Method for sequencing glycan

Abstract

Provided in the present invention is a method for sequencing a glycan, the method comprising: linking a glycan to a linker molecule to obtain a linker molecule-glycan conjugate; applying a voltage across a nanopore to allow the glycan to pass through a nanopore and traverse a sensing region; and obtaining an electrical signal characteristic of the glycan to determine a glycan sequence. Further provided in the present invention is a method for detecting a glycan. The detection method may be either qualitative or quantitative.

Description

A method for glycan sequencing Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for sequencing glycan chains. Background Technology

[0002] Carbohydrates, as the third class of biological macromolecules besides nucleic acids and proteins, play a vital role in life activities. The rich biological functions of carbohydrate molecules are hidden in their complex structures (including monosaccharide composition and its linkage sequence, monosaccharide configuration, glycosidic bond type, glycan modification sites and number, branching positions and their sequence information). Therefore, analyzing their sequences is an important foundation for understanding their complex biological functions and promoting the development of carbohydrate science.

[0003] Traditional methods for analyzing sugar molecule sequences include mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and related techniques. Before mass spectrometry / NMR analysis, sugar molecules are hydrolyzed, and their monosaccharide composition is determined using methods such as high-performance liquid chromatography (HPLC). For mass spectrometry, sugar molecules (especially highly polymerized sugar chains) are typically fragmented using different methods to obtain multiple sets of fragment units. The fragment ion information generated after fragment unit breakage is analyzed to infer the connection sequence, connection mode, modification sites, and types of monosaccharides within the fragment units, thereby obtaining the sequence information of the fragment units. Finally, bioinformatics methods are used to arrange and align the fragment units to obtain the sequence information of the sugar chain. For NMR spectroscopy, the changes in the chemical shifts and coupling relationships of carbon and hydrogen atoms in the sugar molecule are mainly used to infer the monosaccharide configuration, connection sequence, connection mode, modification sites, and types within the sugar chain, thus obtaining the sequence information of the sugar chain. For example, patent CN114636750A discloses a data parsing device for sugar chain mass spectrometry analysis.

[0004] However, since mass spectrometry primarily relies on the mass-to-charge ratio (the ratio of fragment mass to its charge) of fragment ions for sequence analysis, when the molecular weights of glycan molecules or fragment ions are completely equal (e.g., differences lie only in the monosaccharide configuration or the glycan is composed of repeating units), the differences in information obtained by mass spectrometry are minimal, making it difficult to resolve information such as sugar molecule configuration and repeating unit length. Nuclear magnetic resonance spectroscopy, on the other hand, analyzes information such as the chemical shifts of carbon and hydrogen atoms within a limited range to infer sequence information. As the length of the glycan chain increases, the number of carbon and hydrogen atoms increases, significantly increasing the complexity of this information. Furthermore, both of these techniques suffer from limitations such as high cost, cumbersome operation, long processing time, and difficulty in comprehensively resolving glycan sequence information.

[0005] Nanopore analysis is a low-cost, high-throughput, and easy-to-operate single-molecule detection technique. The basic principle of nanopore detection is that when a voltage is applied across a nanopore in a specific electrolyte medium, ions migrate directionally, generating a current. When the analyte passes through the nanopore, it blocks the pore and interacts with it in complex ways, causing a change in current. The characteristics of this current change directly reflect the composition and structure information of the analyte. Therefore, compared to traditional techniques such as mass spectrometry and NMR, nanopore analysis has greater potential to obtain comprehensive compositional and structural information of sugar molecules. Currently, nanopore analysis has been successfully applied in the field of nucleic acid or peptide sequencing. However, for sugar molecules, in addition to the possible structural differences between them and nucleic acids or peptides, there are still some limitations. 210 Beyond the difference in size, the essential difference lies in the fact that sugar chains are non-template-driven biopolymers, making amplification and scale-up impossible. This hinders the rapid acquisition of large sample volumes and makes it impossible to directly infer sequences from templates. Another crucial difference is that most sugar molecules are electrically neutral, posing significant challenges to their driving, entry, or translocation within nanopores. Therefore, nanopore analysis techniques remain limited to applications in differentiation or detection. For example, non-patent literature (Hagen Bayley et al., Single-Molecule Determination of the Isomers of d-Glucose and d-Fructose that Bind to Boronic Acids, 2018) discloses the use of phenylboronic acid to modify nanopores to differentiate monosaccharide isomers in a mixed system. Another example is non-patent literature (Zhaobing Gao et al., Mapping the Acetylamino and Carboxyl Groups on Glycans by Engineered α-Hemolysin Nanopores, 2023) which discloses modifying nanopores to allow the localization of acetylamino and carboxyl groups on polysaccharides. In summary, current applications of nanopores in sugar molecules are all focused on the overall differentiation or detection of sugar molecules, and their mapping relationship with electrical signals is limited to the entire sugar molecule. However, sugar molecules have complex compositions and structures, and variable conformations, making it difficult to accurately obtain the mapping relationship between sugar molecule sequence and electrical signals through overall detection. Currently, there are no reports of applications that enable sugar chains to translocate at an appropriate rate and sequence them in a chain-like form. Summary of the Invention

[0006] To further analyze sugar molecule sequence information, this application proposes a glycan sequencing method. This method connects adapter molecules to one or both ends of the glycan chain, achieving both glycan chain stretching and, through motor protein regulation of the adapter molecule's movement within the nanopore, indirectly controlling the speed at which the connected glycan chain passes through the nanopore. The method then measures the electrical signal of the glycan chain passing through the sensing region of the nanopore to analyze glycan characteristics. This method offers high sensitivity and accuracy and promises to enable real-time and rapid interpretation of glycan sequence information. The specific scheme is as follows:

[0007] A first aspect of the present invention provides a method for glycan sequencing, the method comprising:

[0008] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0009] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0010] C) Obtain the electrical signal characteristics of the sugar chains;

[0011] D) Determine the sugar sequence by electrical signal characteristics.

[0012] The glycan chains pass through nanopores, with the movement of linker molecules controlled by motor proteins. Preferably, the motor proteins regulate the movement of linker molecules within the nanopores, thereby controlling the speed at which the attached glycan chains pass through the nanopores.

[0013] The connector element is at least one, preferably two or more. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0014] The connector molecules can be charged.

[0015] In one specific embodiment of the present invention, 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 conjugate-linker molecule, preferably a linker molecule is connected to the reducing end to form a linker molecule-sugar chain conjugate.

[0016] In one specific embodiment of the present invention, when one end of the sugar chain to be tested is connected to a connector molecule, the sugar chain to be tested carries at least 4 negative charges.

[0017] In one specific embodiment of the present invention, 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 may be the same or different.

[0018] The linker molecule is connected to the sugar chain via a covalent bond or via a linker group.

[0019] 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.

[0020] In one specific embodiment of the present invention, the thiol group modified on the connector molecule reacts and connects with the maleimide group modified on the sugar chain.

[0021] In one specific embodiment of the present invention, the DBCO modified on the linker molecule reacts and connects with the azide group modified on the sugar chain.

[0022] The electrical signal characteristics mentioned include, but are not limited to, electrical signal waveforms.

[0023] The electrical signal waveform is formed by sensing the sugar chain through nanopores.

[0024] 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.

[0025] Preferably, step D) includes establishing a correspondence between the electrical signal features obtained in step C) and the glycan features.

[0026] Preferably, step D) includes establishing an electrical signal feature-glycan sequence database.

[0027] 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, presence of modifications, type of modifications, location of modifications, number of modifications, presence of branches, location of branches, number of branches, or branch sequence characteristics. The branch sequence 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, presence of modifications, type of modifications, location of modifications, and number of modifications.

[0028] Furthermore, the method also includes performing step signal analysis on the obtained electrical signal characteristics.

[0029] Preferably, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction.

[0030] Preferably, the parameters include, but are not limited to, amplitude, time duration, or standard deviation.

[0031] The parameters can be obtained directly from the electrical signal or obtained through processing by the built-in model. They can be individual parameters or new parameters formed by combining parameters in a certain way.

[0032] In one specific embodiment of the present invention, the analyzed step signal is compared with a known database signal to determine the glycan sequence.

[0033] In one specific embodiment of the present invention, a glycan feature database is constructed, and then the analyzed step signal is compared with the signal in the constructed glycan feature database to determine the glycan sequence.

[0034] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement order of monosaccharides and / or their derivatives in the sugar chain.

[0035] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement order of glycosidic bonds in the sugar chain.

[0036] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0037] In one specific embodiment of the present invention, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0038] In one specific embodiment of the invention, the sugar chain to be tested contains sialic acid. Preferably, sialic acid is modified at the end of the sugar chain. Sugar chain extension is achieved by the electric field force on the negatively charged sialic acid and the linker molecule attached thereto.

[0039] In one specific embodiment of the present invention, the adapter molecule is a biopolymer, including but not limited to nucleic acids or peptides. It is sufficient 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.

[0040] The linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a polypeptide-glycan-polypeptide conjugate, a nucleic acid-glycan-polypeptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

[0041] The adapter molecule interacts with the motor protein (preferably with specific binding) to control the speed at which the adapter molecule passes through the nanopore.

[0042] 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.

[0043] Preferably, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase.

[0044] Further 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, a sequence of Hel308 Mma (Methanosarcina mazei (strain ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88)) or a variant thereof, or a sequence of Hel308 Tga (Thermococcus gammatolerans (strain DSM 15229 / JCM 11827 / EJ3 ... or a The sequence of Mok (Methanothermococcus okinawensis (strain DSM 14208 / JCM 11175 / IH1)) or a variant thereof, the sequence of Hel308 Fac (Ferroplasma acidarmanus Fer1) or a variant thereof, the sequence of Hel308 Csy (Cenarchaeum symbiosum (strain A)) or a variant thereof, the sequence of Hel308 Mhu (Methanospirillum hungatei JF-1 (strain ATCC 27890 / DSM 864 / NBRC 100397 / JF-1)) or a variant thereof, or the sequence of F8813 protein or a variant thereof.

[0045] 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.

[0046] The adapter molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or their variants.

[0047] The polypeptide is an amino acid sequence that can be specifically recognized by unfolded enzymes or has a certain charge distribution.

[0048] Preferably, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme (Caseinolytic protease X).

[0049] The nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0050] The nucleic acid mentioned is DNA, RNA, modified DNA, or modified RNA.

[0051] In one specific embodiment of the present invention, the adapter molecule is DNA, and one end of the glycan chain to be tested is connected to DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate. Preferably, DNA is connected to the reducing end to form a DNA-glycan conjugate.

[0052] The DNA that acts as the adapter molecule can be single-stranded, double-stranded, or partially double-stranded.

[0053] In one specific embodiment of the present invention, 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 the present invention, the DNA2 is single-stranded. For example, the DNA2 can be polyT.

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

[0056] The nanopore is a known nanopore in the prior art, as long as it has the ability to sense sugar chains, such as a biological nanopore or a solid nanopore.

[0057] Preferably, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin (α-HL) nanopores, Mycobacterium smegmatis porin A (MspA) nanopores, Aeromonas hydrolysin (AeL), curli production assembly / transport component (CsgG) nanopores, outer membrane porin F (OmpF) nanopores, cytolysin A (ClyA) nanopores, phi29 nanopores, ferric isohydroxamic acid uptake component A (FhuA) nanopores, pleurodactylysin A (PlyA) nanopores, pleurodactylysin B (PlyB) nanopores, or fragaceatoxin C (FraC) nanopores.

[0058] Preferably, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

[0059] The chain length of the sugar chain is greater than 1.

[0060] The sugar chain can be a straight chain or have at least one branch.

[0061] The sugar chain may be modified, for example, by sulfonation.

[0062] The sugar chain can be charged or electrically neutral.

[0063] The sugar chain is composed of monosaccharides and / or their derivatives linked by glycosidic bonds.

[0064] 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.

[0065] 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.

[0066] The configuration of each anomeric carbon in the sugar chain can be α or β.

[0067] The types of glycosidic bonds in sugar chains 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.

[0068] A second aspect of the present invention provides a method for detecting electrical signals characteristic of sugar chains, the method comprising:

[0069] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0070] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0071] C) Obtain the electrical signal characteristics of the sugar chain.

[0072] The glycan chains pass through nanopores, with the movement of linker molecules controlled by motor proteins. Preferably, the motor proteins regulate the movement of linker molecules within the nanopores, thereby controlling the speed at which the attached glycan chains pass through the nanopores.

[0073] The connector element is at least one, preferably two or more. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0074] The connector molecules described above can be charged.

[0075] In one specific embodiment of the present invention, 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. Preferably, the linker molecule is attached to the reduced end to form a linker molecule-sugar chain conjugate.

[0076] In one specific embodiment of the present invention, 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 may be the same or different.

[0077] The linker molecule is connected to the sugar chain via a covalent bond or via a linker group.

[0078] 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.

[0079] In one specific embodiment of the present invention, the thiol group modified on the connector molecule reacts and connects with the maleimide group modified on the sugar chain.

[0080] In one specific embodiment of the present invention, the DBCO modified on the linker molecule reacts and connects with the azide group modified on the sugar chain.

[0081] The electrical signal characteristics mentioned include, but are not limited to, electrical signal waveforms.

[0082] The electrical signal waveform is formed by sensing the sugar chain through nanopores.

