Multicomponent method for simultaneously detecting surface proteins, cytoplasmic proteins and nuclear proteins of single cells and device for single-cell multicomponent detection

Abstract

The application provides a multi-omics method for simultaneously detecting surface proteins, cytoplasmic proteins and nuclear proteins of single cells and a device for single-cell multi-omics detection. The microfluidic unit of the device comprises a plurality of inlets in communication with a microfluidic channel, which are used to supply cells and a plurality of markers to the microfluidic channel to form microdroplets containing single cells, the plurality of inlets comprise a first antibody inlet, the first antibody can bind to the surface proteins of the cells and at least one selected from cytoplasmic proteins and nuclear proteins, and the first antibody is connected with a first DNA sequence; a separation unit is connected with the microfluidic unit, which is used to separate target substances based on the markers in the microdroplets and establish a text library; an analysis and sequencing unit is connected with the microfluidic unit, which is used to perform single-cell transcriptome sequencing and protein information analysis of the cells based on the text library. The device can be used for single-cell multi-omics detection and can simultaneously detect surface proteins, cytoplasmic proteins and nuclear protein information.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological information, in particular, to a multi-omics method for simultaneously detecting single-cell surface proteins, cytoplasmic proteins and nuclear proteins and a device for single-cell multi-omics detection. BACKGROUND

[0002] Single-cell multi-omics analysis technology is a comprehensive analysis method aimed at studying the molecular composition and function of individual cells. This technology combines multiple different biological techniques to analyze and study individual cells from multiple aspects, thereby obtaining more comprehensive and accurate single-cell data. Single-cell multi-omics technology has a wide range of applications, including immunology, oncology, etc., and can more comprehensively understand the heterogeneity of cells in tissues or organs, thereby revealing changes in cells and key proteins and other substances during disease development, etc. Current single-cell multi-omics analysis technologies mainly include single-cell transcriptome sequencing and single-cell surface protein detection, and CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) is a commonly used method that combines single-cell transcriptome and surface protein detection.

[0003] However, the CITE-seq technology still has the following significant deficiencies: first, this technology can only detect protein expression on the surface of cells: the CITE-seq method can detect the expression of cell surface proteins, while cytoplasmic proteins and nuclear proteins inside the cell play a crucial role in many key biological processes. For example, cytoplasmic proteins are involved in signal transduction, metabolic regulation, and stress response, while nuclear proteins play an indispensable role in gene expression regulation, chromatin remodeling, and DNA repair processes. In tumor cells, abnormal expression or dysfunction of nuclear proteins such as p53 and cytoplasmic proteins such as AKT often are key factors for cancer progression and drug resistance. However, the current CITE-seq technology cannot capture these important protein information inside the cell, thus greatly limiting its application ability in in-depth analysis of cell function and its change process.

[0004] In addition, the omics data integration of the CITE-seq technology also has certain limitations: although CITE-seq can integrate the data of the transcriptome and surface protein, it cannot capture the cytoplasmic and nuclear protein information at the single cell level, resulting in obvious limitations in multi-omics data integration. For example, the changes in the transcriptome cannot necessarily fully reflect the dynamics of the protein level, and the expression and modification of cytoplasmic and nuclear proteins often directly affect the behavior and fate of cells. The inability to comprehensively integrate surface proteins, cytoplasmic proteins, nuclear proteins and transcriptome data seriously limits the understanding of the complex functional network of cells, especially in the processes that need to study cancer progression, cell differentiation or immune response, etc. The single surface protein detection of CITE-seq is far from meeting the research needs.

[0005] Therefore, the existing CITE-seq technology, although to some extent, solves the joint analysis of single cell transcriptome and surface protein, but its limitations make it unable to effectively analyze the dynamic changes of cytoplasmic protein and nuclear protein in cells, limiting its application potential in a wider biomedical field. SUMMARY

[0006] The present application aims to at least partially alleviate or solve at least one of the above-mentioned problems.

