • a transmembrane protein pore preferably enables movement of a polynucleotide or nucleic acid (eg, DNA or RNA) through the pore.
  • Transmembrane protein pores can be monomers or oligomers.
  • the pore preferably consists of several repeating subunits, such as 6, 7 or 8 subunits.
  • the pore is more preferably a heptamer or octamer pore.
  • Transmembrane protein pores typically contain barrels or channels through which ions can flow.
  • the subunits of the pore generally surround a central axis and provide strands for either transmembrane ⁇ barrels or channels or transmembrane ⁇ -helical bundles or channels.
  • the method of the present invention can enrich anchor molecules on an amphiphilic membrane, and further enrich analytes on the amphiphilic membrane by connecting the anchor molecules to analytes.
  • the amphiphilic membrane has a detector inserted into it, so the method of the invention can also concentrate the analyte in the area of the detector.
  • the method of the present invention first uses space-occupying molecules to bind and occupy the lipid membrane, and then binds the anchor molecules added later to the amphiphilic membrane not occupied by the space-occupying molecules, thereby achieving enrichment.
  • the present invention provides a method for enriching anchor molecules on an amphiphilic membrane, including: providing a molecular membrane, the molecular membrane includes an amphiphilic membrane and a lipid membrane, and the amphiphilic membrane is disposed in the lipid membrane; and provide occupying molecules and anchoring molecules, so that the molecular film contacts the occupying molecules and anchoring molecules in sequence, wherein contact with the occupying molecules causes the occupying molecules to bind to the lipid film, and contact with the anchoring molecules causes the anchoring molecules to bind on the amphiphilic membrane, thereby enriching the anchor molecules directly on the amphiphilic membrane.
  • the present invention provides a method for enriching an analyte on an amphiphilic membrane, including: causing an anchor molecule to bind to the amphiphilic membrane; and contacting the analyte with the anchor molecule, such that the analyte Binds to the anchoring molecule, thereby enriching the analyte on the amphiphilic membrane through the anchoring molecule.
  • the present invention provides a method of enriching an analyte in a region of a detector, comprising: enriching the analyte on an amphiphilic membrane with a detector inserted into the amphiphilic membrane; and The region where the analyte is drawn by the anchoring molecule is brought close to the detector inserted in the amphiphilic membrane.
  • a molecular membrane as shown in Figure 1 which molecular membrane includes a detector (eg, a transmembrane pore) inserted into an amphiphilic membrane, an amphiphilic membrane (eg, a lipid bilayer) surrounding the transmembrane pore. and a lipid film surrounding an amphiphilic membrane (such as a film formed by a mixture of silicone oil and phosphatidylcholine).
  • the molecular membrane is attached to the chip matrix, and the amphiphilic membrane communicates with the matrix through the lipid film. fixed.
  • the space-occupying molecules, anchoring molecules and analytes of the present invention can be added from the cis or trans side of the amphiphilic membrane. In a preferred embodiment, the occupying molecules, anchoring molecules and analytes of the present invention can be added from the cis side of the amphiphilic membrane.
  • a spacer molecule (such as DNA linked to a hydrophobic polypeptide) is added from the cis side of the amphipathic membrane. After the spacer molecule contacts the molecular membrane, the hydrophobic polypeptide will bind to the non-amphipathic membrane region of the molecular membrane. On the contrary little or no binding to amphiphilic membranes. Hydrophobic peptides are connected to macromolecules such as DNA, thereby occupying the space on the surface of the oil film, making it difficult for other molecules to attach to the surface of the oil film.
  • anchoring molecules (such as cholesterol-linked DNA) are added from the cis side of the amphiphilic membrane. Cholesterol is more likely to attach to the surface of the amphiphilic membrane rather than the surface of the lipid membrane, thus appearing as an anchor on the amphiphilic membrane. Enrichment.
  • the polynucleotide is ligated to the linker to form the construct by any suitable method known in the art.
  • Linkers can be synthesized separately and then chemically or enzymatically ligated to the polynucleotide. Alternatively, the linker is generated during processing of the polynucleotide.
  • a linker is attached to a polynucleotide at or near one end of the polynucleotide. Any known method can be used to couple the polynucleotide-containing construct to the membrane.
  • the construct is preferably coupled to the amphiphilic membrane via an anchor molecule already attached to the amphiphilic membrane.
  • the linker and the anchoring molecule respectively have nucleotide sequences capable of complementary base pairing, thereby realizing connection between the linker and the anchoring molecule, and thereby connecting the analyte to the amphipathic membrane.
  • the detector (such as a transmembrane pore) is inserted into the amphiphilic membrane, and the analytes enriched on the amphiphilic membrane are drawn close to the area of the transmembrane pore by the anchoring molecules, thereby achieving enrichment of the analyte in the detector area.
  • Some embodiments of the invention include characterizing analytes.
  • the analyte can be characterized as it moves relative to the detector. Movement of the analyte relative to the detector may be driven by any suitable device. In some embodiments, the movement of the analyte is driven by physical or chemical forces (potentials), and in some embodiments the physical forces are provided by electrical (eg, voltage) potentials or temperature gradients, etc.
  • the analyte when a potential is applied across a detector (eg, a transmembrane pore), the analyte moves relative to the detector.
  • the analyte such as a polynucleotide, is negatively charged, so applying a voltage across the nanopore will cause the analyte to move relative to the detector.
  • moving a polynucleotide through a transmembrane pore means moving a polynucleotide from one side of the pore to the other. Movement of the polynucleotide through the pore can be driven or controlled by electrical potential or enzymatic action, or both. Movement may be one-way, or may allow backward and forward movement.
  • Polynucleotide binding proteins are preferably used to control the movement of polynucleotides through the pores.
  • a polynucleotide binding protein may be any protein capable of binding to a polynucleotide and controlling its movement through the pore. Determining whether a protein binds to a polynucleotide is straightforward in the art. Proteins typically interact with polynucleotides and modify at least one of their properties. Proteins can modify polynucleotides by cleaving them to form individual nucleotides or shorter nucleotide chains such as dinucleotides or trinucleotides. The moiety can modify a polynucleotide by positioning or moving the polynucleotide to a specific location (ie, controlling its movement).
  • the characterization method of the present invention can be performed using the molecular membrane of the present invention, and the analytes are enriched near the transmembrane pores through the enrichment method of the present invention.
  • the characterization method may include measuring one, two, three, four or five or more characteristics of the analyte (eg, polynucleotide).
  • the characteristics are preferably 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) polynucleotide Whether the nucleotide is modified.
  • Methods of the invention comprise moving a polynucleotide through a transmembrane pore such that a portion of the nucleotides of the polynucleotide interact with the pore.