[0083] 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.

[0084] 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, presence of modifications, type of modifications, location of modifications, number of modifications, presence of branches, location of branches, number of branches, or branch sequence characteristics. The branch sequence 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, presence of modifications, type of modifications, location of modifications, and number of modifications.

[0085] The method also includes performing step signal analysis on the obtained electrical signal characteristics.

[0086] Preferably, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction.

[0087] Preferably, the parameters include, but are not limited to, amplitude, time duration, or standard deviation.

[0088] The parameters can be obtained directly from the electrical signal or obtained through processing by the built-in model. They can be individual parameters or new parameters formed by combining parameters in a certain way.

[0089] In one specific embodiment of the present invention, the analyzed step signal is compared with a known database signal to determine the glycan sequence.

[0090] In one specific embodiment of the present invention, a glycan feature database is constructed, and then the analyzed step signal is compared with the signal in the constructed glycan feature database to determine the glycan sequence.

[0091] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement order of monosaccharides and / or their derivatives in the sugar chain.

[0092] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement order of glycosidic bonds in the sugar chain.

[0093] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0094] In one specific embodiment of the present invention, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0095] The sugar chain to be tested contains sialic acid. Preferably, sialic acid is modified at the end of the sugar chain. Sugar chain extension is achieved by the electric field force on the negatively charged sialic acid and the linker molecule attached to it.

[0096] The adapter molecules include, but are not limited to, nucleic acids or peptides. It is sufficient 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.

[0097] The linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a polypeptide-glycan-polypeptide conjugate, a nucleic acid-glycan-polypeptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

[0098] The adapter molecule interacts with the motor protein (preferably with specific binding) to control the speed at which the adapter molecule passes through the nanopore.

[0099] 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.

[0100] Preferably, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase.

[0101] More 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.

[0102] Preferably, the polymerase is phi29 polymerase or a variant thereof, pol6 or a variant thereof, or pol7 or a variant thereof.

[0103] The adapter molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or their variants.

[0104] The polypeptide is an amino acid sequence that can be specifically recognized by unfolded enzymes or has a certain charge distribution.

[0105] Preferably, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme.

[0106] The nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0107] The nucleic acid mentioned is DNA, RNA, modified DNA, or modified RNA.

[0108] 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.

[0109] 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.

[0110] In one specific embodiment of the present invention, the DNA2 is single-stranded. For example, the DNA2 can be polyT.

[0111] In one specific embodiment of the present invention, the DNA1 is a single strand, which is annealed and coupled to a partially complementary fragment.

[0112] The nanopore is a known nanopore in the prior art, as long as it has the ability to sense sugar chains, such as a biological nanopore or a solid nanopore.

[0113] Preferably, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin nanopores, MspA nanopores, Aeromonas hydrolysin (AeL), CsgG nanopores, OmpF nanopores, ClyA nanopores, phi29 nanopores, FhuA nanopores, PlyA nanopores, PlyB nanopores, or FraC nanopores.

[0114] Preferably, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

[0115] The chain length of the sugar chain is greater than 1.

[0116] The sugar chain is a straight chain or has at least one branch.

[0117] The sugar chain has been modified, for example, by sulfonation.

[0118] The sugar chain can be charged or electrically neutral.

[0119] The sugar chain is composed of monosaccharides and / or their derivatives linked by glycosidic bonds.

[0120] 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.

[0121] 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.

[0122] The configuration of each anomeric carbon in the sugar chain can be α or β.

[0123] The types of glycosidic bonds in sugar chains 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.

[0124] A third aspect of the present invention provides a method for identifying glycan characteristics, the method comprising:

[0125] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0126] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0127] C) Obtain the electrical signal characteristics of the sugar chains;

[0128] D) Determine the characteristics of sugar chains through electrical signal features.

[0129] Preferably, 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.

[0130] 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.

[0131] Preferably, the glycan feature identification involves qualitative or quantitative analysis of the glycan features.

[0132] In a fourth aspect, the present invention provides an application of the method for detecting electrical signals of glycan characteristics in glycan sequencing or glycan detection.

[0133] Preferably, the detection of glycans includes quantitative and / or qualitative methods.

[0134] A fifth aspect of the present invention provides a method for detecting sugar chains, the detection method comprising:

[0135] A) Connect the sugar chains in the sample to the linker molecules;

[0136] B) Apply a voltage across the nanopore so that the sugar chains in the sample pass through the nanopore and through the sensing area;

[0137] C) Obtain the electrical signal characteristics of the sugar chains;

[0138] D) Determine the characteristics of sugar chains through electrical signal features.

[0139] The detection methods include qualitative and / or quantitative methods.

[0140] It is preferable to perform qualitative and / or quantitative analysis of the sugar chain characteristics.

[0141] In one specific embodiment of the present invention, the sample to be tested contains a sugar chain with a consistent sequence.

[0142] In one specific embodiment of the present invention, the sample to be tested contains two or more sugar chains with different sequences, and the mass or concentration ratios of the different sugar chains are the same or different.

[0143] A sixth aspect of the present invention provides a method for controlling the speed at which sugar chains pass through nanopores, the method comprising:

[0144] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0145] B) By applying a voltage across the nanopore, the motor protein regulates the movement of the linker molecules within the nanopore, thereby controlling the speed at which the connected sugar chains pass through the nanopore.

[0146] The electrical signal characteristics of the sugar chain are obtained by passing through the nanopore sensing region.

[0147] A seventh aspect of the present invention provides a method for constructing a database of electrical signals characteristic of sugar chains. The method includes establishing a correspondence between sugar chain features and electrical signals measured by the sugar chains through a nanopore sensing region, wherein the electrical signals measured by the sugar chains through the nanopore sensing region include:

[0148] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0149] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0150] C) Obtain the electrical signal characteristics of the sugar chain.

[0151] 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, presence of modifications, type of modifications, location of modifications, number of modifications, presence of branches, location of branches, number of branches, or branch sequence characteristics. The branch sequence 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, presence of modifications, type of modifications, location of modifications, and number of modifications.

[0152] The sugar chain features are known, and the obtained electrical signal features are marked as specific sugar chain features.

[0153] The electrical signal characteristics include, but are not limited to, current amplitude, number of steps, step duration, frequency of occurrence of step signals, or standard deviation.

[0154] In an eighth aspect, the present invention provides a database of characteristic electrical signals of sugar chains obtained by the above-described method.

[0155] A ninth aspect of the present invention provides a method for analyzing electrical signals of glycan characteristics to determine glycan sequences, the method comprising performing step signal analysis on the electrical signals obtained by the above method.

[0156] Preferably, the main steps are determined by statistical analysis of the electrical signal, and the step characteristics are determined by parameter analysis of the steps, thereby obtaining the step signal and determining its sequence.

[0157] The step signal analysis is performed using manual methods or algorithms.

[0158] The manual method described can be either entirely manual analysis or analysis performed with the aid of software.

[0159] The algorithm analysis can be based on, but is not limited to, multiple comparison analysis of glycan electrical signals to analyze and extract their electrical signal features. The steps are determined and divided using a built-in statistical model to obtain step signals. Based on the comparison model, the obtained step signals are compared with glycan step signals in the database to determine the glycan sequence.

[0160] The algorithm can be a known algorithm used for nucleic acid and protein sequence alignment and determination, or it can be a newly developed algorithm.

[0161] Preferably, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction.

[0162] Preferably, the parameters include, but are not limited to, amplitude, time duration, or standard deviation.

[0163] The parameters can be obtained directly from the electrical signal or obtained through processing by the built-in model. They can be individual parameters or new parameters formed by combining parameters in a certain way.

[0164] In one specific embodiment of the present invention, the analyzed step signal is compared with a known database signal to determine the glycan sequence.

[0165] In one specific embodiment of the present invention, the analyzed step signal is compared with the database signal obtained above to determine the glycan sequence.

[0166] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement order of monosaccharides and / or their derivatives in the sugar chain.

[0167] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement order of glycosidic bonds in the sugar chain.

[0168] In one specific embodiment of the present invention, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0169] In one specific embodiment of the present invention, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0170] In a tenth aspect of the present invention, a method for analyzing electrical signals of glycan characteristics to quantify glycan characteristics is provided, the method comprising performing step signal analysis on the electrical signals obtained by the above method.

[0171] The eleventh aspect of the present invention provides a method for determining the sequence of monosaccharides and / or their derivatives in a glycan chain, the method comprising:

[0172] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0173] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0174] C) Obtain the electrical signal characteristics of the sugar chains;

[0175] D) Determine the sequence of monosaccharides and / or their derivatives by means of electrical signal characteristics.

[0176] A twelfth aspect of the present invention provides a method for identifying sugar chains or portions thereof, the method comprising:

[0177] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0178] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0179] C) Obtain the electrical signal characteristics of the sugar chains;

[0180] D) Identify sugar chains or parts thereof by electrical signal characteristics.

[0181] The portion of the sugar chain can be a continuous monosaccharide sequence of the sugar chain.

[0182] The sugar chain portion can be one or more monosaccharide residues of the sugar chain.

[0183] The portion of the glycan can be one or more mutation sites in the glycan.

[0184] In a thirteenth aspect of the present invention, a method for sequencing a target analyte, said target analyte comprising a glycan, the method comprising:

[0185] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0186] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0187] C) Obtain the electrical signal characteristics of the sugar chains;

[0188] D) Determine the target analyte sequence by analyzing electrical signal characteristics.

[0189] The target analytes include, but are not limited to, nucleic acids, proteins, or peptides.

[0190] The terms “comprising” or “including” are open-ended descriptions that include the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0191] 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 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.

[0192] 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.

[0193] 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.

[0194] 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 approximately 0.1 nanometers (nm) to approximately 1000 nm. They are present in the detection medium during application, which is typically a salt solution, and its composition and concentration can be arbitrarily varied.

[0195] The term "detection medium" refers to a conductive liquid medium. When a voltage is applied, an ionic current is generated through the nanopore channels, and the target analyte or glycan can be driven from the conductive liquid medium into the nanopores and extended, for example, by electrophoretic forces and / or diffusion. The potential difference may be not less than 20 mV, not less than 40 mV, not less than 60 mV, not less than 80 mV, not less than 100 mV, not less than 120 mV, not less than 140 mV, not less than 160 mV, not less than 180 mV, or not less than 200 mV; or in the range of about 20 mV to 220 mV, about 40 mV to 200 mV, about 60 mV to 180 mV, about 80 mV to 180 mV, about 100 mV to 180 mV, about 120 mV to 180 mV, about 140 mV to 180 mV, or about 160 mV to 180 mV. In the system, the final concentration of the target analyte or glycan can be from about 0.01 μM to about 100 mM, for example from about 0.1 mM to about 50 mM, or for example from about 0.1 mM to about 40 mM. The appropriate concentration can be determined experimentally.

[0196] 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). Attached Figure Description

[0197] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0198] Figure 1 shows the SDS-PAGE gel image obtained after purification of TGA helicase. The first and second lanes are the protein marker and the final purified product, respectively. The band between 100-130 kDa is the TGA helicase band.

[0199] Figure 2 shows the SDS-PAGE gel image of purified M2-MspA protein. The first lane is the protein marker, the second to fourth lanes are the precipitate after bacterial lysis, the supernatant after lysis, and the protein solution after passing through the ion exchange column, respectively, and the fifth and sixth lanes are the protein solutions obtained from the first and second molecular sieve passes, respectively. The band near 100 kDa is the M2-MspA octamer protein.

[0200] Figure 3 shows the mass spectrometry analysis results of the NT89-hepta one-sided coupling product, with the highest peak being the NT89-hepta coupling product.

[0201] Figure 4 shows the mass spectrometry analysis results of the 5T89-nona one-sided coupling product, with the highest peak being the 5T89-nona coupling product.

[0202] Figure 5 shows the mass spectrometry analysis results of the NT89-hepta-3S one-sided coupling product, with the highest peak being the NT89-hepta-3S coupling product.

[0203] Figure 6 shows the mass spectrometry analysis results of the NT89-hepta-5S one-sided coupling product, with the highest peak being the NT89-hepta-5S coupling product.

[0204] Figure 7 shows the mass spectrometry analysis results of the NT89-hepta-3S&5S one-sided coupling product, with the highest peak being the NT89-hepta-3S&5S coupling product.

[0205] Figure 8 shows the mass spectrometry analysis results of the NT89-HP-6 one-sided coupling product, with the highest peak being the NT89-HP-6 coupling product.

[0206] Figure 9 shows the mass spectrometry analysis results of the NT89-HP-8 one-sided coupling product, with the highest peak being the NT89-HP-8 coupling product.

[0207] Figure 10 shows the mass spectrometry analysis results of the NT89-HP-10 one-sided coupling product, with the highest peak being the NT89-HP-10 coupling product.

[0208] Figure 11 shows the mass spectrometry analysis results of the NT89-HA-6 one-sided coupling product, with the highest peak being the NT89-HA-6 coupling product.

[0209] Figure 12 shows the mass spectrometry analysis results of the NT89-HA-8 one-sided coupling product, with the highest peak being the NT89-HA-8 coupling product.

[0210] Figure 13 shows the mass spectrometry analysis results of the NT89-HA-10 one-sided coupling product, with the highest peak being the NT89-HA-10 coupling product.

