An aptamer combination applicable to single-cell sequencing and its application in constructing a cholangiocarcinoma cell atlas

Abstract

The present invention discloses a combination of different nucleic acid aptamers with 291 known targets. By performing sequencing analysis on the nucleic acid aptamer sequences of this combination through single-cell high-throughput sequencing technology, a cell atlas targeting mononuclear cells derived from the peripheral blood of patients with cholangiocarcinoma can be constructed. The nucleic acid aptamers covered by this combination have high specific affinity for their molecular targets. After extending adapters commercialized for sequencing platforms at both ends of the nucleic acid aptamer sequences respectively, by performing high-throughput sequencing on the nucleic acid aptamer sequences with sequencing adapters bound to the surface of mononuclear cells, high-throughput analysis of biomarkers on the surface of mononuclear cells derived from the peripheral blood of patients with cholangiocarcinoma can be achieved, thereby constructing a cell atlas of mononuclear cells derived from the peripheral blood of cholangiocarcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a nucleic acid aptamer combination suitable for single-cell sequencing and its application in constructing a cholangiocarcinoma cell atlas. Background Art

[0002] Cholangiocarcinoma is the second most common primary liver malignancy after hepatocellular carcinoma, including cholangiocarcinoma and gallbladder carcinoma. Cholangiocarcinoma is highly invasive, with insidious early clinical manifestations, rapid disease progression. Surgical resection usually cannot achieve satisfactory therapeutic effects, and it is insensitive to radiotherapy and chemotherapy, resulting in extremely poor prognosis for patients. Moreover, most patients have developed advanced and unresectable diseases at the time of diagnosis. Although certain progress has been made in the current cell atlas of cholangiocarcinoma, there are still defects such as difficulties in sample acquisition, tumor heterogeneity, insufficient research on the immune microenvironment, technical limitations, challenges in data integration and analysis, and heterogeneity of immunotherapy responses. There is an urgent need to develop new cholangiocarcinoma cell atlas construction technologies through technological innovation.

[0003] Single-cell high-throughput sequencing obtains sample information at the single-cell level, solving the problem that traditional sequencing technologies based on population cell sequencing mask cell-to-cell heterogeneity, and refining the research accuracy from population cells to individual cells, which helps to deeply explore cell heterogeneity and life activities. Among them, single-cell transcriptome sequencing (scRNA-seq) is the most widely used single-cell sequencing technology at present. It can reverse transcribe, amplify, and perform high-throughput sequencing on the mRNA of a single cell. According to the similarity of the transcription profiles, different cell types can be distinguished, and even new cell types can be revealed. Among them, cell atlas construction is an important application of scRNA-seq technology development and is a basic prerequisite for studying basic biology, aging, diseases, and treatment responses. From the market perspective, with the increasing demand for single-cell sequencing, many international sequencing companies have successively launched new-generation single-cell sequencing technologies, and there are more and more related research results. Single-cell sequencing technology is becoming a hot spot in gene sequence research. Since 2015, the emergence of technologies such as 10×Genomics, Drop-seq, Micro-well, and Split-seq has greatly reduced the cost of single-cell sequencing. Since then, single-cell high-throughput sequencing technology has been widely applied in basic scientific research and clinical research, which is of great significance for the early diagnosis, tracking, and individualized treatment of cancer.

[0004] However, the widely used scRNA-seq technology is difficult to capture all cells and genes in a tissue or organ, and the obtained data cannot provide complete cell information. At the same time, sequencing depth and scale, biological noise and technical noise will all limit the exploitable nature of scRNA-seq datasets. In addition, the transcriptome is only one of the many regulatory links that define cell types and states and ultimately functions. With the development and progress of technology, more and more omics technologies have been applied in the field of precision medicine. For example, transcriptomics, proteomics, metabolomics, microbiomics, lipidomics, immunomics, etc. can perform multi-omics analysis on single cells to define individual health at the molecular level. At the same time, one of the challenges in the field of precision medicine also includes the parallel detection of cell-to-cell heterogeneity in the genome, epigenome, transcriptome, and proteome. Linking these detection results has the potential to reveal the regulatory and functional mechanisms of cell behavior in healthy development and diseases. Therefore, integrating and analyzing multi-omics single-cell sequencing data can effectively make up for the limitations of single omics, collect more complete cell types and biological information, and help generate a panoramic cell atlas. The study of multi-omics helps to deepen the understanding of pathology and pharmacology, laying a good foundation for biopharmaceutical companies to conduct drug screening and efficacy evaluation.