  • transmembrane pores can be used to distinguish nucleotides with similar structures based on their different effects on the current flow through the pore.
  • Individual nucleotides can be identified at the single-molecule level based on the amplitude of their current when they interact with the pore.
  • a nucleotide is present in the pore if current flows through the pore in a manner specific to that nucleotide (i.e., if a characteristic current associated with that nucleotide is detected flowing through the pore). exist in.
  • the sequential identification of nucleotides in a polynucleotide enables the sequence of the polynucleotide to be estimated or determined.
  • the invention also provides a kit for characterizing analytes, comprising:
  • Molecular membrane which includes an amphiphilic membrane and a lipid membrane.
  • the amphiphilic membrane is arranged in the lipid membrane and has one or more detectors inserted therein;
  • Anchor molecules which are used to bind the analyte to the amphiphilic membrane after the lipid membrane is bound by the occupying molecule, and to bring the analyte close to the detector area.
  • the molecular membrane, space-occupying molecules and anchoring molecules in the kit are as described above.
  • the kit also contains a biochip, which is used to attach the lipid film and support the molecular film.
  • the kit also contains substances known in the art for characterizing the analyte, such as linkers for binding to polynucleotides to form constructs, polynucleotide binding proteins that control the movement of polynucleotides wait.
  • substances known in the art for characterizing the analyte such as linkers for binding to polynucleotides to form constructs, polynucleotide binding proteins that control the movement of polynucleotides wait.
  • the present invention also provides a device for characterizing analytes via nanopores, which device includes a biochip; and the molecular membrane of the present invention.
  • the experimental reagents and instruments used are all conventional commercially available reagents or instruments.
  • the sequences used are synthesized by conventional methods in this field. or commercially available.
  • the synthesized hydrophobic polypeptide and DNA are connected through click chemical linking groups "DBCO” and "N3", and the fluorescent group Cy3 is connected to the 5' end of the DNA.
  • An anchor molecule is designed and synthesized, and its nucleotide sequence is as follows:
  • the 5' end of the nucleotide sequence is connected to cholesterol for binding to the membrane, and the 3' end is connected to the fluorescent group Cy5.
  • the nucleotide sequence can bind to the analyte in a complementary base pairing manner.
  • Example 2 Distribution of space-occupying molecules and anchoring molecules on the chip surface
  • buffer solution 500mM KCl, 20mM HEPES, pH 8.0
  • PC phosphatidylcholine
  • the space-occupying molecules and anchoring molecules prepared in Example 1 were added to the cis-side solution of the chip in the following three different ways, and incubated with the above-mentioned molecular membrane.
  • the three methods are: A) Add individual anchor molecules to the chip at a final concentration of 20nM and incubate at room temperature for 15 minutes; B) Add individual space-occupying molecules to the chip at a final concentration of 200nM and incubate at room temperature for 16 hours; and C ) First add space-occupying molecules to the chip with a final concentration of 200nM and incubate at room temperature for 16 hours. Then add anchor molecules with a final concentration of 20nM and incubate at room temperature for 15 minutes. Using a confocal microscope to take layer scanning photos of the chip and three-dimensional reconstruction, we observed the fluorescence of anchoring molecules and space-occupying molecules in the molecular film in three ways. The light distribution is shown in Figure 2.
  • the fluorescence of individual anchor molecules is evenly distributed across the lipid and amphiphilic membranes of the chip.
  • the fluorescence of individual space-occupying molecules is mainly distributed in the lipid film area on the chip surface and rarely in the amphiphilic membrane area.
  • Example 3 Preparation and distribution of another space-occupying molecule and anchoring molecule
  • the placeholder molecule was synthesized according to the method of Example 1, except that the hydrophobic polypeptide sequence in the placeholder molecule was replaced with: GGGKWWLALALALALALALWWA.
  • the sequence of the synthesized spacer molecule is as follows:
  • the distribution of the space-occupying molecules and anchoring molecules on the molecular film was observed according to the method of Example 2. The results show that there is no significant difference from the results in Example 2.
  • the spacer molecule is added first and then the anchor molecule is added, compared with the case where only the anchor molecule is added, the fluorescence intensity on the amphiphilic membrane/fluorescence intensity in the non-amphiphilic region The ratio has improved significantly.
  • the Y1-S chain contains the leader sequence, iSpC3, denoted as n.
  • the synthesis method is as follows: anneal primers 1, 2, and 3 according to the ratio of 1:2.5:2.5.
  • the final primer annealing concentration is 4 ⁇ M.
  • the annealing program is 98°C for 10min; 6s/-0.1°C, 300 ⁇ Cys; 65°C, 5min; 6s/-0.1°C, 400 ⁇ Cys; 12°C, Hold.
  • the annealed primers were incubated according to the system in Table 1 below.
  • the product is purified with magnetic beads to obtain the ligation product to be sequenced.
  • the results of current changes over time during the sequencing process of the control group and experimental group are shown in Figure 5 and Figure 6.
  • the opening current is about 300-400pA
  • the nucleic acid through-hole current is about 100-200pA.
  • the current drops from the opening to the via hole and then returns to the opening, which is a nucleic acid via hole.
  • the frequency of DNA per unit time in the experimental group samples increased by 3.27 times, indicating that the sequencing sensitivity in the experimental group was also significantly improved.

