Probe and application thereof

Abstract

The invention discloses a probe, the structure of the probe is a lectin-linker-metal chelate, and lectin is selected from one of SNA, MAL-II, PHA-E, PHA-L, PSA, UEA-I, LTL, GSL-II, PNA, VVL, GSL-IB4 and SBA. The invention also provides a probe group. The invention provides an identification method of single-cell glycomics, a detection method of sugar chain distribution on a biological tissue, application of a probe or a probe group in preparation of a kit for detecting single-cell glycomics, application of the probe or the probe group in preparation of a kit for detecting sugar chain distribution and structural characteristics on the biological tissue, and application of the probe or the probe group in preparation of a kit for detecting sugar chain distribution and structural characteristics on the biological tissue. The invention relates to application of a probe or a probe group in preparation of a diagnostic reagent or medicine for diseases caused by abnormal glycosylation.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to a probe and its uses. Background Art

[0002] Glycosylation is an important post-translational modification process. Glycan chains can be modified on proteins, lipids, and RNAs, and are widely involved in various important biological processes. Since glycosylation modifications have significant specific distributions at the tissue and cell levels, single-cell analysis techniques are very important for analyzing the functions of glycan chains. Currently, the most representative high-throughput single-cell techniques are single-cell sequencing technology and mass cytometry technology. Mass cytometry technology consists of two parts: a flow cytometry system and an ICP-MS detection system. After labeling antibodies with metal ion tags, this technology can simultaneously detect dozens of indicators at the single-cell level. However, at the single-cell level, there are few reports on high-throughput glycan analysis techniques. Currently, there are only 5 lectin indicators developed based on mass cytometry technology, and the recognized glycan chains overlap. There are no reports on high-throughput glycan analysis based on mass spectrometry imaging technology. Summary of the Invention

[0003] Based on this, this application provides a probe and a probe group for high-throughput glycan analysis, and uses the probe or probe group to construct a systematic glycan recognition method for single-cell glycomics identification and disease-assisted diagnosis.

[0004] This application provides the following technical solutions.

[0005] 1. A probe, wherein the structure of the probe is lectin-linker-metal chelate,

[0006] Among them, the lectin is selected from AAA, AAL, ABA, ABL, ACG, ACL, AMA, ASA, BanLec, BC2L-A, BC2LCN, BPA, CA, CAA, Calsepa, CGL2, CNL, ConA, CPA, CSA, DBA, Discoidin I, Discoidin II, DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S, Gal7-S, Gal9, GHA, GNA, GRFT, GS-I, GS-II, HAA, HHA, HMA, IRA, Jacalin, LAL, LBA, LCA, LEA, Lentil, Lotus, LPA, LSL-N, MAL-I, MAL-II, Malectin, MNA-G, MNA-M, MOA, MPL, NPA, Orysata, PA-IIL, PA-IL, PALa, PHA-E, PHA-L, PHA-P, PNA, PPL, PSA, PSL1a, PTL-1, PTL-2, PWA, RCA-120, RCA-60, RPA, RS-Fuc, SAMB, SBA, SHA, SJA, SNA, SNA, SSA, STL, TL, UDA, UEA-I, UEA-II, VFA, VRA, VVA, VVA-M, WFA, WGA, or other mannose-binding proteins, galactose-binding proteins, sialic acid-binding proteins, fucose-binding proteins, N-acetylglucosamine-binding proteins, N-acetylgalactosamine-binding proteins, glucose-binding proteins, xylose-binding proteins, uronic acid-binding proteins, oligosaccharide-binding proteins (such as binding lactose, maltose, blood group sugars), polysaccharide-binding proteins (such as binding cellulose, starch, glycosaminoglycan), etc., and one of them.

[0007] 2. The probe according to item 1, wherein the metal chelate has the following structure:

[0008]

[0009] M is Eu, La, Pr, Pm, Gd, Cd, Y, Bi, Pt, In, Sm, Zr, Ag, Ta, Nd, Ho, Tb, Gd, Dy, Yb, Tm, Pd, Cs, Co, Sr, Mo or Er.

[0010] 3. The probe according to item 1, wherein the linker has the following structure:

[0011]

[0012] 4. The probe according to item 1, wherein the probe has the following structure:

[0013] where M is 151 Eu, 138 La, 140 Pr, 157 Gd, 112 Cd, 88 Y, 208 Bi, 195 Pt, 114 In, 151 Sm, 91 Zr, 108 Ag, 181 Ta, 146 Nd, 165 Ho, 167 Er, 159 Tb, 156 Gd, 163 Dy, 153 Eu, 160 Gd, 173 Yb, 169 Tm or 170 Er.

[0014] 5. A probe group, comprising at least two probes, wherein the probe is the probe according to any one of items 1-4.

[0015] 6. The probe group according to item 5, wherein the metals in each of the probes in the probe group are all different.

[0016] 7. The probe group according to item 5, comprising:

[0017] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0018] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, or

[0019] SNA-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0020] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0021] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0022] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0023] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0024] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0025] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0026] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0027] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0028] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, SBA-linker-metal chelate, or

[0029] MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate; or

[0030] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate; or

[0031] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, or

[0032] SNA-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, PNA-linker-metal chelate.

[0033] 8. A method for identifying single-cell glycomics, comprising the following steps:

[0034] Staining cells with the probe described in any one of Items 1-4 or with the probe group in any one of Items 5-7 to obtain a sample to be detected;

[0035] Detecting the sample to be detected by mass cytometry or mass spectrometry imaging technology.

[0036] 9. A method for detecting the glycan distribution on biological tissues, comprising the following steps:

[0037] Staining biological tissues with the probe described in any one of Items 1-4 or with the probe group in any one of Items 5-7 to obtain a sample to be detected;

[0038] Detecting the sample to be detected by mass cytometry or mass spectrometry imaging technology.

[0039] 10. Use of the probe described in any one of Items 1-4 or the probe group in any one of Items 5-7 in the preparation of a kit for detecting single-cell glycomics.

