Multi-omics method for simultaneously detecting surface protein, cytoplasm protein and nucleoprotein of single cell and device for multi-omics detection of single cell

Abstract

The invention provides a multi-omics method for simultaneously detecting surface protein, cytoplasm protein and nucleoprotein of a single cell and a device for multi-omics detection of the single cell. A micro-channel unit of the device comprises a plurality of inlets communicated with a micro-channel and used for supplying cells and various markers to the micro-channel to form micro-droplets containing single cells, the plurality of inlets comprise first antibody inlets, first antibodies can be combined with cell surface proteins, and second antibodies can be combined with cell surface proteins. The first antibody is selected from at least one of cytoplasm protein and nucleoprotein, and the first antibody is connected with a first DNA (Deoxyribose Nucleic Acid) sequence; the separation unit is connected with the micro-channel unit, so as to obtain a target substance based on the separation of the marker in the micro-droplet, and is used for establishing a text library; the analysis and sequencing unit is connected with the micro-channel unit and is used for carrying out 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 detect cell surface protein, cytoplasm protein and nucleoprotein information at the same time.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics, and 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 Art

[0002] Single-cell multi-omics analysis technology is a comprehensive analysis method that aims to study the molecular composition and function of single cells. This technology combines a variety of different biological techniques to conduct multi-faceted analysis and research on single cells, thereby obtaining more comprehensive and accurate single-cell data. Single-cell multi-omics technology has a wide range of applications, including immunology, oncology, etc. It can provide a more comprehensive understanding of the heterogeneity of cells in tissues or organs, and then reveal the changes in cells and key proteins and other substances during processes such as disease development. Current single-cell multi-omics analysis technologies mainly include single-cell transcriptome sequencing and single-cell surface protein detection. CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) is a common method that combines single-cell transcriptome with surface protein detection.

[0003] However, CITE-seq technology still has the following significant shortcomings: First, the technology can only detect protein expression on the cell surface: The CITE-seq method can only detect the expression of cell surface proteins, while cytoplasmic proteins and nuclear proteins in cells play a vital 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. In tumor cells, abnormal expression or dysfunction of nuclear proteins such as p53 and cytoplasmic proteins such as AKT are often key factors in cancer progression and drug resistance. However, the current CITE-seq technology cannot capture important protein information inside these cells, which greatly limits its application in in-depth analysis of cell functions and their changes.

[0004] In addition, the integration of omics data using CITE-seq technology also has certain limitations: although CITE-seq can integrate transcriptome and surface protein data, it cannot capture cytoplasmic and nuclear protein information at the single-cell level, resulting in obvious limitations in the integration of multi-omics data. For example, changes in the transcriptome may not fully reflect the dynamics of protein levels, while the expression and modification of cytoplasmic and nuclear proteins often directly affect cell behavior and fate. The inability to fully integrate surface proteins, cytoplasmic proteins, nuclear proteins and transcriptome data has severely limited our understanding of the complex functional networks of cells, especially in the process of studying cancer progression, cell differentiation or immune response. CITE-seq's single surface protein detection is far from sufficient to meet research needs.

[0005] Therefore, although the existing CITE-seq technology has solved the joint analysis of single-cell transcriptomes and surface proteins to a certain extent, its limitations make it unable to effectively analyze the dynamic changes of cytoplasmic proteins and nuclear proteins in cells, limiting its application potential in a wider range of biomedical fields. Summary of the invention

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

[0007] In view of the above-mentioned technical problems, the present application proposes a multi-omics analysis method that can simultaneously detect single-cell surface proteins, cytoplasmic proteins and nuclear proteins. 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, thereby improving the comprehensiveness of the omics data.

[0008] In view of this, in one aspect of the present application, the present invention proposes a device for single-cell multi-omics detection. The device includes: a microfluidic unit, the microfluidic unit includes a plurality of inlets connected to the microfluidic channel, the plurality of inlets are 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 include a first antibody inlet, the first antibody inlet is used to supply the microfluidic unit with a first antibody capable of binding to the cell surface protein and at least one selected from cytoplasmic proteins and nuclear proteins, the first antibody is connected to a first DNA sequence, and the first DNA sequence is configured to be capable of reverse transcription and amplification in the microdroplet; a separation unit, the separation unit is connected to the microfluidic unit, to separate the target substance based on the marker in the microdroplet and to establish a text library; an analysis and sequencing unit, the analysis and sequencing unit is connected to the microfluidic unit, and the cell is subjected to single-cell transcriptome sequencing and protein information analysis based on the text library. The device can be used for single-cell multi-omics detection and can simultaneously detect cell surface proteins, cytoplasmic proteins and nuclear protein information. Therefore, it can better realize multi-omics analysis at the single-cell level, which is conducive to more comprehensive acquisition of biological information within single cells and promote the advancement of related research.

