Hydrogels embedded with biomaterials

Abstract

This application provides a method for constructing single-cell libraries of biomaterials embedded in hydrogels, which can be used for constructing libraries of mitochondrial DNA and / or open chromatin regions and / or 3' transcriptome (RNA).

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the application of a hydrogel embedded with biomaterials. More specifically, this application relates to a method for constructing a single-cell library using biomaterials embedded in a hydrogel, and the use of the hydrogel embedded with biomaterials in the construction of a single-cell library. Background Technology

[0002] The cell is the most basic unit of life. Currently, in the fields of oncology, immunology, development, and neuroscience, detection technologies, represented by single-cell sequencing, have expanded the breadth and depth of human understanding of life processes. Single-cell sequencing technology analyzes the genome or transcriptome at the single-cell level, comprehensively reconstructing cellular characteristics and differences between populations, and more accurately analyzing the heterogeneity of cell populations. Simultaneously, single-cell sequencing can analyze life processes at single-cell resolution through both temporal and spatial dimensions, deconstructing life with big data. At the tissue, organ, and individual levels, it enables the study of life processes from "breaking down the whole into parts" to "unifying the parts," extracting profound insights from complex biological samples.

[0003] Single-cell sample processing in single-cell multi-omics sequencing refers to the process of preparing single-cell multi-omics libraries by performing biochemical reactions such as cell lysis on intact cell mixtures or pre-extracted nucleus mixtures for downstream flow cytometry sorting into microplates or by forming microdroplets using droplet microfluidics. Pre-extracted nucleus mixtures naturally lose biomolecules from the cytoplasm, resulting in a lack of information in the natural single-cell multi-omics library. Intact cell mixtures, due to the characteristics of cell membranes—they are easily ruptured—lead to significant cross-contamination between cells in the single-cell multi-omics library. Summary of the Invention

[0004] This application is inspired by the widespread compartmentalization phenomenon in nature. For example, cells rely on permeable cell membranes and various organelles to compartmentalize biomolecules and regulate their diffusion, thereby enabling complex life activities while ensuring the exchange of substances with the external environment. A hydrogel with different molecular pore sizes on the inside and outside (i.e., a heterogeneous hydrogel) was designed:

[0005] The technical solution of this application is as follows:

[0006] 1. A method for constructing a single-cell library of biomaterials embedded in a hydrogel, comprising:

[0007] Hydrogels containing embedded biomaterials were treated using transposases;

[0008] Flow cytometry was used to sort hydrogels containing embedded biomaterials that had been treated with transposases.

[0009] The sorted hydrogels embedded with biomaterials were then labeled.

[0010] Library construction was performed on the labeled biological materials.

[0011] 2. The method according to item 1, wherein,

[0012] The transposase is selected from any one of Tn5, Mu, and Vibrio.

[0013] 3. The method according to item 1, wherein the microspheres used for labeling are selected from any one of polystyrene (PS) microspheres, polymethyl methacrylate (PMMA) microspheres, polyethylene microspheres, and agarose soft microspheres.

[0014] 4. The method according to any one of items 1-3, wherein the library construction includes any one, two, or three of the following:

[0015] (i) Constructing a library of mitochondrial DNA;

[0016] (ii) Constructing a library from open chromatin regions;

[0017] (iii) Construct a library from the 3' end transcriptome (RNA).

[0018] 5. The method according to any one of items 1-4, wherein the hydrogel embedded with biomaterial comprises a core gel material embedded with biomaterial, and the biomaterial is a permeable biomaterial.

[0019] 6. The method according to item 5, wherein the hydrogel embedded with biomaterial further includes an outer shell layer capable of encapsulating the core gel material embedded with biomaterial, the outer shell layer having a thickness of 1-2 μm.

[0020] 7. The method according to item 6, wherein the outer shell layer has a porous structure, and the pore size of the porous structure of the outer shell layer is smaller than the average size of the biomaterial.

[0021] 8. The method according to claim 5, wherein the biological material is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells, as well as complexes formed therefrom, preferably the biological material being cells.

[0022] 9. The method according to item 5, wherein the permeation-treated biomaterial is a slightly permeable biomaterial or a strongly permeable biomaterial;

[0023] Preferably,

[0024] The slightly permeable biomaterial is a biomaterial that allows small molecules and some larger molecules to freely enter and exit without cell lysis or destruction of the internal organic structure of the cell.

[0025] The highly permeable biomaterial is a biomaterial that releases cell contents by disrupting the cell membrane.

[0026] 10. The method according to item 5, wherein the slight permeation treatment refers to a low-temperature treatment in a solution containing a nonionic surfactant, the pH of which is 7-8.

[0027] 11. The method according to item 10, wherein the solution containing the nonionic surfactant further comprises one or more of a salt, a buffer solution, and bovine serum albumin.

[0028] 12. The method according to item 10, wherein the temperature of the low-temperature treatment is (-10℃ to 10℃).

[0029] 13. The method according to claim 10, wherein the nonionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPAL CA-630, and Octyl Glucoside.

[0030] 14. The method according to item 5, wherein the core gel material has a porous structure;

[0031] Preferably,

[0032] Biomaterials can be embedded within the porous structure of the core gel material;

[0033] More preferably,

[0034] The porous structure of the core gel material has a pore size of 2-5 μm, and the porous structure of the outer shell layer has a pore size of 24 nm-86 nm.

[0035] 15. The use according to item 5, wherein the core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose;

[0036] Preferably,

[0037] The molecular weight of the core gel material is 0.18kDa-800kDa.

[0038] More preferably,

[0039] The outer shell layer comprises a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, which gives the outer shell layer a porous structure.

[0040] More preferably,

[0041] The hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate (PEGDA), polypropylene glycol, ethylene oxide, and propylene oxide.

[0042] 16. The use according to item 5, wherein the mass ratio of the core gel material to the outer shell layer in the hydrogel is (2-25):1, preferably (5-20):1.

[0043] 17. The use according to item 5, wherein the method for preparing the hydrogel comprises the following steps:

[0044] Biomaterials are encapsulated within a core gel material phase;

[0045] Hydrogels are generated by controlling the curing or semi-curing of the core gel material phase, the outer shell phase, and the oil phase using microfluidic manipulation:

[0046] The hydrogel is obtained by permeation treatment of biomaterial hydrogels;

[0047] The core gel material phase is a solution of the core gel material; the outer shell phase is a solution of the outer shell material.

[0048] 18. The use according to item 17, wherein, before the biomaterial is encapsulated in the core gel material phase, the core gel material phase and the outer shell phase are premixed and then subjected to liquid-liquid separation treatment to obtain the separated core gel material phase and outer shell phase.

[0049] 19. The use as described in item 17, wherein,

[0050] The concentration range of the core gel material is 2%-50%.

[0051] 20. The use according to item 18, wherein the concentration of the high molecular weight hydrophilic polymer in the outer shell phase ranges from 3% to 50%.

[0052] 21. Applications of hydrogels embedded with biomaterials in the construction of single-cell libraries.

[0053] 22. The use according to item 21, wherein the single-cell library construction comprises any one, two, or three of the following:

[0054] (i) Constructing a library of mitochondrial DNA;

[0055] (ii) Constructing a library from open chromatin regions;

[0056] (iii) Construct a library from the 3' end transcriptome (RNA).

[0057] 23. Application of hydrogels embedded with biological materials in single-cell copy number variation sequencing.

[0058] 24. The use of hydrogels embedded with biomaterials for transposase treatment.

[0059] 25. Applications of hydrogels embedded with biomaterials in flow cytometry.

[0060] 26. Applications of hydrogels embedded with biomaterials in single-cell tagging.

[0061] 27. The use according to any one of items 21-26, wherein the biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel involved in any one of items 1-20.

[0062] Compared with the prior art, the beneficial effects of this application are as follows:

[0063] The outer shell is a small-pore artificial membrane, enabling controlled exchange of substances (enzymes, primers, and PCR amplification products, etc.); the core gel material is a non-hollow, large-pore matrix, which can support biomolecular membrane systems and reduce the diffusion efficiency of biomolecules; the core gel material contains biomolecules that can be permeated: the cell permeation step will remove biomolecular membrane liposomes, thereby allowing larger molecules such as antibodies to enter the interior of biomolecules, while better preserving the physiological characteristics of biomolecules.