[0040] 11. Use of the probe according to any one of items 1 - 4 or the probe group in any one of items 5 - 7 in the preparation of a kit for detecting the glycan distribution and structural characteristics on in vivo or ex vivo biological tissues.

[0041] 12. Use of the probe according to any one of items 1 - 4 or the probe group in any one of items 5 - 7 in the preparation of a diagnostic reagent or drug for diseases caused by abnormal glycosylation.

[0042] 13. The use according to item 12, wherein the diseases are immune system diseases such as systemic lupus erythematosus, rheumatoid arthritis, hematological diseases such as leukemia, lymphoma, solid tumors such as liver cancer, pancreatic cancer, lung cancer, respiratory system diseases such as asthma, chronic obstructive pulmonary disease, digestive system diseases such as gastric ulcer, colitis, cholecystitis, endocrine system diseases such as diabetes, obesity, thyroid diseases, nervous system diseases such as cerebral infarction, neurodegenerative diseases, cardiovascular diseases such as atherosclerosis, coronary heart disease, infectious diseases such as bacterial, viral, fungal, parasitic, mycoplasma infections, etc., urinary system diseases such as nephritis, IgA nephropathy, reproductive system diseases such as infertility, endometriosis, inflammatory diseases such as osteoarthritis, colitis, cancer, genetic diseases related to sugar synthesis and degradation, etc.

[0043] 14. The probe according to any one of items 1 - 4 or the probe group in any one of items 5 - 7 is used in combination with the detection of other omics.

[0044] Further, the other omics are single - cell transcriptome, single - cell epigenome, proteome, metabolome, spatial transcriptome, spatial metabolome.

[0045] For the probe provided in the present application, since the lectin in its structure can specifically recognize glycans, and the metal in its structure can be detected by mass cytometry technology and mass spectrometry imaging technology, the probe can be used for single - cell glycomics identification and can also detect the glycan distribution at different biological tissue levels.

[0046] For the probe group provided in the present application, the lectins in the probe group can recognize different glycans, and there is no interference between the lectins. It can not only be used for single - cell glycomics identification, but also, by combining with mass cytometry imaging technology, can systematically and comprehensively display the glycan distribution differences at different tissue levels in the human body. Through the mass cytometry imaging system, it is found that glycans can distinguish different important structures in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 a shows the preparation flow chart of the lectin - metal probe.

[0048] Figure 1b shows the box plot of the metal intensity of different lectins.

[0049] Figure 2 a shows the N-glycan processing pathway in mammalian cells.

[0050] Figure 2 b shows the correlation heat map of the lectin binding signal and Z-score of 293T cells after treatment with glycosylation inhibitors and neuraminidase.

[0051] Figure 2 c shows the density plot of the lectin intensity of 293T cells.

[0052] Figure 3 Co-staining with lectin does not hinder the antibody signal intensity.

[0053] Figure 4 Cluster analysis of mouse immune cells using lectin and antibody is shown.

[0054] Figure 5 The widespread distribution of glycans in various tissue types is shown.

[0055] Figure 6 The results of the UMAP comprehensive analysis performed with 12 lectins are shown.

[0056] Figure 7 HE images and imaging mass spectrometry images of cervical squamous epithelium (a-c), low-grade squamous intraepithelial lesion (d-f), high-grade squamous intraepithelial lesion (g-i), and carcinoma (j-l) of different patients are shown.

[0057] Figure 8 The heat map of the glycan array of 16 lectins is shown.

[0058] Figure 9 The situation of the direct binding crosstalk of lectins investigated by the lectin array chip is shown.

[0059] Figure 10 It is shown that after blocking with mannan, the binding of PSA to other lectins is weakened.

[0060] Figure 11 The high labeling efficiency of lectin is shown.

[0061] Figure 12 The heat map of the lectin binding intensity of different cell lines is shown.

[0062] Figure 13 The t-SNE map of immune cells in the mouse spleen is shown.

[0063] Figure 14 The heat map of the lectin intensity of different tissue structures is shown.

[0064] Figure 15 It shows the distribution pattern of sialylation in various organs.

[0065] Figure 16 It shows the glycan characteristics and distribution in skin tissue.

[0066] Figure 17 It shows the glycan characteristics and distribution in kidney tissue.

[0067] Figure 18 It shows the glycan characteristics and distribution in mammary gland tissue.

[0068] Figure 19 It shows the glycan characteristics and distribution in bronchial tissue.

[0069] Figure 20 It shows the glycan characteristics and distribution in colon tissue.

[0070] Figure 21 It shows the glycan characteristics and distribution in cervical squamous epithelial tissue.

[0071] Figure 22 It shows the UMAP analysis of different cell groups of different patients.

[0072] Figure 23 It shows the cell clustering in low-grade squamous intraepithelial lesion tissue.

[0073] Figure 24 It shows the cell clustering in high-grade squamous intraepithelial lesion tissue. Detailed implementation manners

[0074] The following describes exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding. It should be considered merely exemplary. Therefore, those of ordinary skill in the art should recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, the description of well-known functions and structures is omitted below.

[0075] The present application provides a probe, wherein the structure of the probe is a lectin-linker-metal chelate,

[0076] Among them, the lectin is selected from AAA, AAL, ABA, ABL, ACG, ACL, AMA, ASA, BanLec, BC2L-A, BC2LCN, BPA, CA, CAA, Calsepa, CGL2, CNL, ConA, CPA, CSA, DBA, Discoidin I, Discoidin II, DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S, Gal7-S, Gal9, GHA, GNA, GRFT, GS-I, GS-II, HAA, HHA, HMA, IRA, Jacalin, LAL, LBA, LCA, LEA, Lentil, Lotus, LPA, LSL-N, MAL-I, MAL-II, Malectin, MNA-G, MNA-M, MOA, MPL, NPA, Orysata, PA-IIL, PA-IL, PALa, PHA-E, PHA-L, PHA-P, PNA, PPL, PSA, PSL1a, PTL-1, PTL-2, PWA, RCA-120, RCA-60, RPA, RS-Fuc, SAMB, SBA, SHA, SJA, SNA, SNA, SSA, STL, TL, UDA, UEA-I, UEA-II, VFA, VRA, VVA, VVA-M, WFA, WGA, or other mannose-binding proteins, galactose-binding proteins, sialic acid-binding proteins, fucose-binding proteins, N-acetylglucosamine-binding proteins, N-acetylgalactosamine-binding proteins, glucose-binding proteins, xylose-binding proteins, uronic acid-binding proteins, oligosaccharide-binding proteins (such as binding lactose, maltose, blood group sugars), polysaccharide-binding proteins (such as binding cellulose, starch, glycosaminoglycan), etc., and one of them.

