A method for increasing resolution of glycan sequencing by electroosmotic flow

Abstract

The application relates to the technical field of sugar chain sequencing, in particular to a method for improving the resolution of sugar chain sequencing by electroosmotic flow. The method comprises the following steps: applying a voltage to make a to-be-detected sugar chain of a coupling linker molecule translocate from a first fluid chamber to a second fluid chamber through a nanopore, the first fluid chamber and the second fluid chamber contain a buffer, an electroosmotic flow is formed between the first fluid chamber and the second fluid chamber, and the nanopore is arranged between the first fluid chamber and the second fluid chamber. The method realizes the direct analysis of the sugar chain by the nanopore technology based on the stretching effect of the electroosmotic flow and the speed control effect of the motor protein, retains complete structure information, and avoids the derivative side reaction of traditional technology.

Description

Technical Field

[0001] This application relates to the field of glycan sequencing technology, specifically to a method for improving the resolution of glycan sequencing using electroosmotic flow. Background Technology

[0002] Polysaccharides are the third class of biological macromolecules, after nucleic acids and proteins. The complexity and diversity of polysaccharide structures determine their close connection to a variety of biological functions. Analyzing the composition, linkage patterns, branching structures, and functional group modifications of monosaccharide units in polysaccharides is of great significance for fields such as pharmaceutical research and development, synthetic biology, microbiology, and food science.

[0003] Traditional polysaccharide sequencing methods include enzymatic digestion-based mass spectrometry, nuclear magnetic resonance (NMR) fingerprinting, and fluorescent labeling-capillary electrophoresis. These methods are costly, complex, and time-consuming. Therefore, there is an urgent need for a low-cost, simple, high-throughput method that can detect polysaccharides in real time.

[0004] Nanopore sequencing is a low-cost, simple, efficient, and label-free single-molecule detection technology that has been successfully applied in the fields of nucleic acid and protein sequencing. This sequencing technology primarily relies on the electrophoretic effect to drive single nucleic acid molecules or nucleic acid-protein complexes through nanopores for sequencing.

[0005] Nanopore technology enables high-resolution sequencing of nucleic acids with single-nucleotide precision. This is because the uniform negative charge of nucleic acid molecules causes them to experience a directional electric field in ionic solutions, resulting in a "straightened" state. Unlike nucleic acid molecules, polysaccharides have a more complex charge distribution. A random single-stranded polysaccharide molecule does not have a uniform charge, and therefore experiences a non-uniform electric field. Consequently, when passing through the contraction region of a nanopore, it cannot exhibit the same "straightened" state as nucleic acid molecules, and the measured current signal shows less pronounced "step-like" changes. Summary of the Invention

[0006] To address the problems of existing technologies, this application provides a method for improving the resolution of glycan nanopore sequencing. The method introduces divalent and / or trivalent cations into a fluid chamber to form an electroosmotic flow, stretching and immobilizing the glycan molecules, thereby significantly enhancing the "step-like" changes in the current signal and significantly increasing the number of "steps." This method provides high resolution for glycan sequencing. The specific scheme is as follows: A first aspect of this application provides a method for sequencing glycans, the sequencing method comprising: applying a voltage to cause the glycan to be tested, coupled to a head molecule, to translocate from a first fluid chamber through a nanopore to a second fluid chamber, wherein the first and second fluid chambers contain buffer solutions to form an electroosmotic flow between the first and second fluid chambers, and wherein the nanopore is disposed between the first and second fluid chambers.

[0007] Preferably, the buffer solution contains cations. The cations may be monovalent, divalent, and / or trivalent cations.

[0008] Preferably, the monovalent cation includes potassium ions (K ions). + ), ammonium ions (NH4+) + Sodium ions (Na) + ), cuprous ions (Cu) + Lithium ion (Li) + ) or silver ions (Ag) + One or more of the following.

[0009] Preferably, the divalent cation contains magnesium ions (Mg). 2+ ), calcium ions (Ca 2+ Barium ions (Ba) 2+ ), manganese ions (Mn) 2+ ), copper ions (Cu) 2+ ), ferrous ions (Fe 2+ ), zinc ions (Zn 2+ Nickel ions (Ni) 2+ ), cobalt ions (Co) 2+ ) or lead ions (Pb 2+ One or more of the following.

[0010] Preferably, the trivalent cation includes aluminum ions (Al). 3+ ), chromium ions (Cr 3+ ), thallium ions (Ti 3+ Gallium ions (Ga) 3+ Indium ions (In) 3+ ), cobalt ions (Co) 3+ ), lanthanum ion (La) 3+ ), cerium ions (Ce) 3+ ), neodymium ions (Nd) 3+ ) or iron ions (Fe 3+ One or more of the following.

[0011] More preferably, the cation contains K + Mg 2+ Ca 2+ Ba2+ Mn 2+ or Fe 3+ One or more of them.

[0012] Preferably, the cation concentration in the buffer solution is greater than or equal to 5 mM, for example, 5-10000 mM, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 mM, etc., preferably 5-2000 mM.

[0013] Preferably, the buffer solution in the first fluid chamber is the same as or different from that in the second fluid chamber.

[0014] Preferably, the cations in the buffer solutions in the first fluid chamber and the second fluid chamber can be the same or different.

[0015] Preferably, the cation concentrations in the buffer solutions in the first fluid chamber and the second fluid chamber can be the same or different.

[0016] Preferably, the cations in the first fluid chamber include K + Mg 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of the following, with Mg being the preferred choice. 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of the following, with Mg being more preferred. 2+ Ca 2+ or Fe 3+ One or more of them.

[0017] Preferably, the cation concentration in the first fluid chamber is 100-10000 mM, such as 100, 200, 272, 300, 400, 500, 539, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 mM, more preferably 100-2000 mM or 200-1000 mM, and even more preferably 272-539 mM.

[0018] Preferably, the concentration of divalent and / or trivalent cations in the first fluid chamber is 100-10000 mM, such as 100, 200, 272, 300, 400, 500, 539, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 mM, more preferably 100-2000 mM or 200-1000 mM, and even more preferably 272-539 mM.

[0019] In one specific embodiment of the present invention, the buffer solution of the first fluid chamber comprises: a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000mM FeCl3; preferably 272-539mM FeCl3.

[0020] Preferably, the cations in the second fluid chamber include K. + Mg 2+ Ca 2+ Or Ba 2+ One or more of the following; Mg is further preferred. 2+ Ca 2+ Or Ba 2+ Further optimization of Mg 2+ or Ca 2+ .

[0021] Preferably, the concentration of divalent and / or trivalent cations in the second fluid chamber is greater than or equal to 5 mM, for example greater than or equal to 5, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, greater than or equal to 25, greater than or equal to 30, greater than or equal to 35, greater than or equal to 40, greater than or equal to 45, greater than or equal to 50, greater than or equal to 55, greater than or equal to 60, greater than or equal to 65, greater than or equal to 70, greater than or equal to 75, greater than or equal to 80, greater than or equal to 85, greater than or equal to 90, greater than or equal to 100, greater than or equal to 1000, greater than or equal to 10000 mM, for example 5-10000 mM, preferably 5 mM-80 mM, and further 10 mM-40 mM.

[0022] In some embodiments, the buffer solution in the second fluid chamber contains Mg. 2+ And other cations.

[0023] In some embodiments, the buffer solution in the second fluid chamber contains 1) Mg 2+ , and 2)K + Ca 2+ Or Ba 2+ One or more of the following. Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 5-80mM MgCl2; B) 400-800mM KCl and 5-80mM CaCl2; C) 400-800mM KCl and 5-80mM BaCl2; D) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM CaCl2; or, E) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM BaCl2.

[0024] Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 10-40mM MgCl2; B) 400-800mM KCl and 10-40mM CaCl2; C) 400-800mM KCl and 10-40mM BaCl2; D) 400-800mM KCl, 5-40mM MgCl2 and 10-40mM CaCl2; or, E) 400-800mM KCl, 5-40mM MgCl2 and 10-40mM BaCl2.

[0025] In one specific embodiment of this application, the buffer solution in the second fluid chamber comprises 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the first fluid chamber comprises a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000 mM FeCl3; preferably 272-539 mM FeCl3.

[0026] In one specific embodiment of this application, the buffer solution in the first fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the second fluid chamber contains A) 400-800 mM KCl and 5-80 mM MgCl2; B) 400-800 mM KCl and 5-80 mM CaCl2; C) 400-800 mM KCl and 5-80 mM BaCl2; D) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM CaCl2; or, E) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM BaCl2.

[0027] Preferably, the buffer solution also contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0028] In some embodiments, the buffer solution in the second fluid chamber further contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0029] In some embodiments, the buffer solution in the first fluid chamber further contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0030] In some embodiments, the first fluid chamber may be cis-side and the second fluid chamber may be trans-side.

[0031] In some embodiments, the second fluid chamber may be the cis-side and the first fluid chamber may be the trans-side.

[0032] The pH of the buffer solution is neutral, weakly acidic, or weakly alkaline.