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Abstract

本发明涉及富集方法、表征分析物的方法及其装置，更具体地涉及将锚定分子富集在两亲膜上的方法、将分析物富集在两亲膜上的方法、将分析物富集在检测器的区域中的方法、表征分析物的方法、表征分析物的试剂盒、纳米孔表征分析物用的分子膜、纳米孔表征分析物的装置及应用。本发明可以实现低浓度分析物的富集，提高检测灵敏度。

Description

富集方法、表征分析物的方法及其装置
相关申请的交叉引用
本申请要求享有于2022年8月16日提交的名称为“富集方法、表征分析物的方法及其装置”的中国专利申请202210982932.5的优先权，该申请的全部内容通过引用并入本文中。
技术领城
本申请属于生物分析检测的技术领域，具体涉及富集方法、表征分析物的方法及其装置，更具体地涉及将锚定分子富集在两亲膜上的方法、将分析物富集在两亲膜上的方法、将分析物富集在检测器的区域中的方法、表征分析物的方法、表征分析物的试剂盒、纳米孔表征分析物用的分子膜、纳米孔表征分析物的装置及应用。
背景技术
现有纳米孔测序中，在充满电解液的腔内，带有检测器如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，分析物可穿过小孔，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。
在纳米孔测序过程中，有效地捕获待测物对于提高检测灵敏度非常重要。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，而检测器如纳米孔仅设置在分子膜的两亲膜处，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
本发明人出乎意料地发现当使用占位分子结合在油脂膜的非两亲膜区域上之后，再添加锚定分子，可以实现锚定分子在两亲膜上的富集，进而实现分析物在两亲膜中的检测器的区域的富集，提高检测灵敏度。
因此，本发明的第一方面涉及一种将锚定分子富集在两亲膜上的方法，所述方法包括：
(a)提供分子膜，所述分子膜包括两亲膜和油脂膜，所述两亲膜设置在所述油脂膜中，使得形成的分子膜为包含两亲膜和围绕两亲膜周围的油脂膜的结构；和
(b)提供占位分子和锚定分子，使所述分子膜依次与所述占位分子和所述锚定分子接触，其中与所述占位分子接触使得所述占位分子结合在所述油脂膜上，与所述锚定分子接触使得所述锚定分子结合在所述两亲膜上。
优选地，所述占位分子在所述分子膜的顺侧或反侧与所述分子膜接触，更优选地，所述占位分子在所述分子膜的顺侧与所述分子膜接触。
优选地，所述占位分子包含第一占位分子和可选的一个或多个第二占位分子，所述第一占位分子用于与所述油脂膜连接，所述一个或多个第二占位分子中的至少一个用于与所述第一占位分子连接。
优选地，所述第一占位分子包含疏水分子，优选疏水多肽或疏水高分子聚合物，更优选地，所述疏水高分子聚合物选自聚噻吩、聚苯乙炔和聚菲乙炔；
所述第二占位分子包含亲水大分子，优选DNA、PEG、聚丙烯酰胺，聚丙烯酸或纤维素。
优选地，所述锚定分子包含膜结合部分和分析物结合部分，所述膜结合部分用于结合在所述两亲膜上，所述分析物结合部分用于结合分析物。
优选地，所述膜结合部分选自脂质、脂肪酸、固醇、碳纳米管或氨基酸；所述分析物结合部分为核酸序列。
优选地，该方法还包括提供生物芯片，所述生物芯片用于附着所述油脂膜并支撑所述分子膜。
优选地，该方法还包括将一个或多个检测器插入到所述两亲膜中。
优选地，所述检测器包含纳米孔，包括固态孔和/或生物孔，所述生物孔包括跨膜蛋白孔。
优选地，所述两亲膜是脂双层膜，所述油脂膜是硅油、磷脂酰胆碱、磷脂酰乙醇氨、磷脂酰甘油中的任一种或多种形成的混合物膜。
本发明的第二方面涉及一种将分析物富集在两亲膜上的方法，包括：使锚定分子，优选锚定分子的膜结合部分结合在两亲膜上；
以及使分析物与所述锚定分子接触，使所述分析物结合在所述锚定分子上，优选结合在所述锚定分子的分析物结合部分上。
本发明的第三方面涉及一种将分析物富集在检测器的区域中的方法，包括：使分析物富集在两亲膜上，所述两亲膜中插入有检测器；以及使所述分析物在所述锚定分子的牵引下靠近所述检测器的区域。
本发明的第四方面涉及一种表征分析物的方法，包括将分析物富集在检测器的区域中，并且在分析物相对于检测器移动时进行一次或多次测量，其中所述一次或多次测量指示一种分析物的一个或多个特征，从而在分析物相对于所述检测器移动时对其进行表征。
优选地，该方法用于表征多种分析物。
优选地，所述分析物或每种分析物选自多核苷酸、多肽、多糖和脂质中的一种或多种，优选为多核苷酸，所述多核苷酸包括DNA和/或RNA。
本发明的第五方面涉及一种用于表征分析物的试剂盒，包含：
(a)分子膜，所述分子膜包括两亲膜和油脂膜，所述两亲膜设置在所述油脂膜中并插入有一个或多个检测器，使得形成的分子膜为包含插入有检测器的两亲膜和围绕两亲膜周围的油脂膜的结构；
(b)占位分子，所述占位分子用于结合在所述油脂膜上；以及
(c)锚定分子，所述锚定分子用于在所述油脂膜被所述占位分子结合后将分析物结合在所述两亲膜上，并使所述分析物靠近所述检测器的区域。
优选地，所述占位分子用于结合在所述油脂膜的顺侧。
优选地，所述占位分子包含第一占位分子和可选的一个或多个第二占位分子，所述第一占位分子用于与所述油脂膜连接，所述一个或多个第二占位分子中的至少一个用于与所述第一占位分子连接。
优选地，所述第一占位分子包含疏水分子，优选疏水多肽或疏水高分子聚合物，更优选地，所述疏水高分子聚合物选自聚噻吩、聚苯乙炔和聚菲乙炔；
所述第二占位分子包含亲水大分子，优选DNA、PEG、聚丙烯酰胺，聚丙烯酸或纤维素。
优选地，所述锚定分子包含膜结合部分和分析物结合部分，所述膜结合部分用于结合在所述两亲膜上，所述分析物结合部分用于结合分析物。
优选地，所述膜结合部分选自脂质、脂肪酸、固醇、碳纳米管或氨基酸；所述分析物结合部分为核酸序列。
优选地，所述试剂盒还包括生物芯片，所述生物芯片用于附着所述油脂膜并支撑所述分子膜。
本发明的第六方面涉及一种纳米孔表征分析物用的分子膜，其中所述分子膜包括两亲膜和油脂膜，所述两亲膜设置在所述油脂膜中并插入有一个或多个检测器，所述油脂膜上结合有占位分子，所述两亲膜上结合有锚定分子。
优选地，所述占位分子结合在所述油脂膜的顺侧。