[0077] Furthermore, the metal chelate has the following structure:

[0078] M is Eu, La, Pr, Pm, Gd, Cd, Y, Bi, Pt, In, Sm, Zr, Ag, Ta, Nd, Ho, Tb, Gd, Dy, Yb, Tm, Pd, Cs, Co, Sr, Mo or Er.

[0079] Furthermore, the linker has the following structure:

[0080]

[0081] Furthermore, the probe has the following structure:

[0082] Where M is M is 151 Eu,138 La, 140 Pr, 157 Gd, 112 Cd, 88 Y, 208 Bi, 195 Pt, 114 In, 151 Sm, 91 Zr, 108 Ag, 181 Ta, 146 Nd, 165 Ho, 167 Er, 159 Tb, 156 Gd, 163 Dy, 153 Eu, 160 Gd, 173 Yb, 169 Tm or 170 Er.

[0083] This application provides a probe group, which includes at least two probes, and the probes are the probes described in any one of items 1-4.

[0084] Furthermore, the metals in each of the probes in the probe group are all different.

[0085] Furthermore, the probe group includes:

[0086] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0087] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, or

[0088] SNA-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0089] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0090] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0091] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0092] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0093] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0094] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0095] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0096] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or

[0097] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, SBA-linker-metal chelate, or

[0098] MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate; or

[0099] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate; or

[0100] SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, or

[0101] SNA-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, PNA-linker-metal chelate.

[0102] This application provides a method for identifying single-cell glycomics, including the following steps:

[0103] Staining cells with the aforementioned probe or the aforementioned probe group to obtain a sample to be detected;

[0104] Detecting the sample to be detected using mass cytometry or mass spectrometry imaging technology.

[0105] This application provides a method for detecting the glycan distribution on biological tissues, which includes the following steps:

[0106] Staining biological tissues with the aforementioned probe or the aforementioned probe group to obtain a sample to be detected;

[0107] Detecting the sample to be detected using mass cytometry or mass spectrometry imaging technology.

[0108] Hyperion Tissue Mass Spectrometry Imaging System

[0109] This system uses cutting-edge high-tech technology - Imaging Mass Cytometry technology to maximize the advantages of the ultra-high detection channel number of mass cytometry (CyTOF) in tissue imaging research, and its performance far exceeds that of traditional immunohistochemistry or immunofluorescence technology. Hyperion TM The tissue mass spectrometry imaging system has up to 135 detection channels. Currently, through a single scan, image data of 4 - 37 glycan and protein markers on tissue section samples can be obtained, accurately reflecting comprehensive information in many aspects such as the phenotypes and mutual relationships of glycans, proteins, and corresponding cells in the tissue microenvironment. Especially for some precious FFPE samples or frozen tissue sections, more in-depth analysis can be carried out at the subcellular level.

[0110] Mass cytometry (CyTOF) uses lectins conjugated with metal isotopes as tags. Lectins can specifically recognize glycans, and the metals in their structures can be detected by mass cytometry and mass spectrometry imaging techniques. Therefore, the probes can be used for single-cell glycomics identification and can also detect the glycan distribution at different biological tissue levels. Compared with traditional fluorescence flow cytometry, the advantages of mass cytometry are as follows: 1. Interference between channels can be ignored, and no compensation calculation is required. 2. More than 40 glycan and protein indicators can be detected in a single run, enabling fine cell typing.

[0111] This application provides the use of the aforementioned probe or probe group in the preparation of a kit for detecting single-cell glycomics.

[0112] This application provides the use of the aforementioned probe or probe group in the preparation of a kit for detecting the glycan distribution and structural characteristics on in vivo or ex vivo biological tissues.

[0113] This application provides the use of the aforementioned probe or probe group in the preparation of a diagnostic reagent or drug for diseases caused by abnormal glycosylation.

[0114] The diseases include immune system diseases, hematological diseases, solid tumors, respiratory diseases such as asthma and chronic obstructive pulmonary disease, digestive system diseases such as gastric ulcer, colitis, and cholecystitis, endocrine system diseases such as diabetes, obesity, and thyroid diseases, nervous system diseases such as cerebral infarction and neurodegenerative diseases, cardiovascular diseases such as atherosclerosis and coronary heart disease, infectious diseases such as bacterial, viral, fungal, parasitic, and mycoplasma infections, urinary system diseases such as nephritis and IgA nephropathy, reproductive system diseases such as infertility and endometriosis, inflammatory diseases such as osteoarthritis and colitis, cancers, and genetic diseases related to sugar synthesis and degradation.

[0115] The immune system is selected from rheumatoid arthritis, systemic lupus erythematosus, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, IgG4-related pancreatitis, cholangitis, kidney diseases, autoimmune hematological diseases, autoimmune skin disease areas psoriasis, vitiligo, or autoimmune vascular system diseases.

[0116] Hematological diseases such as leukemia and lymphoma.