[0007] In view of the above technical problems, the present application proposes a multi-omics analysis method capable of simultaneously detecting single cell surface protein, cytoplasmic protein and nuclear protein. Through the combination of microfluidic droplet technology and labeled antibodies, the detection of cytoplasmic and nuclear proteins is realized, and through high-throughput detection, the integration of cytoplasmic and nuclear protein information at the single cell level is realized, improving the comprehensiveness of omics data.

[0008] In view of this, in one aspect of the present application, a device for single-cell multi-omics detection is provided. The device comprises: a microfluidic unit comprising a plurality of inlets in communication with a microfluidic channel, the plurality of inlets being configured to supply cells and a plurality of markers to the microfluidic channel to form a microdroplet containing single cells, the plurality of inlets comprising a first antibody inlet configured to supply a first antibody capable of binding to the cell surface protein and at least one selected from cytoplasmic protein and nuclear protein to the microfluidic unit, the first antibody being connected to a first DNA sequence configured to be capable of reverse transcription and amplification within the microdroplet; a separation unit connected to the microfluidic unit to separate target substances based on the markers in the microdroplet and to establish a text library; and an analysis and sequencing unit connected to the microfluidic unit to perform single-cell transcriptome sequencing and protein information analysis on the cells based on the text library. The device can be used for single-cell multi-omics detection and can simultaneously detect cell surface protein, cytoplasmic protein and nuclear protein information, thus better achieving multi-omics analysis at the single-cell level, facilitating more comprehensive acquisition of biological information in single cells and promoting the advancement of related research.

[0009] According to an embodiment of the present application, the microfluidic unit further comprises a second antibody inlet configured to supply a second antibody capable of binding to the protein or antigen to be detected to the microfluidic unit, the second antibody being connected to a magnetic bead, and the separation unit is a magnetic bead separation unit. In this way, the first antibody-second antibody complex labeled with a specific protein can be easily separated out for analysis to establish a text library.

[0010] According to an embodiment of the present application, the microfluidic unit further comprises a Barcode inlet configured to supply a Barcode gel ball to the microfluidic unit, the Barcode gel ball comprising a Barcode sequence. In this way, the information of single cells can be better labeled in the subsequent high-throughput detection process, thereby facilitating the detection and integration of protein information at the single-cell level.

[0011] According to an embodiment of the present application, the analysis and sequencing unit is a high-throughput sequencing unit.

[0012] In another aspect of the present application, a multi-omics method for simultaneously detecting single-cell surface protein, cytoplasmic protein and nuclear protein is provided. The multi-omics method comprises:

[0013] (1) forming a single-cell microdroplet by using a microfluidic unit, the single-cell microdroplet including a single cell to be tested, a plurality of markers including a first antibody to which a first DNA sequence is attached, a reverse transcription reagent set, and an amplification reagent set, the first antibody being capable of binding to a cell surface protein, a cytoplasmic protein, and a nuclear protein, so that the first DNA sequence is subjected to reverse transcription and amplification in the microdroplet;

[0014] (2) separating target substances in the microdroplet based on a separation unit and establishing a text library;

[0015] (3) performing single-cell transcriptome sequencing and protein information analysis on the cell based on the text library, the protein information including a cell surface protein, a cytoplasmic protein, and a nuclear protein.

[0016] The method can conveniently obtain cell surface proteins, cytoplasmic proteins, and nuclear protein information, thereby obtaining more comprehensive and accurate single-cell data.

[0017] According to an embodiment of the present application, the markers further include a second antibody configured to be capable of binding to the protein or antigen to be tested, and the second antibody is attached to a magnetic bead, and the separation unit is a magnetic bead separation unit. Thus, the first antibody-second antibody complex labeled with a specific protein can be conveniently separated for establishing a text library.

[0018] According to an embodiment of the present application, the markers further include a Barcode gel bead including a Barcode sequence.

[0019] According to an embodiment of the present application, the Barcode gel bead includes a gel bead, and a first primer sequence, a Read1 sequence, a Barcode sequence, and a PolydT sequentially attached to the gel bead.