[0005] Single-cell multi-omics has revolutionized the study of complex biological systems. By routinely measuring gene expression in thousands of cells, a cell atlas of the entire organism can be constructed. Currently, there is no research on the nucleic acid aptamer-based single-cell high-throughput sequencing technology to establish a large-scale correlation between transcriptome, metabolome, and microbiome data with treatment response and other clinical data, develop a multi-omics prediction model for the efficacy of immunotherapy combined with cholangiocarcinoma, and analyze the mechanisms of the identified key targets through in vitro and in vivo models.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide a nucleic acid aptamer combination suitable for single-cell high-throughput sequencing. Using this nucleic acid aptamer combination, multiple molecular targets on the cell membrane surface can be specifically recognized, thereby constructing a single-cell atlas based on nucleic acid aptamer recognition. In addition, in complex clinical blood samples, taking the peripheral blood mononuclear cells (PBMC) of cholangiocarcinoma patients as a model, this nucleic acid aptamer combination can achieve highly specific recognition of multiple biomarkers of cells in the blood, and is expected to provide research ideas for the molecular typing of complex blood samples in clinical practice and a basis for companion diagnosis based on peripheral blood.

[0008] The technical solution adopted by the present invention is specifically as follows:

[0009] In a first aspect, the present invention provides a nucleic acid aptamer combination suitable for single-cell high-throughput sequencing, and its core sequences include nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.291.

[0010] A nucleic acid aptamer is an oligonucleotide sequence about 15 - 60 nt in length, which has a series of advantages such as a wide target range, high affinity, strong specificity, easy synthesis, etc., and can accurately recognize metal ions, small molecules, amino acids, polypeptides, proteins and cells, and is known as the "antibody of chemists". As a class of recognition molecules, nucleic acid aptamers can be applied to molecular medicine to recognize disease markers and identify diseased cells, and are suitable for molecular probes and therapeutic drugs for clinical applications.

[0011] Compared with antibodies, nucleic acid aptamers have the following characteristics: (1) Nucleic acid aptamers are nucleic acid sequences and can directly chemically synthesize PCR primers and capture sequences for matching single-cell sequencing platforms, while antibodies require chemical coupling of DNA sequences; (2) The preparation ratio of antibody-DNA sequences is uncontrollable and the efficiency is uneven; (3) Nucleic acid aptamers have a low cost, while antibodies and coupling costs are relatively high; (4) Nucleic acid aptamers have a small molecular weight and almost no steric hindrance effect, while antibodies have a relatively large molecular weight and a steric hindrance effect; (5) Some membrane proteins are difficult to purify and corresponding antibodies cannot be prepared, but it is relatively easy to screen nucleic acid aptamers; (6) The nucleic acid aptamer enrichment library obtained by screening for specific samples has broad application prospects in the field of disease diagnosis and treatment.

[0012] The present invention uses single-cell high-throughput sequencing of nucleic acid aptamers bound to the cell membrane surface, aiming to establish a technology that can associate transcriptome, metabolome, and microbiome data with treatment responses and other clinical data with more than 290 targets, and realize the construction of a cell atlas. On the basis of the patent CN117552114A already applied in this laboratory, the coverage range of target detection is expanded, and a nucleic acid aptamer combination at the scale of hundreds is provided. The core sequences of the above nucleic acid aptamer combination include nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.291. Each nucleic acid aptamer has a high specific affinity for its target, and a clinical blood cell atlas can be constructed using the above nucleic acid aptamers.

[0013] For the design of the above nucleic acid aptamer combination, the difficulty lies in not only requiring each sequence to have a high specific recognition ability for its target, but also needing to overcome the problem of intermolecular interference in the scale of hundreds. Therefore, the inventor preferably combines the above nucleic acid aptamers, and the detection targets corresponding to each nucleic acid aptamer are as follows:

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020] In some embodiments, the nucleic acid aptamer combination further includes single-stranded DNA or its derivatives with nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.291 that have undergone substitution and / or deletion and / or addition of one or several nucleotides and have an identity of more than 80% with SEQ ID NO.1 to SEQ ID NO.291.

[0021] In some embodiments, the structure of each nucleic acid aptamer in the nucleic acid aptamer combination is: 5'-adapter sequence - core sequence - 3'-adapter sequence, and the core sequence is the nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.291.