[0117] Solid tumors such as, but not limited to, adrenocortical carcinoma, carcinoma of unknown primary site in adults, adult malignant mesothelioma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytoma (cerebellar astrocytoma, cerebral astrocytoma, childhood cerebral astrocytoma, pineal astrocytoma), basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumors, fallopian tube cancer, brain cancer, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, carcinoma of unknown primary, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epitheloid hemangioendothelioma (EHE), esophageal cancer, Ewing tumor sarcoma family, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, colorectal cancer, head and neck cancer, heart cancer, liver cancer, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphoblastic leukemia, chronic lymphocytic leukemia (also known as chronic lymphoid leukemia), chronic myelogenous leukemia (also known as chronic myeloid leukemia), hairy cell leukemia, lip cancer, liposarcoma, lung cancer (non-small cell lung cancer, small cell lung cancer), lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma, AIDS-related lymphoma, Burkitt lymphoma, central nervous system lymphoma, cutaneous T-cell lymphoma), macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, Merkel cell carcinoma, occult primary metastatic squamous carcinoma of the neck, multiple endocrine neoplasia syndrome, myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myxoma, nasal and paranasal sinus cancer, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer (hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer), pheochromocytoma, pineal germinoma, pineoblastoma, supratentorial primitive neuroectodermal tumor, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, vaginal cancer, vulvar cancer.

[0118] The present application also provides a combined application of the aforementioned probe or the aforementioned probe group with other omics detections.

[0119] The other omics are single-cell transcriptome, single-cell epigenome, proteome, metabolome, spatial transcriptome, spatial metabolome.

[0120] Examples

[0121] The materials and test methods used in the examples of this application are described generally and / or specifically. In the following examples, unless otherwise specified, % represents wt%, that is, weight percentage. Reagents or instruments without the manufacturer indicated are all conventional reagent products that can be obtained through commercial purchase.

[0122] Materials and Reagents: Lectins (SNA, MAL-II, PHA-E, PHA-L, ConA, PSA, UEA-I, LTL, GSL-II, PNA, VVL, AAL, GSL-IB4, WGA, SBA, ECL) were purchased from Vectorlab; X8 antibody labeling kit, cell staining buffer, and Perm and Fixed buffers were purchased from Fluidigm (USA); antibodies were purchased from Biolegend (USA); SATA (N-succinimidyl S-acetylthioacetate) was purchased from DB (China). Neuraminidase (Arthrobacter ureafaciens NanH) was obtained from Escherichia coli.

[0123] High Performance Liquid Chromatography Analysis: High performance liquid chromatography (HPLC) (waters XBridge BEH 200 ASEC 3.5um 7.8×300) was used for labeling efficiency analysis. The mobile phase consisted of a buffer solution containing 100 mM sodium phosphate and 150 mM sodium chloride, and the pH was adjusted to 6.8. Samples with a concentration of 1 mg / ml were prepared, and an injection volume of 20 μl was used for each analysis. The flow rate was set at 0.8 ml / min, and throughout the analysis, the column temperature was maintained at 30 °C. Isocratic elution was used for 60 minutes. An UV detector was used to detect the analyte at a wavelength of 215 nm.

[0124] Example 1 Preparation of Lectin Probes

[0125] Example 1-1

[0126] Figure 1 a is the flow chart for the preparation of lectin probes, Figure 1 b is the box plot of the metal intensity of 15 different lectins. Figure 8 is the heat map of the glycan array of 16 lectins. As shown in Figure 1 a, N-succinimidyl S-acetylthioacetate (SATA) introduced a thiolacetate group onto the lysine residue of the lectin. After deacetylation, the lectin was covalently bound to the metal-loaded polymer through the reaction of the thiol group and maleimide. This process successfully labeled various lectin species with different metals.

[0127] To prepare the SATA-modified lectin, first, the lectin (SNA) was diluted to a working concentration of 10 μM and dissolved in 1 mL of the prepared lectin buffer. In the buffer, 500 μM of N-Succinimidyl-S-acetylthioacetate (SATA) was added, and a coupling reaction was carried out at room temperature for 2 hours to ensure the effective binding between the lectin and SATA. Using a 30 kDa Biomax Ultrafree molecular weight ultrafiltration tube and combined with three buffer washes, the unreacted SATA was thoroughly removed, thus ensuring the specificity of the reaction and the high purity of the final product. Then, to introduce thiol groups, 1 mL of the lectin-SATA solution was mixed with 100 μL of 0.5 M hydroxylamine for deacetylation. Finally, using the X8 antibody labeling kit provided by Fluidigm Corporation, efficient coupling of the modified lectin with metal chelates was achieved, thus obtaining the lectin probe described in this application, and its structure is as follows:

[0128] The lectin is SNA, and M is 142 Nd.

[0129] Using Fluidigm's X8 antibody labeling kit, IgG antibodies or the lectin of this application can be coupled with specific metal isotopes, such as 142 Nd. In this example, the lectin-SATA was used as an intermediate product, and a stable binding with metal chelates was formed through the method provided by the kit, thus obtaining the probe for CyTOF analysis. Metal chelates generally refer to complexes formed through the coordination between metal ions and ligands. These ligands can be various organic molecules, such as nitrogen-containing, oxygen-containing, or sulfur-containing compounds, which can form multiple coordination bonds with metal ions. Specifically, which metal's metal chelate it is depends on the types of ligands and metal ions used. For example, it can be chelates formed by metal ions such as copper, nickel, iron, or zinc with corresponding ligands.

[0130] The metal tag in the probe endows the probe with a unique signal in CyTOF analysis. In this way, the probe can be used to detect and quantify various proteins, sugars, and other biomolecules on the cell surface or inside at the single-cell level, thus providing detailed information about the cell state and function.

[0131] The difference between Example 1-2 and Example 1-1 is that the types of lectin and the metal in the metal chelate are different. The lectin in this example is MAL-II, and the metal in the metal chelate is 146 Nd.

[0132] Examples 1-3 are different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is PHA-E, and the metal in the metal chelate is 165 Ho

[0133] Example 1-4 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is PHA-L, and the metal in the metal chelate is 167 Er.

[0134] Example 1-5 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is PSA, and the metal in the metal chelate is 159 Tb.

[0135] Example 1-6 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is UEA-I, and the metal in the metal chelate is 156 Gd.

[0136] Example 1-7 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is LTL, and the metal in the metal chelate is 163 Dy.