[0009] According to an embodiment of the present application, the microfluidic unit further includes a second antibody inlet, which is used to supply a second antibody that can bind to the protein or antigen to be detected to the microfluidic unit, the second antibody is connected to magnetic beads, and the separation unit is a magnetic bead separation unit. Thus, the first antibody and the second antibody complex labeled with a specific protein can be easily separated for analysis and establishment of a text library.

[0010] According to an embodiment of the present application, the microfluidic unit further includes a barcode inlet, which is used to supply barcode colloid balls to the microfluidic unit, and the barcode colloid balls include barcode sequences. Thus, the information of single cells can be better marked in the subsequent high-throughput detection process, which is conducive to the detection and integration of protein and other information at the single cell level.

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

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

[0013] (1) forming a single-cell micro-droplet using a microfluidic unit, wherein the single-cell micro-droplet includes a single cell to be tested, a plurality of markers, a reverse transcription reagent set, and an amplification reagent set, wherein the plurality of markers includes a first antibody connected to a first DNA sequence, so that the first DNA sequence is reverse transcribed and amplified in the micro-droplet, and the first antibody can bind to the cell surface protein, cytoplasmic protein, and nuclear protein;

[0014] (2) separating the target substance in the microdroplet based on the separation unit and using it to establish a text library;

[0015] (3) Based on the text library, single-cell transcriptome sequencing and protein information analysis are performed on the cells, wherein the protein information includes cell surface proteins, cytoplasmic proteins and nuclear proteins.

[0016] This method can easily obtain information including cell surface proteins, cytoplasmic proteins and nuclear proteins, thereby obtaining more comprehensive and accurate single-cell data.

[0017] According to an embodiment of the present application, the marker further includes a second antibody, the second antibody is configured to be able to bind to the protein or antigen to be detected, and the second antibody is connected to magnetic beads, and the separation unit is a magnetic bead separation unit. Thus, the first antibody and the second antibody complex labeled with a specific protein can be easily separated for establishing a text library.

[0018] According to an embodiment of the present application, the marker further includes a Barcode gum ball, and the Barcode gum ball includes a Barcode sequence.

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

[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 is complementary to the first primer sequence.

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

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

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

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

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

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

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

[0028] According to an embodiment of the present application, the microfluidic unit includes a microfluidic channel and a plurality of inlets connected to 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 present application does not specifically limit the specific structure of the microfluidic unit, and those skilled in the art can design and adjust it according to actual conditions, such as the type and size of the single cell to be tested, and the specific composition of the plurality of markers. Specifically, the acquisition of single-cell microdroplets can be achieved 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, oil cutting technology can be used to obtain single-cell microdroplets in a microfluidic system. Figure 3, the microfluidic unit can supply the solution containing the cells to be tested and the aforementioned multiple markers to the microfluidic system, and supply the oil phase through different microfluidics to "cut" the solution mixed with the markers and the cells to be tested to form oil-in-water emulsion droplets. By designing the flow rate of the material, the position and shape of the oil phase inlet, etc., micro droplets containing only a single cell can be "cut": the cell fluid can be supplied to the microfluidic channel 1 through the cell fluid inlet 2, and the required reagents are 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 mixed liquid inlet 3, forming a single cell droplet 7 downstream of the microfluidic channel 1. In some embodiments, the cell fluid may contain a cell lysis solution, so that the cells can be lysed after the single cell droplet is formed to expose the intracellular proteins, thereby realizing the simultaneous detection of cell surface and internal proteins.

[0030] It should be noted that in this application, the term "marker" should be understood in a broad sense. For example, the marker includes the aforementioned first antibody for binding to a specific protein to be tested, and may also include reagents required for DNA transcription and amplification processes, such as amplification mixtures of raw materials, probes, enzymes and other substances. Although the above substances are not "markers" in the traditional sense, they are used to assist in obtaining multi-omics transcriptome sequencing and protein information in the method proposed in this application. Therefore, in this 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 in the microdroplet. Thus, transcription and amplification processes can be performed in the microdroplet.