[0022] In some embodiments, the 5'-adapter sequence is as shown in SEQ ID NO.292: TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTaaa; the 3'-adapter sequence is as shown in SEQ ID NO.293: AAAAAAAAAAAAAAAAAAAAAAAAA.

[0023] For the adapter sequences, in order to adapt to the BGI high-throughput sequencing platform, the above adapter sequences are selected in the present invention. However, it should be noted that the adapter sequences included in the present invention are not limited to the above sequences, and also include adapter sequences adapted to other platforms, which can be adjusted by those skilled in the art according to different platforms.

[0024] In a second aspect, the present invention also provides a kit, which includes the above nucleic acid aptamer combination.

[0025] In a third aspect, the present invention also provides the use of the above nucleic acid aptamer or kit in the analysis of cell membrane surface molecule expression levels or the construction of cell atlases.

[0026] In some embodiments, the above cells include human and murine cholangiocarcinoma cells.

[0027] Fourthly, the present invention also provides a method for detecting the expression level of cell surface proteins for non-diagnostic purposes, which includes: incubating a biological sample with the above nucleic acid aptamer combination, performing high-throughput single-cell sequencing on the transcriptome and nucleic acid aptamer library of the reaction system after sufficient reaction, and then performing bioinformatics analysis to obtain the molecular expression levels on the surfaces of different cell types.

[0028] In some embodiments, the above biological sample includes peripheral blood mononuclear cells of cholangiocarcinoma.

[0029] The present invention has the following beneficial effects:

[0030] The present invention provides a large-scale nucleic acid aptamer combination suitable for single-cell high-throughput sequencing. The preferably selected nucleic acid aptamers in this combination have high specific affinity for their targets, enhancing the reliability and accuracy of detection. In addition, combined with single-cell high-throughput sequencing technology, it can perform high-throughput specific recognition of different cell targets in complex clinical samples, thereby constructing a clinical blood cell map based on hundreds of nucleic acid aptamers at the scale level. The nucleic acid aptamer combination based on single-cell high-throughput sequencing used in the present invention is easy to synthesize and modify, can be mass-produced and applied, and has a lower synthesis cost and a wider application range compared with antibodies. Therefore, the nucleic acid aptamer single-cell high-throughput sequencing technology adopted has the characteristics of simple operation, low cost, and wide application range, which is of great significance for cell map construction, molecular diagnosis, research and development of targeted therapeutic drugs, and even clinical applications. Brief Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a schematic diagram of the principle of the single-cell proteomics analysis technology based on nucleic acid aptamer high-throughput sequencing of the present invention;

[0033] Figure 2 It is the total number of nucleic acid aptamers measured in each group (a), and the number of nucleic acid aptamers in each cell on average in each group (b) after incubating with nucleic acid aptamer combinations at different concentrations such as 0, 2, 20, 200, 1000, 2000 nM in Example 1;

[0034] Figure 3 It is the number of multiple nucleic acid aptamers that can specifically bind to cholangiocarcinoma cell lines analyzed in Example 1 when the incubation concentration is 200 nM;

[0035] Figure 4 For Example 2, 6 cases of peripheral blood mononuclear cells (PBMCs) from patients with cholangiocarcinoma were treated with the above nucleic acid aptamer combination, and up to 2,634 nucleic acid aptamers could be identified in each group;

[0036] Figure 5 For different cell clustering analysis (a) of PBMCs from 6 cholangiocarcinoma patients analyzed in Example 2; cell subset annotation (b); proportional bar chart showing differences in proportions between different cell types (c); the nucleic acid aptamer combination mainly binds to the MAIT T cell subset (d);

[0037] Figure 6 For the number of nucleic acid aptamers that can specifically bind to PBMCs of cholangiocarcinoma patients analyzed in Example 2. Detailed implementation mode

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0039] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0040] Example 1

[0041] This example provides a method for analyzing cell surface proteins using a nucleic acid aptamer-based single-cell high-throughput sequencing technology, and the schematic principle diagram is as Figure 1 shown. The specific operation method is as follows:

[0042] The isolated cholangiocarcinoma cell line was collected in a 15 mL centrifuge tube, centrifuged at 500 g for 5 min, the supernatant was discarded, and then washed twice with 1×DPBS. Finally, it was resuspended in Binding Buffer, and the viable and dead cells were counted using a cell counting chamber to determine the cell viability and cell concentration.