[0020] According to an embodiment of the present application, the first DNA sequence includes an antibody tag and a second primer sequence, and the second primer sequence and the first primer sequence are complementary.

[0021] According to an embodiment of the present application, the multi-omics method is performed by using the device described above. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A structural schematic diagram of a device for single-cell multi-omics detection according to an embodiment of the present application is shown;

[0023] Figure 2 A flowchart of a detection method according to an embodiment of the present application is shown;

[0024] Figure 3A structural schematic diagram of a microfluidic system for single-cell multi-omics detection according to an embodiment of the present application is shown.

[0025] Figure 4 A microfluidic system of Example 1 of the present application and formed single-cell droplets are shown. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar notations represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0027] In one aspect of the present application, the present application provides a device for single-cell multi-omics detection. Referring to Figure 1 , the device comprises a microfluidic unit, a separation unit and an analysis and sequencing unit. The microfluidic unit is used to obtain microdroplets containing single cells and a plurality of markers, the separation unit is used to capture the complex of the first antibody and the second antibody labeled with the target protein to realize the establishment of the text library. The analysis and sequencing unit can obtain information at the level of single cells through high-throughput sequencing, which includes transcriptome information of single cells and protein information, wherein the protein information includes cell surface proteins, cytoplasmic proteins and nuclear proteins. Thus, the device can simultaneously detect single-cell surface proteins, cytoplasmic proteins and nuclear proteins based on multi-omics analysis methods by combining microfluidic droplet technology with labeled antibodies, realizes the integration of cytoplasmic and nuclear protein information at the level of single cells, and improves the comprehensiveness of omics data.

[0028] According to an embodiment of the present application, the microfluidic unit comprises a microfluidic channel and a plurality of inlets in communication with the microfluidic channel. The plurality of inlets are used to supply cells and a plurality of markers to the microfluidic channel to form microdroplets containing single cells. The specific structure of the microfluidic unit is not particularly limited in the present application, and those skilled in the art can design and adjust it according to the actual situation, such as the type and size of the single cells to be tested, and the specific composition of the plurality of markers. Specifically, the single-cell microdroplets can be obtained by designing the structure of the microfluidic channel, such as the volume of the channel, the position of the plurality of inlets, and the shape of the channel.

[0029] For example, the oil cutting technique can be used to obtain single-cell microdroplets in the microfluidic system. Referring to Figure 3The microfluidic unit can supply a solution containing the cells to be tested and the various markers described above to the microfluidic system, and supply the oil phase through different microfluidic channels to "cut" the solution mixed with the markers and the cells to be tested, forming water-in-oil emulsion droplets. By designing the flow rate of the material, the position and shape of the oil phase inlet, etc., microdroplets containing only a single cell can be "cut": the cell solution can be supplied to the microfluidic channel 1 through the cell solution inlet 2, and the required reagents can be supplied to the microfluidic channel 1 through the first antibody inlet 4, the second antibody inlet 5, the cutting oil inlet 6, and the amplification mixture inlet 3, forming single-cell droplets 7 downstream of the microfluidic channel 1. In some embodiments, the cell solution can contain a cell lysis solution, so that the cells can be lysed after the formation of single-cell droplets, exposing the intracellular proteins, thereby enabling simultaneous detection of cell surface and internal proteins.

[0030] It should be particularly noted that in the present application, the term "marker" should be understood broadly. For example, the markers include the first antibodies described above for binding to specific proteins to be tested, and can also include reagents required for DNA transcription and amplification processes, such as raw materials, probes, enzymes, and other substances in the amplification mixture. Although the above-mentioned substances are not "markers" in the traditional sense, they are all used to assist in obtaining transcriptome sequencing and protein information in the method proposed in the present application, and therefore in the present application, they are supplied to the microfluidic system through multiple inlets of the microfluidic system and are "cut" together with a single cell to be tested inside the microdroplet. Thus, transcription and amplification processes can be carried out inside the microdroplet.