优选地，所述占位分子包含第一占位分子和可选的一个或多个第二占位分子，所述第一占位分子与所述油脂膜连接，所述一个或多个第二占位分子中的至少一个与所述第一占位分子连接。
优选地，所述第一占位分子包含疏水分子，优选疏水多肽或疏水高分子聚合物，更优选地，所述疏水高分子聚合物选自聚噻吩、聚苯乙炔和聚菲乙炔；
所述第二占位分子包含亲水大分子，优选DNA、PEG、聚丙烯酰胺，聚丙烯酸或纤维素，更优选为DNA。
优选地，所述锚定分子包含膜结合部分和分析物结合部分，所述膜结合部分用于结合在所述两亲膜上，所述分析物结合部分用于结合分析物。
优选地，所述膜结合部分选自脂质、脂肪酸、固醇、碳纳米管或氨基酸；所述分析物结合部分为核酸序列。
本发明的第七方面涉及一种纳米孔表征分析物的装置，所述装置包括：
生物芯片；和
如第六方面提供的分子膜。
本发明的第八方面涉及如第一至第四方面提供的方法、如第五方面提供的试剂盒、如第六方面提供的分子膜或如第七方面提供的装置在制备表征分析物的产品或表征分析物中的应用。
本发明的技术方案取得了以下技术效果：
本发明实现了通过使用占位分子结合在油脂膜的非两亲膜区域上之后，再添加锚定分子，提高锚定分子在两亲膜上的富集，进而提高分析物在两亲膜的检测器区域的有效富集，尤其是在分析物的浓度较低时，例如在分析物浓度低至20pM时，可以高效地富 集低浓度分析物。本发明可以降低或避免锚定分子在芯片支撑结构表面的附着，从而提高富集效果。
本发明可以提高对靶多核苷酸等待测分析物的检测灵敏度。
附图说明
图1示出了本发明的一个实施方案中表征分析物的装置的部分结构示意图。其中，10、油脂膜；20、两亲膜；30、跨膜孔；40、芯片基质。
图2示出了A)单独的锚定分子、B)单独的占位分子和C)先添加占位分子再添加锚定分子在分子膜中的荧光分布情况。在A)中，单独的锚定分子的荧光在芯片的油脂膜和两亲膜上均匀分布；在B)中，单独的占位分子的荧光主要分布在芯片表面的油脂膜区域，而很少分布在两亲膜区域；在C)中，锚定分子的荧光在两亲膜区域上较其他区域更强。
图3示出了图2的A)和C)的情况下锚定分子的荧光强度比(两亲膜区/非两亲膜区)。其中先加入占位分子再加入锚定分子的情况下荧光强度比(两亲膜区/非两亲膜区)明显提高，约为仅添加锚定分子的1.5倍。
图4示出了接头结合酶的结构，其中酶结合在接头的Y1-S单链的虚线区域上，接头与待测多核苷酸序列连接。
图5示出了对照组(即仅加入锚定分子)中对ssDNA进行纳米孔测序的信号图。
图6示出了实验组(即先加入占位分子，再加入锚定分子)中对ssDNA进行纳米孔测序的信号图。
序列表说明
SEQ ID NO.：1示出了实施例1的占位分子的DNA的序列
5’-GGTCGGTGCTGGACTTTTTTTTTTTTTTTTTTTTT-3’
SEQ ID NO.：2示出了实施例1的占位分子的疏水多肽的序列
GGGKKLALA LALALALALALKKA
SEQ ID NO.：3示出了实施例1的锚定分子的核酸序列
5’-TTTTTT TTTTT TTTTT TTTTT CTCCGCTCGCCAGTT-3’
SEQ ID NO.：4示出了实施例3的占位分子的疏水多肽的序列
GGGKWWLALALALALALALWWA
SEQ ID NO.：5示出了实施例4的接头的Y1-S核酸序列
5’-nnnnn nnnnn nnnnn nnnnn ATCCT TTTTA GAATT TTAGA GAT TTTTT TTTTT AGAGA TTCAG AGATT CAGAG ATTCA GAG-3’，其中n表示iSpC3
SEQ ID NO.：6示出了实施例4的接头的Y2-S核酸序列
5’-ATCTC TAAAA TTCTA AAAAG GAT-3’
SEQ ID NO.：7示出了实施例4的接头的Y-Bottom-S核酸序列
5’-CTCTG AATCT CTGAA TCTCT GAATC TCT AACTG GCGAG CGGAG A-3’
具体实施方式
应理解，所公开的产物和方法的不同应用可根据本领域内的具体需要进行调整。还应理解，本文所用的术语只是为了描述本发明的具体实施方案的目的，而非意图进行限制。
本文中——无论在上文还是下文——引用的所有出版物、专利和专利申请以引用的方式全文纳入本文。
定义
为了更清楚地解释本发明的实施方式，本文中使用了一些科学术语和专有名词。除非在本文中进行了明确定义，所有这些术语和名词应当被理解为具有本领域技术人员所通常理解的含义。为了更清楚起见，对于本文中使用的某些术语进行了以下定义。
分子膜
如图1所示，基于跨膜孔测序技术进行生物分子表征时，需要在基因芯片的基质40上制备分子膜，分子膜包括混合分布的油脂膜10和两亲膜20，两亲膜20设置在油脂膜10中，使得形成的分子膜为包含两亲膜20和围绕两亲膜20周围的油脂膜10的结构。两亲膜20为两亲分子形成的膜结构，两亲膜20上设置有检测器，如跨膜孔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膜20具体可以采用何种形式的膜结构，通常为脂双层。脂双层是细胞膜的模型，并为一系列的实验性研究充当极好的平台。例如，脂双层可用于使用单通道记录的膜蛋白的体外研究。或者，脂双层可用作生物传感器来检测一系列物质的存在。所述脂双层可为任何脂双层。合适的脂双层包括但不限于平面的脂双层、支撑的双层或脂质体。与两亲膜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可以稳定的附着在基质40上，两亲膜20可以通过油脂膜10与基质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。跨膜孔30为沿两亲膜20厚度方向设置的通孔，以连通两亲膜20两侧。在电势的驱动下，水合离子可以从两亲膜20的一侧流到另一侧。跨膜孔30具体可以是蛋白质孔、多核苷酸孔或固态孔等，本申请不限定跨膜孔30的具体形成形式。在电势差的作用下，分析物可以依次从跨膜孔30通过，分析物从跨膜孔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分析物的表征信息。例如根据电信号变化信息得到分析物的尺寸信息、序列信息、同一性信息、修饰信息等。
在一个优选的实施方式中，本发明的分子膜为纳米孔表征分析物用的分子膜，其中分子膜包括两亲膜和油脂膜，两亲膜设置在油脂膜中并插入有一个或多个检测器，油脂膜上结合有占位分子，两亲膜上结合有锚定分子。