[0137] Example 1-8 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is GSL-II, and the metal in the metal chelate is 153 Eu.

[0138] Example 1-9 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is PNA, and the metal in the metal chelate is 160 Gd.

[0139] Example 1-10 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is VVL, and the metal in the metal chelate is 173 Yb.

[0140] Example 1-11 is different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is GSL-IB4, and the metal in the metal chelate is 169 Tm.

[0141] Examples 1-12 are different from Example 1-1 in that the types of lectins and metals in the metal chelates are different. The lectin in this example is SBA, and the metal in the metal chelate is 170 Er.

[0142] From Figure 1 b and Figure 11 it can be seen that various lectin species labeled with metals reached an intensity of more than 5×10 6 , demonstrating the high labeling efficiency of lectins.

[0143] Specificity of the probe in Example 2

[0144] The lectin array chip can examine the direct binding crosstalk of lectins. Figure 9 a is the heat map of the lectin array chip, Figure 9 b is the bar chart of the lectin array chip. As Figure 9 the results show, lectins PSA, AAL, and ConA can recognize a variety of other lectins, suggesting that these lectins carry high-mannose-type sugar chains with core α1,3-fucose, and thus are recognized, which affects the application of these lectins as a probe set. To reduce the influence, mannan was used as a blocking agent for blocking treatment. Figure 10 The results show that after blocking with mannan, the binding of PSA to other lectins is weakened.

[0145] To clarify the specificity of the binding of metal-labeled lectins, glycosylation inhibitors and neuraminidase were used to treat 293T cells to change the sugar chain structure for analysis. By using the inhibitors kifunesine and Swainshine of mannosidase I and mannosidase II to reduce complex N-glycans, including N-glycans containing GlcNAc and three-arm and four-arm branches ( Figure 2 a). To inhibit glycosylation, the 293T cell line was incubated with DMEM containing kifunesine (1 μg / ml), Swainshine (1 μ / ml), or 2-deoxy-2-fluoro-L-fucose (1 μg / ml) for 48 hours. For neuraminidase treatment, the cells were incubated in 20 μg / ml neuraminidase in PBS at 37 °C for 2 hours. Subsequently, the cells were collected, washed with PBS, stained with the lectin probes prepared in Examples 1-1, 1-3 to 1-5, 1-9, and analyzed by machine. The results are as Figure 2 b. From Figure 2 a- Figure 2 b, it can be seen that treatment with kifunesine and Swainshine can significantly lead to a significant decrease in the binding signals of PHA-E (recognizing GlcNAc bisecting on N-glycans) and PHA-L (targeting three-arm and four-arm N-glycans) (Figure 2 b). Kifunensine effectively blocked the synthesis of α2,6-sialic acid (detected by SNA) and core fucose (detected by PSA), while Swainshine had no effect on sialic acid and fucose modifications ( Figure 2 a-b). In addition, inhibition of fucosyltransferase using 2F-peracetylated fucose (a cell-permeable fluorinated fucose derivative) led to a significant reduction in core fucose signal (detected by PSA) ( Figure 2 a-b). Treatment with sialidase removed α2,6-sialic acid from the glycan structure, reducing SNA recognition and increasing the terminal galactose recognized by PNA Figure 2 b), thus, this method showed high specificity in glycan analysis.

[0146] To evaluate the impact of glycan stability on analysis during cell cryopreservation, 293T cells were cryopreserved at -80 °C at different time intervals and analyzed simultaneously. The research results are as Figure 2 c. After cryopreservation for one month, the SNA signal (a marker of α2,6-sialic acid) decreased slightly, but there was no significant difference between one month and three months of storage. The signals of PSA, PHA-E, PHA-L, SBA, and VVL decreased slightly after cryopreservation for one month, however, the signal decrease was more significant after three months. These results emphasize that glycan analysis of cryopreserved cell samples within one month can ensure data accuracy as much as possible.

[0147] Example 3 Glycome Study

[0148] Cell sample preparation: The glycan characteristics of cells were identified by the lectin probes of this application. All cell lines, including RAW264.7, 293T, HeLa, A549, HepG2, 4T1, Vero, THP-1, B16, and Hepa-1G, were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. Subsequently, they were stained with the lectin probes prepared in Example 1 and analyzed by machine. To isolate immune cells from mice, the animals were euthanized using an appropriate method, and the spleens were removed using sterile instruments. The spleens were placed in a sterile petri dish and rinsed with sterile PBS. The spleens were transferred to a 50 mL conical tube and connected to a 75 μm filter. The tissue was ground while adding 10 mL of DMEM for washing. The cell suspension was centrifuged at 400×g at 4 °C for 10 minutes, and the supernatant was carefully discarded. The cell pellet was resuspended in 5-10 mL of PBS. To determine the number of cells obtained, a hemocytometer or an automatic cell counter was used for counting. The isolated immune cells were used for subsequent lectin and antibody staining.

[0149] Cell staining and CyTOF loading procedure: 5 million cells were washed twice with metal-free PBS (biosharp), and then resuspended in PBS containing 25 μM cisplatin. The cells were incubated with cisplatin for 2 minutes, and then 2 ml of cell staining buffer (purchased from Fludigm) was added. The cells were washed three times with the cell staining buffer, and the cells were blocked with 50 μL of glycan blocking solution (0.1% Mannan and 0.1% BSA in the cell staining buffer solution) for 10 minutes. The cells were stained with 50 μL of lectin mixture (each lectin 1 μL diluted to a final volume of 50 μL in the cell staining buffer) for 30 minutes. For co-staining with antibodies, the cells were washed twice with the cell staining buffer, and then resuspended in 50 μL of blocking buffer (0.1% BSA and 0.1% IgG in the cell staining buffer solution) and blocked for 10 minutes. 50 μL of antibody mixture (1 μL of each antibody in the cell staining buffer, with a total volume of 50 μL) was added to the cell suspension and stained for 30 minutes. The cells were washed three times with the cell staining buffer, and then resuspended in 250 nM iridium internal standard compound (Fluidigm) in the fixation buffer at room temperature for 30 minutes or overnight at 4 °C. After that, the cells were washed twice with the cell staining buffer and water. Finally, the cells were resuspended in a cell collection solution at a concentration of 10 6 cells / ml. Before collecting CyTOF data, 1 / 9 volume of EQ calibration beads (Fluidigm) was added to the cell suspension. Cells were collected at a rate of 200 - 400 / s on a Helio-upgraded CyTOF2 instrument (Fluidigm).