[0211] Figure 14 shows the mass spectrometry analysis results of the two-sided coupling product of NT89-hepta-polyT(DBCO), with the highest peak being the coupling product of NT89-hepta-polyT(DBCO).

[0212] Figure 15 shows the results of 15% Urea-PAGE gel analysis of bilaterally coupled DNA products.

[0213] Figure 16 shows the results of 15% Urea-PAGE gel analysis of bilaterally coupled DNA products.

[0214] Figure 17 shows the results of 15% Urea-PAGE gel analysis of bilaterally coupled DNA products.

[0215] Figure 18 shows representative signals of NT89.

[0216] Figure 19 shows a representative signal of NT89-2.

[0217] Figure 20 shows a representative signal of NT89-polyT.

[0218] Figure 21 shows a representative signal of 5T89.

[0219] Figure 22 shows a representative signal of NT89-hepta.

[0220] Figure 23 shows a representative signal of NT89-nona.

[0221] Figure 24 shows a representative signal of NT89-hepta-3S.

[0222] Figure 25 shows a representative signal of NT89-hepta-5S.

[0223] Figure 26 shows representative signals of NT89-hepta-3S & 5S.

[0224] Figure 27 shows representative signals of NT89-hepta-1S, 3S, and 5S.

[0225] Figure 28 shows a representative signal of NT89-HP-6.

[0226] Figure 29 shows a representative signal of the NT89-HP-8.

[0227] Figure 30 shows a representative signal of NT89-HP-10.

[0228] Figure 31 shows a representative signal of NT89-HA-6.

[0229] Figure 32 shows a representative signal of NT89-HA-8.

[0230] Figure 33 shows a representative signal of NT89-HA-10.

[0231] Figure 34 shows the NT89-HP-8 ladder signal.

[0232] Figure 35 shows the NT89-HP-10 ladder signal.

[0233] Figure 36 shows the NT89-HA-8 ladder signal.

[0234] Figure 37 shows the NT89-HA-10 ladder signal.

[0235] Figure 38 shows the superimposed sugar ladder signal of NT89-HP and HA series.

[0236] Figure 39 is a schematic diagram of the bilaterally coupled DNA glycan nanopore sequencing principle.

[0237] Figure 40 shows a representative signal of NT89-hepta-N3-DBCO-polyT.

[0238] Figure 41 shows a representative signal of NT89-hepta-SMCC-SH-polyT.

[0239] Figure 42 shows the NT89-hepta-SMCC-SH-polyT ladder signal.

[0240] Figure 43 shows a representative signal of NT89-hepta-N3-DBCO-polyT(10nt).

[0241] Figure 44 shows a representative signal of NT89-hepta-N3-DBCO-polyT(30nt).

[0242] Figure 45 shows a representative signal of NT89-mono-polyT.

[0243] Figure 46 shows a representative signal of NT89-tri-polyT.

[0244] Figure 47 shows a representative signal of NT89-penta-polyT.

[0245] Figure 48 shows a representative signal of 5T89-nona-polyT.

[0246] Figure 49 shows the NT89-mono-polyT ladder signal.

[0247] Figure 50 shows the NT89-tri-polyT step signal.

[0248] Figure 51 shows the NT89-penta-polyT step signal.

[0249] Figure 52 shows the 5T89-nona-polyT step signal.

[0250] Figure 53 shows a representative signal of NT89-hepta-1S-polyT.

[0251] Figure 54 shows the NT89-hepta-1S-polyT step signal.

[0252] Figure 55 shows a representative signal of NT89-hepta-3S-polyT.

[0253] Figure 56 shows the NT89-hepta-3S-polyT step signal.

[0254] Figure 57 shows a representative signal of NT89-hepta-5S-polyT.

[0255] Figure 58 shows the NT89-hepta-5S-polyT step signal.

[0256] Figure 59 shows representative signals of NT89-hepta-3S & 5S-polyT.

[0257] Figure 60 shows the NT89-hepta-3S&5S-polyT step signal.

[0258] Figure 61 shows a representative signal of 5T89-nona-1S-polyT.

[0259] Figure 62 shows the 5T89-nona-1S-polyT step signal.

[0260] Figure 63 shows a representative signal of 5T89-nona-3S-polyT.

[0261] Figure 64 shows the 5T89-nona-3S-polyT step signal.

[0262] Figure 65 shows a representative signal of 5T89-nona-5S-polyT.

[0263] Figure 66 shows the 5T89-nona-5S-polyT step signal.

[0264] Figure 67 shows a representative signal of 5T89-nona-7S-polyT.

[0265] Figure 68 shows the 5T89-nona-7S-polyT step signal.

[0266] Figure 69 shows a representative signal of NT89-hepta-isomer-polyT.

[0267] Figure 70 shows the NT89-hepta-isomer-polyT step signal.

[0268] Figure 71 shows the sequencing analysis results of hepta-1S:hepta-3S:-hepta-5S = 1:1:1, where "(m)" refers to "mixture", that is, the corresponding single component in the mixture. The absence of "m" indicates a pure single component.

[0269] Figure 72 shows the sequencing analysis results of hepta-3S:-hepta-3S&5S=3:1, where "(m)" refers to "mixture", that is, the corresponding single component in the mixture. The absence of "m" indicates a pure single component.

[0270] Figure 73 shows the sequencing analysis results of hepta-3S:-hepta-3S&5S=1:1, where "(m)" refers to "mixture", that is, the corresponding single component in the mixture. The absence of "m" indicates a pure single component.

[0271] Figure 74 shows the sequencing analysis results of hepta-3S:-hepta-3S&5S=1:3, where "(m)" refers to "mixture", that is, the corresponding single component in the mixture. The absence of "m" indicates a pure single component.

[0272] Figure 75 is a schematic diagram of pynanolab fitting the hepta step signal.

[0273] Figure 76 shows representative signals of Pep-a-hepta-Pep-b.

[0274] Figure 77 shows the Pep-a-hepta-Pep-b step signal.

[0275] Figure 78: Glycan sequence, where 6S represents sulfonation modification. Detailed Implementation

[0276] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0277] The following are some specific embodiments of this application:

[0278] In the first specific embodiment, a method for glycan sequencing is provided, the method comprising:

[0279] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0280] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0281] C) Obtain the electrical signal characteristics of the sugar chains;

[0282] D) Determine the sugar sequence by electrical signal characteristics.

[0283] In the second embodiment, according to the first embodiment, the connector molecule is able to pull the sugar chain through the nanopore.

[0284] In a third embodiment, according to the first or second embodiment, the glycans are moved by motor proteins through nanopores, controlling the movement of the linker molecules.

[0285] In the fourth embodiment, according to any one of the first to third embodiments, the motor protein regulates the movement of the adaptor molecules in the nanopore, thereby controlling the speed at which the connected glycan chains pass through the nanopore.

[0286] In the fifth embodiment, according to any one of the first to fourth embodiments, there is at least one connector molecule, preferably two or more.

[0287] In the sixth embodiment, according to any one of the first to fifth embodiments, 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. Preferably, the linker molecule is connected to the reducing end to form a linker molecule-sugar chain conjugate.

[0288] In the seventh embodiment, according to any one of the first to fifth embodiments, 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.

[0289] In the eighth embodiment, according to any one of the first to seventh embodiments, the linker molecule is connected to the sugar chain by a covalent bond or by a linking group.

[0290] In the ninth embodiment, according to any one of the first to eighth embodiments, the linker molecule is linked to the sugar chain by 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.

[0291] In the 10th embodiment, according to any one of the 1-9 embodiments, the thiol group modified on the connector molecule reacts and connects with the maleimide group modified on the sugar chain.

[0292] In the 11th embodiment, according to any one of the 1-9 embodiments, the DBCO modified on the linker molecule is reactively linked to the azide group modified on the sugar chain.

[0293] In the 12th embodiment, according to any one of the 1-11 embodiments, the electrical signal characteristics include, but are not limited to, electrical signal waveforms.

[0294] In the 13th embodiment, according to any one of the 1-12 embodiments, 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.

[0295] In the 14th embodiment, according to any one of the 1-13 embodiments, step D) includes: establishing a correspondence between the electrical signal features obtained in step C) and the glycan features.

[0296] In the 15th embodiment, according to the 14th embodiment, 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, position of modification, number of modifications, whether there are branches, position of branches, number of branches or branch sequence characteristics.

[0297] In the 16th embodiment, according to the 15th embodiment, the branched sequence features include, but are not limited to, the composition of monosaccharide units, the configuration of monosaccharide units, the number of monosaccharide units, the number of repeating units, the type of glycosidic bond, the number of glycosidic bonds, the order of monosaccharide units, whether there is modification, the type of modification, the position of modification, and the number of modifications.

[0298] In the 17th embodiment, according to any one of the 1-16 embodiments, the method further includes performing step signal analysis on the obtained electrical signal features.

[0299] In the 18th embodiment, according to the 17th embodiment, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction.

[0300] In the 19th embodiment, according to the 18th embodiment, the parameters include, but are not limited to, amplitude, time course, or standard deviation.

[0301] In the 20th embodiment, according to the 17th embodiment, the analyzed step signal is compared with a known database signal to determine the glycan sequence.

[0302] In the 21st embodiment, according to the 17th embodiment, a glycan feature database is constructed, and then the analyzed step signal is compared with the signal in the constructed glycan feature database to determine the glycan sequence.

[0303] In the 22nd embodiment, according to any one of the 1-21 embodiments, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives in the sugar chain.

[0304] In the 23rd embodiment, according to any one of the 1-21 embodiments, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0305] In the 24th embodiment, according to any one of the 1-21 embodiments, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0306] In the 25th embodiment, according to any one of the 1-24 embodiments, the test sugar chain has sialic acid.

[0307] In the 26th embodiment, according to any one of the 1-25 embodiments, the adapter molecule includes, but is not limited to, nucleic acids or peptides.

[0308] In the 27th embodiment, according to any one of the 1-26 embodiments, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

[0309] In the 28th embodiment, according to any one of the 1-27 embodiments, 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.

[0310] In the 29th embodiment, according to the 28th embodiment, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase.

[0311] In the 30th embodiment, according to the 28th or 29th embodiment, the helicase or a variant thereof is a sequence of Hel308Tga or a variant thereof, a sequence of Hel308 Mbu or a variant thereof, a sequence of Hel308 Pfu or a variant thereof, a sequence of Hel308 Mma or a variant thereof, a sequence of Hel308 Mok or a variant thereof, a sequence of Hel308 Fac or a variant thereof, a sequence of Hel308 Csy or a variant thereof, a sequence of Hel308 Mhu or a variant thereof, or a sequence of F8813 protein or a variant thereof.

[0312] In the 31st embodiment, according to the 28th embodiment, the polymerase is phi29 polymerase or a variant thereof, pol6 or a variant thereof, pol7 or a variant thereof.

[0313] In the 32nd embodiment, according to any one of the 1-31 embodiments, the adapter molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or variants thereof.

[0314] In the 33rd embodiment, according to the 32nd embodiment, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme.

[0315] In the 34th embodiment, according to any one of the 26th-28th embodiments, the nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0316] In the 35th embodiment, according to any one of the 26th-34th embodiments, the nucleic acid is DNA, RNA, modified DNA, or modified RNA.

[0317] In the 36th embodiment, according to any one of embodiments 1-35, the adapter molecule is DNA, and one end of the glycan chain to be tested is linked to DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate. Preferably, DNA is linked to the reducing end to form a DNA-glycan conjugate.

[0318] In the 37th embodiment, according to any one of the 1-35 embodiments, 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.

[0319] In the 38th embodiment, according to any one of the 1-37 embodiments, the nanopore is a biological nanopore or a solid nanopore.

[0320] In the 39th embodiment, according to the 38th embodiment, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin nanopores, MspA nanopores, AeL nanopores, CsgG nanopores, OmpF nanopores, ClyA nanopores, phi29 nanopores, FhuA nanopores, PlyA nanopores, PlyB nanopores, or FraC nanopores.

[0321] In the 40th embodiment, according to the 38th embodiment, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

[0322] In the 41st embodiment, according to any one of the 1-40 embodiments, the chain length of the sugar chain is greater than 1.

[0323] In the 42nd embodiment, according to any one of the 1-41 embodiments, the sugar chain may be a straight chain or have at least one branch.

[0324] In the 43rd embodiment, according to any one of the 1-42 embodiments, the sugar chain may be modified, for example, by sulfonation.

[0325] In the 44th embodiment, according to any one of the 1-43 embodiments, the sugar chain may be charged or electrically neutral.

[0326] In the 45th embodiment, according to any one of the 1-44 embodiments, the sugar chain is composed of monosaccharides and / or their derivatives linked by glycosidic bonds.

[0327] In the 46th embodiment, according to the 45th embodiment, the monosaccharide includes, but is not limited to, pentose sugars, hexose sugars, and monosaccharides with six or more carbon atoms.

[0328] In the 47th embodiment, according to the 45th embodiment, the monosaccharide derivative includes, but is not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides.

[0329] In the 48th embodiment, according to any one of the 1-47 embodiments, the configuration of each anomeric carbon in the sugar chain can be α or β.

[0330] In the 49th embodiment, according to any one of the 1-48 embodiments, the type of glycosidic bond in the sugar chain includes, but is not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4.