[0043] Take 1 million biliary cancer cells according to the concentration measured from the above samples and place them in 400 μL of Binding Buffer. Then add aptamer combinations with different total concentrations of 0, 2, 20, 200, 1000, 2000 nM, etc. (the 291 aptamers contained are at equimolar concentration), and immediately incubate on ice for 30 min, gently inverting the mixture up and down at intervals during this period. Subsequently, centrifuge at 800 g for 3 min at room temperature, discard the supernatant, wash with Washing Buffer, resuspend the cells with 300 μL of Washing Buffer, and count the live and dead cells using a cell counting chamber to determine the cell viability and cell concentration. Finally, take 22,000 cells according to the measured cell concentration for library preparation and high-throughput sequencing. The detailed steps are carried out according to the DNBSeq C series high-throughput single-cell RNA library preparation kit set provided by BGI and the instruction manual of the BGI sequencer.

[0044] It can be known through bioinformatics analysis that: as Figure 2 shown, in the group with an incubation concentration of 200 nM, 2634 aptamers can be identified in a single cell. Based on the above optimal incubation concentration, as Figure 3 shown (only part of the identification results are shown in the figure), multiple aptamers that can specifically bind to the biliary cancer cell line can be identified. The top 4 corresponding targets are: CD49c, PTPRF, alkaline phosphatase heterodimers, PTK7.

[0045] Example 2

[0046] This example provides a method for detecting the protein expression level of PBMC in a blood sample based on aptamers. The specific operation method is as follows:

[0047] 1. Isolation of PBMC in the blood sample

[0048] Centrifuge the fresh whole blood in the anticoagulant tube at 3000 rpm / min for 10 min, discard the upper plasma, supplement with 2% FBS DPBS to 20 mL, gently invert and mix, and then slowly add the diluted sample to the SepMate TM through the central small hole of the plug-in of the density gradient centrifugation liquid (Ficoll) TMIn a test tube (the upper end of the density gradient centrifugation solution should just cover the insert), centrifuge at 1200 g for 10 min at room temperature (for samples stored for more than 24 h, the recommended centrifugation time is 20 min). Then quickly transfer the buffy coat layer to a new test tube. This layer is rich in PBMC. Wash the PBMC with 10 mL of 2% FBS DPBS, centrifuge at 300 g for 8 min at room temperature, and discard the supernatant. If the above precipitate is light pink, red blood cell lysis may not be necessary. If the color is dark red, red blood cell lysis is required: Add 1×DPBS and red blood cell lysate (Solarbio) in a ratio of 1:3, gently pipette and mix well with a pipette tip, let stand on ice for 15 min, and gently invert the tube up and down at intervals. Centrifuge at 500 g for 5 min at 4°C to remove the supernatant; resuspend with an appropriate amount of Binding Buffer according to the cell density, and count the live and dead cells using a cell counting chamber to determine the cell viability and cell concentration.

[0049] 2. Incubation and loading of nucleic acid aptamers

[0050] Take 1 million cells in a 400 μL system according to the concentration measured above for the sample, add 200 nM nucleic acid aptamer aptamer (same as in Example 1), incubate on ice for 30 min, gently invert the tube up and down at intervals, then centrifuge at 800 g for 3 min at room temperature, discard the supernatant, wash once with Washing Buffer, resuspend the cells with an appropriate amount of Washing Buffer, and count the live and dead cells using a cell counting chamber to determine the cell viability and cell concentration. Take 22,000 cells for loading test. Subsequently, the following steps are carried out according to the instructions of the DNBSeq C Series High-throughput Single-cell RNA Library Preparation Kit Set provided by BGI and the BGI sequencer manual.

[0051] Through bioinformatics analysis, it can be known that after treating 6 peripheral blood mononuclear cells (PBMC) from patients with cholangiocarcinoma with the above nucleic acid aptamer combination, as Figure 4 shown, up to 2634 nucleic acid aptamers can be identified in these 6 groups. Subsequently, through cluster analysis of different subsets of PBMC, as Figure 5 shown, there are differences in the proportion of cell subsets among different patients. In addition, the nucleic acid aptamers mainly bind to the MAIT T cell subset. Among them, Figure 6 (only part of the detection results are shown in the figure) shows that multiple nucleic acid aptamers that can specifically bind to cholangiocarcinoma PBMC can be identified. The top 10 corresponding targets are: Endothelium-and Platelet-selectins, Integrinα6β4, CCKBR, Neu5Ac, TfR CD71, Zika NS1, ABF1 protein, Insulin.