[0064] This application encapsulates biomolecules within a hydrogel, and permeation treatment is then performed on this hydrogel to increase the strength of biomolecule permeability and compatibility with various biological reactions. Under mild permeation conditions (i.e., weak or slight permeation treatment), the inner hydrogel provides enhanced support for biomembrane molecules, maximizing the preservation of the biomembrane structure. Under stronger permeation conditions, the inner hydrogel reduces the diffusion efficiency of biomolecules; simultaneously, the outer hydrogel membrane provides selective permeability. In summary, hydrogels with different pore sizes on the inner and outer layers allow for high-throughput reagent addition or reduced addition while preventing cross-contamination of biomolecules.

[0065] This application utilizes a hydrogel platform with different molecular pore sizes inside and out, breaking through the current limitations of water-in-oil microdroplet systems internationally, and enabling high-throughput single-cell multi-omics library construction; this application is applicable to any particles containing biofilms.

[0066] The hydrogel embedded with biomaterials in this application is used to construct a library of mitochondrial DNA. This mitochondrial DNA library construction can be used to study mitochondrial genetic variations in cells and to understand the structure and function of mitochondrial DNA; it can also be used to explore mitochondrial-related diseases, as mitochondrial DNA variations are associated with a variety of diseases (such as mitochondrial diseases, neurodegenerative diseases, etc.), and library construction helps to study the mechanisms of these diseases; it can also be used for evolutionary research, as mitochondrial DNA plays an important role in evolutionary research, and through library construction, the differences in mitochondrial DNA between species can be understood.

[0067] The hydrogel embedded with biomaterials in this application enables the construction of libraries for open chromatin regions, which can be used to study gene regulation. The construction of open chromatin region libraries helps to identify and study gene regulatory regions, including promoters and enhancers; it can be used for functional annotation: by analyzing open regions, gene function and regulatory mechanisms can be predicted; it can also identify potential regulatory elements: helping to identify potential regulatory elements related to cell specificity, developmental processes or diseases.

[0068] The hydrogel embedded with biological materials in this application is used to construct 3' end transcriptome (RNA) libraries, which can be used to study gene expression: 3' end transcriptome library construction is used to study gene expression patterns in cells in depth, especially focusing on 3' end RNA; it can be used for single-cell analysis: suitable for single-cell RNA sequencing, it can reveal changes in gene expression at the single-cell level. It can also be used to study transcription endpoint regulation: it helps to understand the regulation of RNA processing, splicing, and stability. Attached Figure Description

[0069] Figure 1 The following image shows a bright-field microscope image of the hydrogel in the examples and comparative examples.

[0070] Figure 2 The structural diagram of the hydrogel of this application is shown;

[0071] Figure 3 A schematic diagram of the droplet microfluidic chip for generating hydrogels according to this application is shown; where 1 represents the outer shell material phase inlet, 2 represents the core gel material phase inlet, 3 represents the oil phase inlet, and 4 represents the hydrogel collection outlet.

[0072] Figure 4 Images of cells before and after rupture are shown.

[0073] Figure 5 An image showing the average diameter of the hydrogel is displayed.

[0074] Figure 6 This image shows a scanning electron microscope (SEM) image of the interior of the hydrogel in Example 1; the porous structure of the core gel material. Figure 6 In section A, the porous structure of the outer shell has the following pore sizes: Figure 6 As shown in B.

[0075] Figure 7 The study demonstrated that the hydrogel could significantly retain DNA molecules larger than 968 bp.

[0076] Figure 8 An image of the hydrogel under slightly permeable conditions is shown.

[0077] Figure 9 An image of a hydrogel under highly permeable conditions is shown.

[0078] Figure 10 An image showing a cell nucleus encapsulated in a hydrogel is shown.

[0079] Figure 11 The image shows cells encapsulated in a hydrogel.

[0080] Figure 12 The image shows cells encapsulated in a hydrogel.

[0081] Figure 13 The human-mouse mixed cross-contamination rate in single-cell scATAC-seq data is shown: Figure 13 Group A consists of intact cells (a mixture of human 293T cells and mouse 3T3 cells in equal proportions). Figure 13 In the middle group B, intact cells (a mixture of human 293T cells and mouse 3T3 cells in equal proportions) are encapsulated in hydrogels with different molecular pore sizes inside and outside.

[0082] Figure 14 An image of the hydrogel in Comparative Example 1 is shown.

[0083] Figure 15 An image of the hydrogel in Comparative Example 2 is shown.

[0084] Figure 16 An image of the hydrogel in Comparative Example 3 is shown.

[0085] Figure 17 An image showing the thickness of the hydrogel shell layer of this application is displayed.

[0086] Figure 18 The fragment distribution after the Tn5-tagged reaction is shown.

[0087] Figure 19 This paper presents the results of our laboratory's self-assembled Tn5 enzyme and its activity verification. Among them, Figure 19 A: Tn5 tagging reaction of 50 ng HEK293T genomic DNA with Tn5S5 / S7 was performed, followed by PCR amplification; B: Agarose gel electrophoresis was used to detect the amplification products.

[0088] Figure 20 The results show that human 293T cells encapsulated in a hydrogel, after Tn5 tagging and nucleic acid dyeing, can be sorted by flow cytometry. The sorting strategy is as follows: Figure 20 In section A, hydrogels that tested positive (containing cells) were sorted out as follows: Figure 20 B;

[0089] Figure 21 The figure shows the average sequencing depth of mtDNA, using a single-cell mtDNA library construction in three hydrogels as an example.

[0090] Figure 22 The diagram shows the results of library construction for cellular-level mitochondrial DNA and chromatin accessibility simultaneously using human 293T cells and mouse 3T3 cells encapsulated in a hydrogel.

[0091] Figure 23 The experiment demonstrates the construction of mtDNA and chromatin accessibility libraries in hydrogels (based on the Tn5 S5 / S7 library construction assay).

[0092] Figure 24 The diagram shows the results of three-end transcriptome sequencing performed in permeable membrane droplets (based on Tn5 S5 / S7 library construction followed by in situ reverse transcription, and then library construction using Tn5 S7 / S7 (tagged)).

[0093] Figure 25 The results show the microfluidic platform co-encapsulated with hydrogel droplets and single-cell-tagged microspheres containing the Nextera capture sequence.

[0094] Figure 26 The figure shows the results of high-throughput deep sequencing of mitochondrial DNA, chromatin accessibility, and 3' transcriptome simultaneously at the single-cell level using a self-developed hydrogel droplet microfluidic platform with different inner and outer molecular pore sizes. Detailed Implementation

[0095] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0096] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0097] In this application, hydrogels with different molecular pore sizes on the inside and outside also refer to hydrogels that are not homogeneous on the inside and outside.

[0098] This application provides a hydrogel embedded with biomaterial, the hydrogel comprising a core gel material embedded with biomaterial, wherein the biomaterial is a biomaterial that has undergone permeation treatment.

[0099] In some embodiments, the hydrogel containing the embedded biomaterial further includes an outer shell layer capable of encapsulating the core gel material containing the embedded biomaterial, the outer shell layer having a thickness of 1-2 μm; for example, the thickness of the outer shell layer can be 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm or any range thereof.

[0100] The hydrogel of this application comprises: an external small-pore artificial membrane for controllable exchange of substances (enzymes, primers, and PCR amplification products, etc.); an internal non-hollow large-pore matrix for supporting the biomolecular membrane system and reducing the diffusion efficiency of biomolecules; and internally containing biomolecules that can be permeated: the cell permeation step will remove biomolecular membrane liposomes, thereby allowing larger molecules such as antibodies to enter the biomolecule interior while better preserving the physiological characteristics of the biomolecule.

[0101] In some embodiments, the outer shell layer has a porous structure, and the pore size of the porous structure of the outer shell layer is smaller than the average size of the biomaterial.

[0102] In this application, the average size of the biological material refers to the average diameter of the biological material.

[0103] In some embodiments, the biomaterial is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and cells, as well as complexes formed therefrom, with cells being the preferred biomaterial.

[0104] When the biological material of this application is protein, after the permeation treatment of this application, when the protein is fixed in the gel material, it can be used for protein purification, as follows: The hydrogel in this application can be used as a medium for protein separation and purification, and the protein purity can be improved by selective protein adsorption and elution. At the same time, encapsulating the protein can improve its stability during the purification process, thereby helping to prepare more stable protein drugs and biological products.

[0105] When the biological material of this application is nucleic acid, after the permeation treatment of this application, the nucleic acid is fixed in the gel material and can be used for molecular diagnostics, as follows: the nucleic acid fixed in the hydrogel of this application can be used for molecular diagnostics and can be detected by PCR amplification of specific genes or pathogens.