[0331] In the 50th embodiment, a method for detecting electrical signals characteristic of glycan chains is provided, the method comprising:

[0332] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0333] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0334] C) Obtain the electrical signal characteristics of the sugar chain.

[0335] In the 51st embodiment, according to the 50th embodiment, the connector molecule is capable of pulling the sugar chain through the nanopore.

[0336] In the 52nd embodiment, according to the 50th or 51st embodiment, the glycans are moved by motor proteins controlling the movement of linker molecules through nanopores.

[0337] In the 53rd embodiment, according to any one of the 50th-52nd embodiments, the motor protein regulates the movement of the adapter molecules in the nanopore, thereby controlling the speed at which the connected glycan chains pass through the nanopore.

[0338] In the 54th embodiment, according to any one of the 50th-53rd embodiments, there is at least one connector molecule, preferably two or more.

[0339] In the 55th embodiment, according to any one of embodiments 50-54, 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. Preferably, the linker molecule is connected to the reducing end to form a linker molecule-sugar chain conjugate.

[0340] In the 56th embodiment, according to any one of the 50th-54th embodiments, 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.

[0341] In the 57th embodiment, according to any one of the 50th-56th embodiments, the linker molecule is covalently linked to the sugar chain or is linked by a linking group.

[0342] In the 58th embodiment, according to any one of the 50th-57th embodiments, the linker molecule is linked to the sugar chain by 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.

[0343] In the 59th embodiment, according to any one of the 50-58 embodiments, the thiol group modified on the connector molecule reacts and connects with the maleimide group modified on the sugar chain.

[0344] In the 60th embodiment, according to any one of the 50th-58th embodiments, the DBCO modified on the linker molecule is reactively linked to the azide group modified on the sugar chain.

[0345] In the 61st embodiment, according to any one of the 50th-60th embodiments, the electrical signal characteristics include, but are not limited to, electrical signal waveforms.

[0346] In the 62nd embodiment, according to any one of the 50th-61st embodiments, 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.

[0347] In the 63rd embodiment, according to any one of the 50th-62nd embodiments, 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, position of modification, number of modifications, whether there are branches, position of branches, number of branches or branch sequence characteristics.

[0348] In the 64th embodiment, according to the 63rd embodiment, 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, position of modification, and number of modifications.

[0349] In the 65th embodiment, according to any one of the 50th-64th embodiments, the test sugar chain has sialic acid.

[0350] In the 66th embodiment, according to any one of the 50th-65th embodiments, the adapter molecule includes, but is not limited to, nucleic acids or peptides.

[0351] In the 67th embodiment, according to any one of the 50th-66th embodiments, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate.

[0352] In the 68th embodiment, according to any one of the 50-67 embodiments, 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.

[0353] In the 69th embodiment, according to the 68th embodiment, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase.

[0354] In the 70th embodiment, according to the 68th embodiment, the helicase or a variant thereof is a sequence of Hel308 Tga or a variant thereof, a sequence of Hel308 Mbu or a variant thereof, a sequence of Hel308 Pfu or a variant thereof, a sequence of Hel308 Mma or a variant thereof, a sequence of Hel308 Mok or a variant thereof, a sequence of Hel308 Fac or a variant thereof, a sequence of Hel308 Csy or a variant thereof, a sequence of Hel308 Mhu or a variant thereof, or a sequence of F8813 protein or a variant thereof.

[0355] In the 71st embodiment, according to the 68th embodiment, the polymerase is phi29 polymerase or a variant thereof, pol6 or a variant thereof, pol7 or a variant thereof.

[0356] In the 72nd embodiment, according to any one of the 50th-71st embodiments, the adapter molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or variants thereof.

[0357] In the 73rd embodiment, according to the 72nd embodiment, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme.

[0358] In the 74th embodiment, according to any one of the 66th-73rd embodiments, the nucleic acid is single-stranded, double-stranded, or partially double-stranded.

[0359] In the 75th embodiment, according to any one of the 66-74 embodiments, the nucleic acid is DNA, RNA, modified DNA, or modified RNA.

[0360] In the 76th embodiment, according to any one of embodiments 50-75, the adapter molecule is DNA, and one end of the glycan chain to be tested is linked to DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate. Preferably, DNA is linked to the reducing end to form a DNA-glycan conjugate.

[0361] In the 77th embodiment, according to any one of the 50th-75th embodiments, 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.

[0362] In the 78th embodiment, according to any one of the 50-77 embodiments, the nanopore is a biological nanopore or a solid nanopore.

[0363] In the 79th embodiment, according to the 78th embodiment, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin nanopores, MspA nanopores, AeL nanopores, CsgG nanopores, OmpF nanopores, ClyA nanopores, phi29 nanopores, FhuA nanopores, PlyA nanopores, PlyB nanopores, or FraC nanopores.

[0364] In the 80th embodiment, according to the 78th embodiment, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores.

[0365] In the 81st embodiment, according to the 49th embodiment, the chain length of the sugar chain is greater than 1.

[0366] In the 82nd embodiment, according to any one of the 50th-81st embodiments, the sugar chain may be a straight chain or have at least one branch.

[0367] In the 83rd embodiment, according to any one of the 50-82 embodiments, the sugar chain may be modified, for example, by sulfonation.

[0368] In the 84th embodiment, according to any one of the 50-83 embodiments, the sugar chain may be charged or electrically neutral.

[0369] In the 85th embodiment, according to any one of the embodiments 50-84, the sugar chain is composed of monosaccharides and / or their derivatives linked by glycosidic bonds.

[0370] In the 86th embodiment, according to the 85th embodiment, the monosaccharide includes, but is not limited to, pentose sugars, hexose sugars, and monosaccharides with six or more carbon atoms.

[0371] In the 87th embodiment, according to the 85th embodiment, the monosaccharide derivative includes, but is not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides.

[0372] In the 88th embodiment, according to any one of the embodiments 50-87, the configuration of each anomeric carbon in the sugar chain can be α or β.

[0373] In the 89th embodiment, according to any one of the embodiments 50-88, the type of glycosidic bond in the sugar chain includes, but is not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4.

[0374] In the 90th embodiment, a method for identifying glycan characteristics is provided, the method comprising:

[0375] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0376] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0377] C) Obtain the electrical signal characteristics of the sugar chains;

[0378] D) Determine the characteristics of sugar chains through electrical signal features.

[0379] In the 91st embodiment, according to the 90th embodiment, 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, position of modification, number of modifications, whether there are branches, position of branches, number of branches or branch sequence characteristics.

[0380] In the 92nd embodiment, according to the 91st embodiment, the branched sequence features include, but are not limited to, the composition of monosaccharide units, the configuration of monosaccharide units, the number of monosaccharide units, the number of repeating units, the type of glycosidic bond, the number of glycosidic bonds, the order of monosaccharide units, whether there is modification, the type of modification, the position of modification, and the number of modifications.

[0381] In the 93rd specific embodiment, the glycan feature identification is a qualitative or quantitative assessment of the glycan features.

[0382] In the 94th embodiment, the method for detecting electrical signals of glycan characteristics as described in any of embodiments 1-92 is applied in glycan sequencing or glycan detection, preferably, the glycan detection includes quantitative and / or qualitative methods.

[0383] In the 95th embodiment, a method for detecting sugar chains is provided, the detection method comprising:

[0384] A) Connect the sugar chains in the sample to the linker molecules;

[0385] B) Apply a voltage across the nanopore so that the sugar chains in the sample pass through the nanopore and through the sensing area;

[0386] C) Obtain the electrical signal characteristics of the sugar chains;

[0387] D) Determine the characteristics of sugar chains through electrical signal features.

[0388] In the 96th embodiment, according to the 95th embodiment, the detection method includes qualitative and / or quantitative analysis; preferably, qualitative and / or quantitative analysis of glycan characteristics is performed.

[0389] In the 97th embodiment, according to the 95th or 96th embodiment, the sample to be tested contains a sequence-consistent glycan chain.

[0390] In the 98th embodiment, according to the 95th or 96th embodiment, the sample to be tested contains two or more sugar sequences, and the mass or concentration ratios of the different sugar sequences are the same or different.

[0391] In the 99th embodiment, a method for controlling the speed at which sugar chains pass through nanopores is provided, the method comprising:

[0392] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0393] B) By applying a voltage across the nanopore, the motor protein regulates the movement of the linker molecules within the nanopore, thereby controlling the speed at which the connected sugar chains pass through the nanopore.

[0394] In the 100th embodiment, a method for constructing a database of glycan feature electrical signals is provided. The method includes establishing a correspondence between glycan features and electrical signals measured by the glycans through a nanopore sensing region. The electrical signals measured by the glycans through the nanopore sensing region include:

[0395] A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates;

[0396] B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region;

[0397] C) Obtain the electrical signal characteristics of the sugar chain.

[0398] In the 101st embodiment, according to the 100th embodiment, 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, position of modification, number of modifications, whether there are branches, position of branches, number of branches or branch sequence characteristics.

[0399] In the 102nd embodiment, according to the 101st embodiment, the branched sequence features include, but are not limited to, the composition of monosaccharide units, the configuration of monosaccharide units, the number of monosaccharide units, the number of repeating units, the type of glycosidic bond, the number of glycosidic bonds, the order of monosaccharide units, whether there is modification, the type of modification, the position of modification, and the number of modifications.

[0400] In the 103rd embodiment, according to any one of the 100-102 embodiments, the glycan feature is known, and the obtained electrical signal feature is marked as a specific glycan feature.

[0401] In the 104th embodiment, according to any one of the 100-103 embodiments, 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.

[0402] In the 105th embodiment, a database of characteristic electrical signals of sugar chains obtained in any of the 100-104 embodiments is provided.

[0403] In the 106th embodiment, a method is provided for analyzing electrical signals of glycan characteristics to determine glycan sequences, the method comprising performing step signal analysis on electrical signals obtained in any of the 1-92 embodiments.

[0404] In the 107th embodiment, according to the 106th embodiment, the step signal analysis is performed using manual methods or algorithms.

[0405] In the 108th embodiment, according to the 107th embodiment, the manual method can be either completely manual analysis or analysis performed with the aid of software.

[0406] In the 109th embodiment, according to the 107th embodiment, the algorithm analysis is based on multiple comparison analysis of glycan electrical signals, analyzing and extracting their electrical signal features, determining and classifying the steps through a built-in statistical model to obtain step signals, and comparing the obtained step signals with glycan step signals in the database based on the comparison model to determine the glycan sequence.

[0407] In the 110th embodiment, according to the 106th embodiment, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction.

[0408] In the 111th embodiment, according to the 110th embodiment, the parameters include, but are not limited to, amplitude, time course, or standard deviation.

[0409] In the 112th embodiment, according to the 106th embodiment, the analyzed step signal is compared with a known database signal to determine the glycan sequence.

[0410] In the 113th embodiment, according to the 106th embodiment, the analyzed step signal is compared with the glycan feature electrical signal database obtained in the 105th embodiment to determine the glycan sequence.

[0411] In the 114th embodiment, according to any one of the 106-113 embodiments, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives in the sugar chain.

[0412] In the 115th embodiment, according to the 106th-113th embodiments, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain.

[0413] In the 116th embodiment, according to the 106th-113th embodiments, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan.

[0414] In the 117th embodiment, a method is provided for analyzing electrical signals of glycan characteristics to quantify glycan characteristics, the method comprising performing step signal analysis on the electrical signals obtained in any of the embodiments described in 1-92.

[0415] Example 1: Obtaining components in a motor protein-driven glycan sequencing system

[0416] 1. Experimental Methods

[0417] 1.1 Preparation of TGA helicase

[0418] The plasmid (NCBI: ACS33372.1) containing the TGA helicase coding sequence was used to transform BL21 competent cells. The transformed bacterial culture was plated on LB agar containing ampicillin and incubated overnight at 37°C. Then, single colonies were picked from the LB plates and inoculated into a small amount of LB liquid medium containing ampicillin. After incubation overnight at 37°C, the bacterial culture was transferred to a large amount of LB liquid medium containing ampicillin. OD 600Once the pH reached 0.6-0.8, 0.5 mM IPTG was added to induce TGA helicase protein expression. The next day, the bacterial culture was removed, centrifuged, and the bacterial pellet was resuspended in lysis buffer. The resuspended bacterial culture was then lysed using a high-pressure homogenizer. After lysis, the culture was centrifuged, and 60% ammonium chloride solution was added to the supernatant. The protein was precipitated at 4°C for 2 hours. The protein pellet was then centrifuged at 18000g for 40 minutes, resuspended, and transferred to a nickel column for elution with different concentrations of imidazole to obtain the TGA helicase protein.