[0033] Preferably, the pH of the buffer solution is 6-9, and more preferably 7.5-8.5.

[0034] The nanopores are inserted into the lipid bilayer.

[0035] The measurement cell is divided into two fluid chambers, and materials known in the prior art can be used. The material can be of any shape, as long as it has suitable pores into which nanopores can be inserted. For example, the material can be a membrane or a tube. The material is an insulating material.

[0036] The material contains pores of 30-70 μm.

[0037] A lipid bilayer is formed on the pores.

[0038] The membrane is an insulating material, such as an organic membrane or a synthetic membrane, such as a polytetrafluoroethylene membrane or a plastic membrane.

[0039] The tube is made of insulating material, such as a plastic tube. The connector has one or more components, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 10.

[0040] Preferably, a linker molecule is attached to one end of the sugar chain to be tested to form a linker molecule-sugar chain conjugate, or a sugar chain-linker molecule conjugate. In one specific embodiment of the invention, a linker molecule is attached to the reduced end of the sugar chain to be tested.

[0041] Preferably, a connector molecule is connected to both ends of the sugar chain to be tested to form a connector molecule 1-sugar chain-connector molecule 2 coupling, wherein connector molecule 1 and connector molecule 2 may be the same or different. In a specific embodiment of the present invention, a connector molecule is connected to both the reduced and non-reduced ends of the sugar chain to be tested.

[0042] The linker molecule is connected to the sugar chain via a covalent bond or via a linking group. Preferably, the linker molecule is connected to the sugar chain via an oxime bond, an amide bond, a thioether bond, a disulfide bond, a phosphoryl bond, a hydrazone bond, an acylurea bond, or a ring bond formed by a click reaction.

[0043] In one specific embodiment of this application, an azide group is modified at one end of the sugar chain, and a DBCO group is modified at one end of the linker molecule, forming a linker molecule-sugar chain conjugate or a sugar chain-linker molecule conjugate through a click chemical reaction.

[0044] In one specific embodiment of this application, an azide group is modified at one end of the sugar chain, and an amino group is modified at the other end. One end of the linker molecule 1 is modified with a DBCO group, and one end of the linker molecule 2 is modified with a thiol group. The linker molecule 1-sugar chain-linker molecule 2 is formed through a click chemical reaction between DBCO and azide, and a reaction between amino and thiol groups.

[0045] In one specific embodiment of this application, the test sugar chain of the coupling head molecule is displaced from the first fluid chamber to the second fluid chamber through nanopores under the action of electrophoretic force and electroosmotic flow.

[0046] In some embodiments, the glycan chain of the coupled connector molecule is displaced from the first fluid chamber to the second fluid chamber through nanopores under the influence of electrophoretic force, electroosmotic flow, and motor protein pull.

[0047] The adapter molecule is a biopolymer, including but not limited to nucleic acids or peptides. It is sufficient to ensure that at least one adapter molecule can interact with (preferably specifically bind to) the motor protein. Its sequence can be any known or interesting sequence. The length can also be arbitrarily adjusted.

[0048] Preferably, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a glycan-nucleic acid conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a glycan-peptide conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

[0049] In one specific embodiment of this application, the adapter molecule is a nucleic acid.

[0050] The nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0051] The nucleic acid mentioned is DNA, RNA, modified DNA, or modified RNA.

[0052] In one specific embodiment of this application, the adapter molecule is DNA, and one end of the glycan chain to be tested is linked to the DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate. Preferably, the DNA is linked to the reducing end to form a DNA-glycan conjugate.

[0053] In one specific embodiment of this application, the adapter molecule is DNA, and both ends of the glycan chain to be tested are connected to DNA to form a DNA1-glycan-DNA2 conjugate, wherein DNA1 and DNA2 may have the same sequence or different sequences.

[0054] In one specific embodiment of this application, the DNA2 is single-stranded. For example, the DNA2 can be polyT.

[0055] In one specific embodiment of this application, the DNA1 is a single strand, which is annealed and coupled to a partially complementary fragment.

[0056] The motor proteins mentioned include, but are not limited to, nucleases, preferably including, but not limited to, helicases or variants thereof, polymerases or variants thereof, or transloses or variants thereof.

[0057] Preferably, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase.

[0058] Preferably, the helicase or its variant is a sequence of Hel308 Tga (Thermococcus gammatolerans (strain DSM 15229 / JCM 11827 / EJ3)) or a variant thereof, a sequence of Hel308 Mbu (Methanococcoides burtonii (strain DSM 6242 / NBRC 107633 / OCM 468 / ACE-M)) or a variant thereof, a sequence of Hel308 Pfu (Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)) or a variant thereof, or a sequence of Hel308 Mma (Methanosarcina mazei (strain ATCCBAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM)) or a variant thereof. The sequence of Hel308Mok (Methanothermococcus okinawensis (strain DSM 14208 / JCM 11175 / IH1)) or its variant, the sequence of Hel308Fac (Ferroplasma acidarmanus Fer1) or its variant, the sequence of Hel308Csy (Cenarchaeum symbiosum (strain A)) or its variant, the sequence of Hel308Mhu (Methanospirillumhungatei JF-1 (strain ATCC 27890 / DSM 864 / NBRC 100397 / JF-1)) or its variant, or the sequence of F8813 protein or its variant.

[0059] Preferably, the polymerase is Bacillus phage φ29 (phi29) polymerase or a variant thereof, Clostridium phage phiCPV4 (pol6, GenBank: AFH27113.1) or a variant thereof, Actinomycete phage Av-1 (pol7, GenBank: ABR67671.1) or a variant thereof.

[0060] In one specific embodiment of this application, the linker molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or variants thereof; the polypeptide is an amino acid sequence that can be specifically recognized by an unfolded enzyme or has a certain charge distribution. Preferably, the unfolded enzyme includes, but is not limited to, Caseinolytic protease X (ClpX).

[0061] The nanopores mentioned are those known in the prior art, as long as they have the ability to sense sugar chains.

[0062] Preferably, the nanopores are biological nanopores or solid nanopores. More preferably, the biological nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin (α-HL) nanopores or variants thereof, Mycobacterium smegmatis porin A (MspA) nanopores or variants thereof, Aeromonas hydrolysin (AeL) or variants thereof, curli production assembly / transport component (CsgG) nanopores or variants thereof, outer membrane porin F (OmpF) nanopores or variants thereof, cytolysin A (ClyA) nanopores or variants thereof, phi29 nanopores or variants thereof, ferric isohydroxamate uptake component A (FhuA) nanopores or variants thereof, pleurotomycin A (PlyA) nanopores or variants thereof, pleurotomycin B (PlyB) nanopores or variants thereof, or fragaceatoxin C (FraC) nanopores or variants thereof. More preferably, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

[0063] Preferably, the sequencing method further includes obtaining electrical signal characteristics by passing the glycan chain to be tested of the coupled head molecule through the sensing region, and determining the glycan chain sequence through the electrical signal characteristics.

[0064] The electrical signal characteristics include, but are not limited to, electrical signal waveforms.

[0065] The electrical signal waveform is formed by sensing the sugar chain through nanopores.

[0066] Preferably, the electrical signal characteristics include, but are not limited to, current amplitude, number of steps, step duration, frequency of occurrence of step signal, or standard deviation.

[0067] In one specific embodiment of this application, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives in the sugar chain.

[0068] In one specific embodiment of this application, the sugar chain sequence is the arrangement order of glycosidic bonds in the sugar chain.

[0069] In one specific embodiment of this application, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0070] In one specific embodiment of this application, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0071] The glycan characteristics include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there are modifications, type of modifications, location of modifications, number of modifications, whether there are branches, location of branches, number of branches, or branch sequence characteristics.

[0072] Preferably, the branched sequence features include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there is modification, type of modification, location of modification, and number of modifications.

[0073] The chain length of the sugar chain to be tested is greater than 1.

[0074] The sugar chain to be tested can be a straight chain or have at least one branch.

[0075] The sugar chain to be tested may be modified, such as by sulfonation.

[0076] The sugar chain to be tested can be charged or electrically neutral.

[0077] The glycan chain to be tested is composed of monosaccharides and / or their derivatives linked by glycosidic bonds.

[0078] Preferably, the monosaccharide includes, but is not limited to, pentose, hexose, and monosaccharides with six or more carbon atoms. Examples of the monosaccharide include, but are not limited to, one or more of the following: sialic acid, galactose, glucose, N-acetylgalactose, N-acetylglucose, fucose, mannose, lysose, fructose, sorbitol, tagatose, rhamnose, arabinose, erythrose, xylose, glucuronic acid, and iduronic acid. The monosaccharide can be D-type or L-type.

[0079] Preferably, the monosaccharide derivatives include, but are not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides.

[0080] Preferably, the configuration of each anomeric carbon in the sugar chain can be α or β.

[0081] Preferably, the types of glycosidic bonds in the sugar chain include, but are not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4.