[0150] Glycome study of different cells: Next, the lectome maps of 10 common cell lines (B16, 293T, HeLa, HepG2, RAW264.7, THP-1, A549, Hepa 1-6, Vero, 4T1) were analyzed using LectoScape. These cell lines exhibited different glycan expressions on their surfaces ( Figure 12 ). Notably, in specific human cancer cell lines, especially in the cervical cancer cell line HeLa, the lung adenocarcinoma cell line A549, and the hepatocellular carcinoma cell line HepG2, increased levels of complex N-glycans, core fucose, and sialylation were observed ( Figure 12)。Increased fucosylation and sialylation are associated with cell and immune evasion and promotion of metastasis in cancer. In single-cell glycomics, co-staining of lectins and antibodies is essential because antibodies enhance cell population separation and facilitate analysis of the glycan composition of immune cells. However, due to their glycoprotein nature and interaction with lectins, antibodies may introduce biases. To address this issue, cells are first stained with lectins, and then IgG proteins are used to block the free carbohydrate recognition domains on the lectins to ensure accuracy before antibody staining. Eleven immune cell markers and twelve lectins are used to characterize the immune cell glycome. The results are as Figure 3 shown, co-staining with lectins does not hinder antibody signal intensity.

[0151] Cluster analysis of mouse immune cells using lectins and antibodies is as Figure 4 and Figure 13 : Figure 4 a is a heatmap showing the peak intensities of clusters of mouse immune cells identified by SNN clustering. Figure 4 b and Figure 13 are both t-SNE plots showing different cell populations clustered by k-means. Subsequently, lectin signals are integrated to analyze different immune cell glycomes ( Figure 4 ). Lectins with high affinity for N-glycans (PHA-E, PHA-L, PSA, and SNA) show substantial binding to mouse immune cells. Notably, SNA has limited binding to Cd11b+ leukocytes (myeloid cells, dendritic cells), while PHA-E shows strong affinity for them ( Figure 4 a). This supports previous findings on the role of GlcNAc N-glycans in dendritic cell, neutrophil maturation, and immune responses. In addition, sialic acid on B cells plays a key role in their survival and anti-apoptosis. Ly-6G+ cells show high expression of terminal GalNAc and α2,3-sialylated Galβ1,3GalNAc ( Figure 4 a). When immune cells are grouped using lectin and antibody data, a unique B cell population with high levels of terminal GalNAc is identified, which is not obvious when using antibodies alone ( Figure 4 b). This suggests that certain B cells may produce IgA27 during differentiation, and the B cell group showing high expression of terminal GalNAc with N-glycosylation and mucin-type O-glycosylation is worthy of further study, highlighting the dynamic changes of glycans and their biological functions during immune cell differentiation.

[0152] Example 4 Glycan Distribution on the Surface of Biological Tissues

[0153] Sample preparation steps:

[0154] Paraffin-embedded tissue sections: Prepared in accordance with the principles outlined in the Declaration of Helsinki and its subsequent amendments. Informed consent for participation in this study was provided by all human subjects, and the research protocol was approved by the Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University (IRB number: 2017-11). First, the tissues were fixed in formalin, dehydrated in alcohol, and embedded in paraffin, and then tissue arrays were constructed. Then, the paraffin-embedded tissue blocks were trimmed to the required size for sectioning. The paraffin blocks were mounted on a microtome, and thin sections (4 μm thick) were cut using a sharp blade. Then, the sections were collected on glass slides, and the paraffin was removed by immersing the slides in xylene or other clearing agents.

[0155] HE staining: The tissue sections were placed in a laboratory oven at 65 °C for 1 hour. The slides were placed in xylene for 5 minutes and repeated 3 times to deparaffinize the sections. Then, the tissue sections were rehydrated in 100% ethanol, 95% ethanol, 80% ethanol for 3 minutes, and finally rehydrated in deionized water for 5 seconds. The sections were stained with hematoxylin for 3 minutes and washed with water for 5 minutes. And the sections were immersed in acidic ethanol 10 times. The section was rinsed 4 times with water. The sections were stained with eosin for 30 seconds. The slides were dehydrated in 95% ethanol, 100% ethanol for 5 minutes, and then placed in xylene for 15 minutes, 3 times in total. Finally, the section was covered with a coverslip by using each support.

[0156] Tissue preparation and antigen retrieval: After HE staining following dewaxing and rehydration, heat-induced epitope retrieval of the slides was performed in Tris-EDTA buffer (pH 9.2) using a pressure cooker (Medite) at 95 °C for 30 minutes or 80 °C for 80 minutes. The slides were cooled in Tris-EDTA buffer for 20 minutes, and then transferred to Tris-buffered saline (TBS) at room temperature for at least 20 minutes.

[0157] Mass spectrometry imaging: Subsequently, they were blocked with 0.1% Mannan, 0.1% BSA blocking buffer for 30 minutes. Then, the blocked samples were stained overnight at 4 °C with a 1:100 diluted mixture in the same blocking buffer. After staining, the samples were washed three times in PBST, 5 - 10 minutes for each wash. Subsequently, briefly incubated with iridium embedding agent diluted 1:500 in PBS for 2 - 5 minutes. After that, the tissue sections were washed three additional times in PBST, 5 minutes for each wash. Then, the samples were washed in ultrapure water and air-dried in preparation for IMC (Imaging Mass Cytometry) acquisition.