[0106] When the biomaterial of this application is sugar, after the permeation treatment of this application, when the sugar is fixed in the gel material, it can be used for glycobiology research, specifically as follows: Encapsulating sugar molecules helps to study the interaction of sugar molecules on the cell surface, which is very important for studying cell adhesion, immune response, and the recognition of pathogens of infectious diseases.

[0107] When the biomaterial of this application is lipid, after the permeation treatment of this application, when the lipid is fixed in the gel material, it can be used for drug delivery, specifically as follows: lipids encapsulated in hydrogel can be used to improve drug delivery, and by preparing lipid nanoparticles, the solubility and bioavailability of drugs can be improved.

[0108] When the biomaterial of this application is a metabolite, and after the permeation treatment of this application, the metabolite is immobilized in the gel material, it can be used for drug screening and toxicity assessment, specifically as follows: Immobilizing the metabolite in a hydrogel can be used for drug screening and toxicity assessment. This provides a new approach for evaluating the impact of new drugs on metabolic pathways and studying the potential toxicity of drugs, thus benefiting drug development and toxicity research.

[0109] When the biomaterial of this application is a polypeptide, and after the permeation treatment of this application, the polypeptide is fixed in the gel material, it can be used for drug development, specifically as follows: encapsulating polypeptides in hydrogel can improve the stability and targeting of drugs, which is beneficial for drug development and therapeutic research.

[0110] When the biological material of this application is bacteria and / or virus, after the permeation treatment of this application, when the bacteria and / or virus are fixed in the gel material, the encapsulation of bacteria or virus in the hydrogel is beneficial for the analysis of cell genome or viral genetic material at the single-cell level, which is beneficial for vaccine development, and can also be used for pathogen research.

[0111] When the biological material of this application is a cell nucleus, after the permeation treatment of this application, the cell nucleus is fixed in the gel material. The cell nucleus is encapsulated in the hydrogel and after permeation treatment, chromatin openness measurement at the single-cell level can be performed, thereby providing profound insights into epigenetics.

[0112] When the biomaterial of this application consists of two or more types of cells or their complexes, and after being permeable to the gel material according to this application, the two or more types of cells or their complexes are fixed in the gel material, which helps to study and understand the interactions between cells and opens up profound insights for cell biology research.

[0113] In some embodiments, the permeable biomaterial is either slightly permeable or strongly permeable; wherein, the slightly permeable biomaterial is one that allows small molecules and some larger molecules to freely enter and exit without cell lysis or destruction of the internal organic structure of the cell; and the strongly permeable biomaterial is one that releases cell contents through cell membrane disruption.

[0114] In some implementations, slight permeation treatment refers to low-temperature treatment in a solution containing a nonionic surfactant at a pH of 7-8.

[0115] In some embodiments, the solution containing the nonionic surfactant also includes one or more of the following: salt, buffer solution, and bovine serum albumin.

[0116] In some embodiments, the salt is selected from one or more of sodium chloride, magnesium chloride, sodium sulfate, and magnesium sulfate.

[0117] In some implementations, the buffer solution is selected from one or more of Tris-HCl, phosphate buffer, acetate buffer, and HEPES buffer.

[0118] In some embodiments, the nonionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPA CAL-630, and Octyl Glucoside.

[0119] In some embodiments, the temperature for cryogenic treatment is (-10℃ to 10℃). For example, the temperature for cryogenic treatment can be -10℃, -8℃, -6℃, -4℃, -2℃, 0℃, 2℃, 4℃, 6℃, 8℃, 10℃ or any range thereof.

[0120] In some embodiments, the enhanced permeability treatment refers to treatment in a solution containing protease, or treatment in a solution containing SDS, or treatment using an acoustic disruptor to rupture cells using acoustic energy.

[0121] In one embodiment, the strong permeation treatment refers to treatment in a solution containing a protease, wherein the protease is selected from one or more of Proteinase K, trypsin, chymotrypsin, elase, and pepsin. The mild permeation treatment (or weak permeation treatment) refers to treating the hydrogel containing biological material with a mild permeation reagent composed of 10 mM Tris-HCl (ph 7.4), 10 mM NaCl, 3 mM MgCl2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40, followed by incubation on ice for 3-5 minutes. The strong permeation treatment refers to treating the hydrogel containing biological material with a strong permeation reagent composed of 0.1 M NaCl2, 1 mM CaCl2, and 0.05 μg / μL Proteinase K, followed by incubation at 55°C for 30 minutes, and then at 95°C for 10 minutes.

[0122] The slightly permeable hydrogel can be used to immobilize biomaterials within the hydrogel described in this application, and may have the following applications:

[0123] (1) Bioseparation and dialysis:

[0124] It can be used to separate biomolecules and particles, such as proteins, DNA, and RNA. This facilitates applications in bioseparation techniques, such as electrophoresis, dialysis, and filtration, to purify and analyze biological samples.

[0125] (2) Cell sorting and enrichment:

[0126] By immobilizing different types of cells in hydrogels and slightly permeating them, they can be used for labeling cell-specific molecules, which can then be used for cell sorting and enrichment. This helps to separate specific cell subpopulations or single cells for single-cell research or cell therapy.

[0127] (3) Biosensors:

[0128] Immobilizing cells in hydrogels can be used to create biosensors that detect specific biomolecules, cytokines, or photosensitizing signals. This is highly beneficial for medical diagnostics, environmental monitoring, and biosensing applications.

[0129] Single-cell whole-genome sequencing has always been a challenge because genes are embedded within chromosomes. Traditional methods require highly permeable lysis buffers, which limits the simultaneous high-permeability treatment of cells within a microfluidic system. The hydrogel technology proposed in this application can highly permeable treat cells and encapsulate them together with single-cell barcode-encoded microspheres, thereby simplifying single-cell whole-genome sequencing and overcoming the limitations of traditional methods. This innovative approach promises to enable highly efficient single-cell genome analysis.

[0130] In one specific embodiment, the thickness of the outer shell layer is 1-2 μm, obtained by setting a flow rate ratio during the droplet microfluidic hydrogel generation. The flow rate ratio (in μL / min) is as follows: the flow rate ratio of the outer shell material phase to the biomolecule-containing core gel material phase is 1:(1-2). For example, the outer shell material phase is controlled at 1.11 μL / min, and the biomolecule-containing core gel material phase is controlled at 1.55 μL / min to generate the hydrogel. The above flow rates for the outer shell material phase and the biomolecule-containing core gel material phase are merely an example; those skilled in the art will understand that adjustments can be made when operating with different equipment.

[0131] In one embodiment, the resulting hydrogel, when observed under a bright-field microscope, showed an outer shell layer thickness of 1-2 μm. For example... Figure 17 As shown, the thickness of the outer shell layer of the hydrogel is in the range of 1-2 μm.

[0132] In this application, Figure 2 This diagram illustrates biomolecules encapsulated within a hydrogel with a difference in pore size between the inner and outer layers. The resulting hydrogel can be placed in large quantities into 1.5 mL centrifuge tubes for permeation treatment, solution addition, and removal. The pore size of the hydrogel shell is smaller than the average size (diameter) of the internal biomaterial, thus providing selective permeability. The hydrogel core material is a non-hollow, large-pore matrix, which supports the biomolecular membrane system and reduces the diffusion efficiency of biomolecules.

[0133] In some embodiments, the core gel material in the above-described hydrogel has a porous structure; biomaterials can be embedded within the porous structure of the core gel material.

[0134] In some embodiments, the pore size of the porous structure of the core gel material is 2-5 μm; for example, the pore size of the porous structure of the core gel material can be any range from 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, 3 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm, 3.7 μm, 3.8 μm, 3.9 μm, 4 μm, 4.1 μm, 4.2 μm, 4.3 μm, 4.4 μm, 4.5 μm, 4.6 μm, 4.7 μm, 4.8 μm, 5 μm, or between these values.

[0135] In some embodiments, the core gel material in the above-described hydrogel is a hydrophilic polymer; the core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose.

[0136] In some embodiments, the molecular weight of the core gel material is 0.18 kDa to 800 kDa; for example, the molecular weight of the core gel material can be 0.18 kDa, 0.2 kDa, 0.3 kDa, 0.4 kDa, 0.5 kDa, 0.6 kDa, 0.7 kDa, 0.8 kDa, 0.9 kDa, 1 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, or any range thereof.

[0137] In some embodiments, in the two hydrogels described above, the outer shell layer comprises a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, which gives the outer shell layer a porous structure.