[0031] According to embodiments of the present application, the multiple inlets include a first antibody inlet. The first antibody inlet is used to supply a first antibody to the microfluidic unit, and the first antibody is capable of binding to the cell surface protein and at least one selected from the cytoplasmic protein and the nuclear protein. The first antibody is connected to a first DNA sequence, and the first DNA sequence is configured to enable reverse transcription and amplification inside the microdroplet.

[0032] In some embodiments, the first antibody can include one or more antibodies. The first antibody is an antibody capable of labeling the cell surface protein and selected from the cytoplasmic protein and the nuclear protein, for labeling the protein to be tested in the single-cell microdroplet. As described above, the cytoplasmic protein and the nuclear protein play a crucial role in many key biological processes, and therefore labeling of the above-mentioned proteins facilitates the acquisition of biological information related to the proteins. For example, according to some specific embodiments of the present application, the first antibody can include an antibody for labeling the surface protein CD44, an antibody for labeling the cytoplasmic protein GAPDH, and an antibody for labeling the nuclear protein histone H3.

[0033] In some embodiments, the first DNA sequence can be a specific DNA sequence for subsequent detection and amplification. For example, the first DNA sequence can be a small oligonucleotide sequence that is different from the DNA sequence in the cell to be detected.

[0034] According to embodiments of the present application, the microfluidic unit can further comprise a Barcode inlet. The Barcode inlet is used to supply the microfluidic unit with Barcode beads, which comprise Barcode sequences. The main function of the Barcode beads is to realize single-cell level analysis and detection. Those skilled in the art can select Barcode sequences according to actual needs. Specifically, the Barcode beads carry unique Barcode sequences to mark single-cell information and ensure single-cell resolution in a multi-cell sample. For example, specifically, the Barcode beads can comprise a bead and a small piece of first primer sequence, Readl sequence (sequence combined with the predicted required starting sequence), and Barcode sequence connected thereto. The Barcode sequence can be further linked with PolydT, which can capture cell mRNA. After capture, the sequence of the mRNA can be reversed to form cDNA. In other embodiments, other sequences that can capture specific sequence DNA or RNA can be used instead of PolydT to capture specific sequences in cells. The Barcode sequence connected to each bead is different, and each single-cell droplet can contain only one Barcode bead. Thus, single cells can be labeled based on Barcode beads.

[0035] In some specific embodiments, the first DNA sequence described above can comprise an antibody tag and a second primer sequence. Specifically, an antibody tag with a known sequence can be used, and the second primer sequence can be a sequence complementary to the first primer sequence. Thus, after amplification, the Barcode sequence with single-cell characteristics can be connected to the first antibody and the first DNA sequence.

[0036] According to embodiments of the present application, the microfluidic unit further comprises a second antibody inlet. The second antibody inlet is used to supply the microfluidic unit with a second antibody. The second antibody is connected with magnetic beads for subsequent separation, and functions to form a first and second antibody complex labeled with target proteins (cell surface proteins, cytoplasmic proteins, nuclear proteins) by binding to the proteins or antigens to be detected. The complex can be separated by a separation unit, so as to separate the complex containing protein information and mRNA information from the microdroplet, and then realize the establishment of an antibody tag and a cell mRNA library and / or the sequencing process through reverse transcription and amplification.

[0037] According to an embodiment of the present application, the separation unit and the microfluidic unit are connected to separate target substances based on the markers in the microdroplets and to establish a library. For example, the markers can be magnetic beads that can be separated from the microdroplets by the separation unit. When the markers are magnetic beads, the separation unit can be a magnetic bead separation system for capturing the first and second antibody complexes labeled with the target proteins, and the subsequent library establishment and sequencing can be performed after separation by a magnetic force stand. In the present application, the specific structure of the magnetic bead separation system is not particularly limited, and those skilled in the art can determine the specific structure of the magnetic bead separation system according to the specific circumstances of the first and second antibodies, the first DNA sequence connected to the first antibody, and the magnetic beads, to improve the efficiency and effect of separation.