[0031] According to an embodiment of the present application, the plurality of inlets include a first antibody inlet. The first antibody inlet is used to supply the first antibody to the microfluidic unit, the first antibody being capable of binding to the cell surface protein and at least one selected from the group consisting of a cytoplasmic protein and a nuclear protein. The first antibody is connected to a first DNA sequence, which is configured to be capable of reverse transcription and amplification in the microdroplet.

[0032] In some embodiments, the first antibody may include one or more antibodies. The first antibody is an antibody capable of marking cell surface proteins, and is selected from cytoplasmic proteins and nuclear proteins, and is used to mark the protein to be tested in the microdroplets of a single cell. As mentioned above, cytoplasmic proteins and nuclear proteins play a vital role in many key biological processes, so the labeling of the above proteins is conducive to obtaining biological information related to the protein. For example, according to some specific embodiments of the present application, the first antibody may include an antibody for marking the surface protein CD44, an antibody for marking the cytoplasmic protein GAPDH, and an antibody for marking the nuclear protein histone H3.

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

[0034] According to an embodiment of the present application, the microfluidic unit may further include a Barcode inlet. The Barcode inlet is used to supply the Barcode colloid ball to the microfluidic unit, and the Barcode colloid ball includes a Barcode sequence. The main function of the Barcode colloid ball is to realize the analysis and detection at the single cell level. Those skilled in the art can select the Barcode sequence according to actual needs. Specifically, the Barcode colloid ball carries a unique Barcode sequence for marking single cell information to ensure single cell analysis in multi-cell samples. For example, specifically, the Barcode colloid ball may include a colloid ball and a small first primer sequence connected to it, a Read1 sequence (a sequence combined with the starting sequence required for prediction) and a Barcode sequence, and a PolydT may also be linked after the Barcode sequence, which can capture cell mRNA, and the sequence of the mRNA may be reversed after capture to form cDNA. In other embodiments, other sequences with the ability to capture specific sequence DNA or RNA may be used instead of PolydT to capture specific sequences in cells. The Barcode sequence connected to each colloid ball is different, and each single cell droplet may contain only one Barcode colloid ball. Thus, single cells can be marked based on Barcode colloid balls.

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

[0036] According to an embodiment of the present application, the microfluidic unit further includes a second antibody inlet. The second antibody inlet is used to supply the second antibody to the microfluidic unit. The second antibody is connected to a magnetic bead for subsequent separation, which functions to form a first and second antibody complex labeled with a target protein (cell surface protein, cytoplasmic protein, nuclear protein) by binding to the protein or antigen to be tested. The complex can be separated by a separation unit, thereby separating the complex containing protein information and mRNA information from the microdroplet, and then through reverse transcription and amplification, the establishment and / or sequencing process of the text library of the antibody label and the cell mRNA is realized.

[0037] According to an embodiment of the present application, the separation unit is connected to the microfluidic unit to separate the target substance based on the marker in the microdroplet and use it to establish a text library. For example, the marker can be a substance that can be separated from the microdroplet based on the separation unit, such as the aforementioned magnetic beads connected to the second antibody. When the marker is a magnetic bead, the separation unit can be a magnetic bead separation system for capturing the first and second antibody complexes labeled with the target protein, and after separation by a magnetic frame, subsequent library construction and sequencing can be performed. In the present application, the specific structure of the magnetic bead separation system is not particularly limited. Those skilled in the art can determine the specific structure of the magnetic bead separation system based on the specific circumstances of the first and second antibodies, as well as the first DNA sequence and magnetic beads connected to the first antibody, to improve the efficiency and effect of separation.

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

[0039] In the embodiments of the present application, the specific conditions of the analysis and sequencing unit can be designed according to the single-cell micro-droplets to be analyzed. Since the micro-droplets integrated with the first antibody capable of labeling the internal protein of the cell have been formed in the microfluidic unit, high-throughput sequencing is performed in the analysis and sequencing unit to simultaneously obtain the transcriptome information and protein information of the single cell.

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

[0041] Multi-omics detection is more comprehensive, improving the defect of related technologies that multi-omics can only detect cell surface proteins. Through the design of specific antibodies, the detection of cytoplasmic proteins and nuclear proteins is achieved, expanding the research scope of single-cell multi-omics.

[0042] It can realize multi-dimensional analysis at the single-cell level. Through the gel ball barcode labeling and reverse transcription amplification process in the droplet, it realizes the integrated analysis of transcriptome and proteome data and conducts multi-dimensional research at the single-cell level.