[0138] In this application, a high molecular weight hydrophilic polymer refers to a polymer with a weight-average molecular weight greater than about 6,000 Daltons (kDa), and a low molecular weight hydrophilic polymer refers to a polymer with a weight-average molecular weight less than about 6,000 Daltons (kDa).

[0139] In some embodiments, the hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate (PEGDA), polypropylene glycol (PPG), ethylene oxide, and propylene oxide.

[0140] In some embodiments, the molecular weight of the high molecular weight hydrophilic polymer is 6kDa-20kDa; for example, the molecular weight of the high molecular weight hydrophilic polymer can be 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa, 12kDa, 13kDa, 14kDa, 15kDa, 16kDa, 17kDa, 18kDa, 19kDa, 20kDa or any range thereof.

[0141] In some embodiments, the molecular weight of the low molecular weight hydrophilic polymer is 0.2 kDa-6 kDa. For example, the molecular weight of the low molecular weight hydrophilic polymer can be 0.2 kDa, 0.3 kDa, 0.4 kDa, 0.5 kDa, 0.6 kDa, 0.7 kDa, 0.8 kDa, 0.9 kDa, 1.0 kDa, 1.5 kDa, 2.0 kDa, 2.5 kDa, 3.0 kDa, 3.5 kDa, 4.0 kDa, 4.5 kDa, 5.0 kDa, 5.5 kDa, 6.0 kDa, or any range thereof.

[0142] In some embodiments, the pore size of the porous structure of the outer shell layer is 24nm-86nm; for example, the pore size of the porous structure of the outer shell layer is 24nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 86nm or any range thereof.

[0143] In some embodiments, the porosity of the porous structure of the outer shell layer can be 80%, 81%, 83%, 85%, 87%, 89%, 90%, or any range thereof.

[0144] In some embodiments, the mass ratio of the core gel material to the outer shell layer in the hydrogel is (2-25):1; for example, the mass ratio of the core gel material to the outer shell layer is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1 or any range between them.

[0145] In some embodiments, the mass ratio of the core gel material to the outer shell layer in the hydrogel is (5-20):1.

[0146] In some embodiments, in the two hydrogels described above, the mass of the low molecular weight hydrophilic polymer is not higher than the mass of the high molecular weight hydrophilic polymer; preferably, the mass ratio of the high molecular weight hydrophilic polymer to the low molecular weight hydrophilic polymer is (1-2):1; for example, the mass ratio of the high molecular weight hydrophilic polymer to the low molecular weight hydrophilic polymer can be 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1 or any range therebetween.

[0147] This application provides a method for preparing the above-mentioned hydrogel, which includes the following steps: encapsulating biomaterial in a core gel material phase; generating a hydrogel by controlling the solidification or semi-solidification of the core gel material phase, the outer shell phase, and the oil phase using microfluidic manipulation; permeation treatment of the biomaterial hydrogel to obtain the hydrogel; the core gel material phase being a solution of the core gel material; and the outer shell phase being a solution of the outer shell material.

[0148] In some embodiments, before the biomaterial is encapsulated in the core gel phase, the core gel phase and the outer shell phase are premixed and then subjected to liquid-liquid separation to obtain the separated core gel phase and outer shell phase.

[0149] In some embodiments, the concentration range of the core gel material is 2%-50%; for example, the concentration range of the core gel material can be 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or any range between therewith.

[0150] The concentration here refers to the concentration obtained by dividing mass (g) by volume (mL), and the concentration includes biological materials.

[0151] In some embodiments, the concentration of the high molecular weight hydrophilic polymer in the outer shell phase ranges from 3% to 50%; for example, the concentration range of the high molecular weight hydrophilic polymer can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or any range between therewith.

[0152] This application provides the application of the above-described hydrogel or the hydrogel prepared by the above method in single-cell multi-omics library construction.

[0153] In one embodiment of this application, a certain amount of PEGDA (MW8kDa), a certain amount of PEGDA (MW575 Da) and a certain amount of Dextran (MW500kDa) are taken to prepare a hydrogel mixture, which is then brought to a final volume of 1mL. After being mixed evenly, the mixture is centrifuged at 16000g for 30 minutes in a benchtop centrifuge to induce liquid-liquid phase separation.

[0154] After centrifugation, a clear separation boundary was observed between the upper PEGDA-rich phase and the lower dextran-rich phase, and droplets of the upper and lower phases were respectively aspirated into the corresponding centrifuge tubes.

[0155] Prepare biomolecules by resuspending HEK293T cells in 1 mL of DPBS containing 0.04% BSA at 300 g for 3 min at 4 °C. Add 1.0 mL of DPBS containing 0.04% BSA to resuspend the biomolecules, and pipette 10 μL onto a counting plate. Take approximately 1 million biomolecules and resuspend them in 300 μL of Lextran-Rich solution.

[0156] Use a 3 mL BD syringe to hold 2% FS10 in HFE-7500; a 1 mL BD syringe to hold PEGDA-rich; and a 1 mL BD syringe to hold dextran-rich biomolecular suspensions.

[0157] Place the syringe filled with reagents into the syringe pump and collect the droplets using a 1.5 mL EP tube. Set the microfluidic flow rate as follows: oil phase 6.67 μL / min; PEGDA-rich phase 1.11 μL / min; Dextran-rich phase 1.55 μL / min.

[0158] Place approximately 200 μL of the collected droplet in a 1.5 mL centrifuge tube under a UV lamp and irradiate for 2 min to solidify the PEGDA in the droplet into a gel.

[0159] Pipette 10 μL of liquid onto a Countess slide to observe the formation of reaction compartments, and observe whether the reaction compartments are uniform in size and whether there is any aggregation.

[0160] Add droplets to 500 μL of HFE-7500 containing 20% ​​(vol / vol) perfluorooctanoic acid and centrifuge for 5 seconds. Remove the oil from the bottom of the centrifuge tube, add 500 μL of DPBS buffer containing 0.1% (vol / vol) Pluronic F-68, mix well by pipetting, and centrifuge for 5 seconds. Discard the supernatant; the lower layer, the hydrogel with different pore sizes on the inside and outside, should be collected in a 1.5 mL centrifuge tube.

[0161] Slight permeability of biomolecules can be achieved in hydrogels.

[0162] The specific conditions for slight permeation are as follows: hydrogels containing biological materials were treated with a slight permeation reagent consisting of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40 and incubated on ice for 3 minutes.

[0163] This application utilizes the complementary compatibility of natural polymer dextran and polyethylene glycol diacrylate (PEGDA) to prepare a hydrogel sample processing system with good hydrophilicity and biocompatibility by means of droplet microfluidics in a two-phase aqueous system (one phase solution is dextran, the other phase solution is PEGDA, and the biomolecules are resuspended in the dextran phase).

[0164] This application provides a method for constructing a single-cell library of biomaterials embedded in a hydrogel, specifically a method for constructing a single-cell multi-omics library based on hydrogels with different molecular pore sizes inside and outside the cell. The method includes: treating the hydrogel containing biomaterials with a transposase; sorting the transposase-treated hydrogel containing biomaterials with a flow cytometry; tagging the sorted hydrogel containing biomaterials; and constructing a library from the tagged biomaterials.

[0165] Those skilled in the art will understand that transposase processing, flow cytometry sorting, labeling, and library construction are common methods in the field, as long as they meet their objectives.

[0166] Hydrogels embedded with biomaterials can be either slightly permeable or highly permeable.

[0167] In this application, tagging refers to the step of cutting a DNA molecule with a transposon complex including a transposase and inserting a specific DNA fragment at a specific location. After a purification step to remove the transposase, the additional sequence is added to the end of the inserted fragment by PCR, ligation, or any other suitable method known to those skilled in the art.

[0168] In some embodiments, the transposase is selected from any one of Tn5, Mu, and Vibrio.

[0169] Next-generation sequencing (NGS) library construction involves fragmenting or screening DNA samples into target sequences of specified lengths, and then adding oligonucleotide sequencing adapters P5 and P7 for subsequent sequencing. Traditional library construction methods require steps such as DNA fragmentation, end repair, adapter ligation, library amplification, and multiple purification and sorting processes, which is time-consuming. This application utilizes transposases such as Tn5 in sequencing library construction, transforming the multi-step reactions of DNA fragmentation, end repair, and adapter ligation into a single step, significantly shortening library construction time and improving efficiency. All NGS library construction methods described in this application are based on transposase-based methods.

[0170] In some embodiments, the microspheres used for labeling are selected from any one of polystyrene (PS) microspheres, polymethyl methacrylate (PMMA) microspheres, polyethylene microspheres, and agarose soft microspheres.