[0419] 1.2 Preparation of M2-MspA nanopores

[0420] The M2-MspA plasmid (NCBI:CAB56052.1) was used to transform E. coli BL21(DE3) competent cells. The transformed bacterial culture was plated on LB agar containing kanamycin and incubated overnight at 37°C. Then, single colonies were picked from the LB plates and inoculated into a small amount of LB liquid medium containing kanamycin. After incubation overnight at 37°C, the bacterial culture was transferred to a large amount of LB liquid medium containing kanamycin. OD 600 Once the pH reached 0.6-0.8, IPTG was added to induce M2-MspA protein expression. The next day, the bacterial culture was centrifuged, and the precipitate was resuspended in AEX-A buffer (containing 2% Triton X-100) at a ratio of 1:9. Subsequently, the resuspended bacterial culture was autoclaved at low temperature. The lysed product was centrifuged, filtered, and the supernatant of the lysed product was initially purified using an anion exchange column. The eluent with UV reduced to 335 mAU was collected. Saturated ammonium sulfate was added to the obtained eluent to precipitate the product. After precipitation, the supernatant was removed by centrifugation to obtain a crude protein extract. The crude protein extract was redissolved at a ratio of 1:3 and incubated overnight at 37°C to stabilize the protein structure. The next day, insoluble impurities and potentially denatured proteins were removed by centrifugation and heating. The supernatant was separated and concentrated using an ultrafiltration tube. Finally, the concentrated supernatant was passed through an SD200 molecular sieve to obtain high-purity M2-MspA protein. After determining the protein concentration, glycerol was added in proportion and stored at -20°C for later use.

[0421] 1.3 ssDNA-glycan coupling

[0422] 1.3.1 Coupling of ssDNA to glycans (unilateral coupling)

[0423] The 5' end of handleDNA was modified with DBCO and then dissolved in ddH2O (final concentration 1 mM). The glycans were dissolved in N,N-dimethylacetamide (DMA, CAS No. 124-40-3) (final concentration 5 mM). The two were mixed at a 1:1.1 equivalent ratio and reacted at room temperature for 2 h. After the reaction was completed, 10% of the total volume of the reaction system was added with 5M NaCl and 5% ice-cold ethanol to precipitate the handleDNA-glycan conjugate. After centrifugation to remove the supernatant, the precipitate was lyophilized to obtain the handleDNA glycan conjugate.

[0424] 1.3.2 Coupling of ssDNA to glycans (bilateral coupling)

[0425] 1.3.2.1 Coupling of handleDNA-5'-DBCO, leadDNA-3'-DBCO to glycans

[0426] DBCO was modified at the 5' end of handle DNA, which was then dissolved in ddH2O (final concentration 1 mM). The glycan was dissolved in DMA (final concentration 5 mM). The two were mixed at a 1:1.1 equivalent ratio and reacted at room temperature for 2 h. After the reaction was completed, 10% of the total reaction volume of 5M NaCl and 5% ice-cold ethanol were added to precipitate the handle DNA-glycan conjugate. After centrifugation to remove the supernatant, the precipitate was lyophilized to obtain the handle DNA-glycan conjugate.

[0427] Next, an appropriate amount of handleDNA-glycan conjugate was dissolved in phosphate buffer at pH 8.4 (final concentration 0.5 mM) and reacted with benzenesulfonyl azide at a 1:80 equivalent ratio at 80°C for 2 h. After the reaction was completed, 10% of the total reaction volume of 5M NaCl and 5% ice-cold ethanol were added to precipitate handleDNA-glycan conjugate-N3. After centrifugation to remove the supernatant, the precipitate was lyophilized to obtain handleDNA-glycan conjugate-N3.

[0428] HandleDNA-glycan conjugate-DBCO-modified leadDNA at its N3 and 3' ends was dissolved in ddH2O (final concentration 1 mM), and the two were mixed at a 1:1.2 equivalent ratio and reacted at 50°C for 2 h. After the reaction was completed, 10% of the total reaction volume of 5M NaCl and 5% ice-cold ethanol were added to precipitate the handleDNA-glycan-leadDNA conjugate. After centrifugation to remove the supernatant, the precipitate was lyophilized to obtain handleDNA-glycan-leadDNA.

[0429] 1.3.2.2 Coupling of handleDNA-5'-DBCO, leadDNA-3'-SH to glycans

[0430] 3 nmol of handle DNA modified with DBCO at its 5' end was reacted with 6 nmol of glycan solution at 29 °C and 600 rpm for 12 h to obtain handle DNA-glycan conjugate. 2 μL of 15 nmol / μL SMCC (N-Succinimidyl 4-(N-maleimidomethyl)cycl, CAS No. 64987-85-5) solution was added to the handle DNA-glycan solution and reacted at room temperature and 600 rpm for 1 h. The product was then purified by desalting column chromatography to obtain handle DNA-glycan-Mal (maleimide group). 120 μL of 0.1 nmol / μL lead DNA-3'-SH was added to handle DNA-glycan-Mal and reacted at room temperature and 600 rpm for 12 h. The product was purified by 15% urea-polyacrylamide gel electrophoresis followed by gel extraction to obtain handle DNA-glycan-lead DNA conjugate.

[0431] 1.4 Establishment of the testing system

[0432] 1.4.1 Preparation of polytetrafluoroethylene membrane with uniform small pores of 30-70 μm in the center

[0433] A suitably sized polytetrafluoroethylene (PTFE) membrane is placed on the platform between the electrodes of an EDM drill, with the center of the membrane close to the upper and lower electrodes. A high voltage of approximately 160 kV is then applied to break down the center of the membrane, creating micropores. The shape and size of the micropores are observed under a microscope; only those with uniform shapes and pore diameters of 30-70 μm can be used for subsequent experiments.

[0434] 1.4.2 Assembly of Nanopore Detection Cells

[0435] Take two nanopore sample loading slots and evenly apply silicone grease to the edges of the small holes on the splicing surfaces. Then, attach the previously perforated PTFE membrane to the splicing surface of either sample loading slot (make sure the small holes on the PTFE membrane overlap with the small holes in the sample loading slot as much as possible; if the edge of the membrane extends beyond the edge of the splicing surface, cut off a portion to make the edges align). You can smooth the membrane surface from the center outwards. Next, place the two sample loading slots in the detection cell base with their splicing surfaces facing each other. After aligning the splicing surfaces, tighten the screws on the side of the base to ensure the splicing surfaces of the two sample loading slots are tightly pressed together, thus completing the assembly of the nanopore detection cell.

[0436] 1.4.3 Construction of Nanoporous Planar Lipid Bilayer

[0437] The planar lipid bilayer detection system mainly includes a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell is placed within the system and divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm. Each chamber is connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode are defined as the cis side and the trans side, respectively. Unless otherwise specified, 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) is typically added to each side. 0.5 μl of diaphytylphosphatidylcholine (DPhPC, 25 mg / ml) is added to the surface of one side of the electrolyte solution. Using a dip-coating method, DPhPC forms a phospholipid bilayer on the 30-70 μm pore in the center of the PTFE membrane. At this point, the system is in an open-circuit state, resulting in a detected current value near 0. Next, a voltage of 300-400mV is applied to the lipid bilayer, which is ideal for breaking down the lipid bilayer (i.e., the detection current becomes infinite, at which point the lipid bilayer is both stable and suitable for protein coating). If it can still break down after one repetition, then it is assumed that the thickness of the lipid bilayer formed in the next lifting is appropriate, thus completing the construction of the nanoporous planar lipid bilayer.

[0438] 1.4.4 Determination of pore openings on the membrane of porins and analysis of analytes

[0439] After a stable planar lipid bilayer is formed, 0.5-1 μl of M2-MspA porin (0.25 mg / ml) is added to the cis side. When a stable current of approximately 170 pA is detected, it indicates that M2-MspA has been stably inserted into the lipid bilayer. At this point, the analyte is added to the cis side, and the current signal is continuously recorded using Smartnano software. Unless otherwise specified, the sampling frequency is generally 50 kHz with a 5 kHz low-pass filter.

[0440] 2. Experimental Results

[0441] Figure 1 shows the gel image obtained after purification of TGA helicase. The first and second lanes are the protein marker and the final purified product, respectively. The band between 100-130 kDa is the TGA helicase band.

[0442] Figure 2 shows the purified gel image of M2-MspA. The first lane is the protein marker, the second to fourth lanes are the precipitate after bacterial lysis, the supernatant after lysis, and the protein solution after passing through the ion exchange column, respectively, and the fifth and sixth lanes are the protein solutions obtained from the first and second molecular sieve passes, respectively. The band near 100 kDa is the M2-MspA octamer protein.

[0443] Figures 3 and 5-13 show the mass spectrometry results of the one-sided coupling products of NT89 with hepta, hepta-3S, hepta-5S, hepta-3S&5S, HP-6, HP-8, HP-10, HA-6, HA-8, and HA-10, respectively. The highest peak is the NT89-glycan coupling product. Figure 4 shows the mass spectrometry results of the one-sided coupling product of 5T89 with nona, with the highest peak being the 5T89-glycan coupling product. Figure 14 shows the mass spectrometry results of the two-sided coupling products of NT89, polyT, and hepta (prepared according to 1.3.2.1), with the highest peak being each NT89-glycan-polyT coupling product.

[0444] Figures 15-17 show the analytical results of 15% Urea-page gels of the bilateral coupling products of NT89-5'-DBCO (or 5T89-5'-DBCO or 6T89-5'-DBCO), polyT-3'-SH and hepta, nona, hepta-5S, hepta-3S, hepta-3S&5S, hepta-1S, hepta-isomer, nona-1S, nona-3S, nona-5S, nona-7S, mono, tri, and penta (prepared according to 1.3.2.2).

[0445] 3. Experimental Materials

[0446] The DNA sequence is shown in Table 1, and the glycan sequence is shown in Figure 78.

[0447] Table 1

[0448] Example 2: Detection of ssDNA using a motor protein-driven nanopore

[0449] 1. Experimental Methods

[0450] NT89, NT89-2, NT89-polyT, 5T89, and 6T89 from Example 1 were annealed and coupled with tether DNA (T1C) at a 1:1 molar ratio to form double-stranded structures. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the ssDNA-tetherDNA annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0451] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the annealing product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0452] 2. Experimental Results

[0453] In this embodiment, the ssDNA electrochemical signal was validated to determine the integrity of the glycan chain. As shown in Figures 18-21, a sudden drop in the current signal detected by the nanopore at +180mV, accompanied by a stepwise change, was observed. This is the characteristic current signal generated by ssDNA passing through the nanopore under the action of helicase. In particular, the first half of the NT89-polyT signal is accompanied by four rising high-amplitude current steps, while the second half shows a long plateau signal with a blockage rate of about 20%. This feature is consistent with the nucleotide sequence in Figure 41—the signals of the front handle DNA and the rear lead DNA passing through the pore. After changing the handle DNA sequence, the characteristic signal mainly shows changes in the number and form of the terminal steps. Compared to NT89, NT89-2 only lacks the two terminal steps, while 5T89 shows changes in the stepwise form of the two terminal steps and the overall time course, thus completing the validation of the DNA electrochemical signal.

[0454] Example 3: Detection of ssDNA-glycan conjugates (one-sided conjugation) using a motor protein-driven nanopore.

[0455] 1. Experimental Methods

[0456] NT89-hepta, NT89-nona, NT89-hepta-3S, NT89-hepta-5S, NT89-hepta3S&5S, NT89-hepta-1S&3S&5S, NT89-HP-6, NT89-HP-8, NT89-HP-10, NT89-HA-6, NT89-HA-8, and NT89-HA-10, prepared according to the method in Example 1, were annealed and coupled with tether DNA (T1C) at a 1:1 molar ratio to form a double-stranded structure. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0457] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the annealing product, the ice-incubated product of the helicase TGA, and 5 μl of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0458] 2. Experimental Results

[0459] In this embodiment, we attempted to detect glycan signals after ligating DNA to the reduced ends of the glycan chains. As shown in Figures 22-23, the signal characteristics of NT89-hepta and NT89-nona are consistent with those of NT89 shown in Figure 18, meaning that only the characteristic signal of NT89 can be observed, indicating that the characteristic glycan signal cannot be obtained at this time. Considering that hepta and nona carry relatively little charge, they may not be able to be sufficiently stretched under the action of an electric field, thus resulting in the inability to generate a distinguishable glycan signal. Therefore, negatively charged sulfonic acid groups were introduced into the sugar units at positions 3, 5, 3&5, and 1&3&5 of the hepta glycan chain (starting from the reduced end), and then coupled with NT89 respectively to obtain NT89-hepta-3S, NT89-hepta-5S, NT89-hepta-3S&5S, and NT89-hepta-1S&3S&5S, with 2, 2, 3, and 4 charges on the four glycan chains, respectively. As shown in Figures 24-27, the signal characteristics of NT89-hepta-3S, NT89-hepta-5S, and NT89-hepta-3S & 5S are still consistent with those of NT89 shown in Figure 18. However, NT89-hepta-1S & 3S & 5S showed characteristic glycan signals, indicating that when DNA is coupled to only one side in this detection system, it is necessary for the glycan to carry at least 4 charges to obtain a recognizable glycan signal.

[0460] Based on this, naturally occurring negatively charged glycosaminoglycans—hyaluronic acid (HA-6, HA-8, HA-10, corresponding to hexasaccharides, octasaccharides, and decasaccharides, respectively, carrying 3, 4, and 5 charges) and short chains of heparin precursors (HP-6, HP-8, HP-10, corresponding to hexasaccharides, octasaccharides, and decasaccharides, respectively)—were selected for further analysis. As shown in Figures 28-30, the signal characteristics of NT89-HA-6 are consistent with those of NT89 shown in Figure 18, while NT89-HA-8 and NT89-HA-10 exhibit their own characteristic signals. The signal pattern of NT89-HP is similar to that of NT89-HA. As shown in Figures 31-33, the signal characteristics of NT89-HP-6, which carries 3 charges on the sugar chain, are consistent with those of NT89 shown in Figure 18, while NT89-HP-8 and NT89-HP-10 exhibit their own characteristic signals. The signal characteristics of NT89-HA-8, NT89-HA-10, NT89-HP-8, and NT89-HP-10 were analyzed using the method described in Example 10, as shown in Figures 34-38. It can be seen that the four different lengths and linkages of glycans differ in the number and amplitude of steps, demonstrating the feasibility of using this system for model and naturally occurring glycan sequence analysis. However, with unilaterally coupled DNA, the glycan signal duration is short and the signal resolution is low. Therefore, bilaterally coupled DNA was subsequently used to promote glycan extension and improve signal resolution.