[0082] A second aspect of the present invention provides a method for improving glycan sequencing resolution or glycan detection by electroosmotic flow, the method comprising applying a voltage to cause the glycan to be detected by coupling a head molecule to translocate from a first fluid chamber through a nanopore to a second fluid chamber, wherein the first and second fluid chambers contain buffer solutions to form an electroosmotic flow between the first and second fluid chambers, and wherein the nanopore is disposed between the first and second fluid chambers.

[0083] Preferably, the buffer solution contains cations. The cations may be monovalent, divalent, and / or trivalent cations.

[0084] Preferably, the monovalent cation contains K + NH4 + Na + Cu + Li + or Ag + One or more of them.

[0085] Preferably, the divalent cation comprises Mg 2+ Ca 2+ Ba 2+ Mn 2+ Cu 2+ Fe 2+ Zn 2+ Ni 2+ Co 2+ or Pb 2+ One or more of them.

[0086] Preferably, the trivalent cation comprises Al 3+ Cr 3+ Ti 3+ Ga 3+ In 3+ Co 3+ La 3+ Ce 3+ 、Nd 3+ or Fe 3+ One or more of them.

[0087] More preferably, the cation contains K + Mg 2+ Ca 2+ Ba 2+ Mn 2+or Fe 3+ One or more of them.

[0088] Preferably, the cation concentration in the buffer solution is greater than or equal to 5 mM, for example, 5-10000 mM, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 mM, etc., preferably 5-2000 mM.

[0089] Preferably, the buffer solution in the first fluid chamber is the same as or different from that in the second fluid chamber.

[0090] Preferably, the cations in the buffer solutions in the first fluid chamber and the second fluid chamber can be the same or different.

[0091] Preferably, the cation concentrations in the buffer solutions in the first fluid chamber and the second fluid chamber can be the same or different.

[0092] Preferably, the cations in the first fluid chamber include K + Mg 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of the following, with Mg being the preferred choice. 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of the following, with Mg being more preferred. 2+ Ca 2+ or Fe 3+ One or more of them.

[0093] Preferably, the cation concentration in the first fluid chamber is 100-10000 mM, such as 100, 200, 272, 300, 400, 500, 539, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 mM, more preferably 100-2000 mM or 200-1000 mM, and even more preferably 272-539 mM.

[0094] Preferably, the concentration of divalent and / or trivalent cations in the first fluid chamber is 100-10000 mM, such as 100, 200, 272, 300, 400, 500, 539, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 mM, more preferably 100-2000 mM or 200-1000 mM, and even more preferably 272-539 mM.

[0095] In one specific embodiment of this application, the buffer solution in the first fluid chamber comprises: a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000mM FeCl3; preferably 272-539mM FeCl3.

[0096] Preferably, the cations in the second fluid chamber include K. + Mg 2+ Ca 2+ Or Ba 2+ One or more of the following; Mg is further preferred. 2+ Ca 2+ Or Ba 2+ Further optimization of Mg 2+ or Ca 2+ .

[0097] Preferably, the concentration of divalent and / or trivalent cations in the second fluid chamber is greater than or equal to 5 mM, for example greater than or equal to 5, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, greater than or equal to 25, greater than or equal to 30, greater than or equal to 35, greater than or equal to 40, greater than or equal to 45, greater than or equal to 50, greater than or equal to 55, greater than or equal to 60, greater than or equal to 65, greater than or equal to 70, greater than or equal to 75, greater than or equal to 80, greater than or equal to 85, greater than or equal to 90, greater than or equal to 100, greater than or equal to 1000, greater than or equal to 10000 mM, for example 5-10000 mM, preferably 5 mM-80 mM, and further 10 mM-40 mM.

[0098] In some embodiments, the buffer solution in the second fluid chamber contains Mg. 2+ And other cations.

[0099] In some embodiments, the buffer solution in the second fluid chamber contains 1) Mg 2+ , and 2)K + Ca 2+ Or Ba 2+ One or more of them.

[0100] Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 5-80mM MgCl2; B) 400-800mM KCl and 5-80mM CaCl2; C) 400-800mM KCl and 5-80mM BaCl2; D) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM CaCl2; or, E) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM BaCl2.

[0101] Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 10-40mM MgCl2; B) 400-800mM KCl and 10-40mM CaCl2; C) 400-800mM KCl and 10-40mM BaCl2; D) 400-800mM KCl, 5-40mM MgCl2 and 10-40mM CaCl2; or, E) 400-800mM KCl, 5-40mM MgCl2 and 10-40mM BaCl2.

[0102] In one specific embodiment of this application, the buffer solution in the second fluid chamber comprises 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the first fluid chamber comprises a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000 mM FeCl3; preferably 272-539 mM FeCl3.

[0103] In one specific embodiment of this application, the buffer solution in the first fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the second fluid chamber contains A) 400-800 mM KCl and 5-80 mM MgCl2; B) 400-800 mM KCl and 5-80 mM CaCl2; or, C) 400-800 mM KCl and 5-80 mM BaCl2; D) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM CaCl2; or, E) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM BaCl2.

[0104] Preferably, the buffer solution also contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0105] In some embodiments, the buffer solution in the second fluid chamber further contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0106] In some embodiments, the buffer solution in the first fluid chamber further contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0107] In some embodiments, the first fluid chamber may be cis-side and the second fluid chamber may be trans-side.

[0108] In some embodiments, the second fluid chamber may be the cis-side and the first fluid chamber may be the trans-side.

[0109] The pH of the buffer solution is neutral, weakly acidic, or weakly alkaline.

[0110] Preferably, the pH of the buffer solution is 6-9, and more preferably 7.5-8.5.

[0111] The nanopores are inserted into the lipid bilayer.

[0112] The measurement cell is divided into two fluid chambers, and materials known in the prior art can be used. The material can be of any shape, as long as it has suitable pores into which nanopores can be inserted. For example, the material can be a membrane or a tube. The material is an insulating material.

[0113] The material contains pores of 30-70 μm.

[0114] A lipid bilayer is formed on the pores.

[0115] The membrane is an insulating material, such as an organic membrane or a synthetic membrane, such as a polytetrafluoroethylene membrane or a plastic membrane.

[0116] The tube is made of insulating material, such as a plastic tube.

[0117] The connector molecules are one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0118] Preferably, a linker molecule is attached to one end of the sugar chain to be tested to form a linker molecule-sugar chain conjugate, or a sugar chain-linker molecule conjugate. In one specific embodiment of the invention, a linker molecule is attached to the reduced end of the sugar chain to be tested.

[0119] Preferably, a connector molecule is connected to both ends of the sugar chain to be tested to form a connector molecule 1-sugar chain-connector molecule 2 coupling, wherein connector molecule 1 and connector molecule 2 may be the same or different. In a specific embodiment of the present invention, a connector molecule is connected to both the reduced and non-reduced ends of the sugar chain to be tested.

[0120] The linker molecule is connected to the sugar chain via a covalent bond or via a linking group. Preferably, the linker molecule is connected to the sugar chain via an oxime bond, an amide bond, a thioether bond, a disulfide bond, a phosphoryl bond, a hydrazone bond, an acylurea bond, or a ring bond formed by a click reaction.

[0121] In one specific embodiment of this application, an azide group is modified at one end of the sugar chain, and a DBCO group is modified at one end of the linker molecule, forming a linker molecule-sugar chain conjugate or a sugar chain-linker molecule conjugate through a click chemical reaction.

[0122] In one specific embodiment of this application, an azide group is modified at one end of the sugar chain, and an amino group is modified at the other end. One end of the linker molecule 1 is modified with a DBCO group, and one end of the linker molecule 2 is modified with a thiol group. The linker molecule 1-sugar chain-linker molecule 2 is formed through a click chemical reaction between DBCO and azide, and a reaction between amino and thiol groups.

[0123] In some embodiments, the glycan chain of the coupled head molecule is displaced from the first fluid chamber to the second fluid chamber through nanopores under the action of electrophoretic force and electroosmotic flow.

[0124] In some embodiments, the glycan chain of the coupled connector molecule is displaced from the first fluid chamber to the second fluid chamber through nanopores under the influence of electrophoretic force, electroosmotic flow, and motor protein pull.

[0125] The adapter molecule is a biopolymer, including but not limited to nucleic acids or peptides. It is sufficient to ensure that at least one adapter molecule can interact with (preferably specifically bind to) the motor protein. Its sequence can be any known or interesting sequence. The length can also be arbitrarily adjusted.

[0126] Preferably, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a glycan-nucleic acid conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a glycan-peptide conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

[0127] In one specific embodiment of this application, the adapter molecule is a nucleic acid.

[0128] The nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0129] The nucleic acid mentioned is DNA, RNA, modified DNA, or modified RNA.

[0130] In one specific embodiment of this application, the adapter molecule is DNA, and one end of the glycan chain to be tested is linked to the DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate. Preferably, the DNA is linked to the reducing end to form a DNA-glycan conjugate.