[0171] The tagged microspheres carry unique sequence identifiers. This application enables the unique labeling of each cell by co-encapsulating different cells with uniquely identified tagged microspheres. The advantage of this approach is that it allows for the simultaneous analysis of a large number of individual cells, providing high-throughput single-cell data and facilitating a more comprehensive understanding of cell population heterogeneity, such as for studying differences between cell subtypes, identifying rare cell types, and exploring cellular responses under different physiological conditions.

[0172] Flow cytometry is a highly precise biological analysis technique used to analyze and classify mixtures of single cells or microparticles. Single-cell separation can be achieved by sorting cell-containing hydrogels into 96-well plates (independent reaction chambers), and single-cell analysis can be realized by combining this technique with subsequent reaction steps. The advantages are as follows: most research institutions are equipped with flow cytometers, meaning most laboratories can use this method for single-cell analysis. Cells can be pre-stained with certain biomarkers, and then specific cells can be sorted by flow cytometry for subsequent single-cell analysis experiments.

[0173] In some implementations, the library construction includes any one, two, or three of the following: (i) library construction of mitochondrial DNA; (ii) library construction of open chromatin regions; and (iii) library construction of 3' transcriptome (RNA).

[0174] The hydrogel embedded with biomaterials in this application for constructing mitochondrial DNA libraries has the following beneficial effects:

[0175] 1) Studying mitochondrial genetics: The construction of mitochondrial DNA libraries can be used to study mitochondrial genetic variations in cells and to understand the structure and function of mitochondrial DNA.

[0176] 2) Exploring mitochondrial-related diseases: Mitochondrial DNA variations are associated with a variety of diseases (such as mitochondrial diseases, neurodegenerative diseases, etc.), and library construction helps to study the mechanisms of these diseases.

[0177] 3) Evolutionary research: Mitochondrial DNA plays an important role in evolutionary research. Through library construction, we can understand the differences in mitochondrial DNA among species.

[0178] The hydrogel embedded with biomaterials in this application has the following effects on library construction of open chromatin regions:

[0179] 1) Studying gene regulation: Constructing open chromatin libraries helps to identify and study gene regulatory regions, including promoters and enhancers.

[0180] 2) Functional annotation: By analyzing open regions, gene function and regulatory mechanisms can be predicted. Identification of potential regulatory elements: Helps identify potential regulatory elements related to cell specificity, developmental processes, or diseases.

[0181] The hydrogel embedded with biological materials in this application has the following effects on the construction of 3' end transcriptome (RNA) libraries:

[0182] 1) Studying gene expression: 3' transcriptome library construction is used to study gene expression patterns in cells in depth, especially focusing on 3' RNA.

[0183] 2) Single-cell analysis: Applicable to single-cell RNA sequencing, it can reveal changes in gene expression at the single-cell level.

[0184] 3) Studying transcription endpoint regulation: This helps to understand the regulation of RNA processing, splicing, and stability.

[0185] This application provides the use of the above-mentioned hydrogels embedded with biological materials in single-cell library construction. The single-cell library construction includes any one, two, or three of the following: (i) library construction of mitochondrial DNA; (ii) library construction of open chromatin regions; and (iii) library construction of 3' end transcriptome (RNA).

[0186] This application provides the use of the above-mentioned hydrogels embedded with biological materials in single-cell copy number variation sequencing.

[0187] This application provides the use of the above-mentioned hydrogel embedded with biological material in transposase treatment.

[0188] This application provides the use of the above-mentioned hydrogels embedded with biomaterials in flow cytometry.

[0189] This application provides the use of the above-mentioned hydrogels embedded with biomaterials in single-cell tagging.

[0190] Example 1

[0191] A hydrogel mixture was prepared by taking 0.036 g of PEGDA (MW8kDa), 0.024 g of PEGDA (MW575Da), and 0.6 g of Dextran (MW500kDa). The volume was adjusted to 1 mL, and the mixture was thoroughly mixed. The mixture was then centrifuged at 16000 g for 30 minutes in a benchtop centrifuge to induce liquid-liquid phase separation. The mass ratio of the high molecular weight hydrophilic polymer to the low molecular weight hydrophilic polymer was 3:2, and the mass ratio of the core gel material to the outer shell was 10:1. The core gel material refers to Dextran, and the outer shell refers to PEGDA (MW8kDa) and PEGDA (MW575Da).

[0192] After centrifugation, a clear separation boundary was observed between the upper PEGDA-rich phase and the lower dextran-rich phase, and droplets of the upper and lower phases were respectively aspirated into the corresponding centrifuge tubes.

[0193] Prepare the biomolecules by resuspending HEK293T cells in 1 mL of DPBS containing 0.04% BSA at 1800 rpm for 3 min at 4°C. Add 1.0 mL of DPBS containing 0.04% BSA to resuspend the biomolecules, and pipette 10 μL onto a counting plate. Take approximately 1 million biomolecules and resuspend them in 300 μL of Lextran-Rich solution.

[0194] Use a 3 mL BD syringe to hold 2% FS10 in HFE-7500; a 1 mL BD syringe to hold PEGDA-rich; and a 1 mL BD syringe to hold dextran-rich biomolecular suspensions.

[0195] Place the syringe filled with reagents into the syringe pump and collect the droplets using a 1.5 mL EP tube. Set the microfluidic flow rate as follows: oil phase 6.67 μL / min; PEGDA-rich phase 1.11 μL / min; Dextran-rich phase 1.55 μL / min.

[0196] Place approximately 200 μL of the collected droplet in a 1.5 mL centrifuge tube under a UV lamp and irradiate for 2 min to solidify the PEGDA in the droplet into a gel.

[0197] Pipette 10 μL of liquid onto a Countess slide to observe the formation of reaction compartments, and observe whether the reaction compartments are uniform in size and whether there is any aggregation.

[0198] Add 500 μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanoic acid to the centrifuge tube and centrifuge for 5 seconds. Remove the oil from the bottom of the centrifuge tube, add 500 μL of DPBS buffer containing 0.1% (vol / vol) Pluronic F-68, mix well by pipetting, and centrifuge for 5 seconds. Discard the supernatant from the centrifuge tube; the lower layer, i.e., the hydrogel with different molecular pore sizes on the inside and outside, should be collected in a 1.5 mL centrifuge tube.

[0199] Slight permeability of biomolecules can be achieved in hydrogels. The specific conditions for slight permeability are as follows: hydrogels containing biological materials are treated with a slight permeability reagent consisting of 10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40, and incubated on ice for 3 minutes.

[0200] The pore size of the porous structure of the core gel material was determined by cryo-scanning electron microscopy analysis, such as... Figure 6 The pore size shown in Figure A is 2-5 μm. The pore size of the porous structure of the outer shell was determined by transmission electron microscopy analysis, as follows: Figure 6 The aperture shown in B is 24nm-86nm.

[0201] The thickness of the outer shell layer is 1.5-2 μm.

[0202] Example 2

[0203] The only difference between Example 2 and Example 1 is that Dextran is 0.3g and the mass ratio of core gel material to outer shell is 5:1; the rest are the same.

[0204] Example 3

[0205] The only difference between Example 3 and Example 1 is that Dextran is 0.9g and the mass ratio of core gel material to outer shell is 15:1; the rest are the same.

[0206] Example 4

[0207] The only difference between Example 4 and Example 1 is that Dextran is 1.2g and the mass ratio of core gel material to outer shell is 20:1, otherwise they are the same.

[0208] Example 5

[0209] The only difference between Example 5 and Example 1 is that Dextran is 1.5g and the mass ratio of core gel material to outer shell is 25:1; the rest are the same.

[0210] Example 6

[0211] The only difference between Example 6 and Example 1 is that Dextran is 1.8g and the mass ratio of core gel material to outer shell is 30:1; the rest are the same.

[0212] Example 7

[0213] The only difference between Example 7 and Example 1 is that Dextran is 0.12g and the mass ratio of core gel material to outer shell is 2:1; the rest are the same.

[0214] Example 8

[0215] The only difference between Example 8 and Example 1 is that Dextran is 0.06g and the mass ratio of the core gel material to the outer shell is 1:1; the rest are the same.

[0216] Example 9

[0217] The only difference between Example 9 and Example 1 is that Dextran is replaced with polyvinyl alcohol; otherwise, they are the same.

[0218] Example 10

[0219] The only difference between Example 10 and Example 1 is that PEGDA (MW8kDa) is replaced with polypropylene glycol (MW8kDa), otherwise they are the same.