[0461] Example 4: Detection of hepta-ssDNA conjugates linked by different linkers (bilateral conjugates)

[0462] 1. Experimental Methods

[0463] The hepta-bilateral DNA conjugate NT89-hepta-polyT from Example 1 was annealed and conjugated with T1C at a 1:1 molar ratio to form a double-stranded structure. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0464] The experimental principle is shown in Figure 39. The electrophysiological detection device used includes a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell is divided into two chambers by a polytetrafluoroethylene membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode are defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) is added to each side. DPhPC (25 mg / ml) is added to the surface of the electrolyte solution on one side. Using a dip-coating method, DPhPC forms a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the polytetrafluoroethylene membrane. At this point, the system is in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein is added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the annealing product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0465] 2. Experimental Results

[0466] In this embodiment, two hepta-DNA conjugates with different linkers (bilateral conjugation, prepared according to the method in Example 1) were designed and tested.

[0467] 1) Signal analysis of each sugar chain

[0468] For hepta-bilateral DNA conjugates linked by different linkers, as shown in Figure 40 (the box shows the signal magnification during glycan perforation; unless otherwise specified, the following analysis only considers this signal segment), NT89-hepta-N3-DBCO-polyT exhibits the following characteristics: ① Transition phase: A long or sloping plateau is followed by a high-density, low-amplitude spike signal; ② Rise phase: A step or gradual rise to the highest plateau, with the highest plateau current amplitude (I0)... res / I0:36-41%); ③ Falling phase. The current drops from the highest plateau through a small plateau or directly back to the polyT blockage level. As shown in Figure 41 (the box shows an enlarged view of the signal when the sugar chain passes through the via), the NT89-hepta-SMCC-HS-polyT exhibits similar three-stage characteristics: ① Transition phase. A relatively stable long plateau is accompanied by a short plateau signal, without low-amplitude spike signals; ② Rising phase. It reaches the highest plateau in a step-like manner, and the current amplitude of the highest plateau (I0:36-41%) is... res / I0:43%); ③ Decline phase. After a slightly longer plateau, it declines to the lowest point, and then rises back to the polyT blockage level.

[0469] 2) Comparison and analysis of glycan signals.

[0470] NT89-hepta-SMCC-HS-polyT highest plateau current amplitude (I res / I0:43%), NT89-hepta-N3-DBCO-polyT highest platform current amplitude (I res The percentage ( / I0:36-41%) is presumably due to the influence of different linkers on the different steric hindrance effects of the glycans. This indicates that the aforementioned ladder signal may also be contributed by the linker, and this system can distinguish signal differences caused by different linkers. Further signal analysis and data processing were performed on the NT89-hepta-SMCC-HS-polyT ladder signal using the method of Example 10, and the results are shown in Figure 42. Unless otherwise specified, SMCC will be used as the linker to couple ssDNA and the glycans in subsequent analyses.

[0471] Example 5: Detection of hepta-DNA conjugates (bilateral conjugates) with lead DNA of different lengths

[0472] 1. Experimental Methods

[0473] The NT89-hepta-N3-DBCO-polyT (10nt) and NT89-hepta-N3-DBCO-polyT (30nt) from Example 1 were annealed and coupled with tether DNA (T1C) at a 1:1 molar ratio to form a double-stranded structure. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0474] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the annealing product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0475] 2. Experimental Results

[0476] In this embodiment, two different lengths of polyT were designed as lead DNA strand guide coupling pores and to indicate glycan integrity signals. 1) Analysis of each glycan signal.

[0477] As shown in Figures 43-44, following the event signal's decline from the opening current, the initial stage of NT89-hepta-N3-DBCO-polyT exhibits a characteristic signal similar to that in Figure 18, indicating that this part represents the rate-controlled passage stage of handle DNA under the action of helicase. Accompanying the NT89 signal's termination, the current signal sequentially exhibits a three-stage characteristic: a slow current increase followed by a long transitional plateau, a step-like rise, and a step-like decline. After the transition and rise phases, the current increases to I... resThe highest plateau is approximately 38%. Given that the highest current blockage rate of ssDNA under the same testing conditions is below 20%, this portion represents the current signal generated when the glycans enter the narrowest part of the nanopore under the action of helicase on the ssDNA. Since the diameter of monosaccharide molecules in the glycans is smaller than the diameter of nucleotides composed of multiple structural units, the blockage amplitude is smaller, and the through-pore current is larger. When the current signal in the descent phase returns to a stable current level, a characteristic current signal similar to the latter half of Figure 20 can be displayed, namely the lead DNA (polyT) signal. This signal, which can indicate the glycan-related signal between handle DNA and lead DNA, is a signal associated with glycans.

[0478] 2) Comparison and analysis of glycan signals.

[0479] For hepta-DNA conjugates (bilateral conjugations) of different polyT lengths, both NT89-hepta-N3-DBCO-polyT (10nt) and NT89-hepta-N3-DBCO-polyT (30nt) returned to DNA signal levels as the glycan signal decreased in a stepwise manner, with the only difference being the time course of polyT blocking the long plateau. This example confirms that both 10nt and 30nt polyTs can function as indicators of the end of glycan signaling.

[0480] Example 6: Detection of ssDNA conjugates of different lengths by a motor protein-driven nanopore (bilateral conjugation)

[0481] 1. Experimental Methods

[0482] The NT89-mono-polyT, NT89-tri-polyT, NT89-penta-polyT, NT89-hepta-polyT, and 5T89-nona-polyT from Example 1 were annealed and coupled with tether DNA (T1C) at a 1:1 molar ratio to form double-stranded structures. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0483] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the annealed product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0484] 2. Experimental Results

[0485] In this embodiment, six sugar chains of different lengths were designed and synthesized as model sugar chains, namely monosaccharides, trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides.

[0486] 1) Glycan signal analysis.

[0487] Figure 18 shows the handle DNA signal before the mono, tri, penta, and hepta glycan chains, while Figure 21 shows the handle DNA signal before nona. When the glycan-DNA conjugate enters the nanopore, the current decreases from the opening current. The initial stage of the conjugates of ssDNA with glycan chains of different lengths (bilateral coupling) all show characteristic signals similar to those in Figure 18, indicating that this part is the rate-controlled pore-passing stage of handle DNA under the action of helicase.

[0488] As shown in Figure 45, after the NT89 signal ends, several short-duration rising steps can be seen, transitioning to the highest platform of approximately 41% of the current amplitude. Subsequently, several short-duration falling steps can be seen, recovering to a stable current level of approximately 20% after the lowest amplitude step.

[0489] As shown in Figure 46, after the NT89 signal ends, several short-duration rising steps can be seen, transitioning to the highest platform of approximately 43% of the current amplitude. Subsequently, several short-duration falling steps can be seen, returning to a stable current level of approximately 20% of the amplitude after the lowest amplitude step.

[0490] As shown in Figure 47, the NT89-penta-polyT signal exhibits a step-like change pattern after the NT89 signal ends, rising first and then falling. It then rises through several steps to a maximum platform with an amplitude of approximately 42%, followed by a short-duration decline to the lowest step, and finally returns to a stable current level with an amplitude of approximately 20%.

[0491] As shown in Figure 48, the 5T89-nona-polyT signal transitions from DNA signal to glycan signal as a sloping plateau signal that first rises slowly and then falls slowly (peak signal I). res / I0 is approximately 23%, then a stepwise rise reaches I res / I0 is approximately 42% of the highest plateau. When the current signal decreases, it can step back to a long plateau signal higher than the polyT blocking current.

[0492] 2) Comparison and analysis of glycan signals.

[0493] Compared to NT89-hepta-polyT, the glycan signals of NT89-penta-polyT and 5T89-nona-polyT show differences in both the step pattern and the number of glycan signals. In terms of the step pattern, unlike the hepta (hereafter referred to by glycan name in the conjugate detection results) transition phase signal which rises slowly with a long plateau, the penta and nona terminal signals often decline. Secondly, the hepta signal often drops to the lowest plateau before rising back to the polyT level, while the nona signal continues to decline until it recovers to the polyT current level.

[0494] For mono and tri, no long plateau signals were observed. In addition, all six sugar chains showed differences in current amplitude, time history, and number of steps in the step signal. The step signals of mono, tri, penta, and nona were analyzed and processed using the method of Example 10, and the results are shown in Figures 49-52.

[0495] 3) The relationship between signal differences and sugar sequence.

[0496] In general, ssDNA bilateral conjugates of monosaccharides, trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides can be distinguished from each other in terms of the step pattern and amplitude of the current signal. For the three-stage signal of the glycan, since the highest plateau blockage rate is greater than the highest current blockage rate of ssDNA under the same testing conditions, this part represents the pure sugar current signal generated when the glycan enters the narrowest part of the nanopore under the action of helicase on the ssDNA. Because the diameter of monosaccharide molecules in the glycan is smaller than the diameter of nucleotides composed of multiple structural units, the blockage amplitude is smaller, and the permeation current is larger. The step signals before and after the highest plateau are caused by the combined action of the linker and the glycan. In conclusion, the above results demonstrate that this method has a certain ability to distinguish glycan sequences of different lengths.

[0497] Example 7: Detection of conjugates (bilateral conjugation) of sugar chains with different sulfonation modifications to ssDNA

[0498] 1. Experimental Methods

[0499] The sulfonated sugar chains prepared in Example 1, section 1.3.2.2, and their bilateral conjugates with ssDNA, hepta-1S, hepta-3S, hepta-5S, hepta-3S & 5S (all hepta-saccharides), nona-1S, nona-3S, nona-5S, and nona-7S (all nona-saccharides), were annealed with tetherDNA (T1C) at a 1:1 molar ratio to form double-stranded structures. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0500] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the annealing product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0501] 2. Experimental Results

[0502] In this embodiment, a total of eight sulfonated sugar chains are used, of which four are hepta-sugar chains, namely hepta-1S, hepta-3S, hepta-5S, and hepta-3S&5S, respectively, at positions 1, 3, 5, and 3 & 5; and four are nona-sugar chains, namely nona-1S, nona-3S, nona-5S, and nona-7S, respectively, at positions 1, 3, 5, and 7.

[0503] 1) Signal analysis of sulfonated heptaglycolic chains

[0504] As shown in Figures 53-60, along with the handleDNA perforation signal end, hepta-1S, hepta-3S, hepta-5S, and hepta-3S&5S all exhibit a "three-stage" characteristic current signal similar to that in Figure 41 (hepta), which successively manifests as a long transitional plateau, a step-like rise, and a step-like fall.

[0505] The sulfonated hepta-1S at position 1 reaches a long plateau through several steps, then rises through several steps to a maximum plateau with an amplitude of approximately 42%. Subsequently, it reaches a minimum plateau through several steps, and finally returns to the polyT current level.

[0506] 3-sulfonated hepta-3S was subjected to a long plateau with slow current increase (the long plateau was divided into three segments, with current amplitude I). res / I o After being 22%, 21%, and 24% respectively, it jumped to I. res / I o The highest plateau was approximately 43%. This is because the highest I value for ssDNA sequences near the glycan chain occurred under the same testing conditions. res / I o All values ​​are below 20%. This portion should be the current signal generated when the glycans enter the narrowest part of the nanopore under the action of helicase on ssDNA. Since the diameter of monosaccharide molecules in the glycans is smaller than the diameter of nucleotides composed of multiple structural units, the blockage amplitude is smaller, and the through-pore current is larger. After the current drops abruptly from the highest plateau to the lowest point, the signal will rise again to the polyT blockage level.

[0507] When hepta-5S, modified with sulfonation at position 5, transitions from DNA signaling to glycan signaling, it exhibits an approximate "asymmetric hill" signal (peak signal I) that initially rises slowly and then falls slowly. res / I o Approximately 22%) accompanied by long plateau signals (I res / I o Approximately 17%), then a step jump to I res / I o The highest plateau is approximately 41%. When the current signal decreases, it reaches the lowest plateau via a stepped path before rising back to the polyT blocking signal.

[0508] Hepta-3S & 5S, with sulfonation modification at both positions 3 and 5, exhibited a transition signal that rose sharply to a peak signal at a blockage rate of approximately 22%, followed by a slow decline, accompanied by a long plateau signal (I). res / I o Approximately 18%), after which one ascends the steps to the highest platform (I). res / I o (Approximately 43%), then stepped to the lowest plateau, and subsequently rose back to the polyT blockage level.