在本文中，“顺侧”指分析物的进入侧，分析物或修饰的分析物通过该侧进入纳米孔的开口；“反侧”指分析物的流出侧，分析物或修饰的分析物通过该侧流出纳米孔的开口。例如，顺侧位于芯片的顶部，反侧位于芯片的底部。在一些情况下，分子从膜或孔的顺侧向反侧穿过孔。
占位分子优选结合在油脂膜的顺侧。
占位分子
本发明的占位分子为在基于跨膜孔测序技术进行生物分子表征时，在生物分子与跨膜孔周围的两亲膜结合之前，先结合在非两亲膜区域(即油脂膜上未设置两亲膜的区域)上的物质。该物质占位在油脂膜上的非两亲膜区域，以使其他物质无法结合在该区域，因而称为占位分子。
在本发明的一个实施方案中，占位分子包含第一占位分子和可选的一个或多个第二 占位分子或由第一占位分子和可选的一个或多个第二占位分子组成。第一占位分子用于与油脂膜，优选油脂膜上的非两亲膜区域连接，一个或多个第二占位分子中的至少一个用于与第一占位分子连接。第一占位分子包含或是疏水分子，优选疏水多肽或疏水高分子聚合物，更优选地，疏水高分子聚合物选自聚噻吩、聚苯乙炔和聚菲乙炔；第二占位分子包含或是亲水大分子，优选DNA、PEG、聚丙烯酰胺，聚丙烯酸或纤维素。在一个实施方案中，占位分子为疏水多肽。在另一个实施方案中，占位分子为疏水多肽连接DNA的结构。
在一个实施方案中，占位分子中的第一占位分子通过点击化学反应与第二占位分子连接。点击化学反应(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。在优选的实施方案中，第一占位分子上连接有第一连接基团，第二占位分子上连接有第二连接基团，通过第一连接基团和第二连接基团发生点击化学反应，实现第一占位分子和第二占位分子的快速连接。第一连接基团和第二连接基团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：第一连接基团和第二连接基团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，本实施方案不限定第一连接基团和第二连接基团具体为哪种可发生点击化学反应的基团。
锚定分子
本发明的锚定分子为在基于跨膜孔测序技术进行生物分子表征时，在占位分子占据在非两亲膜区域上之后，结合到两亲膜上的物质。该锚定分子锚定在两亲膜上，在分析物与锚定分子结合之后，使分析物在锚定分子的牵引下靠近两亲膜中的检测器的区域。
在本发明的一个实施方案中，锚定分子包含膜结合部分和分析物结合部分或由膜结合部分和分析物结合部分组成。膜结合部分用于结合在两亲膜上，分析物结合部分用于结合分析物。锚定分子是连接分析物与两亲膜的分子。膜结合部分选自脂质、脂肪酸、固醇、碳纳米管或氨基酸；分析物结合部分优选为核酸序列。该核酸序列可与分析物，如多核苷酸通过碱基互补配对方式连接。
在一个实施方案中，锚定分子为胆固醇连接核酸的结构。
在一个实施方案中，锚定分子中的膜结合部分通过点击化学反应与分析物结合部分连接。点击化学反应如上所述。在优选的实施方案中，膜结合部分上连接有第一连接基 团，分析物结合部分上连接有第二连接基团，通过第一连接基团和第二连接基团发生点击化学反应，实现膜结合部分和分析物结合部分的快速连接。
分析物
分析物可以是任何物质。适合的分析物包括但不限于，金属离子、无机盐、聚合物例如聚合的酸或碱、染料、漂白剂、药物、诊断剂、休闲类药物、爆炸物和环境污染物。
分析物可以是由细胞分泌的分析物。或者，分析物可以是存在于细胞内的分析物，这样使得在实施本发明之前必须从细胞提取分析物。
分析物选自多核苷酸、多肽、多糖和脂质中的一种或多种。分析物优选地为多核苷酸例如核酸，包括脱氧核糖核酸(DNA)和/或核糖核酸(RNA)。多核苷酸可以是单链或双链。多核苷酸可以是环状的。多核苷酸可以是适体，与微RNA杂交的探针或微RNA本身。多核苷酸可为任意长度。例如，多核苷酸可为至少10个，至少50个，至少100个，至少150个，至少200个，至少250个，至少300个，至少400个或至少500个核苷酸对的长度。多核苷酸可为1000或更多个核苷酸对，5000或更多个核苷酸对的长度或100000或更多个核苷酸对的长度。
分析物可存在于任何适合的样品中。本发明通常在已知含有或怀疑含有分析物的样品上实施。本发明可以在含有一种或多种种类未知的分析物的样品上实施。或者，本发明可以在样品上实施以确认已知或预期存在于所述样品中的一种或多种分析物的种类。
检测器
检测器可以是响应分析物的存在、不存在或特征而提供可读信号的任何结构。检测器可以是响应分析物的存在、不存在或特征而提供可读信号的任何结构。适合的检测器在本领域中已知，包含纳米孔，包括固态孔和/或生物孔。它们包括但不限于跨膜孔、隧道电极、经典电极、纳米管、FET(场效晶体管)和光检测器例如原子力显微镜(AFM)和扫描隧道显微镜(STM)。
在优选实施方案中，检测器用电的方式检测分析物。可以使用标准的单通道记录设备进行电测量，如Stoddart D et al.,Proc Natl Acad Sci,12；106(19):7702-7,Lieberman KR et al,J Am Chem Soc.2010；132(50):17961-72和国际申请WO-2000/28312所述。或者，可以使用多通道系统进行电测量，例如如国际申请WO-2009/077734和国际申请WO-2011/067559所述。
在其他优选实施方案中，检测器不用荧光方式检测分析物。
跨膜孔是容许外加电位驱动水合离子从膜的一侧流动到膜另一侧的结构。
跨膜孔优选地为跨膜蛋白孔。跨膜蛋白孔是容许水合离子(例如分析物)从膜的一侧流动到膜另一侧的多肽或多肽集合。在本发明中，跨膜蛋白孔能够形成容许外加电位驱动水合离子从膜的一侧流动到另一侧的孔。跨膜蛋白孔优选地容许分析物(例如核苷酸)从膜(例如脂双层)的一侧流动到另一侧。跨膜蛋白孔优选地使得多核苷酸或核酸(例如DNA或RNA)能够移动穿过所述孔。
跨膜蛋白孔可以是单体或寡聚体。所述孔优选地由若干个重复亚基例如6、7或8个亚基组成。所述孔更优选地为七聚体或八聚体孔。
跨膜蛋白孔通常包含离子可以流过的桶或通道。所述孔的亚基通常围绕一个中心轴并为跨膜β桶或通道或者跨膜α-螺旋束或通道提供链。
富集
本发明的方法可将锚定分子富集在两亲膜上，进而通过将锚定分子连接分析物而将分析物富集在两亲膜上。两亲膜中插入有检测器，因此本发明的方法还可将分析物富集在检测器的区域中。本发明的方法通过首先使用占位分子结合并占据在油脂膜上，然后使后加入的锚定分子结合在占位分子未占据的两亲膜上，从而实现富集。