[0052] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid aptamer combination applicable to single-cell high-throughput sequencing, characterized in that, The nucleic acid aptamer combination includes nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.

291.

2. The nucleic acid aptamer combination according to claim 1, wherein The structure of each nucleic acid aptamer in the nucleic acid aptamer combination is: 5'-adapter sequence - core sequence - 3'-adapter sequence, and the core sequence is the nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.

291.

3. The nucleic acid aptamer combination according to claim 2, wherein The 5'-adapter sequence is as shown in SEQ IDNO.292; the 3'-adapter sequence is as shown in SEQ ID NO.

293.

4. The nucleic acid aptamer combination according to any one of claims 1-3, characterized in that, The targets of the nucleic acid aptamer combination include: emodin, mature 3T3-L1 adipocytes, CTLA-4, H37Rv, PCNA and KI13B, CD8, Annexin A2, CD44, E. coli 11775, type 11 of Streptococcus pyogenes, AXL tyrosine kinase receptor, glioblastoma multiforme, OMVs of multiple Gram-negative bacteria, Integrin α6β4, murine c-kit receptor, Siglec-5 protein, Listeria monocytogenes ATCC19115, pneumonia, Yersinia enterocolitica, CD20, virus-encoded hemagglutinin, PL45, CD109, Staphylococcus epidermidis, Endothelium- and Platelet-selectins, Bone marrow mesenchymal stem cells, mIgM, hepatocellular carcinoma, mouse liver hepatoma cell line BNL 1ME A.7R.1 cells, stress-induced phosphoprotein 1, V.fischeri, IgE, CD71, PD-L1, Proprotein convertase subtilisin / kexin 9, integrin β1, MR / DC-SIGN peptide, galectin-7, CD63 protein, human CD4protein, CD73, CD117, CD133, CD33, CD70, CD19, CD-31 and Actin, CD25, H9N2 AIVpurified haemagglutinin, human CRP, Myoglobin, H2A Protein, Bacterial RNApolymerase, malarial antigen PfLDH, YBX1, SARS-CoV-2 RBD, vimentin, Zika NS1, cTnI, Sephadex, G-protein-coupled cholecystokinin B receptor, hepatitis B surfaceantigen, TGF-β receptor II, Epithelial cell adhesion molecule, transcriptionfactor protein AP-1, sepiapterin, Anti-hIL-17A, Gremlin-1, Human lipocalin 6, Rhesus D, Mas-related G-protein coupled receptor X2, aspartyl proteases b-siteAPP-cleaving enzyme 1, MUP13, integrin β3, Connective tissue growth factor, Non-structural protein 1, Recombinant influenza A mini-hemagglutinin protein, Carcinoembryonic antigen, intercellular adhesion molecule-1, Ca. 2+ bindingprotein S100P, HspX, Ole e 1, COVID-19 nucleocapsid protein, Ras protein, α4integrin, LipL32, β-lactoglobulin, β-conglutin, angiotensin II peptide, Kallikrein-related peptidase 6, Fc region of mouse IgG, protective antigen 63, H9 peptide, transcription factor double homeobox protein 4, protein domain III, IGFIIR, Orai1, sCD80, Cε3-Cε4 protein, alkaline phosphatase (AP) heterodimers, recombinant human erythropoietin-α, Human Rad51, HCV RNA polymerase, Taq DNA polymerase, Neuropeptide Y, thrombin, ABF1 protein, rat liver X receptors, HIV-RT, platelet-derived growth factor, Globo H, HPV, proneurotensin / neuromedin N, Anti-FLAG M2 Antibody, a-synucleinoligomers, a-Bungarotoxin, Antigen binding fragmentof antivesicular stomatitis virus rabbit polyclonal antibodies, CD8a, CA125, Thymidine Kinase 1, Nucleolin, Aflatoxin B1, Amplified in breast cancer 1, Prostate Specific Membrane Antigen, Alpha-fetoprotein, Golgi Protein 73, beta1-Adrenoceptor Autoantibodies, phospholamban, DC-specific intercellular adhesionmolecule (ICAM)-3grabbing non-integrin、HA1 proteins of the H5N1 influenzavirus、Rat Brain Tumor Microvessels、Receptor Binding Region of the ViralHemagglutinin、the core protein of HBV、SMV-VLPs、Her-2、Mouse