[0158] Data acquisition was performed on a Helios time-of-flight mass spectrometer (CyTOF) coupled with a Hyperion imaging system (Fluidigm). The selected ablation area was larger than the actual required area to account for the loss of overlapping area between slices due to cumulative rotation. The selected ablation area for each section was less than 0.5 mm 2 . Laser ablation was performed at a frequency of 200 Hz with a resolution of approximately 1 μm. All data were collected using commercial Fluidigm-CyTOF software.

[0159] Statistical analysis: The data were normalized to EQ calibration beads and then exported as FCS files. The CyTOF datasets were transformed by using arcsinh and cofactor 5. Cells obtained by CyTOF were identified by ordered gating using FlowJo software. Cells in IMC imaging were segmented using CellProfiler software. TSNE and UMAP analyses were performed using the R package Seurat. Cell clusters were identified by a clustering algorithm based on modular optimization of shared nearest neighbors (SNN). Hierarchical clustering was performed after the peak of the cell clusters, and k-means clustering was subsequently applied for further classification.

[0160] Imaging of different human tissues: The probes in Example 1 were used to analyze the glycome of tissue sections in different human organs, including colon, cervical squamous epithelium, skin, lung, kidney, breast, heart, liver, and brain. As Figure 5 revealed the widespread distribution of glycans in various tissue types and highlighted the changes in glycan patterns among them. Here, lectins that bind tightly to specific structures or cells within the tissue are highlighted and distinguished by different colors.

[0161] Higher lectin signals were observed in epithelial tissues compared to parenchymal tissues, as shown in Figure 14 , and epithelial cells have multiple functions and shapes, exhibiting a dense glycosylation coat. A cell subset with elevated terminal GalNAc expression was found, which showed an affinity for SBA binding and was represented in stone gray. These cells were present in various tissue types, including the granular layer of the skin ( Figure 5 a), intermediate cells in cervical squamous epithelial tissue ( Figure 5 b), epithelial cells in the mucosal lining of the colon ( Figure 5 c), ciliated cells in small airways ( Figure 5 d), distal convoluted tubules ( Figure 5 e), and glandular cells in breast tissue ( Figure 5 f). In stratified epithelia, such as skin and cervical tissues, the basal layer was found to highly express α2,3-sialylated Galβ1,3GalNAc on O-glycans (binding to MAL-II, Figure 5(highlighted in red in a-b). This finding generally indicates that glycosylation changes during the transition from the basal layer to the middle layer. The application of the lectin probe prepared in Example 1 in tissue sections effectively differentiates various structures within epithelial tissues and can serve as a biomarker for specific structures. Additionally, high expression of α1,2-fucose was observed in synapses of the brain tissue ( Figure 5 i), which is thought to regulate the release of neurotransmitters at synapses by preventing the rapid degradation of these proteins.

[0162] Sialylation is an important terminal modification that directly affects inflammation, immune responses, and susceptibility to pathogen infections. Inspired by these complex relationships, the distribution patterns of sialylation in various organs were explored through research, and the results are as Figure 14 - Figure 15 shown. Not all tissues show significant sialylation abundance. Compared with connective tissues and muscle cells in the liver, heart, and brain, epithelial cells, including those in the skin, colon, and lung, tend to exhibit a large amount of sialylation. Notably, human bronchioles are characterized by the presence of α2,6-linked sialic acid, which is a key receptor for the H5N1 virus. Additionally, examination of colon specimens showed higher concentrations of α2,6-linked sialic acid in columnar absorptive cells, while the abundance of α2,3-sialylated Galβ1,3GalNAc increased in the glandular lumen. The complex interaction between the gut microbiota and colon sialic acid levels has attracted extensive attention in the industry.

[0163] Use in disease diagnosis in Example 5

[0164] Distribution of glycans in disease diagnosis: To study the potential application of the lectin probe prepared in Example 1 in disease diagnosis, as Figure 16 - 21 shown, the image data was processed into a single-cell dataset, and the cells were divided into different subpopulations. Notably, the research results are as Figure 6 shown. The lectin can effectively distinguish various cell types present in cervical squamous epithelium, colon, and kidney tissue specimens. More specifically, the lectin probe prepared in Example 1 can clearly distinguish the basal layer within the cervical epithelial tissue ( Figure 6 a-c, Figure 21 ), an area of considerable significance in cervical cancer screening and diagnosis. Additionally, glandular cells in the colon tissue can be clearly distinguished ( Figure 6 d-f, Figure 20 ), and these cells are key indicators for the early detection of colorectal tumors. In kidney tissue, the glomerular region shows significant differences from other structural components ( Figure 6 g-i, Figure 17)。The glomerulus is often associated with various pathological conditions, such as glomerulonephritis, which is a key clinical and pathological manifestation of organ damage. Therefore, the lectin probe prepared in Example 1 has the ability to distinguish key anatomical features within the human body organs and is expected to be used for disease prediction and ultra-early diagnosis.

[0165] To further verify the application of the lectin probe prepared in Example 1 in disease diagnosis, patient samples were collected for analysis. Normal cervical squamous epithelial tissue sections from three different patients were analyzed to determine the glycan patterns therein. Through lectin signal examination, the results were as Figure 22 shown, and the cells were divided into four groups: basal cells, parabasal cells, intermediate cells, and superficial cells. The basal cells showed an increased expression level of α2,3-sialylated Galβ1,3GalNAc but a decreased terminal GalNAc. In contrast, the intermediate cells within the cervical epithelial tissue showed the highest abundance of terminal GalNAc glycans. Cervical intraepithelial neoplasia (CIN) is divided into three grades according to the degree of abnormal cell infiltration into the cervical epithelial tissue. Currently, the grading of CIN largely relies on histological assessment, which is prone to subjectivity in the judgment of pathologists. CIN 1 (mild dysplasia) is characterized by low-grade structural abnormalities involving atypical cells confined to the lower third of the epithelium, while CIN 2 and CIN 3 are associated with atypical squamous cells, affecting more than half of the epithelium.