[0131] In one specific embodiment of this application, the adapter molecule is DNA, and both ends of the glycan chain to be tested are connected to DNA to form a DNA1-glycan-DNA2 conjugate, wherein DNA1 and DNA2 may have the same sequence or different sequences.

[0132] In one specific embodiment of this application, the DNA2 is single-stranded. For example, the DNA2 can be polyT.

[0133] In one specific embodiment of this application, the DNA1 is a single strand, which is annealed and coupled to a partially complementary fragment.

[0134] The motor proteins mentioned include, but are not limited to, nucleases, preferably including, but not limited to, helicases or variants thereof, polymerases or variants thereof, or transloses or variants thereof.

[0135] Preferably, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase.

[0136] Preferably, the helicase or its variant is a sequence of Hel308 Tga (Thermococcus gammatolerans (strain DSM 15229 / JCM 11827 / EJ3)) or a variant thereof, a sequence of Hel308 Mbu (Methanococcoides burtonii (strain DSM 6242 / NBRC 107633 / OCM 468 / ACE-M)) or a variant thereof, a sequence of Hel308 Pfu (Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)) or a variant thereof, or a sequence of Hel308 Mma (Methanosarcina mazei (strain ATCCBAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM)) or a variant thereof. The sequence of Hel308Mok (Methanothermococcus okinawensis (strain DSM 14208 / JCM 11175 / IH1)) or its variant, the sequence of Hel308Fac (Ferroplasma acidarmanus Fer1) or its variant, the sequence of Hel308Csy (Cenarchaeum symbiosum (strain A)) or its variant, the sequence of Hel308Mhu (Methanospirillumhungatei JF-1 (strain ATCC 27890 / DSM 864 / NBRC 100397 / JF-1)) or its variant, or the sequence of F8813 protein or its variant.

[0137] Preferably, the polymerase is Bacillus phage φ29 (phi29) polymerase or a variant thereof, Clostridium phage phiCPV4 (pol6, GenBank: AFH27113.1) or a variant thereof, Actinomycete phage Av-1 (pol7, GenBank: ABR67671.1) or a variant thereof.

[0138] In one specific embodiment of the present invention, the linker molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or variants thereof; the polypeptide is an amino acid sequence that can be specifically recognized by an unfolded enzyme or has a certain charge distribution. Preferably, the unfolded enzyme includes, but is not limited to, Caseinolytic protease X (ClpX).

[0139] The nanopores mentioned are those known in the prior art, as long as they have the ability to sense sugar chains.

[0140] Preferably, the nanopores are biological nanopores or solid nanopores. More preferably, the biological nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin (α-HL) nanopores or variants thereof, Mycobacterium smegmatis porin A (MspA) nanopores or variants thereof, Aeromonas hydrolysin (AeL) or variants thereof, curli production assembly / transport component (CsgG) nanopores or variants thereof, outer membrane porin F (OmpF) nanopores or variants thereof, cytolysin A (ClyA) nanopores or variants thereof, phi29 nanopores or variants thereof, ferric isohydroxamate uptake component A (FhuA) nanopores or variants thereof, pleurodactylysin A (PlyA) nanopores or variants thereof, pleurodactylysin B (PlyB) nanopores or variants thereof, or fragaceatoxin C (FraC) nanopores or variants thereof. More preferably, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

[0141] Preferably, the method further includes the process of the glycan chain to be tested of the coupled head molecule passing through the sensing region to obtain electrical signal characteristics, and determining the glycan chain sequence through the electrical signal characteristics.

[0142] The electrical signal characteristics include, but are not limited to, electrical signal waveforms.

[0143] The electrical signal waveform is formed by sensing the sugar chain through nanopores.

[0144] Preferably, the electrical signal characteristics include, but are not limited to, current amplitude, number of steps, step duration, frequency of occurrence of step signal, or standard deviation.

[0145] In one specific embodiment of this application, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives in the sugar chain.

[0146] In one specific embodiment of this application, the sugar chain sequence is the arrangement order of glycosidic bonds in the sugar chain.

[0147] In one specific embodiment of this application, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0148] In one specific embodiment of this application, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0149] The glycan characteristics include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there are modifications, type of modifications, location of modifications, number of modifications, whether there are branches, location of branches, number of branches, or branch sequence characteristics.

[0150] Preferably, the branched sequence features include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there is modification, type of modification, location of modification, and number of modifications.

[0151] The chain length of the sugar chain to be tested is greater than 1.

[0152] The sugar chain to be tested can be a straight chain or have at least one branch.

[0153] The sugar chain to be tested may be modified, such as by sulfonation.

[0154] The sugar chain to be tested can be charged or electrically neutral.

[0155] The glycan chain to be tested is composed of monosaccharides and / or their derivatives linked by glycosidic bonds.

[0156] Preferably, the monosaccharide includes, but is not limited to, pentose, hexose, and monosaccharides with six or more carbon atoms. Examples of the monosaccharide include, but are not limited to, one or more of the following: sialic acid, galactose, glucose, N-acetylgalactose, N-acetylglucose, fucose, mannose, lysose, fructose, sorbitol, tagatose, rhamnose, arabinose, erythrose, xylose, glucuronic acid, and iduronic acid. The monosaccharide can be D-type or L-type.

[0157] Preferably, the monosaccharide derivatives include, but are not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides.

[0158] Preferably, the configuration of each anomeric carbon in the sugar chain can be α or β.

[0159] Preferably, the types of glycosidic bonds in the sugar chain include, but are not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4.

[0160] The resolution includes more valid events and / or more steps.

[0161] The method for detecting glycans can be qualitative or quantitative. The detection can determine the presence, number, or concentration of glycan characteristics within the glycan chain.

[0162] The glycan characteristics include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there are modifications, type of modifications, location of modifications, number of modifications, whether there are branches, location of branches, number of branches, or branch sequence characteristics.

[0163] Preferably, the branched sequence features include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there is modification, type of modification, location of modification, and number of modifications.

[0164] In one specific embodiment of this application, the detection can detect the presence, number, or concentration of a specific monosaccharide unit in the glycan chain, the presence, location, number, or concentration of a specific mutation type, the presence or number of a specific mutation location, or the presence, location, number, or concentration of a specific modification type, etc.

[0165] A third aspect of this application provides a reagent for glycan nanopore sequencing, the reagent comprising a buffer solution serving as a first fluid chamber and a buffer solution serving as a second fluid chamber in nanopore sequencing, placed in the fluid chambers to form an electroosmotic flow; the buffer solution contains cations.

[0166] The cation can be a monovalent cation, a divalent cation, and / or a trivalent cation.

[0167] Preferably, the monovalent cation contains K + NH4 + Na + Cu + Li + or Ag + One or more of them.

[0168] Preferably, the divalent cation comprises Mg 2+ Ca 2+ Ba 2+ Mn 2+ Cu 2+ Fe2+ Zn 2+ Ni 2+ Co 2+ or Pb 2+ One or more of them.

[0169] Preferably, the trivalent cation comprises Al 3+ Cr 3+ Ti 3+ Ga 3+ In 3+ Co 3+ La 3+ Ce 3+ 、Nd 3+ or Fe 3+ One or more of them.

[0170] More preferably, the cation contains K + Mg 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of them.

[0171] Preferably, the cation concentration in the buffer solution is greater than or equal to 5 mM, for example, 5-10000 mM, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 mM, etc., preferably 5-2000 mM.

[0172] Preferably, the buffer solution in the first fluid chamber is the same as or different from that in the second fluid chamber.

[0173] Preferably, the cations in the buffer solutions in the first fluid chamber and the second fluid chamber can be the same or different.

[0174] Preferably, the cation concentrations in the buffer solutions in the first fluid chamber and the second fluid chamber can be the same or different.

[0175] Preferably, the cations in the first fluid chamber include K + Mg 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of the following, with Mg being the preferred choice. 2+ Ca 2+ Ba2+ Mn 2+ or Fe 3+ One or more of the following, with Mg being more preferred. 2+ Ca 2+ or Fe 3+ One or more of them.

[0176] Preferably, the cation concentration in the first fluid chamber is 100-10000 mM, such as 100, 200, 272, 300, 400, 500, 539, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 mM, more preferably 100-2000 mM or 200-1000 mM, and even more preferably 272-539 mM.

[0177] Preferably, the concentration of divalent and / or trivalent cations in the first fluid chamber is 100-10000 mM, such as 100, 200, 272, 300, 400, 500, 539, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 mM, more preferably 100-2000 mM or 200-1000 mM, and even more preferably 272-539 mM.

[0178] In one specific embodiment of the present invention, the buffer solution of the first fluid chamber comprises: a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000mM FeCl3; preferably 272-539mM FeCl3.

[0179] Preferably, the cations in the second fluid chamber include K. + Mg 2+ Ca 2+ Or Ba 2+ One or more of the following; Mg is further preferred.2+ Ca 2+ Or Ba 2+ Further optimization of Mg 2+ or Ca 2+ .