tumor endothelialcells、T-cell Receptor-cluster of differentiation 3、hnRNP A1、CD4、high densitylipoprotein binding protein、CD24、Carbonic anhydrase IX、E-and P-Selectin、Chloramphenicol、Concanavalin A、bovine catalase、ricin toxin、Cellobiose、cyanotoxin cylindrospermopsin、CD30、EGFR、Digoxin、Activator protein-1 familycJun / cJun homodimer、N-cadherin、DEK、ATP、Alpha-methylacyl CoA racemase、K88、Estrogen receptor alpha、Staphylococcus aureus Enterotoxin B、EpCAM、prothrombin、Glucagon receptor、Neu5Ac、C-MET、CA125、CA19-9、tenascin-C、Reactivegreen 19、6XHis-tag、H4N-terminaltail、HIV-1RT、VEGF、Insulin、type-1 trans -HIV-1 activation-responsive RNA element, (R)- or (S)-ibuprofen, metastasis-related molecules, sialic-acid-binding immunoglobulin-like lectins, MUC1, Hen egg-white lysozyme and recombinant human lysozyme, Lysergamine, metergoline, and other small ergot alkaloids, L-Selectin, L-Arginine, utilizing N ε - p -nosyl- L -lysine, Matrix metalloproteinase 14, alpha-synuclein, lipopolysaccharide, NMUC1, 8-OHdG, Plasmodium vivax lactatedehydrogenase, Hemin, Pro-gas trinreleasing peptide, Protein A, protein kinase C-d, L-Tyrosinamide, P.mirabilis, Retinol-bindingprotein-4, response regulatorPhoP, Leishmania infantum H2A, Sialyllactose, EGFRvIII, Sulforhodamine B, T-2toxin, S. enteritidis, S. typhimurium, Tumor necros is factor-alpha, biogenicamine tyramine, L-tryptophan, abrin toxin, transforming growth factor-β1, VEGF, Vaspin, RNase H1, Vasopressin, PCa cell line PC-3, Vibrio parahaemolyticus, L1cell adhesion molecule, Prostate specific antigen, β-Bungarotoxin, TStreptavidin, 17β-estradiol, florfenicol and florfenicol amine, 8-oxoguanine DNAglycosylase, Low-density lipoprotein receptor-related protein 6, PTK7, MET, PRNP, CD49C, PTPRF, CD318, ZNRF3, antigen MAGE-A3111–125, Fractalkine, VCAM-1, Thyroglobulin, ephrin type-A receptor 2 / CD98 heavy chain, brain-derivedneurotrophic factor, receptor-type tyrosine-protein phosphatase F, Cellularprion protein, F.nucleatum, Zonula Occludens-1, M-Ig, Human epididymis protein4, ribosomal protein S7, Indoleamine 2,3-dioxygenase 1, F. bozemanae, Human neutrophil elastase, fibroblast activation protein, C-reactive Proteins, N-Cadherin Protein, Rituximab, Thy-1 membrane glycoprotein, Immunoglobulin heavy muchain, glycated human serum albumin, hyaluronic acid binding domain of CD44, Heparanase, CatB, CD16, CD123, CTLA4, TNF-α, IFN-γ, PD-1 and prohibitin 2.

5. A kit, characterized in that, It includes the nucleic acid aptamer combination according to any one of claims 1-4.

6. Use of the nucleic acid aptamer combination according to any one of claims 1-4 or the kit according to claim 5 in the analysis of the expression level of cell membrane surface molecules or the construction of a cell atlas, characterized in that, The application is for non-diagnostic purposes.

7. The application according to claim 6, wherein The cells include: human and murine cholangiocarcinoma cell lines.

8. A method for detecting the expression level of cell surface molecules for non-diagnostic purposes, characterized in that, The method includes: mixing a biological sample with the nucleic acid aptamer group according to any one of claims 1-4, performing single-cell high-throughput sequencing of the transcriptome and nucleic acid aptamer library on the reaction system after sufficient reaction, and then performing bioinformatics analysis to obtain the molecular expression levels on the surfaces of different cell types.

9. The method according to claim 8, wherein The biological sample includes peripheral blood mononuclear cells of cholangiocarcinoma.

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