在一个实施方案中，本发明提供一种将锚定分子富集在两亲膜上的方法，包括：提供分子膜，分子膜包括两亲膜和油脂膜，两亲膜设置在油脂膜中；和提供占位分子和锚定分子，使分子膜依次与占位分子和锚定分子接触，其中与占位分子接触使得占位分子结合在油脂膜上，与锚定分子接触使得锚定分子结合在两亲膜上，从而使锚定分子直接富集在两亲膜上。
在一个实施方式中，本发明提供一种将分析物富集在两亲膜上的方法，包括：使得锚定分子结合在两亲膜上；以及使分析物与锚定分子接触，使得分析物结合在锚定分子上，从而使分析物通过锚定分子富集在两亲膜上。
在一个实施方式中，本发明提供一种将分析物富集在检测器的区域中的方法，包括：使分析物富集在两亲膜上，所述两亲膜中插入有检测器；以及使分析物在锚定分子的牵引下靠近插入两亲膜中的检测器的区域。
在本发明的方法中，提供如图1所示的分子膜，该分子膜包含插入两亲膜中的检测器(例如跨膜孔)、围绕跨膜孔的两亲膜(例如脂双层)以及围绕两亲膜的油脂膜(例如硅油和磷脂酰胆碱混合形成的膜)。该分子膜附着在芯片基质上，两亲膜通过油脂膜与基质 固定。本发明的占位分子、锚定分子以及分析物可从两亲膜的顺侧或反侧加入。在优选的实施方式中，本发明的占位分子、锚定分子以及分析物可从两亲膜的顺侧加入。
在一些实施方案中，将占位分子(比如疏水多肽连接的DNA)从两亲膜的顺侧加入，占位分子与分子膜接触之后，疏水多肽会结合在分子膜的非两亲膜区域，相反很少结合或不结合在两亲膜上。疏水多肽与DNA等大分子相连，从而占据油脂膜表面的空间，使油脂膜表面不易附着其他分子。此时，再加将锚定分子(比如胆固醇连接的DNA)从两亲膜的顺侧加入，胆固醇更容易附着在两亲膜表面而不是油脂膜的表面，从而表现为在两亲膜上的富集。
当存在分析物(例如多核苷酸)时，通过本领域已知的任何合适方法将多核苷酸与接头连接形成构建体。接头可单独合成，然后化学连接或酶连接到多核苷酸上。或者，接头在处理多核苷酸的过程中产生。接头在多核苷酸的一个末端或其附近连接至多核苷酸上。可使用任何已知的方法将包含多核苷酸的构建体偶联到膜上。构建体优选地是通过已经连接在两亲膜上的锚定分子偶联到两亲膜上。优选地，接头与锚定分子上分别具有可碱基互补配对的核苷酸序列，从而实现接头与锚定分子的连接，进而将分析物与两亲膜连接。
检测器(例如跨膜孔)插入到两亲膜中，富集在两亲膜上的分析物在锚定分子的牵引下靠近跨膜孔的区域，从而实现分析物在检测器区域的富集。
分析物相对于检测器移动
本发明的一些实施方案包括表征分析物。分析物可以在其相对于检测器移动时被表征。分析物相对于检测器的移动可以由任何合适的装置驱动。在一些实施方案中，分析物的移动由物理或化学力(电位)驱动，在一些实施方案中，物理力由电(例如电压)电位或温度梯度等提供。
在一些实施方案中，当在检测器(例如跨膜孔)上施加电势时，分析物相对于检测器移动，分析物例如多核苷酸带负电荷，因此在纳米孔上施加电压将导致分析物在施加的电压电位的影响下相对于纳米孔移动。例如，如果将正电压施加到纳米孔的反侧，那么这将诱导带负电荷的分析物从纳米孔的顺侧移动到纳米孔的反侧。
在本发明的方法中，使多核苷酸移动通过跨膜孔是指使多核苷酸从孔的一侧移动到另一侧。多核苷酸通过孔的移动可受电势或酶促作用或电位和酶促作用驱动或控制。移动可以是单向的，或可允许向后和向前移动。
优选地使用多核苷酸结合蛋白来控制多核苷酸移动通过孔。多核苷酸结合蛋白可以是能够与多核苷酸结合并且控制其通过孔的移动的任何蛋白质。在本领域中确定蛋白质是否与多核苷酸结合是很简单的。蛋白质通常与多核苷酸相互作用并且修饰其至少一种性质。蛋白质可以通过裂解多核苷酸以形成单独的核苷酸或如二核苷酸或三核苷酸的更短的核苷酸链来修饰多核苷酸。所述部分可通过将多核苷酸定位或移动到特异性位置(即控制其移动)来修饰多核苷酸。
表征分析物的方法
本发明的表征方法可使用本发明的分子膜进行，通过本发明的富集方法将分析物富集到跨膜孔附近。
所述表征方法可以包括测量分析物(例如多核苷酸)的一个、两个、三个、四个或五个或更多个特征。所述特征优选选自(i)多核苷酸的长度，(ii)多核苷酸的同一性，(iii)多核苷酸的序列，(iv)多核苷酸的二级结构，以及(v)多核苷酸是否被修饰。
本发明的方法包含移动多核苷酸通过跨膜孔，使得多核苷酸的一部分核苷酸与孔相互作用。
这些方法是可能的，因为跨膜孔可用于区分具有相似结构的核苷酸，这是基于它们对通过所述孔的电流具有不同的效应。可根据各个核苷酸与孔相互作用时它们的电流振幅，在单分子水平上鉴定各个核苷酸。如果电流以对某种核苷酸特异性的方式流经所述孔(即如果检测到与该核苷酸相关的特征性电流流过所述孔)，那么该核苷酸就在所述孔中存在。连续鉴定多核苷酸中的核苷酸，使得能够估计或确定多核苷酸的序列。
试剂盒
本发明还提供了表征分析物的试剂盒，包含：
(a)分子膜，分子膜包括两亲膜和油脂膜，两亲膜设置在油脂膜中并插入有一个或多个检测器；
(b)占位分子，占位分子用于结合在油脂膜上；以及
(c)锚定分子，锚定分子用于在油脂膜被占位分子结合后将分析物结合在两亲膜上，并使所分析物靠近检测器的区域。
在一个实施方案中，试剂盒中的分子膜、占位分子和锚定分子如前所述。
在一个优选的实施方案中，试剂盒还包含生物芯片，所述生物芯片用于附着油脂膜并支撑分子膜。
在一个优选的实施方案中，试剂盒还包含本领域已知的用于表征分析物的物质，例如用于与多核苷酸结合形成构建体的接头、控制多核苷酸移动的多核苷酸结合蛋白等。
本发明还提供纳米孔表征分析物的装置，所述装置包括生物芯片；和本发明的分子膜。
实施例
以下各实施例中未具体注明的实验操作细节可以参考本文所引用的参考文献，所采用的实验试剂和仪器设备均为常规商业可得的试剂或仪器，所采用的序列由本领域常规方法合成或为商业可得的。
实施例1：占位分子和锚定分子的制备
设计并合成占位分子，该占位分子包含疏水多肽和DNA，该占位分子的序列如下：
Cy3-GGTCGGTGCTGGACTTTTTTTTTTTTTTTTTTTTT-DBCO-N3-GGGKKLALALALALALALALKKA
将合成的疏水多肽和DNA通过点击化学连接基团“DBCO”和“N3”连接，并在DNA的5’端连接有荧光基团Cy3。
设计并合成锚定分子，其核苷酸序列如下：
5’-chol-TTTTTT TTTTT TTTTT TTTTT CTCCGCTCGCCAGTT-Cy5-5’