[0166] Subsequently, the lectin probe of Example 1 was used to study cervical squamous cell carcinoma. As Figure 22 - 24 shown, terminal GalNAc and α2,3-sialylated Galβ1,3GalNAc represent the intermediate layer and basal cells within the cervical squamous epithelial cells, respectively. In Figure 7 , the distributions of terminal GalNAc and α2,3-sialylated Galβ1,3GalNAc at different stages of the cervical squamous epithelial tissue were shown. The basal cells are characterized by high expression of α2,3-sialylated Galβ1,3GalNAc and low expression of terminal GalNAc, and are mainly present in low-grade squamous intraepithelial lesions (LSILs), affecting the lower third of the epithelium ( Figure 7 d-f). In contrast, high-grade squamous intraepithelial lesions (HSILs) show the presence of basal cells, affecting more than half of the epithelium ( Figure 7 g-i). Cervical squamous cancer cells also express α2,3-sialylated Galβ1,3GalNAc and terminal GalNAc, thereby identifying them as parabasal-like cells, basal-like cells, and intermediate-like cells ( Figure 7j-l). Additionally, within basal cells, compared to LSIL (also known as CIN1), α2,3-sialylated Galβ1,3GalNAc, α2,6-sialic acid, and terminal GalNAc were significantly downregulated in HSIL ( Figure 7 m). Two glycan signals capable of differentiating various cells within cervical squamous epithelial tissue have been identified through research. These glycans were significantly downregulated in HSIL tissue compared to LSIL tissue. This finding implies that the probe of Example 1 can effectively distinguish different subtypes of CIN1, contributing to the identification of cases requiring early intervention.

[0167] Although the embodiments of the present application have been described above in conjunction with the accompanying drawings, the present application is not limited to the above specific embodiments and application fields. The above specific embodiments are merely illustrative and guiding, rather than restrictive. Those of ordinary skill in the art can also make many forms under the inspiration of this specification and without departing from the scope protected by the claims of the present application, and all of these fall within the scope of protection of the present application.

Claims

1. A probe, wherein, The structure of the probe is lectin-linker-metal chelate, wherein the lectin is selected from AAA, AAL, ABA, ABL, ACG, ACL, AMA, ASA, BanLec, BC2L-A, BC2LCN, BPA, CA, CAA, Calsepa, CGL2, CNL, ConA, CPA, CSA, DBA, Discoidin I, Discoidin II, DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S, Gal7-S, Gal9, GHA, GNA, GRFT, GS-I, GS-II, HAA, HHA, HMA, IRA, Jacalin, LAL, LBA, LCA, LEA, Lentil, Lotus, LPA, LSL-N, MAL-I, MAL-II, Malectin, MNA-G, MNA-M, MOA, MPL, NPA, Orysata, PA-IIL, PA-IL, PALa, PHA-E, PHA-L, PHA-P, PNA, PPL, PSA, PSL1a, PTL-1, PTL-2, PWA, RCA-120, RCA-60, RPA, RS-Fuc, SAMB, SBA, SHA, SJA, SNA, SNA, SSA, STL, TL, UDA, UEA-I, UEA-II, VFA, VRA, VVA, VVA-M, WFA, WGA, or other mannose-binding proteins, galactose-binding proteins, sialic acid-binding proteins, fucose-binding proteins, N-acetylglucosamine-binding proteins, N-acetylgalactosamine-binding proteins, glucose-binding proteins, xylose-binding proteins, uronic acid-binding proteins, oligosaccharide-binding proteins (such as those binding lactose, maltose, blood group sugars), polysaccharide-binding proteins (such as those binding cellulose, starch, glycosaminoglycan), etc., and one of them.

2. The probe according to claim 1, wherein The metal chelate has the following structure: M is Eu, La, Pr, Pm, Gd, Cd, Y, Bi, Pt, In, Sm, Zr, Ag, Ta, Nd, Ho, Tb, Gd, Dy, Yb, Tm, Pd, Cs, Co, Sr, Mo or Er.

3. The probe according to claim 1, wherein The linker has the following structure:

4. The probe according to claim 1, wherein, The probe has the following structure: where M is 151 Eu, 138 La, 140 Pr, 157 Gd, 112 Cd, 88 Y, 208 Bi, 195 Pt, 114 In, 151 Sm, 91 Zr, 108 Ag, 181 Ta, 146 Nd, 165 Ho, 167 Er, 159 Tb, 156 Gd, 163 Dy, 153 Eu, 160 Gd, 173 Yb, 169 Tm or 170 Er.

5. A probe group, wherein, Comprising at least two probes, and the probe is the probe described in any one of claims 1-4.

6. The probe group according to claim 5, wherein, The metals in each of the probes in the probe group are all different.

7. The probe group according to claim 5, wherein Comprising: SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, or SNA-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate, or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, SBA-linker-metal chelate, or MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate, GSL-IB4-linker-metal chelate, SBA-linker-metal chelate; or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, VVL-linker-metal chelate; or SNA-linker-metal chelate, MAL-II-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, UEA-I-linker-metal chelate, LTL-linker-metal chelate, GSL-II-linker-metal chelate, PNA-linker-metal chelate, or SNA-linker-metal chelate, PHA-E-linker-metal chelate, PHA-L-linker-metal chelate, PSA-linker-metal chelate, PNA-linker-metal chelate.

8. A method for identifying single-cell glycomics, wherein, Comprising the following steps: Staining cells with the probe described in any one of claims 1-4 or with a probe group in any one of claims 5-7 to obtain a sample to be detected; Detecting the sample to be detected by mass cytometry or mass spectrometry imaging technology.

9. A method for detecting the sugar chain distribution on biological tissues, wherein, Comprising the following steps: Staining a biological tissue with the probe described in any one of claims 1-4 or with a probe group in any one of claims 5-7 to obtain a sample to be detected; Detecting the sample to be detected by mass cytometry or mass spectrometry imaging technology.

10. Use of the probe described in any one of claims 1-4 or the probe group in any one of claims 5-7 in the preparation of a kit for detecting single-cell glycomics.

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