[0180] Preferably, the concentration of divalent and / or trivalent cations in the second fluid chamber is greater than or equal to 5 mM, for example greater than or equal to 5, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, greater than or equal to 25, greater than or equal to 30, greater than or equal to 35, greater than or equal to 40, greater than or equal to 45, greater than or equal to 50, greater than or equal to 55, greater than or equal to 60, greater than or equal to 65, greater than or equal to 70, greater than or equal to 75, greater than or equal to 80, greater than or equal to 85, greater than or equal to 90, greater than or equal to 100, greater than or equal to 1000, greater than or equal to 10000 mM, for example 5-10000 mM, preferably 5 mM-80 mM, and further 10 mM-40 mM.

[0181] In some embodiments, the buffer solution in the second fluid chamber contains Mg. 2+ And other cations.

[0182] In some embodiments, the buffer solution in the second fluid chamber contains 1) Mg 2+ , and 2)K + Ca 2+ Or Ba 2+ One or more of the following. Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 5-80mM MgCl2; B) 400-800mM KCl and 5-80mM CaCl2; C) 400-800mM KCl and 5-80mM BaCl2; D) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM CaCl2; or, E) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM BaCl2.

[0183] Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 10-40mM MgCl2; B) 400-800mM KCl and 10-40mM CaCl2; C) 400-800mM KCl and 10-40mM BaCl2; D) 400-800mM KCl, 5-40mM MgCl2 and 10-40mM CaCl2; or, E) 400-800mM KCl, 5-40mM MgCl2 and 10-40mM BaCl2.

[0184] In one specific embodiment of this application, the buffer solution in the second fluid chamber comprises 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the first fluid chamber comprises a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000 mM FeCl3; preferably 272-539 mM FeCl3.

[0185] In one specific embodiment of this application, the buffer solution in the first fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the second fluid chamber contains A) 400-800 mM KCl and 5-80 mM MgCl2; B) 400-800 mM KCl and 5-80 mM CaCl2; C) 400-800 mM KCl and 5-80 mM BaCl2; D) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM CaCl2; or, E) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM BaCl2.

[0186] Preferably, the buffer solution also contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0187] In some embodiments, the buffer solution in the second fluid chamber further contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0188] In some embodiments, the buffer solution in the first fluid chamber further contains 5-20 mM HEPES, such as 5, 10, 15 or 20 mM HEPES.

[0189] In some embodiments, the first fluid chamber may be cis-side and the second fluid chamber may be trans-side.

[0190] In some embodiments, the second fluid chamber may be the cis-side and the first fluid chamber may be the trans-side.

[0191] The pH of the buffer solution is neutral, weakly acidic, or weakly alkaline.

[0192] Preferably, the pH of the buffer solution is 6-9, and more preferably 7.5-8.5.

[0193] In a fourth aspect, the present invention provides a kit or apparatus comprising the above-described reagents.

[0194] A fifth aspect of this application provides the use of the reagent described in the third aspect as a fluid chamber buffer in glycan nanopore sequencing, preferably in improving the resolution of glycan nanopore sequencing.

[0195] The beneficial effects of this application are: 1) Under the action of electroosmotic flow, electrophoretic force and motor protein, the glycan chain is stretched and fixed, the glycan blockage current value increases, the number of steps increases, and high-resolution glycan sequencing is achieved.

[0196] 2) Ion-mediated electroosmosis in the fluid chamber can modulate the nanopore single-molecule sensing platform, revealing the key role of the buffer in the fluid chamber for glycan nanopore sequencing, and obtaining the optimal ionic environment for nanopore glycan sequencing through screening of specific cations.

[0197] 3) In an optimized ionic environment, ten sugar chains exhibited a discrete current step architecture directly related to their chemical structure, and different sulfation modes and glycosidic bond linkages were analyzed.

[0198] 4) The method of this application is label-free and hydrolysis-free. Traditional methods require chemical derivatization or enzymatic pretreatment (e.g., mass spectrometry requires methylation labeling, and existing nanopore technology for polysaccharide detection requires glycoside hydrolase treatment). Attached Figure Description

[0199] The embodiments of this application will now be described in detail with reference to the accompanying drawings, wherein: Figure 1 : The sequence of the polysaccharide, where 6S represents sulfonation modification and pro represents trimethylene.

[0200] Figure 2 This study compares and statistically analyzes well events in sequencing buffers and sequencing complexes using KCl and CaCl2 (539 mM) systems. In this analysis, A represents the corresponding current trajectory under the sequencing complex in the KCl system sequencing buffer, B represents the corresponding current trajectory under the sequencing complex in the CaCl2 (539 mM) system sequencing buffer, and C and D represent the current t-phase events in the KCl system sequencing buffer. off With ton Statistical graphs, E and F represent sequencing buffer t in the CaCl2 system (539 mM). off With t on Statistical chart.

[0201] Figure 3 Polysaccharide sequencing signals, average relative blocking current (D), and step division (E) under KCl system sequencing buffer (A), CaCl2 system (272 mM) (B), and CaCl2 system (539 mM) sequencing buffer (C) conditions.

[0202] Figure 4 This is a schematic diagram illustrating the technical principle of nanopore polysaccharide sequencing. A) represents that during polysaccharide sequencing, almost no electroosmotic flow occurs on the cis and trans sides. The glycan chains pass through the nanopore mainly under the influence of TGA traction force (motor protein pull) and EPF (electrophoretic force), and the glycan chains are in a coiled state. B) represents that during polysaccharide sequencing, enhanced electroosmotic flow occurs on the cis and trans sides. The glycan chains pass through the nanopore under the combined influence of TGA traction force (motor protein pull), EPF (electrophoretic force), and EOF (electrophoretic flow), and the glycan chains are in a straightened state.

[0203] Figure 5 For trans-chamber ion specificity studies, including K + (A) Mg 2+ (B) Ca 2+ (C) Ba 2+ (D) Mn 2+ (E), Fe 3+ (F).

[0204] Figure 6 For the study of ion specificity in CIS chambers, containing Mg 2+ (A) Ca 2+ (B) Ba 2+ (C).

[0205] Figure 7 Mg for cis chamber 2+ The effects of concentration gradients on sequencing signals include 5mM (A), 10mM (B), 20mM (C), 40mM (D), and 80mM (E).

[0206] Figure 8The sequencing signals and step divisions of the heptaose series G1 (A), G1-1S (B) and G1-5S (C) with different sulfation modes were compared under optimized ion conditions (D, E).

[0207] Figure 9 The sequencing signals and step divisions of the heptasaccharide series G1, G1-3S, and G1-3S, and 5S under optimized ion environment were compared for different sulfation modes.

[0208] Figure 10 Comparison of sequencing signals and step divisions of nonaglycones G2, G2-1S, and G2-5S with different sulfation modes under optimized ion environment.

[0209] Figure 11 Comparison of step signals for heptases with different sulfation modes.

[0210] Figure 12 Comparison of step signals for heptase and nonaose (G1-5S and G2-5S) sulfated at position 5. Detailed Implementation

[0211] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0212] The term "electroosmotic flow" refers to the fluid flow caused when a voltage is applied to both ends of a porous medium, microchannel, or other fluid conduit.

[0213] The term "nucleic acid" includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences. Examples include DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, and tRNA. Modifications can include 5' end modification, 3' end modification, introduction of non-natural nucleotides, base modification, sugar ring modification, and phosphate backbone modification. Base modifications include, but are not limited to, one or more of the following: 5-position pyrimidine modification, 8-position purine modification, or 5-bromouracil substitution. Sugar ring modifications include, but are not limited to, the 2'-OH group being replaced by one or more of the following groups: H, OZ, Z, halo, SH, SZ, NH2, NHZ, NZ2, or CN, where Z is an alkyl group. Phosphate backbone modifications include, but are not limited to, thiophosphate modification. Non-natural nucleotides include, but are not limited to, one or more of the following: nucleotides having non-natural bases, non-natural sugar moieties, or non-natural backbones. The non-natural bases mentioned include, but are not limited to, 2-aminoadenine-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-aminopropyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazoguanine, 3-deazoadenine, 4-thiouracil, 4-thiothymidine, uracil-5-yl, and hypoxanthine. -9-yl(I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromouracil, 5-trifluoromethyluracil, 5-bromocytosine, 5-trifluoromethylcytosine, 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted pyrimidine, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, cyclocytosine, cytarabine, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-Dihydrocytosine, 5-Iodocytosine, Hydroxyurea, Iodouracil, 5-Nitrocytosine, 5-Bromouracil, 5-Chlorouracil, 5-Fluorouracil, 5-Iodouracil, 6-alkyl derivatives of adenine and guanine, 6-Azuridine, 6-Azouracil, 6-Azocytosine, Azuridine, 6-Azothymidine, 6-Thioguanine, 7-Methylguanine, 7-Methyladenine, 7-Denitroguanine, 7-Denitroguanosine, 7-Denitroguanosine, 7-Denitro-Adenine, 7-Denitro-8-Azuridine, 8-Azuridine, 8-Azuridine, 8-Azuridine, 8-Azuridine, 8-Aminoadenine, 8-Aminoguanine, 8-Thiol Adenine, 8-Thiol Guanine, 8-Thioalkyl Adenine, 8-thioalkylguanine, 8-hydroxyadenine, 8-hydroxyguanine, N4-ethylcytosine, N-2-substituted purine, N-6-substituted purine, O-6-substituted purine, fluorinated nucleic acid, tricyclic pyrimidine, phenoxazincytidine ([5,4-b][l,4]benzoxazin-2(3H)-one), phenthiazincytidine (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps, phenoxazincytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4... [5-b]indole-2-one), pyridoindolecytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylpiperidine, inosine, N6-isopentene adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine 5-Methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylpiperidine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenteneadenine, uracil-5-oxyacetic acid, weidooxyglycoside, pseudouracil, piperidine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil or 2,One or more of 6-diaminopurines. The non-natural sugar moiety includes, but is not limited to, modifications at the 2' position of the following groups: OH; substituted lower alkyl, alkylaryl, aralkyl, O-alkylaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-ynyl, S-ynyl, N-ynyl, O-alkyl-O-alkyl, 2'-F, 2'-OCH3, 2'-O(CH2)2OCH3, wherein the alkyl, alkenyl, and ynyl groups can be substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 ynyl, -O[(CH2), n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON[(CH2)] n CH3)]2, wherein n and m are from 1 to 10; and / or one or more of the following group of modifications selected at the 5' position: 5'-vinyl, 5'-methyl (R or S), and at the 4' position: 4'-S, heterocyclic alkyl, heterocyclic aryl, aminoalkylamino, polyalkylamino, or substituted silyl.