核苷酸序列的5’端连接胆固醇用于结合膜，3’端连接荧光基团Cy5，核苷酸序列可与分析物以碱基互补配对方式结合。
实施例2：占位分子和锚定分子在芯片表面的分布
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1所示。
将实施例1制备的占位分子和锚定分子采用以下三种不同的方式添加到芯片的顺侧溶液中，与上述分子膜进行孵育。三种方式分别为：A)将单独的锚定分子加入芯片中，终浓度20nM，室温孵育15分钟；B)将单独的占位分子加入芯片中，终浓度200nM，室温孵育16小时；以及C)向芯片中先添加占位分子，终浓度200nM，室温孵育16小时，再添加锚定分子，终浓度20nM，室温孵育15分钟。用共聚焦显微镜对芯片进行层扫拍照并三维重构，观察到锚定分子和占位分子在三种方式中在分子膜中的荧 光分布情况如图2所示。
A)单独锚定分子在芯片表面上的分布
单独的锚定分子的荧光在芯片的油脂膜和两亲膜上均匀分布。
B)单独占位分子在芯片表面上的分布
单独的占位分子的荧光主要分布在芯片表面的油脂膜区域，而很少分布在两亲膜区域。
C)加入占位分子后，锚定分子在芯片表面上的分布
在B)的基础上，加入锚定分子，可以看到锚定分子的荧光在两亲膜区域上较其他区域更强。
对图2的荧光结果进行定量分析，计算锚定分子在两亲膜区荧光强度/非两亲膜区域荧光强度，结果如图3所示。可以看出先加入占位分子再加入锚定分子的情况，较仅添加锚定分子的情况，两亲膜区荧光强度/非两亲膜区荧光强度的比率明显提高，约为1.5倍。
实施例3：另一占位分子与锚定分子的制备及分布
按照实施例1的方法合成占位分子，不同之处在于：将占位分子中的疏水多肽序列替换为：GGGKWWLALALALALALALWWA。合成的占位分子的序列如下：
Cy3-GGTCGGTGCTGGACTTTTTTTTTTTTTTTTTTTTT-DBCO-N3-GGGKWWLALALALALALALWWA。
按照实施例2的方法观察该占位分子和锚定分子在分子膜上的分布。结果显示，与实施例2中的结果无显著差异，先加入占位分子再加入锚定分子的情况，较仅添加锚定分子的情况，两亲膜上荧光强度/非两亲区域荧光强度的比率明显提高。
实施例4：对分析物进行纳米孔测序
(1)设置对照组和实验组，对照组和实验组中，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)在对照组中，只加入锚定分子；在实验组中，先加入实施例1制备的占位分子，再加入实施例1制备的锚定分子。
(3)在对照组和实验组中均加入相同浓度(20pM)的待测样品，使用齐碳科技有 限公司的基因测序仪QNome-9604对样品中的DNA(1kb定长文库，即待测多核苷酸序列)进行纳米孔测序，获得过孔信号图。
纳米孔测序方法具体如下：
接头结构的序列如下：
①Y1-S：5’-nnnnn nnnnn nnnnn nnnnn ATCCT TTTTA GAATT TTAGA GAT TTTTT TTTTT AGAGA TTCAG AGATT CAGAG ATTCA GAG-3’；
其中，Y1-S链包含前导序列，即iSpC3，表示为n。
②Y2-S：5’-ATCTC TAAAA TTCTA AAAAG GAT-3’
③Y-Bottom-S：5’-P-CTCTG AATCT CTGAA TCTCT GAATC TCT AACTG GCGAG CGGAG A-3’
使用以上序列，合成如图4所示的接头。合成方法如下所示：将引物①、②、③按照1:2.5:2.5比例退火，引物退火终浓度为4μM，退火程序98℃ 10min；6s/-0.1℃，300×Cys；65℃，5min；6s/-0.1℃，400×Cys；12℃，Hold。
退火后的引物按照如下表1中的体系进行孵育。
表1：退火后的引物的孵育体系
将按照上表的孵育体系混合的样品加入1.5mL低吸附离心管中(锡纸包裹避光)，轻柔混匀(不可以使用涡旋震荡仪)，放入30℃金属浴30min。最后，孵育后的产物进行磁珠纯化，获得结合解旋酶的接头复合物。
使用合成的接头进行测序：
按照下表2配制连接反应体系，瞬时离心，室温静置10min。
表2：连接反应的体系：

将产物进行磁珠纯化，获得待测序的连接产物。
使用QNome9604测序平台，分别按实施例2中A)和C)的方式(即对照组和实验组)添加占位分子和锚定分子到测序buffer(600mM KCl、10mM HEPES pH8.0、3mM MgCl2、3mM ATP)中；然后取上述制备好的连接产物加入到测序buffer中，轻轻颠倒混匀，瞬时离心，将该混合液加入到测序芯片中静置15min之后在35℃的条件下进行测试。
对照组和实验组测序过程中电流随时间变化结果参阅图5和图6。开孔电流约为300-400pA，核酸过孔电流约为100-200pA。电流由开孔下降到过孔再恢复到开孔为一次核酸过孔。相比于对照组，实验组样品中DNA的单位时间过孔频率提高了3.27倍，说明实验组中测序灵敏度也得到了明显的提高。

Claims (24)

  1. 一种将锚定分子富集在两亲膜上的方法，包括：
    (a)提供分子膜，所述分子膜包括两亲膜和油脂膜，所述两亲膜设置在所述油脂膜中；和
    (b)提供占位分子和锚定分子，使所述分子膜依次与所述占位分子和所述锚定分子接触，其中与所述占位分子接触使得所述占位分子结合在所述油脂膜上，与所述锚定分子接触使得所述锚定分子结合在所述两亲膜上。
  2. 根据权利要求1所述的方法，所述占位分子在所述分子膜的顺侧或反侧与所述分子膜接触，优选地，所述占位分子在所述分子膜的顺侧与所述分子膜接触。
  3. 根据权利要求1或2所述的方法，其中所述占位分子包含第一占位分子和可选的一个或多个第二占位分子，所述第一占位分子用于与所述油脂膜连接，所述一个或多个第二占位分子中的至少一个用于与所述第一占位分子连接。
  4. 根据权利要求3所述的方法，其中所述第一占位分子包含疏水分子，优选地，所述疏水分子选自疏水多肽或疏水高分子聚合物；更优选地，所述疏水分子选自疏水多肽；更优选地，所述疏水高分子聚合物选自聚噻吩、聚苯乙炔或聚菲乙炔；
    所述第二占位分子包含亲水大分子，优选地，所述亲水大分子选自DNA、PEG、聚丙烯酰胺、聚丙烯酸或纤维素，更优选地，所述亲水大分子选自DNA。