[0038] According to an embodiment of the present application, the analysis and sequencing unit and the microfluidic unit are connected to perform single-cell transcriptome sequencing and protein information analysis based on single-cell microdroplets. Specifically, the analysis and sequencing unit can be a high-throughput sequencing unit.

[0039] In an embodiment of the present application, the specific circumstances of the analysis and sequencing unit can be designed according to the single-cell microdroplets to be analyzed. Since the microdroplets integrated with the first antibodies capable of labeling the proteins inside the cells have been formed in the microfluidic unit, high-throughput sequencing can be performed in the analysis and sequencing unit, so that the transcriptome information and protein information of the single cells can be obtained at the same time.

[0040] In general, the device proposed in the present application can have at least one of the following advantages:

[0041] The multi-omics detection is more comprehensive, which improves the defect in the related art that multi-omics can only detect cell surface proteins. Through specific antibody design, detection of cytoplasmic proteins and nuclear proteins is realized, and the research range of single-cell multi-omics is expanded.

[0042] Multi-dimensional analysis at the single-cell level can be realized, and through the gel ball Barcode labeling and the reverse transcription amplification process in the droplets, integrated analysis of transcriptome and proteome data is realized, and multi-dimensional research at the single-cell level is performed.

[0043] Data accuracy and uniformity. The content uniformity of each droplet formed by the microfluidic unit of the present application is high, which reduces the variation in the experimental process, and makes the detection of transcriptome and proteome more accurate and reliable.

[0044] In another aspect of the present application, a multi-omics method for simultaneously detecting single-cell surface proteins, cytoplasmic proteins and nuclear proteins is proposed. The method can be performed by using the device described above. Specifically, referring to Figure 2 The method can include the following steps:

[0045] (1) Forming single-cell microdroplets:

[0046] In this step, single-cell microdroplets are first formed by using the microfluidic unit. The single-cell microdroplets include a single target cell, a plurality of markers, a reverse transcription reagent set, and an amplification reagent set. Specifically, the plurality of markers include a first antibody to which a first DNA sequence is attached.

[0047] The first DNA sequence and the first antibody have been described in detail above and will not be repeated here. The first antibody can bind to the cell surface protein, the cytoplasmic protein, and the nuclear protein, and the first DNA sequence can be reverse transcribed and amplified in the microdroplet.

[0048] As described above, the method proposed in the present application can include one or more first antibodies. For example, in one embodiment, the first antibodies include antibodies capable of specifically labeling the cell surface protein, the cytoplasmic protein, and the nuclear protein. Each antibody can be attached to a first DNA sequence. Similarly, the first DNA sequence can also include a plurality of first DNA sequences for distinguishing the cell surface protein, the cytoplasmic protein, and the nuclear protein labeled by the corresponding first antibodies.

[0049] As described above, in order to achieve reverse transcription and amplification of the DNA, the microdroplet can further include a reverse transcription reagent set and an amplification reagent set.

[0050] In order to achieve separation of target substances and establishment of a library in the subsequent steps, the single-cell microdroplet can further include markers for separation, such as the second antibody attached to the magnetic beads as described above. The second antibody and the magnetic beads have been described in detail above and will not be repeated here.

[0051] In addition, the markers can further include Barcode beads, which include a Barcode sequence. The Barcode beads can be used to label single-cell information and ensure single-cell resolution in a multi-cell sample.

[0052] (2) Separating target substances in the microdroplet:

[0053] In this step, a separation unit is used to separate the target substances in the microdroplet and to establish a library. Specifically, the target substances can be first and second antibody complexes attached to magnetic beads, and the first antibody and / or the second antibody labels the target protein through specific binding sites on the surface of the target protein. In some embodiments, the target protein can include at least one of the cell surface protein, the cytoplasmic protein, and the nuclear protein. Specifically, the target protein can include the cell surface protein, the cytoplasmic protein, and the nuclear protein.

[0054] Specifically, the Barcode bead comprises a bead, and a first primer sequence, a Read1 sequence, a Barcode sequence and a PolydT connected in sequence on the bead. The PolydT can be combined with the PolydA of the mRNA of the cell as a primer for reverse transcription of the mRNA.