[0220] Example 11

[0221] The only difference between Example 11 and Example 2 is that the slight permeation condition is replaced with a strong permeation condition. The strong permeation condition is as follows: the hydrogel containing biological material is treated with a strong permeation reagent consisting of 0.1M NaCl2, 1mM CaCl2, and 0.05μg / μL Proteinase K at a final concentration and incubated at 55°C for 30 minutes, and then incubated at 95°C for 10 minutes.

[0222] Comparative Example 1

[0223] The only difference between Comparative Example 1 and Example 1 is that no slight permeation treatment was performed.

[0224] Comparative Example 2

[0225] The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain Dextran (MW 500kDa), otherwise they are the same.

[0226] Comparative Example 3

[0227] The only difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not contain PEGDA (MW8 kDa) and PEGDA (MW575 Da), otherwise they are the same.

[0228] Table 1

[0229]

[0230] Note: The outer shell thickness shown in Table 1 refers to the result of measuring the outer shell thickness using the microscope's detection module. Due to the existence of errors, the outer shell thickness data in Examples 1-11 are usually within ±10% of the target thickness data, which is within the range recognized by those skilled in the art. For example, 1.7 μm is the outer shell thickness, but in actual testing, the outer shell thickness can be 1.53 μm-1.87 μm.

[0231] Note: The outer shell mass refers to the sum of the masses of the high molecular weight hydrophilic polymer and the low molecular weight hydrophilic polymer. The masses in the table above refer to the masses required when the core gel material, high molecular weight hydrophilic polymer, and low molecular weight hydrophilic polymer are brought to a final volume of 1 mL.

[0232] Examples 12-19

[0233] The preparation method is the same as in Example 1, except for the slight permeation treatment conditions in Table 2.

[0234] Table 2

[0235]

[0236] Hydrogels were prepared according to Tables 1 and 2 above. The collected hydrogels were placed on a glass slide and observed under a microscope to examine their structure. The results are as follows: Figure 1 As shown, Examples 1-7, Examples 9-11, Examples 12-19, and Comparative Example 1 can all form hydrogels with complete structures (circular without gaps when viewed from the plane), while the hydrogel formed in Example 8 has gaps, and Comparative Examples 2 and 3 cannot form hydrogel structures. The inventors selected the hydrogel of Example 1 or referred to Example 1 for subsequent experiments.

[0237] Experimental Example

[0238] Experiment Example 1: Hydrogel Cell Loss Test

[0239] 1. Using microfluidic manipulation, the generated water-in-oil droplets were collected into 1.5 mL centrifuge tubes. Six separate 10 μL droplets were placed onto glass slides to observe the percentage of cell-containing droplets. The average percentage of cell-containing droplets across the six measurements was then calculated. (The percentages of cell-containing droplets in the six measurements were: 0.1157, 0.1114, 0.1037, 0.1051, 0.1012, and 0.125, respectively.)

[0240] 2. Place approximately 200 μL of the collected droplet in a 1.5 mL centrifuge tube under a UV lamp for 2 min to solidify the outer shell material into a gel. After gel formation, add 500 μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanoic acid and centrifuge for 5 s. Remove the oil from the bottom of the centrifuge tube, add 500 μL of DPBS buffer containing 0.1% (w / v) Pluronic F-68, mix well by pipetting, and centrifuge for 5 s. Discard the supernatant in the centrifuge tube; the lower layer, i.e., the hydrogel with different molecular pore sizes inside and out, should be collected in a 1.5 mL centrifuge tube.

[0241] 3. The collected hydrogel from Example 1 was taken in six separate measurements (10 μL each time) and placed on glass slides to observe the percentage of cell-containing hydrogels. The average percentage of cell-containing hydrogels in the total hydrogel was then calculated. (The percentages of cell-containing hydrogels in the six measurements were: 0.1034, 0.1096, 0.1143, 0.1, 0.0952, and 0.1105, respectively.)

[0242] The results are as follows Figure 4 As shown. Figure 4 As shown, the hydrogel prepared in Example 1 was tested and found that there was no significant loss of cells before demulsification (in the state of water-in-oil droplets) and after demulsification (in the state of hydrogel) (there was no statistically significant difference in cell percentage, and the independent samples t-test P value was 0.3254).

[0243] Experiment Example 2: Diameter Testing of Hydrogels with Different Inner and Outer Molecular Pore Sizes

[0244] The hydrogel from Example 1 was aspirated onto a glass slide, and its diameter was measured under a microscope (a total of 64 hydrogels were measured, with diameters of: 61.105, 61.105, 61.105, 61.105, 59.974, 59.974, 58.842, 58.842, 58.842, 58.842, 57.711, 57.711, 57.711, 57.711, 57.711, 57.711, 57.711, 56.579, 56.579, 56.579, 56.579, 56.579, 56.579, 56.579, 56.579, 56.579, 56.579, 55.447 ... .447, 55.447, 55.447, 55.447, 54.316, 54.316, 54.316, 54.316, 54.316, 54.316, 54.316, 54.316, 54.316, 54.316, 53.184, 53.184, 53.184, 53.184, 5 3.184, 53.184, 53.184, 53.184, 53.184, 53.184, 53.184, 52.053, 52.053, 50.921, 50.921, 49.79, 48.671, 48.658, 46.395, 46.395, 45.263).

[0245] like Figure 5 As shown, the entire hydrogel prepared in Example 1 was tested, and the diameter of the hydrogel was approximately 55 μm. Figure 5 Image A in the image is an example of a hydrogel under a microscope. Figure 5 In section B, the diameters of 64 hydrogels were statistically analyzed (mean and standard deviation).

[0246] Experimental Example 3

[0247] The collected hydrogels were analyzed by cryo-scanning electron microscopy (Cryo-SEM) to observe their cross-sectional morphology.

[0248] like Figure 6 As shown, the hydrogel prepared in Example 1 was tested and found that the core gel embedded with biomaterials had a pore size of approximately 2 μm.

[0249] However, it was found that the hydrogel generated in Comparative Example 1 was eccentric and had gaps, such as Figure 14 As shown, the hydrogel generated in Comparative Example 2 is eccentric and has a gap, as... Figure 15 As shown; the hydrogel generated in Comparative Example 3 ruptured and could not form a normal hydrogel structure, as shown. Figure 16 As shown.

[0250] Experiment Example 4

[0251] Following the preparation method of Example 1, the only difference was that HEK293T cells were replaced with pFB25 plasmid, and the average mass concentration of pFB25 plasmid encapsulated in each hydrogel was 7 nM. The plasmid was encapsulated in the hydrogel, and the prepared hydrogel was then placed in primers containing specific amplification lengths of this plasmid, nucleic acid dyes, and PCR reaction solutions. A total of four primers with specific amplicon lengths were used, corresponding to amplicon lengths of 150 bp, 547 bp, 968 bp, and 1187 bp. After PCR, the fluorescence intensity of the nucleic acid dye in the hydrogel for each amplicon length was statistically analyzed using a fluorescence microscope.

[0252] like Figure 7 As shown, the hydrogel prepared in Example 1 was tested and found that DNA molecules larger than 968 bp could be significantly retained in the hydrogel. Figure 7 In Figure A, the fluorescence intensity of a specific amplicon is shown as an example in four hydrogels (Scale bar: 50 μm). Figure 7 B represents the statistical analysis of the relative fluorescence intensity of a specific amplicon in the hydrogel.

[0253] Experimental Example 5: Slightly Permeable Conditions

[0254] The hydrogels encapsulating HEK293T cells were compared between those in DPBS buffer and those in a mild permeabilization buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (vol / vol) NP-40, and 1% (vol / vol) BSA). The mild permeabilization buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (vol / vol) NP-40, and 1% (vol / vol) BSA) was prepared under the same conditions as the hydrogel in Example 1 (results are shown below). Figure 8 As shown in Figure B), the DPBS buffer conditions were the same as those in Example 1, except that the biomolecules were not slightly permeabilized but only treated with DPBS buffer (results are shown in Figure B). Figure 8 (As shown in Figure A). After incubating on ice for 5 minutes, the hydrogel was placed in DPBS containing nucleic acid dye for staining, and then analyzed under a fluorescence microscope.

[0255] like Figure 8 As shown, Figure 8 Image A shows the hydrogel without permeation treatment. Figure 8 Image B shows a hydrogel treated with slightly permeable conditions: it can be seen that biomolecules are well confined within the hydrogel.