  5. 根据权利要求1或2所述的方法，其中所述锚定分子包含膜结合部分和分析物结合部分，所述膜结合部分用于结合在所述两亲膜上，所述分析物结合部分用于结合分析物。
  6. 根据权利要求5所述的方法，其中所述膜结合部分选自脂质、脂肪酸、固醇、碳纳米管或氨基酸。
  7. 根据权利要求1或2所述的方法，还包括提供生物芯片，所述生物芯片上附着所述油脂膜并用于支撑所述分子膜。
  8. 根据权利要求1或2所述的方法，还包括将检测器插入到所述两亲膜中。
  9. 根据权利要求8所述的方法，其中所述检测器包含纳米孔，所述纳米孔包括固态孔和/或生物孔，所述生物孔包括跨膜蛋白孔。
  10. 根据权利要求1或2所述的方法，所述两亲膜包括脂双层膜，所述油脂膜是包 括硅油、磷脂酰胆碱、磷脂酰乙醇氨、磷脂酰甘油中的任一种或多种形成的混合物膜。
  11. 一种将分析物富集在两亲膜上的方法，包括：执行权利要求1至10中任一项所述的方法，使得所述锚定分子，优选所述锚定分子的膜结合部分结合在所述两亲膜上；
    以及使分析物与所述锚定分子接触，使得所述分析物结合在所述锚定分子上，优选结合在所述锚定分子的分析物结合部分上。
  12. 一种将分析物富集在检测器的区域中的方法，包括：执行权利要求11所述的方法，使得所述分析物富集在所述两亲膜上，所述两亲膜中插入有检测器；以及使所述分析物在所述锚定分子的牵引下靠近所述检测器的区域。
  13. 一种表征分析物的方法，包括使用权利要求12所述的方法将分析物富集在检测器的区域中，并且在分析物相对于检测器移动时进行一次或多次测量，其中所述一次或多次测量指示一种分析物的一个或多个特征，从而在分析物相对于所述检测器移动时对其进行表征。
  14. 根据权利要求13所述的方法，其中所述方法用于表征多种分析物。
  15. 根据权利要求12或13所述的方法，其中所述分析物或每种分析物选自多核苷酸、多肽、多糖和脂质中的一种或多种，优选为多核苷酸，所述多核苷酸包括DNA和/或RNA。
  16. 一种用于表征分析物的试剂盒，包含：
    (a)分子膜，所述分子膜包括两亲膜和油脂膜，所述两亲膜设置在所述油脂膜中并插入有检测器；
    (b)占位分子，所述占位分子用于结合在所述油脂膜上；以及
    (c)锚定分子，所述锚定分子用于在所述油脂膜被所述占位分子结合后将分析物结合在所述两亲膜上，并使所述分析物靠近所述检测器的区域。
  17. 根据权利要求16所述的试剂盒，所述占位分子用于结合在所述油脂膜的顺侧。
  18. 根据权利要求16或17所述的试剂盒，其中所述占位分子包含第一占位分子和可选的一个或多个第二占位分子，所述第一占位分子用于与所述油脂膜连接，所述一个或多个第二占位分子中的至少一个用于与所述第一占位分子连接。
  19. 根据权利要求16或17所述的试剂盒，其中所述试剂盒还包括生物芯片，所述生物芯片用于附着所述油脂膜并支撑所述分子膜。
  20. 一种纳米孔表征分析物用的分子膜，其中所述分子膜包括两亲膜和油脂膜，所 述两亲膜设置在所述油脂膜中并插入有检测器，所述油脂膜上结合有占位分子，所述两亲膜上结合有锚定分子。
  21. 根据权利要求20所述的分子膜，所述占位分子结合在所述油脂膜的顺侧。
  22. 根据权利要求20或21所述的分子膜，其中所述占位分子包含第一占位分子和可选的一个或多个第二占位分子，所述第一占位分子与所述油脂膜连接，所述一个或多个第二占位分子中的至少一个与所述第一占位分子连接。
  23. 一种纳米孔表征分析物的装置，所述装置包括：
    生物芯片；和
    如权利要求20-22中任一项所述的分子膜。
  24. 根据权利要求1-15中任一项所述的方法、或权利要求16-19中所述的试剂盒、权利要求20-22中任一项所述的分子膜或权利要求23所述的装置在制备表征分析物的产品或表征分析物中的应用。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103588940A (zh) * 2013-11-07 2014-02-19 中国科学技术大学 两亲性嵌段聚合物、聚合物囊泡及其制备方法和应用
CN103733063A (zh) * 2011-05-27 2014-04-16 牛津纳米孔技术有限公司 偶联方法
US20170022557A1 (en) * 2014-04-04 2017-01-26 Oxford Nanopore Technologies Ltd. Method of target molecule characterisation using a molecular pore
CN108147990A (zh) * 2016-12-02 2018-06-12 上海中医药大学 一种膜锚定元件及其应用
CN112014369A (zh) * 2020-08-25 2020-12-01 上海市皮肤病医院 超灵敏数字层析快速检测分析物的系统及方法
CN113999291A (zh) * 2021-12-28 2022-02-01 北京齐碳科技有限公司 嵌入接头、锚定分子、分子膜、装置、方法及应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103733063A (zh) * 2011-05-27 2014-04-16 牛津纳米孔技术有限公司 偶联方法
CN103588940A (zh) * 2013-11-07 2014-02-19 中国科学技术大学 两亲性嵌段聚合物、聚合物囊泡及其制备方法和应用
US20170022557A1 (en) * 2014-04-04 2017-01-26 Oxford Nanopore Technologies Ltd. Method of target molecule characterisation using a molecular pore
CN108147990A (zh) * 2016-12-02 2018-06-12 上海中医药大学 一种膜锚定元件及其应用
CN112014369A (zh) * 2020-08-25 2020-12-01 上海市皮肤病医院 超灵敏数字层析快速检测分析物的系统及方法
CN113999291A (zh) * 2021-12-28 2022-02-01 北京齐碳科技有限公司 嵌入接头、锚定分子、分子膜、装置、方法及应用

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