[0055] The first DNA sequence comprises an antibody tag and a second primer sequence, and the second primer sequence is complementary to the first primer sequence. Thus, the sequence with protein information and mRNA information can be separated by protein connection on the magnetic bead for the establishment of the library.

[0056] (3) Perform single-cell transcriptome sequencing and protein information analysis:

[0057] In this step, single-cell transcriptome sequencing and protein information analysis can be performed based on the single-cell microdroplets obtained in the foregoing steps and the established library. For example, the high-throughput sequencing unit in the foregoing device can be used to perform the sequencing analysis.

[0058] Specifically, in this step, the protein information includes information of cell surface proteins, cytoplasmic proteins and nuclear proteins. Since the foregoing proteins have been labeled in the previous steps, the relevant information of the important proteins can be better obtained in the stage of protein information analysis.

[0059] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not indicated in the embodiments, the technology or condition described in the literature in the art or according to the product instruction is used. If the reagent or instrument is not indicated by the manufacturer, it is a conventional product that can be obtained by purchase.

[0060] Example 1

[0061] (1) Labeling cell protein CD44:

[0062] The labeling antibody is anti-mouse CD44 antibody [IM7] (rat monoclonal antibody, IgG2b, Kappa), item number: BD Biosciences #553131.

[0063] The capture antibody is anti-mouse CD44 antibody [KM81] (Syrian hamster monoclonal antibody, IgG), item number: BioLegend #103003.

[0064] IM7 and KM81 bind to different epitopes of CD44, KM81 recognizes the constant region of CD44, and IM7 recognizes different isoforms of CD44 molecules, ensuring detection specificity and sensitivity in double antibody sandwich method. They are suitable for detecting CD44 in various cells, especially immune cells and tumor markers, which can avoid detection problems caused by misidentification of a single epitope.

[0065] (2) Labeling cytoplasmic protein GAPDH

[0066] The labeling antibody is anti-GAPDH antibody [6C5] (mouse monoclonal antibody, IgG1), catalog number: Abeam #ab8245.

[0067] The capture antibody is anti-GAPDH antibody (rabbit polyclonal antibody), catalog number: Cell Signaling Technology #2118.

[0068] The 6C5 monoclonal antibody targets a conserved epitope of GAPDH, ensuring stable binding in multiple species; the rabbit polyclonal antibody can recognize multiple different epitopes of GAPDH, thereby enhancing capture efficiency. GAPDH is often used as an internal reference protein, so these antibody combinations are very suitable for use in quantitative analysis, ensuring stable and accurate detection.

[0069] (3) Labeling nuclear protein: histone H3

[0070] The labeling antibody is anti-histone H3 antibody [D1H2] (rabbit monoclonal antibody, IgG), catalog number: Cell Signaling Technology #4499.

[0071] The capture antibody is anti-histone H3 antibody (mouse monoclonal antibody, IgG1), catalog number: Abeam

[0072] #ab24834.

[0073] D1H2 recognizes specific modifications on histone H3 (such as phosphorylation of lysine 3), which is suitable for use in epigenetic studies; the capture antibody recognizes the N-terminal of histone H3, which is suitable for capturing unmodified histone H3. By combining these two antibodies, histone H3 in different modification states can be distinguished, which helps to study gene expression regulation and chromatin structure changes.

[0074] The microfluidic system as shown in Figure 3 is used to obtain single-cell droplets using the oil cutting technique, and the photo of the single-cell droplets formed is as shown in Figure 4The target substance includes the second antibody, the protein to be detected, the first antibody, and the cDNA segment obtained by reverse transcription of the cell mRNA and connected to the first antibody, and the single cell information is distinguished by the Barcode sequence combined thereon.

[0075] Thus, the information of the cell mRNA and the above three proteins can be obtained simultaneously, and the multi-omics analysis is realized.