[0256] Experimental Example 6: Strong Permeability Conditions

[0257] The hydrogels encapsulating HEK293T cells were compared between those in DPBS buffer and those in a strong permeation buffer (containing 0.1% Triton X-100 and 10 mg / ml protease K). The conditions for the strong permeation buffer (containing 0.1% Triton X-100 and 10 mg / ml protease K) were the same as those for the hydrogel prepared in Example 11 (results are shown below). Figure 9 As shown in Figure B), the DPBS buffer conditions were the same as those in Example 11, except that the biomolecules were not subjected to strong permeation treatment but were simply treated with DPBS buffer (results are shown in Figure B). Figure 9 (As shown in Figure A). After incubating the hydrogel in DPBS buffer on ice for 5 min, the hydrogel was stained with DPBS containing nucleic acid dye and analyzed under a fluorescence microscope. After incubating the hydrogel in strong permeability buffer at 55 degrees Celsius for 30 min, the hydrogel was stained with DPBS containing nucleic acid dye and analyzed under a fluorescence microscope.

[0258] like Figure 9 As shown, Figure 9 Image A shows the hydrogel without permeation treatment. Figure 9 Image B shows a hydrogel treated with strong permeability conditions: it can be seen that biomolecules are well confined within the hydrogel.

[0259] Experiment Example 7:

[0260] After mixing the extracted 293T cell nuclei and the inner core gel material, the resulting droplets were collected into 1.5 mL centrifuge tubes using microfluidic manipulation and placed under a UV lamp for 2 min to solidify the outer shell material into a gel. After gelation, 500 μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanoic acid was added, and the tube was centrifuged for 5 s. The oil at the bottom of the centrifuge tube was removed, and 500 μL of DPBS buffer containing 0.1% (w / v) Pluronic F-68 was added. The mixture was pipetted and centrifuged for 5 s. The supernatant was discarded, and the lower layer, i.e., the hydrogel with different molecular pore sizes inside and out, was collected into a 1.5 mL centrifuge tube (the preparation method is the same as in Example 1, except that the HEK293T cells were replaced with 293T cell nuclei). The collected hydrogel was placed on a glass slide for observation.

[0261] like Figure 10 As shown, the human 293T cell nuclei can be encapsulated in the hydrogel prepared in Example 7 of this experiment.

[0262] Experimental Example 8:

[0263] Following the method described in Example 1, except that 293T cells were replaced with human peripheral blood mononuclear cells (PBMCs), the rest remained the same. The extracted human peripheral blood mononuclear cells (PBMCs) and the core gel material were mixed, and the resulting droplets were collected into 1.5 mL centrifuge tubes using microfluidic manipulation. The tubes were then placed under a UV lamp for 2 minutes to solidify the outer shell material into a gel. After gelation, 500 μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanoic acid was added, and the tubes were centrifuged for 5 seconds. The oil at the bottom of the centrifuge tube was removed, and 500 μL of DPBS buffer containing 0.1% (w / v) Pluronic F-68 was added. The mixture was pipetted and centrifuged for 5 seconds. The supernatant was discarded, and the lower layer, i.e., the hydrogel with different molecular pore sizes inside and out, was collected into a 1.5 mL centrifuge tube. The collected hydrogel was then placed on a glass slide for observation.

[0264] like Figure 11 As shown in the image, human peripheral blood mononuclear cells (PBMCs) can be encapsulated in hydrogels with different molecular pore sizes to undergo multiple biochemical reactions, including permeation.

[0265] Experiment Example 9:

[0266] The hydrogels prepared in Example 1 were divided into four groups and placed in buffer solutions containing 5% dimethyl sulfoxide (Group 1), 25% glycerol (Group 2), 80% ethanol (Group 3), and 0.1% polypropylene glycol and ethylene oxide addition polymer (Group 4), respectively. The hydrogel structures of each group were observed under a microscope after being stored at room temperature (22°C) and at low temperature (-80°C) for 48 hours, respectively.

[0267] like Figure 12 As shown, the hydrogel can be stored in different organic reagents (dimethyl sulfoxide, glycerol, ethanol, and polypropylene glycol with ethylene oxide) or at different temperatures for 48 hours, and the structure remains intact under a microscope.

[0268] Experimental Example 10

[0269] Following the method in Example 1, 293T cells were replaced with a mixture of human 293T cells and mouse 3T3 cells in equal proportions, with half of the cells encapsulated in a hydrogel. The prepared hydrogel (encapsulating a mixture of human 293T cells and mouse 3T3 cells in equal proportions) and the other half of the cell mixture (a mixture of human 293T cells and mouse 3T3 cells in equal proportions) were placed in a mildly permeabilized buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40). After incubation on ice for 5 min, a Tn5 transposase reaction was performed. Following the reaction, the cells were co-encapsulated with single-cell encoded microspheres, followed by demulsification and library construction. The constructed library was subjected to next-generation sequencing and bioinformatics analysis. The results are as follows: Figure 13 As shown.

[0270] Under the same permeation conditions (buffered with 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40), a comparison was made between intact cells and intact cells encapsulated in hydrogels with different inner and outer pore sizes. The results showed that the cross-contamination rate of intact cells was 86.12%. Figure 13 The cross-contamination rate of (A) and intact cells encapsulated in hydrogels with different molecular pore sizes inside and outside was 10.01%. Figure 13 (B) This indicates that under the same permeability treatment conditions, encapsulating intact cells in hydrogels with different molecular pore sizes inside and outside can significantly reduce the cross-contamination rate between cells.

[0271] Experimental Example 11: Tn5 Tagging Reaction of Hydrogels

[0272] Tn5 tagging is a crucial step in high-throughput sequencing library preparation. Ideally, after Tn5 tagging using a hydrogel, DNA molecules are broken down into short fragments of 100 bp to 600 bp, while fragments of 968 bp or greater are retained within the hydrogel system. This means that Tn5-tagged DNA fragments can diffuse freely inside and outside the hydrogel network and can be amplified efficiently. Therefore, theoretically, the fragment distribution of the amplified product should be between 100 bp and 600 bp.

[0273] By performing Tn5 tagging and subsequent insertion sequence amplification experiments on hydrogels containing genomic DNA from 293T cells (each hydrogel containing an average of 1.66 pg of genomic DNA), the inventors discovered that the hydrogels could successfully undergo Tn5 tagging and generate fragments ranging from 100 bp to 600 bp that conform to next-generation sequencing standards. The results are as follows... Figure 18 As shown.

[0274] Experiment Example 12. Library Construction of Mitochondrial DNA at the Single-Cell Level:

[0275] The hydrogel from Example 1 was placed in a 50 μL tagging reaction system containing Tn5 (S5 / S7) at a final concentration of 0.2 μg / μL, 1x tagmentation buffer at a final concentration of 5 mg Cl2, and incubated at 37°C for 30 min.

[0276] The hydrogel after incubation was stained with 1x EvaGreen (YEASEN, 10223ES76) nucleic acid dye.

[0277] Add a total volume of 25 μL of PCR reaction system to a 96-well plate, which includes 1 μM of i5-primer and i7-primer (the i5-primer and i7-primer sequences are from Nextera Index XT Kit v2, FC-131-2001, Illumina) and 1x KAPA HiFi HotStart ReadyMix (KK2600).

[0278] After staining, the hydrogels were flow-cytoscopy-sorted to obtain hydrogels with strong EvaGreen fluorescence signals and placed in 96-well plates pre-filled with PCR reaction system.

[0279] Perform the following PCR reaction

[0280] Step 1: 72℃, 5 min

[0281] Step 2: 98℃, 30s

[0282] Step 3: 98℃, 10s

[0283] Step 4: 63℃, 30s

[0284] Step 5: 72℃, 1 min

[0285] (Repeat step 5, for a total of 8 times)

[0286] Step 6: Maintain at 12℃.

[0287] After PCR, the DNA was purified using 1.2x volume of Vazyme DNA clean beads (N411-01), followed by elution with 30 μL of nuclease-free water. The eluted solution is the final library.

[0288] Relevant data:

[0289] (1) Assembly and activity verification of the Tn5(S5 / S7) transposase used in Experiment Example 12:

[0290] Purchase Tn5 naked enzyme (ABclonal, RM21303) and perform insert sequence assembly. After establishing this system, any DNA fragment sequence can be assembled onto the Tn5 naked enzyme. Taking the assembly of Nextera S5 / S7 as an example, enzyme activity was verified using human 293T cell genomic DNA after assembly. Results are as follows... Figure 19 As shown.