[0509] Analysis of the detection results of hepta-glycan-bilateral DNA conjugates with different sulfonation sites revealed that signal differences were mainly concentrated in the long plateau phase of the transition period. Compared to unsulfonated hepta (hereinafter, the glycan names refer to the glycan signals in the conjugate detection results), the long plateau signal of hepta-3S mainly showed a change in the blocking amplitude—a lower current amplitude (22% vs. 24%), with no significant difference in signal morphology. Hepta-1S, hepta-5S, and hepta-3S & 5S showed differences from hepta in both signal amplitude and morphology. Specifically, regarding the long plateau morphology, unlike hepta and hepta-3S, where the current signal slowly rises to a higher plateau, both hepta-1S and hepta-5S experienced a slow rise followed by a slow decline, and hepta-3S & 5S experienced a step-like rise followed by a slow decline, with a lower long plateau appearing at the end. At the maximum amplitude of the long platform, compared to hepta, the amplitude of hepta-3S, hepta-5S, and hepta-3S & 5S are all smaller.

[0510] From a molecular volume perspective, the decrease in amplitude of the long plateau region in the four sulfonated hepta-saccharide chains is a result of the sulfonic acid groups increasing the local volume of the sugar chain. Furthermore, the order of sulfonation on the sugar chain corresponds well to the order of the affected electrical signal steps; that is, the order in which the steps are affected is basically consistent with the order of sulfonation. In addition, the decrease in amplitude of the long plateau in the disulfonated hepta-3S & 5S is greater than that in the monosulfonated hepta-3S and hepta-5S, suggesting that using a size exclusion model to explain the effect of sulfonation on the step amplitude is reasonable.

[0511] 2) Analysis of sulfonated nonaglycone chains

[0512] As shown in Figures 61-68, along with the handleDNA perforation signal end, nona-1S, nona-3S, nona-5S, and nona-7S all exhibit a "three-stage" characteristic current signal similar to that in Figure 48 (nona), mainly manifested as a long transitional plateau, a step-like rise, and a step-like fall.

[0513] After the 5T89 signal of Nona-1S and nona-3S ended, the amplitude reached the highest point of the long platform (current amplitude of 23% and 22%, respectively) through several steps, then decreased, and then rose to the highest current amplitude step (current amplitude of 40%) before returning to the polyT current level through several steps. However, after the 5T89 signal of nona-5S and nona-7S ended, the amplitude decreased from the long platform through several steps (amplitude of 20% and 18%, respectively) before rising to the highest amplitude step (current amplitude of 42% and 43%, respectively).

[0514] Similar to sulfonated heptaose, the signal differences in the detection results of nonaose chains-bilateral DNA conjugates with different sulfation sites are also concentrated in the long plateau phase of the transition period. The corresponding decrease in the step current amplitude can also conform to the law of the size exclusion model, and the step sequence of the decrease in current amplitude has good consistency with the order of sulfation sites.

[0515] In summary, the above demonstrates that this method can accurately interpret the composition, amount, and location of monosaccharides.

[0516] Example 8: Determination of ssDNA conjugates with different glycosidic bonds (bilateral conjugation)

[0517] 1. Experimental Methods

[0518] The hepta-isomers with different glycosidic bonds prepared in Example 1, section 1.3.2.1, were annealed and coupled with tether DNA (T1C) at a 1:1 molar ratio to form double-stranded structures. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, a 10-fold diluted helicase TGA (1.7 mg / mL) and the annealing product were mixed at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0519] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μL of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μL of the annealing product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0520] 2. Experimental Results

[0521] In this embodiment, glycans with different glycosidic bond replacements are used. Using hepta as a template glycan, the β-1,4 glycosidic bond between the third GlcNAc and the fourth Gal is replaced with a β-1,3 glycosidic bond, resulting in a hepta-isomer glycan.

[0522] 1) Glycan signal analysis.

[0523] As shown in Figures 69-70, analysis of the glycan signals linked by glycosidic bonds reveals that their step signals exhibit characteristics distinct from hepta, including differences in step amplitude and number. When the glycosidic bond linkages within the glycan molecule change, the step signal shows a significant alteration, indicating that the step signal is related to the linkage pattern within the glycan molecule. This demonstrates that this method can differentiate between glycans linked by different glycosidic bonds and ssDNA bilateral conjugate sequences to a certain extent.

[0524] Example 9: Practical Application Scenario: Qualitative and Quantitative Analysis of Sulfonated Heptaose Mixtures

[0525] 1. Experimental Methods

[0526] The sulfonated heptaose conjugates prepared in Example 1, section 1.3.2.2 were annealed and coupled with T1C at a 1:1 molar ratio to form double-stranded structures. The annealing program was 86°C, 76°C, 66°C, 56°C, 46°C, 36°C, 26°C, and 16°C for 1 min each. After annealing, the different sulfonated heptaoses were mixed in different proportions (four mixtures in total: hepta-1S:hepta-3S:hepta-5S = 1:1:1, hepta-3S:hepta-3S&5S = 3:1, hepta-3S:hepta-3S&5S = 1:1, hepta-3S:hepta-3S&5S = 1:3). A 10-fold diluted helicase TGA (1.7 mg / mL) was mixed with each mixture at a 1:1 volume ratio and incubated on ice for at least 10 min.

[0527] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μL of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μL of the annealing product, the ice-incubated product of the helicase TGA, and 5 μL of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0528] 2. Experimental Results

[0529] As shown in Figures 71-74, the sequencing results of the sulfonated heptaose mixture show that the component ratios obtained after signal attribution analysis based on the characteristic steps of each sulfonated heptaose (i.e., after extracting the step signals of the mixture, comparing them with the fifth and seventh steps of each pure sulfonated heptaose, and determining the signal attribution based on the degree of overlap between the two) are in good agreement with the component ratios in the mixture, suggesting that this system has the potential to be applied to the qualitative and quantitative analysis of sulfonated sugar chains in complex systems.

[0530] Example 10: Signal Analysis and Data Processing

[0531] 1. Analytical Methods

[0532] 1) Signal screening and preprocessing

[0533] For each glycan-ssDNA conjugate assay, results from at least three independent measurements were included. All current signals were processed using Clampfit 10.7 and PyNanoLab to extract characteristics of ion current level events. First, all acquired current signals were filtered and noise-reduced using Clampfit 10.7 (8-pole-bessel: 100Hz), and individual single-molecule events were segmented based on the event start time from the point of pore current blockage to the DNA current level and the event end time from the point of recovery of the DNA or glycan signal level back to the pore current level. All single-molecule events were screened, retaining only those with a matching handle DNA signal and those where DNA helicase continued to guide the generation of a different current signal after the handle DNA signal ended. Events containing significant MspA gating and noise, or those containing atypical or incomplete handle DNA signals, were excluded from the screening.

[0534] 2) Event signal ladder division and fitting statistics

[0535] For each glycan type, at least 200 independent single-molecule events were used for statistical analysis of the step current amplitude signal. The step division and fitting of all screened independent single-molecule events were processed using PyNanoLab's "Nanopore Analysis". Specifically, the "multistepspnl" mode was selected for multi-step fitting of the event current signal. By adjusting parameters, a fitted signal close to the actual current signal was obtained. Only fitted signals with "fluctuation2" greater than 1.5 pA, "ignore time less" greater than 5 ms, and a fitting deviation of no more than 1 pA were suitable for step division. The fitted average opening current amplitude of all independent single-molecule events was calculated to obtain the M2-MspA opening current (I0). o The frequency distribution of fittable glycan ladder signals from all independent single-molecule events was statistically analyzed to determine the dominant ladder. Only ladders with an occurrence probability greater than 50% were considered as the dominant ladder signal features of the glycan and used for subsequent signal processing. To avoid deviations between different openings, the relative current amplitude (Ig) was used. res / I o () is the average current for each step event.

[0536] 2. Analysis Results

[0537] This embodiment uses hepta as an example to demonstrate the results of signal preprocessing and data analysis. First, all hepta signals acquired in the experiment underwent noise reduction processing using Clampfit 10.7 with a 100Hz low-pass filter. Independent single-molecule events were defined with the event start time being the transition from pore current blockage to DNA current level, and the event end time being the recovery of the DNA or glycan signal level back to the pore current level. Further signal screening retained only events with matching handle DNA signals, and events where DNA helicase continued to guide the generation of signals different from DNA current signals after the handle DNA signal ended. A total of 354 hepta glycan signal single-molecule events were obtained for subsequent data analysis.

[0538] When analyzing and processing single-molecule event signals, the "multistepspnl" mode of the "Nanopore Analysis" function in PyNanoLab was selected to perform multi-step fitting of the event current signal. By adjusting the parameters, a fitted signal close to the actual current signal was obtained. Only fitted signals with "fluctuation2" greater than 2.5 pA, "ignore time less" greater than 12 ms, and a fitting deviation of no more than 1 pA were suitable for hepta event step division. The average fitting aperture current amplitude for all single-molecule events was calculated to obtain the M2-MspA aperture current I. o The amplitude is approximately 180.44 pA. Statistical analysis of the step signal frequency distribution of 354 hepta single-molecule events revealed 14 levels of fitable glycan ladder signals. Their frequencies (number of occurrences of this ladder / total number of events, ordered chronologically) were 100%, 100%, 98.6%, 28.5%, 97.5%, 100%, 94.6%, 12.4%, 90.4%, 32.5%, 10.7%, 10.7%, 100%, and 100%. Only ladders with a frequency greater than 90% were considered the main hepta ladders, resulting in the 9-step characteristic signal shown in Figure 75. Steps 1 and 9 correspond to the end of handle DNA and the beginning of lead DNA in the conjugate nucleotide sequence, respectively, while steps 2-8 are associated with hepta. The relative current amplitude (IA) was used to determine the hepta characteristic signal. res / I o The current characteristics of each step of the hepta signal are characterized. The average current amplitudes of steps 1 to 9 (named in order of signal occurrence time) are 20%, 24%, 31%, 39%, 43%, 41%, 34%, 17%, and 20%, respectively. This demonstrates that this method can analyze and quantify glycan signals.

[0539] Example 11: Detection of glycan-peptide conjugates using a motor protein-driven nanopore

[0540] 1. Experimental Methods

[0541] The peptide-hepta (peptide sequence shown in Table 2) prepared according to the method in 1.3.2.2 of Example 1 was mixed with the unfolded enzyme ClpX (NCBI: NC_000913.3) and incubated on ice for at least 10 min.

[0542] Table 2

[0543] The electrophysiological detection device used in the experiment included a detection cell, detection electrodes (including a ground electrode and a pressure electrode), a signal amplifier, and an analog-to-digital converter. The detection cell was divided into two chambers by a polytetrafluoroethylene (PTFE) membrane with a central pore of 30-70 μm, each connected to one of the two detection electrodes. The chambers connected to the ground electrode and the pressure electrode were defined as the cis side and the trans side, respectively. 250 μl of electrolyte solution (400 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 8.0) was added to each side. DPhPC (25 mg / ml) was added to the surface of the electrolyte solution on one side, and a dip-coating method was used to form a phospholipid bilayer of suitable thickness on the central 30-70 μm pore of the PTFE membrane. At this point, the system was in an open-circuit state, as indicated by a detected current value near 0. Subsequently, M2-MspA pore protein was added to the cis side. When a stable pore current of approximately 170 pA was observed at a bias voltage of +180 mV, it indicated that the M2-MspA pore protein had stably inserted into the lipid bilayer. At this point, 1 μl of the ice-incubated product and 5 μl of ATP were added to the cis side, and the current signal was continuously recorded using Smartnano software. The sampling frequency was 50 kHz, with a low-pass filter of 5 kHz.

[0544] 2. Experimental Results

[0545] In this embodiment, we attempted to detect glycan signals after linking peptide chains to the reducing ends of glycan chains. As shown in Figures 76-77, the hepta-saccharides of the coupled peptide chains exhibited a ladder-like signal similar to that of the hepta-saccharides of coupled DNA, suggesting the feasibility of glycan sequencing using coupled peptide chains.