[0214] The term "peptide" refers to a chain of two or more amino acid residues linked by peptide bonds. Depending on the number of amino acid residues, peptides can be classified as dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, or peptides composed of more than one amino acid residue. These amino acids can be either natural or non-natural. The non-natural amino acids mentioned include, but are not limited to, one or more of the following: 2-aminoisobutyric acid (Aib), imidazole-4-acetate (IA), imidazole propionic acid (IPA), α-aminobutyric acid (Abu), tert-butylglycine (Tle), 3-aminomethylbenzoic acid, anthranilic acid, deaminohistidine, β-alanine, 2-aminohistidine, β-hydroxyhistidine, homohistidine, Nα-acetylhistidine, α-fluoro-methylhistidine, α-methylhistidine, α,α-dimethylglutamic acid, m-CF3-phenylalanine, α,β-diaminopropionic acid, 3-pyridylalanine, 2-pyridylalanine, 4-pyridylalanine, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl)carboxylic acid.

[0215] The term "derivative" of a substance refers to a derivative that contains the same core chemical structure as the substance, but differs due to at least one structural difference, for example, due to having one or more added and / or removed and / or substituted substituents, and / or due to having one or more atoms substituted by different atoms.

[0216] The term "nanopore" generally refers to a pore, channel, or pathway with a very small diameter on the nanometer scale that extends through a membrane. Nanopores can have a characteristic width or diameter from about 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins. MspA (Mycobacterium smegmatis porin A) is an example of a protein nanopore.

[0217] The term "variant" may have one or more amino acid additions, substitutions, and / or deletions compared to its parent, or may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to its parent, wherein the parent protein or peptide may be a wild-type protein or peptide or its homolog or variant, and retains the parent's function. For example, nanopore variants retain tunnel-forming ability. As another example, helicases or variants thereof, polymerases or variants thereof, transloses or variants thereof, or unfoldases or variants thereof retain the function of interacting with nucleic acids or peptides (preferably specifically binding).

[0218] This application presents a method for classifying current steps in polysaccharide sequencing signals: a threshold-based detection method is used to classify current steps in polysaccharide sequencing. A valid event is defined as a stable current blockage lasting from 200 ms to 2 s. Signal stability is determined by the amplitude of current fluctuations within a segment: the average of the minimum and maximum values ​​of the first 10% of data points in the current segment is calculated; if the difference between the two is ≤1.9 pA, the segment is considered stable and identified as an independent step.

[0219] Example 1: Sequencing Buffer Optimization 1. A sequencing nucleic acid is coupled to one end of the polysaccharide, and a guide nucleic acid is coupled to the other end. 1) Modify one end of the polysaccharide chain with an azide group, and modify the 5' end of the artificially synthesized sequencing nucleic acid (T89) with a DBCO group. Add 45 μL of PBS (pH 7.2-7.4) to a tube of T89 (3 nmol) with a DBCO group modified at the 5' end, add 2 μL of 15 nmol / μL polysaccharide solution (6 nmol), and react at 29℃ and 600 rpm for 12 h. The sequencing nucleic acid and polysaccharide (polysaccharide-T89) are ligated through a click chemical reaction of DBCO and azide.

[0220] 2) Add 2 μL of 15 nmol / μL SMCC solution (30 nmol) to the polysaccharide-T89 solution, react at 600 rpm for 1 h at room temperature, and purify by desalting column to obtain Mal-polysaccharide-T89. Add 120 μL of 0.1 nmol / μL polyT-SH (12 nmol) to Mal-polysaccharide-T89, react at 600 rpm for 12 h at room temperature, run on a urea Page gel, remove from the gel, purify by desalting column to obtain the sequencing nucleic acid-polysaccharide-guide nucleic acid conjugate. The sequences of T89 and polyT are shown in Table 1, and the sequence of the polysaccharide is shown in Table 2. Figure 1 As shown.

[0221] Table 1 2. Detection System The measurement cell employs a classic dual-chamber structure, consisting of a Cis chamber and a Trans chamber. The two chambers are fluidly connected via a polyetheretherketone (PEEK) capillary tube (approximately 25 μm in inner diameter). Before measurement, both chambers are cleaned sequentially with isopropanol, hydrogen peroxide, and Milli-Q water, and then dried with compressed nitrogen. A pair of Ag / AgCl electrodes are inserted into each chamber to conduct current. In principle, the chamber with the grounded electrode is defined as the cis side, and the opposite side as the trans side. Subsequently, the orifice on the plastic tube is treated with 1,2-diphydanyl-sn-glycerol-3-phosphocholine (DPhPC) dissolved in hexane and allowed to air dry. Approximately 0.1 mL of electrolyte buffer is added to each chamber. An air bubble is generated using a pipette and dragged through the orifice, thereby forming a self-assembled phospholipid bilayer on the orifice. MspA M2 nanoporous protein was diluted and added to the cis chamber to obtain spontaneous pore insertion. After observing a signal of single-pore insertion, the cis chamber was immediately perfused with fresh buffer.

[0222] Electrophysiological signals were amplified using an Axopatch 200B patch-clamp amplifier and digitized using a Digidata 1550B digital-to-analog converter. The ion current signal was sampled at 25 kHz, and the output signal was filtered at an angular frequency of 1 kHz.

[0223] 3. Sequencing buffer KCl system: both cis and trans chamber buffers are 400 mM KCl + 5 mM MgCl2 + 10 mM HEPES, pH 8.0.

[0224] The CaCl2 system (272 mM) consists of: cis chamber buffer of 400 mM KCl + 5 mM MgCl2 + 10 mM HEPES (pH 8.0) and trans chamber buffer of 272 mM CaCl2 + 10 mM HEPES (pH 8.0).

[0225] CaCl2 system (539 mM): cis chamber: 800 mM KCl + 5 mM MgCl2 + 10 mM HEPES (pH 8.0); trans chamber: 539 mM CaCl2 + 10 mM HEPES (pH 8.0).

[0226] 4. Sequencing methods First, the sequencing nucleic acid-polysaccharide-guide nucleic acid conjugate is combined with cholesterol-labeled ssDNA oligonucleotides (TTTTTTTTTTTTTTTTgCTACgACCTgCATgAgAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 0.5 µM sequencing complex was prepared by annealing (SEQ ID NO: 3), then mixed with 2 µM TGA helicase (NCBI: ACS33372.1) solution, and 2 µL of this mixture was added to the cis chamber. Sequencing was initiated by adding 2 µL of 100 mM ATP to the cis chamber at 25 °C and 180 mV.

[0227] 5. Results The results are as follows Figure 2-3 As shown, G1-3S and 5S were used as models to analyze glycans.

[0228] like Figure 2 As shown in Figure (A), the translocation current trajectory of the sequencing complex in the KCl system sequencing buffer without TGA helicase rate control illustrates the transient capture event. Labeled parameters: open pore current (I0), residual current (I0). a ), event duration (t) off ), event interval event (t) on ), blocking depth (ΔI = I0-I) a (B) Corresponding current trajectory in sequencing buffer (539 mM) using the CaCl2 system. (CF) Statistical histogram of event duration and interval for sequencing signals recorded over 20 minutes: t off (C,E) and t on(D,F). KCl system sequencing buffer (C,D; n=669 events) vs CaCl2 system (539 mM) (E,F; n=1779 events). Mean duration (τ) off ) and average interval time (τ) on The values ​​were obtained through exponential fitting (y = a·exp(-x / τ)). Comparative analysis showed that calcium ion flow significantly increased the event frequency and prolonged the duration, verifying the generation of electroosmotic flow.