[0043] Data accuracy and uniformity: The content of each droplet formed by the microfluidic unit of the present application is highly uniform, which reduces the variation during the experiment and makes the detection of transcriptome and proteome more accurate and reliable.

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

[0045] (1) Formation of single-cell microdroplets:

[0046] In this step, a single-cell micro-droplet is first formed using a microfluidic unit. The single-cell micro-droplet includes a single cell to be tested, multiple markers, a reverse transcription reagent set, and an amplification reagent set. Specifically, the multiple markers include a first antibody connected to a first DNA sequence.

[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, cytoplasmic protein and nuclear protein, and the first DNA sequence can be reverse transcribed and amplified in the microdroplet.

[0048] As mentioned above, the method proposed in the present application may include one or more first antibodies. For example, in one embodiment, the first antibody includes antibodies that can specifically mark cell surface proteins, cytoplasmic proteins and nuclear proteins. Each antibody can be connected to a first DNA sequence. Similarly, the first DNA sequence can also include multiple, for distinguishing the cell surface proteins, cytoplasmic proteins and nuclear proteins marked by the corresponding first antibodies.

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

[0050] In order to separate the target substance and establish the text library in the subsequent steps, the single cell microdroplet may also include a marker for separation, such as the aforementioned second antibody connected to the magnetic beads. The second antibody and the magnetic beads have been described in detail above and will not be repeated here.

[0051] In addition, the marker may further include a barcode gel ball, wherein the barcode gel ball includes a barcode sequence. The barcode gel ball can be used to mark single cell information to ensure single cell resolution in a multi-cell sample.

[0052] (2) Separation of target substances in microdroplets:

[0053] In this step, a separation unit is used to separate the target substance in the microdroplet, and used to establish a text library. Specifically, the target substance can be a first and second antibody complex connected to magnetic beads, and the first antibody and / or the second antibody can achieve labeling of the protein to be tested through a specific binding site on the surface of the protein to be tested. In certain embodiments, the protein to be tested can include cell surface proteins, and at least one of cytoplasmic proteins and nuclear proteins. Specifically, the protein to be tested can include cell surface proteins, cytoplasmic proteins and nuclear proteins.

[0054] Specifically, the Barcode gel ball includes a gel ball, and a first primer sequence, a Read1 sequence, a Barcode sequence and PolydT sequentially connected to the gel ball. PolydT can be used as a primer to combine with PolydA of mRNA of a cell for reverse transcription of mRNA.

[0055] The first DNA sequence includes an antibody tag and a second primer sequence, and the second primer sequence is complementary to the first primer sequence. Thus, the sequences with protein information and mRNA information can be connected to magnetic beads through proteins to achieve separation and be used for the establishment of a text library.

[0056] (3) 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 previous step and the established text library. For example, the high-throughput sequencing unit in the above-mentioned device can be used to perform the above sequencing analysis.

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

[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 should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0060] Example 1

[0061] (1) Marker cell protein CD44:

[0062] The labeled antibody used was anti-mouse CD44 antibody [IM7] (rat monoclonal antibody, IgG2b, κ), catalog number: BD Biosciences #553131;

[0063] The capture antibody used was anti-mouse CD44 antibody [KM81] (Syrian hamster monoclonal antibody, IgG), catalog 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, thus ensuring detection specificity and sensitivity in the double antibody sandwich method. They are suitable for detecting CD44 in a variety of cells, especially immune cells and tumor markers, and can avoid detection problems caused by misidentification of a single epitope.

[0065] (2) Labeling of cytoplasmic protein GAPDH

[0066] The labeled antibody used was anti-GAPDH antibody [6C5] (mouse monoclonal antibody, IgG1), catalog number: Abcam#ab8245.

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

[0068] The 6C5 monoclonal antibody targets the 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 to ensure stable and accurate detection.

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

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

[0071] Capture antibody: anti-histone H3 antibody (mouse monoclonal antibody, IgG1), catalog number: Abcam

[0072] #ab24834.

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

[0074] Use Figure 3 The microfluidic system shown in the figure uses oil cutting technology to obtain single-cell droplets. The photo of the formed single-cell droplet is shown in Figure 4As shown in . Then, the target substance is separated by magnetic beads and high-throughput sequencing is performed. The target substance includes a second antibody, a protein to be tested, a first antibody, and a cDNA chain obtained by reverse transcription of cell mRNA connected to the first antibody, which is bound to the magnetic beads, and the single cell information is distinguished by the Barcode sequence bound thereto.