[0076] In the description of the embodiments of the present application, unless otherwise explicitly specified and limited, the technical term "connection" should be understood broadly, for example, it can be fixed connection, or detachable connection, or integrated; it can be mechanical connection, or electrical connection; it can be direct connection, or indirect connection through an intermediate medium; it can be internal communication of two elements or interaction relationship between two elements. For those skilled in the art, the specific meaning of the above-mentioned term in the embodiments of the present application can be understood according to the specific circumstances.

[0077] In the description of the present application, the terms "first", "second", etc. are only used to distinguish multiple parameters, and cannot be understood as the limitation of the importance of the multiple parameters and the like.

[0078] In the description of the present application, the terms "top", "bottom", "upper", "lower", etc. indicate the orientation or positional relationship described based on the drawings, and are only for the convenience of describing the present application and do not require the present application to be constructed and operated in a particular orientation, therefore cannot be understood as the limitation of the present application.

[0079] The first feature "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature "above", "over" and "on" the second feature can be that the first feature is directly above or obliquely above the second feature, or only means that the horizontal height of the first feature is higher than that of the second feature. The first feature "below", "under" and "under" the second feature can be that the first feature is directly below or obliquely below the second feature, or only means that the horizontal height of the first feature is lower than that of the second feature.

[0080] In the description of the specification, the description using the terms "one embodiment", "another embodiment", and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment are included in at least one embodiment of the present application. In the specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any suitable manner in one or more embodiments or examples. In addition, the person skilled in the art can combine and modify the different embodiments or examples described in the specification and the features of the different embodiments or examples, without contradiction.

[0081] Although the embodiments of the present application have been shown and described above, the above-described embodiments are exemplary, and it is not understood as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A multi-omics method for simultaneously detecting surface proteins, cytoplasmic proteins and nuclear proteins of single cells, characterized in that, The method is implemented by using a device for single-cell multi-omics detection, the device comprising a microfluidic unit, a separation unit and an analysis and sequencing unit, the method comprising: (1) forming a single-cell microdroplet by using the microfluidic unit, the microfluidic unit comprising a plurality of inlets in communication with a microfluidic channel, the plurality of inlets being used to supply cells and a plurality of markers to the microfluidic channel to form a microdroplet containing a single cell, the plurality of inlets comprising a first antibody inlet and a cell liquid inlet, the cell liquid containing a cell lysis solution, the single-cell microdroplet comprising a single cell to be tested, a plurality of markers, a reverse transcription reagent set and an amplification reagent set, the plurality of markers comprising a first antibody connected with a first DNA sequence, so that the first DNA sequence is subjected to reverse transcription and amplification in the microdroplet, the first antibody being capable of binding to at least one of a cytoplasmic protein and a nuclear protein, and a cell surface protein, (2) separating target substances in the microdroplet based on the separation unit and establishing a text library, the separation unit being connected with the microfluidic unit; (3) performing single-cell transcriptome sequencing and protein information analysis on the cell based on the text library by using the analysis and sequencing unit, the protein information comprising a cell surface protein, a cytoplasmic protein and a nuclear protein, the analysis and sequencing unit being connected with the microfluidic unit.

2. The multi-omic method of claim 1, wherein, The microfluidic unit further comprises a second antibody inlet for supplying a second antibody to the microfluidic unit, the second antibody being configured to be capable of binding to a protein or an antigen to be tested, and the second antibody being connected with a magnetic bead, the separation unit being a magnetic bead separation unit.

3. The multi-omic method of claim 2, wherein, The marker further comprises a Barcode bead, the Barcode bead comprising a Barcode sequence, and the microfluidic unit further comprising a Barcode inlet for supplying the Barcode bead to the microfluidic unit.

4. The multi-omic method of claim 3, wherein, The Barcode bead comprises a bead, and a first primer sequence, a Read1 sequence, a Barcode sequence and a PolydT connected in sequence on the bead.

5. The multi-omic method of claim 4, wherein, The first DNA sequence comprises an antibody tag and a second primer sequence, the second primer sequence being complementary to the first primer sequence.

6. The multi-omic method of claim 1, wherein, The analysis and sequencing unit is a high-throughput sequencing unit.

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