[0229] Further sequencing signal results obtained under TGA helicase-controlled rate control are shown below. Figure 3 As shown. The sequencing signal under KCl system sequencing buffer conditions is as follows. Figure 3 As shown in Figure A, the polysaccharide sequencing signal has a step number of 5 and an average relative blocking current of 0.197; the sequencing signal under CaCl2 system (272 mM) sequencing buffer conditions is as follows: Figure 3 As shown in Figure B, the sequencing signal for the nucleic acid portion remained unchanged, while the step number of the polysaccharide sequencing signal increased to 10, and the average relative blocking current also increased to 0.272; the sequencing signal under further optimized CaCl2 system (539 mM) sequencing buffer conditions is as follows... Figure 3 As shown in Figure C, the sequencing signal for the nucleic acid portion remained unchanged, while the number of steps in the polysaccharide sequencing signal further increased to 14, and the average relative blocking current also further increased to 0.301. The average relative blocking current and median number of steps were statistically analyzed using 20 glycan chains, see [Figure C]. Figure 3 As shown in Figures D and E.

[0230] In summary, after optimizing the sequencing buffer, electroosmotic flow is generated. The polysaccharide, under the combined influence of motor protein tension, electrophoretic force, and electroosmotic flow, is in a straightened and fixed state, thus significantly increasing the number of sequencing signal steps and improving the sequencing resolution of the polysaccharide. The principle is as follows: Figure 4 As shown, under the conditions of KCl system sequencing buffer, the polysaccharide chains are mainly subjected to the pulling force of motor proteins and electrophoretic force, and are in a coiled state; under the conditions of CaCl2 system sequencing buffer, the polysaccharide chains are subjected to the combined effects of the pulling force of motor proteins, electrophoretic force and electroosmotic flow, and are in a straightened and fixed state.

[0231] Example 2: The effect of different divalent and / or trivalent cations on sequencing resolution 1. Study on the ion specificity of trans-chamber Referring to the TGA helicase-controlled sequencing method in Example 1, while keeping the cis chamber buffer (400 mM KCl + 5 mM MgCl2 + 10 mM HEPES, pH 8.0) constant, the trans chamber buffer was varied, with a concentration of 272 mM cation + 10 mM HEPES (pH 8.0). + Ca 2+ Mg 2+ Ba 2+ Mn 2+ Fe 3+ .

[0232] The results are as follows Figure 5 As shown, high concentration of Mg 2+ (B: trans 272 mM MgCl2) increased the relative current blocking value Ires / Iopen to 0.23389 (range: 0.22183–0.24509), improving the resolution to the median 9th order, indicating enhanced glycan stretching. Ca 2+ (C: trans 272 mM CaCl2) produced the highest blocking current (Ires / Iopen = 0.27205) and the highest resolution (median 10 levels), suggesting that this ionic environment strongly generates electroosmotic flow (EOF). Ba 2+ (D) and Mn 2+ (E) also showed improvement (Ires / Iopen: 0.22202 / 0.23111; median resolution: 7 levels), Fe 3+ (F) also increases the current blocking value, while also improving the resolution (median 10 levels).

[0233] 2. Study on the ion specificity of CIS chamber Referring to the TGA helicase-controlled sequencing method in Example 1, while keeping the trans chamber buffer (400 mM KCl + 5 mM MgCl2 + 10 mM HEPES, pH 8.0) constant, the cis chamber buffer was varied as follows: Ca 2+ System: 400mM KCl + 5 mM MgCl2 + 40mM CaCl2 + 10 mM HEPES, pH 8.0; Mg 2+ System: 400mM KCl + 40 mM MgCl2 + 10 mM HEPES, pH 8.0; Ba 2+ System: 400mM KCl + 5 mM MgCl2 + 40mM BaCl2 + 10 mM HEPES, pH 8.0; Mn 2+ System: 400mM KCl + 5 mM MgCl2 + 40mM MnCl2 + 10 mM HEPES, pH 8.0; Fe 3+ System: 400mM KCl + 5 mM MgCl2 + 40mM FeCl3 + 10 mM HEPES, pH 8.0.

[0234] The results are as follows Figure 6 As shown, Ca 2+ System and Mg 2+ The systems all achieve high blocking current values ​​and high resolution for polysaccharide signals. Ba 2+ The system effect was weak, confirming the ion dependence of EOF.

[0235] Example 3: The effect of cis chamber ion concentration on sequencing resolution Referring to the TGA helicase-controlled sequencing method in Example 1, while keeping the trans chamber buffer (400 mM KCl + 5 mM MgCl2 + 10 mM HEPES, pH 8.0) constant, the Mg content of the buffer in the cis chamber was varied. 2+ The concentration of the buffer solution in the CIS chamber was 400 mM KCl + different concentrations of MgCl2 + 10 mM HEPES, pH 8.0.

[0236] The results are as follows Figure 7 As shown, the Mg in the cis chamber is increased. 2+ Gradually increasing the concentration of blocking current: cis chamber 10mM Mg 2+ (B): Ires / Iopen = 0.24314, median number of resolvable steps: 6; cis chamber 40mM Mg 2+ (D): Peak sequencing performance (Ires / Iopen = 0.31082, median number of steps: 10); cis chamber 80mM Mg 2+ (E): The highest blocking current value was reached (Ires / Iopen=0.36578), but the resolution was not further improved (median step: 10 steps).

[0237] Example 4: Glycan sequencing to distinguish the position of sulfate modification The TGA helicase-controlled sequencing method in Example 1 was followed, with the following buffers: cis: 800 mM KCl + 5 mM MgCl2 + 10 mM HEPES, pH 8.0; trans: 539 mM CaCl2 + 10 mM HEPES, pH 8.0.

[0238] The results are as follows Figure 8-11 As shown, using an optimized buffer solution, structurally well-defined glycans achieve unprecedented step-like resolution. Among them, Figure 8 Compared to the template hepta-sugar chain G1, the current at steps 2-7 of G1-1S is reduced, and the current at steps 8-12 of G1-5S is weakened, both of which are due to the steric hindrance of the GlcNAc sulfate group. Figure 9 Compared with the template hepta-sugar chain G1, the current at steps 7-9 of G1-3S is significantly reduced, and the current at steps 9-12 of G1-3S and 5S is significantly weakened, all of which are due to the steric hindrance of the GlcNAc sulfate group. Figure 10 Compared with template G2, G2-1S shows a significant decrease in step current at positions 2-6, similar to G1-1S; G2-5S shows a significant decrease in current at positions 9-13, similar to G1-5S, both of which are due to the steric hindrance of the GlcNAc sulfate group. Figure 11 The results show that sulfation site 1 corresponds to step 3, sulfation site 3 corresponds to step 8, and sulfation site 5 corresponds to step 12, validating single-molecule sequence sequencing of motor proteins under controlled-rate conditions.

[0239] Example 4: Glycan sequencing distinguishes different lengths The TGA helicase-controlled sequencing method in Example 1 was followed, with the following buffers: cis: 800 mM KCl + 5 mM MgCl2 + 10 mM HEPES, pH 8.0; trans: 539 mM CaCl2 + 10 mM HEPES, pH 8.0.

[0240] The results are as follows Figure 12 As shown, G1-5S (heptacarbose) and G2-5S (nonacarbose) share the same steps 1-12, while the unique steps (positions 13-15) of G2-5S correspond to the GlcNAc-sialic acid disaccharide unit. This indicates that the method achieves monosaccharide resolution.

[0241] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0242] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. A method for sequencing glycan chains, characterized in that, The sequencing method includes: A voltage is applied to cause the target sugar chain of the coupling head molecule to translocate from the first fluid chamber to the second fluid chamber through a nanopore. The first and second fluid chambers contain buffer solutions to form an electroosmotic flow between the first and second fluid chambers. The nanopore is disposed between the first and second fluid chambers.

2. The sequencing method according to claim 1, characterized in that, The buffer solution contains cations, which can be monovalent, divalent, and / or trivalent cations.

3. The sequencing method according to claim 2, characterized in that, The monovalent cation contains K + NH4 + Na + Cu + Li + or Ag + One or more of them.

4. The sequencing method according to claim 2, characterized in that, The divalent cation contains Mg 2+ Ca 2+ Ba 2 + Mn 2+ Cu 2+ Fe 2+ Zn 2+ Ni 2+ Co 2+ or Pb 2+ One or more of them.

5. The sequencing method according to claim 2, characterized in that, The trivalent cations include Al 3+ Cr 3+ Ti 3 + Ga 3+ In 3+ Co 3+ La 3+ Ce 3+ 、Nd 3+ or Fe 3+ One or more of them.

6. The sequencing method according to any one of claims 1-5, characterized in that, The cation concentration in the buffer solution is greater than or equal to 5 mM, for example 5-10000 mM, preferably 5-2000 mM.