Claims

A method for glycan sequencing, the method comprising: A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates; B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region; C) Obtain the electrical signal characteristics of the sugar chains; D) Determine the sugar sequence by electrical signal characteristics. According to the method of claim 1, the connector molecule is capable of pulling the sugar chain through the nanopore. According to the method of claim 1 or 2, the glycan chains are moved by motor proteins through nanopores, controlling the movement of the linker molecules. According to any one of claims 1-3, the motor protein regulates the movement of the adapter molecule in the nanopore, thereby controlling the speed at which the attached glycan chains pass through the nanopore. The method according to any one of claims 1-4, wherein there is at least one connector molecule, preferably two or more. According to any one of the methods in claims 1-5, 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 linker molecule is connected to the reducing end to form a linker molecule-sugar chain conjugate. According to the method described in any one of claims 1-5, 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 conjugate, wherein, Connector molecule 1 may be the same as or different from connector molecule 2. According to any one of claims 1-7, the linker molecule is covalently linked to the sugar chain or is linked by a linking group. According to any one of claims 1-8, 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. According to any one of claims 1-9, the thiol group modified on the connector molecule reacts and connects with the maleimide group modified on the sugar chain. According to any one of claims 1-9, the DBCO modified on the linker molecule is reactively linked to the azide group modified on the sugar chain. The method according to any one of claims 1-11, wherein the electrical signal characteristics include, but are not limited to, electrical signal waveforms. According to any one of claims 1-12, 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. According to any one of claims 1-13, step D) comprises: Establish a correspondence between the electrical signal features obtained in step C) and the sugar chain features. According to the method of claim 14, 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. According to the method of claim 15, 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, position of modification, and number of modifications. The method according to any one of claims 1-16 further includes performing step signal analysis on the obtained electrical signal characteristics. According to the method of claim 17, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction. According to the method of claim 18, the parameters include, but are not limited to, amplitude, time course, or standard deviation. According to the method of claim 17, the analyzed step signal is compared with a known database signal to determine the glycan sequence. According to the method of claim 17, a glycan feature database is constructed, and then the analyzed step signal is compared with the signal in the constructed glycan feature database to determine the glycan sequence. According to any one of claims 1-21, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives in the sugar chain. According to any one of claims 1-21, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain. According to any one of the methods of claims 1-21, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan. The method according to any one of claims 1-24, wherein the sugar chain to be tested contains sialic acid. The adapter molecule according to any one of claims 1-25 includes, but is not limited to, nucleic acids or peptides. According to any one of the methods described in claims 1-26, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate. The method according to any one of claims 1-27, wherein 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. According to the method of claim 28, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase. According to the method of claim 28 or 29, the helicase or a variant thereof 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 ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM)) The sequence of Hel308 Mok (Methanothermococcus okinawensis (strain DSM 14208 / JCM 11175 / IH1)) or its variant, the sequence of Hel308 Fac (Ferroplasma acidarmanus Fer1) or its variant, the sequence of Hel308 Csy (Cenarchaeum symbiosum (strain A)) or its variant, the sequence of Hel308 Mhu (Methanospirillum hungatei JF-1 (strain ATCC 27890 / DSM 864 / NBRC 100397 / JF-1)) or its variant, or the sequence of F8813 protein or its variant. According to the method of claim 28, the polymerase is phi29 polymerase or a variant thereof, pol6 or a variant thereof, pol7 or a variant thereof. The method according to any one of claims 1-31, wherein the adapter molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or variants thereof. According to the method of claim 32, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme (Caseinolytic protease X). The nucleic acid described according to any one of claims 26-28 is single-stranded, double-stranded, or partially double-stranded. According to any one of claims 26-34, the nucleic acid is DNA, RNA, modified DNA, or modified RNA. According to any one of claims 1-35, the adapter molecule is DNA, and one end of the glycan chain to be tested is connected to DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate, preferably DNA-glycan conjugate is formed by connecting DNA at the reducing end. According to any one of claims 1-35, the adapter molecule is DNA, and both ends of the glycan chain to be tested are connected to DNA, forming a DNA1-glycan-DNA2 conjugate. DNA1 and DNA2 can have the same sequence or different sequences. The method according to any one of claims 1-37, wherein the nanopore is a biological nanopore or a solid nanopore. According to the method of claim 38, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin nanopores, MspA nanopores, AeL nanopores, CsgG nanopores, OmpF nanopores, ClyA nanopores, phi29 nanopores, FhuA nanopores, PlyA nanopores, PlyB nanopores, or FraC nanopores. According to the method of claim 38, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores. The method according to any one of claims 1-40, wherein the chain length of the sugar chain is greater than 1. According to any one of claims 1-41, the sugar chain may be a straight chain or have at least one branch. The sugar chain may be modified, for example, by sulfonation, according to any one of claims 1-42. According to any one of claims 1-43, the sugar chain may be charged or electrically neutral. According to any one of claims 1-44, the sugar chain is composed of monosaccharides and / or their derivatives linked by glycosidic bonds. According to the method of claim 45, the monosaccharide includes, but is not limited to, pentose sugars, hexose sugars, and monosaccharides with six or more carbon atoms. According to the method of claim 45, the monosaccharide derivative includes, but is not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides. According to any one of claims 1-47, the configuration of each anomeric carbon in the sugar chain can be α or β. According to any one of the methods described in claims 1-48, the type of glycosidic bond in the sugar chain includes, but is not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4. A method for detecting electrical signals characteristic of glycan chains, the method comprising: A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates; B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region; C) Obtain the electrical signal characteristics of the sugar chain. According to the method of claim 50, the connector molecule is capable of pulling the sugar chain through the nanopore. According to the method of claim 50 or 51, the glycan chains are realized through nanopores by motor proteins controlling the movement of linker molecules. According to any one of claims 50-52, the motor protein regulates the movement of the adapter molecule in the nanopore, thereby controlling the speed at which the attached glycan chains pass through the nanopore. The method according to any one of claims 50-53, wherein there is at least one connector molecule, preferably two or more. According to any one of claims 50-54, 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 linker molecule is connected to the reduced end to form a linker molecule-sugar chain conjugate. According to the method described in any one of claims 50-54, 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 conjugate, wherein, Connector molecule 1 may be the same as or different from connector molecule 2. According to any one of claims 50-56, the linker molecule is covalently linked to the sugar chain or is linked by a linking group. According to any one of claims 50-57, 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. According to any one of claims 50-58, the thiol group modified on the connector molecule reacts and connects with the maleimide group modified on the sugar chain. According to any one of claims 50-58, the DBCO modified on the linker molecule is reactively linked to the azide group modified on the sugar chain. The method according to any one of claims 50-60, wherein the electrical signal characteristics include, but are not limited to, electrical signal waveforms. According to any one of claims 50-61, 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. According to any one of the methods described in claims 50-62, 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. According to the method of claim 63, 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, position of modification, and number of modifications. The method according to any one of claims 50-64, wherein the sugar chain to be tested contains sialic acid. The adapter molecule according to any one of claims 50-65 includes, but is not limited to, nucleic acids or peptides. According to any one of claims 50-66, the linker molecule glycan conjugate is a nucleic acid-glycan conjugate, a nucleic acid-glycan-nucleic acid conjugate, a polypeptide-glycan conjugate, a polypeptide-glycan-peptide conjugate, a nucleic acid-glycan-peptide conjugate, or a polypeptide-glycan-nucleic acid conjugate. The method according to any one of claims 50-67, wherein 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. According to the method of claim 68, the helicase is a Hel308 family helicase, Tral helicase, TrwC helicase, RecD helicase, XPD helicase, or Dda helicase. According to the method of claim 68, the helicase or a variant thereof is a sequence of Hel308 Tga or a variant thereof, a sequence of Hel308 Mbu or a variant thereof, a sequence of Hel308 Pfu or a variant thereof, a sequence of Hel308 Mma or a variant thereof, a sequence of Hel308 Mok or a variant thereof, a sequence of Hel308 Fac or a variant thereof, a sequence of Hel308 Csy or a variant thereof, a sequence of Hel308 Mhu or a variant thereof, or a sequence of F8813 protein or a variant thereof. According to the method of claim 68, the polymerase is phi29 polymerase or a variant thereof, pol6 or a variant thereof, pol7 or a variant thereof. The method according to any one of claims 50-71, wherein the adapter molecule is a polypeptide, and the motor protein includes, but is not limited to, unfolded enzymes or variants thereof. According to the method of claim 72, the unfolded enzyme includes, but is not limited to, ClpX unfolded enzyme. The nucleic acid described according to any one of claims 66-73 is single-stranded, double-stranded, or partially double-stranded. According to any one of claims 66-74, the nucleic acid is DNA, RNA, modified DNA, or modified RNA. According to any one of claims 50-75, the adapter molecule is DNA, and one end of the glycan chain to be tested is connected to DNA to form a DNA-glycan conjugate or a glycan-DNA conjugate; preferably, DNA is connected to the reducing end to form a DNA-glycan conjugate. According to any one of claims 50-75, 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. DNA1 and DNA2 can have the same sequence or different sequences. The method according to any one of claims 50-77, wherein the nanopore is a biological nanopore or a solid nanopore. According to the method of claim 78, the bio-nanopores include, but are not limited to, Staphylococcus aureus α-hemolysin nanopores, MspA nanopores, AeL nanopores, CsgG nanopores, OmpF nanopores, ClyA nanopores, phi29 nanopores, FhuA nanopores, PlyA nanopores, PlyB nanopores, or FraC nanopores. According to the method of claim 78, the solid nanopores include, but are not limited to, graphene nanopores, silicon nitride nanopores, titanium dioxide nanopores, or alumina nanopores. According to the method of claim 49, the chain length of the sugar chain is greater than 1. According to any one of claims 50-81, the sugar chain may be a straight chain or have at least one branch. The sugar chain may be modified, for example, by sulfonation, according to any one of claims 50-82. According to any one of claims 50-83, the sugar chain may be charged or electrically neutral. The method according to any one of claims 50-84, wherein the sugar chain is composed of monosaccharides and / or their derivatives linked by glycosidic bonds. According to the method of claim 85, the monosaccharide includes, but is not limited to, pentose sugars, hexose sugars, and monosaccharides with six or more carbon atoms. According to the method of claim 85, the monosaccharide derivative includes, but is not limited to, sugar acids, sugar alcohols, deoxy sugars, amino sugars, methylated sugars, acetylated sugars, fluorinated sugars, glyconucleotides, or glycosides. According to any one of claims 50-87, the configuration of each anomeric carbon in the sugar chain can be α or β. According to any one of claims 50-88, the type of glycosidic bond in the sugar chain includes, but is not limited to, α-1,2, α-1,3, α-1,4, α-1,6, β-1,3, or β-1,4. A method for identifying glycan characteristics, the method comprising: A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates; B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region; C) Obtain the electrical signal characteristics of the sugar chains; D) Determine the characteristics of sugar chains through electrical signal features. According to the method of claim 90, 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, position of modification, number of modifications, whether there are branches, position of branches, number of branches or branch sequence characteristics. According to the method of claim 91, 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, position of modification, and number of modifications. According to the method of claim 91 or 92, the sugar chain feature identification is a qualitative or quantitative assessment of the sugar chain features. The application of the method for detecting electrical signals of glycan characteristics according to any one of claims 1-92 in glycan sequencing or glycan detection, preferably, the glycan detection includes quantitative and / or qualitative methods. A method for detecting sugar chains, the detection method comprising: A) Connect the sugar chains in the sample to the linker molecules; B) Apply a voltage across the nanopore so that the sugar chains in the sample pass through the nanopore and through the sensing area; C) Obtain the electrical signal characteristics of the sugar chains; D) Determine the characteristics of sugar chains through electrical signal features. The detection method according to claim 95 includes qualitative and / or quantitative analysis; preferably, qualitative and / or quantitative analysis of glycan characteristics. According to the detection method of claim 95 or 96, the sample to be tested contains a sugar chain with a consistent sequence. According to the detection method of claim 95 or 96, the sample to be tested contains two or more sugar sequences, and the mass or concentration ratios of the different sugar sequences are the same or different. A method for controlling the speed at which sugar chains pass through nanopores, the method comprising: A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates; B) By applying a voltage across the nanopore, the motor protein regulates the movement of the linker molecules within the nanopore, thereby controlling the speed at which the connected sugar chains pass through the nanopore. A method for constructing a database of electrical signals characteristic of glycans, the method comprising establishing a correspondence between glycan features and electrical signals measured by the glycans through a nanopore sensing region, wherein, The electrical signals measured by the glycan chain through the nanopore sensing region include: A) Connect sugar chains to linker molecules to obtain linker molecule-sugar chain conjugates; B) Apply a voltage across the nanopore to allow the sugar chains to pass through the nanopore and the sensing region; C) Obtain the electrical signal characteristics of the sugar chain. According to the method of claim 100, 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, position of modification, number of modifications, whether there are branches, position of branches, number of branches or branch sequence characteristics. According to the method of claim 101, 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, position of modification, and number of modifications. The method according to any one of claims 100-102, wherein the glycan feature is known, and the obtained electrical signal feature is marked as a specific glycan feature. According to any one of claims 100-103, 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. A database of characteristic electrical signals of sugar chains obtained by the method of any one of claims 100-104. A method for analyzing electrical signals that characterize sugar chains to determine sugar chain sequences, the method comprising performing step signal analysis on the electrical signals obtained by the method of any one of claims 1-92. According to the method of claim 106, the step signal analysis is performed using manual methods or algorithms. According to the method described in claim 107, the manual method can be either completely manual analysis or analysis performed with the aid of software. According to the method of claim 107, the algorithm analysis is based on multiple comparison analysis of glycan electrical signals, analyzes and extracts its electrical signal features, determines and classifies the steps through a built-in statistical model to obtain step signals, and compares the obtained step signals with glycan step signals in the database based on the comparison model to determine the glycan sequence. According to the method of claim 106, the step signal analysis includes, but is not limited to, step division, step feature analysis, or step parameter extraction. According to the method of claim 110, the parameters include, but are not limited to, amplitude, time course, or standard deviation. According to the method of claim 106, the analyzed step signal is compared with a known database signal to determine the glycan sequence. According to the method of claim 106, the analyzed step signal is compared with the glycan characteristic electrical signal database obtained by the method of claim 105 to determine the glycan sequence. According to any one of claims 106-113, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives in the sugar chain. According to the method of claims 106-113, the sugar chain sequence is the arrangement of monosaccharides and / or their derivatives and glycosidic bonds in the sugar chain. According to the method of claims 106-113, the glycan sequence is the arrangement or order of occurrence of glycan features in the glycan. A method for analyzing electrical signals to quantify glycan characteristics, the method comprising performing step signal analysis on the electrical signals obtained by the method of any one of claims 1-92.

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