[0075] In this way, the information of cell mRNA and the above three proteins can be obtained at the same time, realizing multi-omics analysis.

[0076] In the description of the embodiments of the present invention, unless otherwise clearly specified and limited, the technical term "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can also be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0077] In the description of the present invention, the terms "first", "second" and the like are only used to distinguish a plurality of parameters and should not be understood as limiting the importance of the plurality of parameters and the like.

[0078] In the description of the present invention, the orientations or positional relationships indicated by terms such as "top", "bottom", "upper", and "lower" are orientations or positional relationships based on the drawings and are only for the convenience of describing the present invention rather than requiring that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention.

[0079] When a first feature is “above” or “below” a second feature, it can mean 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, when a first feature is “above”, “above” or “above” a second feature, it can mean that the first feature is directly above or obliquely above the second feature, or it can simply mean that the first feature is higher in level than the second feature. When a first feature is “below”, “below” or “below” a second feature, it can mean that the first feature is directly below or obliquely below the second feature, or it can simply mean that the first feature is lower in level than the second feature.

[0080] In the description of this specification, the description with reference to the terms "one embodiment", "another embodiment", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0081] Although the embodiments of the present invention have been shown and described above, the above embodiments are exemplary and should not be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A device for single-cell multi-omics detection, characterized in that: include: A microfluidic unit, the microfluidic unit comprising a plurality of inlets connected to the microfluidic channel, the plurality of inlets being used to supply cells and a plurality of markers to the microfluidic channel to form microdroplets containing single cells, the plurality of inlets comprising a first antibody inlet, the first antibody inlet being used to supply a first antibody capable of binding to the cell surface protein and at least one selected from a cytoplasmic protein and a nuclear protein to the microfluidic unit, the first antibody being connected to a first DNA sequence, the first DNA sequence being configured to be capable of reverse transcription and amplification in the microdroplet; A separation unit, the separation unit is connected to the microfluidic unit, so as to separate the target substance based on the marker in the microdroplet and use it to establish a text library; An analysis and sequencing unit is connected to the microfluidic unit and performs single-cell transcriptome sequencing and protein information analysis on the cells based on the text library.

2. The device according to claim 1, characterized in that The microfluidic unit further comprises a second antibody inlet, the second antibody inlet is used to supply a second antibody capable of binding to the protein or antigen to be detected to the microfluidic unit, the second antibody is connected to a magnetic bead, The separation unit is a magnetic bead separation unit.

3. The device according to claim 1 or 2, characterized in that: The microfluidic unit further includes a barcode inlet, and the barcode inlet is used to supply barcode gum balls to the microfluidic unit, and the barcode gum balls include a barcode sequence.

4. The device according to claim 1, characterized in that The analysis and sequencing unit is a high-throughput sequencing unit.

5. A multi-omics method for simultaneously detecting single cell surface proteins, cytoplasmic proteins and nuclear proteins, characterized in that: include: (1) forming a single-cell micro-droplet using a microfluidic unit, wherein the single-cell micro-droplet includes a single cell to be tested, a plurality of markers, a reverse transcription reagent set, and an amplification reagent set, wherein the plurality of markers includes a first antibody connected to a first DNA sequence, so that the first DNA sequence is reverse transcribed and amplified in the micro-droplet, The first antibody is capable of binding to the cell surface protein, cytoplasmic protein and nuclear protein, (2) separating the target substance in the microdroplet based on the separation unit and using it to establish a text library; (3) Based on the text library, single-cell transcriptome sequencing and protein information analysis are performed on the cells, wherein the protein information includes cell surface proteins, cytoplasmic proteins and nuclear proteins.

6. The multi-omics method according to claim 5, characterized in that The marker further includes a second antibody, the second antibody is configured to be able to bind to the protein or antigen to be detected, and the second antibody is connected to a magnetic bead. The separation unit is a magnetic bead separation unit.

7. The multi-omics method according to claim 6, characterized in that The marker further includes a Barcode gum ball, and the Barcode gum ball includes a Barcode sequence.

8. The multi-omics method according to claim 7, characterized in that The Barcode gumball comprises a gumball, and a first primer sequence, a Read1 sequence, a Barcode sequence and PolydT sequentially connected to the gumball.

9. The method according to claim 8, characterized in that The first DNA sequence includes an antibody tag and a second primer sequence, and the second primer sequence is complementary to the first primer sequence.

10. The multi-omics method according to claim 5, characterized in that The multi-omics method is performed using the device described in any one of claims 1-4.

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