7. The sequencing method according to any one of claims 1-6, characterized in that, The buffer solutions in the first fluid chamber and the second fluid chamber may be the same or different.

8. The sequencing method according to any one of claims 1-7, characterized in that, The concentration of divalent and / or trivalent cations in the first fluid chamber is 100-10000 mM, preferably 100-2000 mM.

9. The sequencing method according to any one of claims 1-8, characterized in that, The cations in the first fluid chamber include K + Mg 2+ Ca 2+ Ba 2+ Mn 2+ or Fe 3+ One or more of them.

10. The sequencing method according to any one of claims 1-9, characterized in that, The buffer solution in the first fluid chamber comprises: a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000mM FeCl3; preferably 272-539mM FeCl3.

11. The sequencing method according to any one of claims 1-10, characterized in that, The concentration of divalent and / or trivalent cations in the second fluid chamber is greater than or equal to 5 mM, preferably 5-10000 mM.

12. The sequencing method according to any one of claims 1-11, characterized in that, The cations in the second fluid chamber include K + Mg 2+ Ca 2+ Or Ba 2+ One or more of them.

13. The sequencing method according to any one of claims 1-12, characterized in that, The buffer solution in the second fluid chamber contains: A) 400-800mM KCl and 5-80mM MgCl2; B) 400-800mM KCl and 5-80mM CaCl2; C) 400-800mM KCl and 5-80mM BaCl2; D) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM CaCl2; or, E) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM BaCl2.

14. The sequencing method according to any one of claims 1-13, characterized in that, The buffer solution in the second fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the first fluid chamber contains a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000 mM FeCl3; preferably 272-539 mM FeCl3.

15. The sequencing method according to any one of claims 1-13, characterized in that, The buffer solution in the first fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the second fluid chamber contains A) 400-800 mM KCl and 5-80 mM MgCl2; B) 400-800 mM KCl and 5-80 mM CaCl2; C) 400-800 mM KCl and 5-80 mM BaCl2; D) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM CaCl2; or, E) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM BaCl2.

16. The sequencing method according to any one of claims 1-15, characterized in that, The connector molecule is one or more; Preferably, a linker molecule is connected to one end of the sugar chain to be tested to form a linker molecule-sugar chain conjugate, or a sugar chain-linker molecule conjugate is formed. Preferably, a connector molecule is connected to both ends of the sugar chain to be tested to form a connector molecule 1-sugar chain-connector molecule 2 coupling, wherein the connector molecule 1 and the connector molecule 2 are the same or different.

17. The sequencing method according to claim 16, characterized in that, The linker molecule is connected to the sugar chain via a covalent bond or via a linking group; Preferably, the linker molecule is linked to the sugar chain via an oxime bond, an amide bond, a thioether bond, a disulfide bond, a phosphoryl bond, a hydrazone bond, an acylurea bond, or a ring formed by a click reaction.

18. The sequencing method according to any one of claims 1-17, characterized in that, Under the influence of electrophoretic force and electroosmotic flow, the test sugar chain of the coupling head molecule is displaced from the first fluid chamber to the second fluid chamber through nanopores; Preferably, the glycan chain of the coupled connector molecule is displaced from the first fluid chamber to the second fluid chamber through nanopores under the action of electrophoretic force, electroosmotic flow and motor protein pull.

19. The sequencing method according to any one of claims 1-18, characterized in that, The linker molecule is a nucleic acid or a polypeptide; Preferably, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a glycan-nucleic acid conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a glycan-peptide conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

20. The sequencing method according to claim 19, characterized in that, The adapter molecule is a nucleic acid, and the motor protein includes, but is not limited to, nucleases, preferably including, but not limited to, helicases or variants thereof, polymerases or variants thereof, or transloses or variants thereof; Preferably, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase; Preferably, the helicase or its variant is a sequence of Hel308 Tga or its variant, Hel308 Mbu or its variant, Hel308 Pfu or its variant, Hel308 Mma or its variant, Hel308 Mok or its variant, Hel308 Fac or its variant, Hel308 Csy or its variant, Hel308 Mhu or its variant, or F8813 protein or its variant. Preferably, the polymerase is phi29 polymerase or a variant thereof, pol6 or a variant thereof, or pol7 or a variant thereof.

21. The sequencing method according to claim 19, characterized in that, The linker molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or their variants. Preferably, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme.

22. The sequencing method according to any one of claims 1-21, characterized in that, The nanopores are biological nanopores or solid nanopores; Preferably, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin nanopores or variants thereof, MspA nanopores or variants thereof, Aeromonas hydrolysin or variants thereof, CsgG nanopores or variants thereof, OmpF nanopores or variants thereof, ClyA nanopores or variants thereof, phi29 nanopores or variants thereof, FhuA nanopores or variants thereof, PlyA nanopores or variants thereof, PlyB nanopores or variants thereof, or FraC nanopores or variants thereof; Preferably, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

23. The sequencing method according to any one of claims 1-22, characterized in that, The sequencing method further includes the process of the glycan chain to be tested of the coupled connector molecule passing through the sensing region to obtain electrical signal characteristics, and determining the glycan chain sequence through the electrical signal characteristics.

24. The sequencing method according to claim 23, characterized in that, The glycan sequence is the arrangement or order of appearance of glycan features in the glycan.

25. The sequencing method according to claim 24, characterized in that, The glycan characteristics include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there is modification, type of modification, location of modification, number of modifications, whether there are branches, location of branches, number of branches or branch sequence characteristics. Preferably, the branched sequence features include, but are not limited to, monosaccharide unit composition, monosaccharide unit configuration, number of monosaccharide units, number of repeating units, glycosidic bond type, number of glycosidic bonds, order of monosaccharide units, whether there is modification, type of modification, location of modification, and number of modifications.

26. The sequencing method according to any one of claims 1-25, characterized in that, The glycan chain to be tested is composed of monosaccharides and / or their derivatives linked by glycosidic bonds; Preferably, the monosaccharides include, but are not limited to, pentose sugars, hexose sugars, and monosaccharides with six or more carbon atoms; Preferably, the monosaccharide derivatives include, but are not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides; Preferably, the types of glycosidic bonds in the sugar chain include, but are not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4.

27. A method for improving glycan sequencing resolution via electroosmosis, characterized in that, The method includes applying a voltage to cause the target sugar chain of the coupling head molecule to translocate from a first fluid chamber through a nanopore to a second fluid chamber, wherein the first and second fluid chambers contain buffer solutions to form an electroosmotic flow between the first and second fluid chambers, and the nanopores are disposed between the first and second fluid chambers.

28. A reagent for glycan nanopore sequencing, characterized in that, The reagent includes a buffer solution for the first fluid chamber and a buffer solution for the second fluid chamber in nanopore sequencing, which are placed in the fluid chambers to form an electroosmotic flow; the buffer solution contains cations.

29. The reagent according to claim 28, characterized in that, The concentration of divalent and / or trivalent cations in the first fluid chamber is 100-10000 mM, preferably 100-2000 mM; Preferably, the buffer solution in the first fluid chamber comprises: a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000mM FeCl3; preferably 272-539mM FeCl3.

30. The reagent according to claim 28 or 29, characterized in that, The concentration of divalent and / or trivalent cations in the second fluid chamber is greater than or equal to 5 mM, preferably 5-10000 mM; Preferably, the buffer solution in the second fluid chamber comprises: A) 400-800mM KCl and 5-80mM MgCl2; B) 400-800mM KCl and 5-80mM CaCl2; C) 400-800mM KCl and 5-80mM BaCl2; D) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM CaCl2; or, E) 400-800mM KCl, 5-80mM MgCl2 and 5-80mM BaCl2.

31. The reagent according to any one of claims 28-30, characterized in that, The buffer solution in the second fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the first fluid chamber contains a) 200-1000 mM KCl; preferably 272-539 mM KCl; b) 200-1000 mM MgCl2; preferably 272-539 mM MgCl2; c) 200-1000 mM CaCl2; preferably 272-539 mM CaCl2; d) 200-1000 mM BaCl2; preferably 272-539 mM BaCl2; e) 200-1000 mM MnCl2; preferably 272-539 mM MnCl2; or f) 200-1000 mM FeCl3; preferably 272-539 mM FeCl3.

32. The reagent according to any one of claims 28-31, characterized in that, The buffer solution in the first fluid chamber contains 400-800 mM KCl and 5-80 mM MgCl2; and the buffer solution in the second fluid chamber contains A) 400-800 mM KCl and 5-80 mM MgCl2; B) 400-800 mM KCl and 5-80 mM CaCl2; C) 400-800 mM KCl and 5-80 mM BaCl2; D) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM CaCl2; or, E) 400-800 mM KCl, 5-80 mM MgCl2 and 5-80 mM BaCl2.

33. The use of the reagent according to any one of claims 28-32 as a fluid chamber buffer in glycan nanopore sequencing, preferably in improving the resolution of glycan nanopore sequencing.

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