[0291] Figure 19 A indicates that the Tn5 enzyme activity was verified by assembling genomic DNA from 293T cells. Tn5 containing the NexteraS5 / S7 sequence can tag genomic DNA. After the reaction fragments are nicked and amplified by sequencing adapter primers, agarose gel electrophoresis shows standard 100bp-700bp diffuse bands. Figure 19 B indicates that, compared to the group without Tn5, the Tn5 enzyme-tagged reaction produces a distinct diffuse band. This proves that the Tn5 naked enzyme and insert sequence assembly system of this application is successful.

[0292] (2) Hydrogels can be adapted for flow cytometry (flow cytometry sorting and gating strategy), and the results are as follows: Figure 20 As shown. Human 293T cells were encapsulated in a hydrogel, and after Tn5 tagging and nucleic acid dyeing, they could be sorted by flow cytometry. The sorting strategy is as follows: Figure 20 In section A, hydrogels that tested positive (containing cells) were sorted out as follows: Figure 20 B.

[0293] (3) Positive hydrogels can be sorted into 96-well plates for mitochondrial DNA sequencing, with the final sequencing coverage as shown in the figure. Figure 21 As shown: The applicant randomly selected three single-cell libraries. Taking the three hydrogels involved in these three single-cell libraries as examples, the average sequencing depths of mtDNA were 38739.78x, 41798.13x, and 48956.15x, respectively. The average sequencing depth refers to how many times each site was sequenced. It can be seen that the single-cell libraries of this application achieved an average of tens of thousands of measurements per site in the mitochondrial genome at the single-cell level (deep sequencing depth). This indicates that the hydrogels used in this application have good sequencing technology for DNA library construction and are suitable for downstream mutation analysis.

[0294] Example 13. Simultaneous library construction using mitochondrial DNA and chromatin accessibility at the single-cell level:

[0295] The hydrogel from Example 10 was used to perform single-cell tagging reaction and droplet PCR using the DNBelab C (BGI contains Tn5) series high-throughput single-cell ATAC library preparation kit from BGI. The droplet PCR product was purified and used for sequencing adapter PCR amplification reaction.

[0296] After PCR, the DNA was purified using 1.2x volume of Vazyme DNA clean beads (N411-01), followed by elution with 30 μL of nuclease-free water. The eluted solution is the final library.

[0297] Sequencing results after simultaneous sequencing of mitochondrial DNA and chromatin openness at the single-cell level, as shown in the figure Figure 22 As shown.

[0298] Human 293T cells and mouse 3T3 cells were encapsulated in hydrogels with different inner and outer molecular pore sizes to simultaneously construct libraries for mitochondrial DNA and chromatin access at the cellular level. The size distribution of the constructed library fragments is shown in the figure. Figure 22 As shown, nuclear base group reads exhibit a high enrichment of transcription start sites (TSS). Figure 22 (B and C), the average mitochondrial genome sequencing depth per cell is approximately 96×, and the coverage is uniform ( Figure 22 (D and E).

[0299] Example 14: Simultaneous library construction of mitochondrial DNA, chromatin openness, and 3' transcriptome at the single-cell level:

[0300] The hydrogel from Example 1 was slightly permeable and subjected to the first tagging reaction to label mitochondrial DNA and open chromatin regions. The tagging reaction system consisted of a total volume of 50 μL containing Tn5 (S5 / S7) at a final concentration of 0.2 μg / μL, 1x tagmentation buffer at a final concentration of 5 mg CL2, and was incubated at 30°C for 30 min.

[0301] RNA reverse transcription is performed in a hydrogel to form heteroduplex RNA and DNA.

[0302] The total volume of the reverse transcription system was 100 μL, containing 2 μM TruseqR1_oligo_dT, 0.5 mM dNTPs, 10 U / μL Maxima H minus Reverse Transcriptase, 2 U / μL RiboLock RNase inhibitor, 0.2 U / μL SUPERaseIn RNase inhibitor, 0.4 U / μL RNaseOUT RNase inhibitor, 1x NaCl RT buffer, and 12% PEG8000.

[0303] Step 1: 10 min, 50℃;

[0304] Step 2: 8℃, 12s;

[0305] Step 3: 15℃, 45s

[0306] Step 4: 20℃, 45s

[0307] Step 5: 30℃, 30s

[0308] Step 6: 42℃, 2min

[0309] Step 7: 50℃, 3 min

[0310] Repeat steps 2-7, for a total of 3 times.

[0311] Step 8: 50℃, 5min

[0312] A second tagging reaction was performed in the hydrogel to label heteroduplex RNA and cDNA. The tagging reaction system consisted of a total volume of 50 μL containing 0.2 μg / μL Tn5 (S7 / S7), 1x tagmentation buffer, and 5 mM MgCl2, and was incubated at 37°C for 30 min.

[0313] Single-stranded nucleotide chain removal and Tn5 transposase tagging nick-filling reactions were performed in a hydrogel. The reaction system consisted of a total volume of 50 μL, containing 0.5 mM dNTPs, 8 U / μL Maxima Hminus Reverse Transcriptase, 2 U / μL Lexol, and 1x NaCl RT buffer. The mixture was incubated at 37 °C for 15 min.

[0314] The single-cell-tagged microspheres (RANbiotech.050.065.2.ATAC) of the Nextera capture sequence were co-encapsulated in droplets within the hydrogel; the microfluidic flow rate was set at 6.67 μL / min for the oil phase, 8 μL / min for the PCRMaster mix phase, 8 μL / min for the hydrogel phase, and 4 μL / min for the single-cell-tagged microsphere phase.

[0315] Purify the droplet PCR product and divide the product into two equal parts by volume. One part is used for PCR amplification of mitochondrial DNA and chromatin open sequencing adapters, and the other part is used for PCR amplification of transcriptome sequencing adapters.

[0316] After PCR, the DNA was purified using 1.2x volumes of Vazyme DNA clean beads (N411-01), followed by elution with 30 μL of nuclease-free water. The eluted solution was the final library.

[0317] Relevant data:

[0318] (1) The hydrogel can be used for mtDNA and chromatin open region tagging reaction (first tagging reaction) with Tn5 S5 / S7 transposase. The results are as follows: Figure 23 As shown. Figure 23 After the A Tn5 S5 / S7 (specific sequence) tagging reaction, standard library construction and indexing PCR were performed, and the average library size was 487 bp, which is consistent with the theoretical value. Figure 23 Analysis of nuclear openness after library construction (as shown in B) indicates that tagging in selectively permeable membrane droplets can preserve chromatin openness information. Figure 23 The analysis of mtDNA sequencing depth and coverage after library construction showed that both coverage and sequencing depth were high (8000x).

[0319] (2) In situ reverse transcription was performed in a hydrogel, followed by RNA / DNA hybrid strand tagging using Tn5 S7 / S7. The results are as follows: Figure 24 As shown. Figure 24 The figure shows the results of three-end transcriptome sequencing performed in permeable membrane droplets (based on Tn5 S5 / S7 library construction followed by in situ reverse transcription, and then library construction using Tn5 S7 / S7 (tagged)). Figure 24 The average size of the library in the A-type database is 350bp, which is consistent with the theoretical value. Figure 24 The B-mode showed that exon regions accounted for 61% of the transcriptome reads; Figure 24 The C-cell matrix shows high coverage at the 3' end of the gene region.

[0320] (3) A microfluidic platform was co-encapsulated with hydrogel droplets of different inner and outer molecular pore sizes and single-cell-tagged microspheres containing the Nextera capture sequence. The results are as follows: Figure 25 As shown. Figure 25 This illustrates a microfluidic platform co-encapsulated with hydrogel droplets and single-cell-tagged microspheres containing the Nexteracapture sequence. Figure 25 ZhongA displays a microfluidic chip design for co-encapsulating droplets; Figure 25 Image B shows a phase image of single-cell labeled microspheres (microspheres are soluble in the presence of DTT); Figure 25 The image shows a chip encapsulated in a C-shaped display. Figure 25 The middle D shows the droplet morphology before PCR; Figure 25 The image shows the droplet morphology after PCR.

[0321] (4) Hydrogels can be used to co-encapsulate single-cell coding microspheres containing Nextera capture sequences into a microarray for simultaneous construction of open mitochondrial DNA and chromatin libraries and 3' transcriptome libraries. Results are as follows: Figure 26 As shown. Figure 26 The results demonstrate the high-throughput deep sequencing of mitochondrial DNA, open chromatin, and 3' transcriptome at the single-cell level using a self-developed hydrogel droplet microfluidic platform with different inner and outer molecular pore sizes: mitochondrial DNA sequencing reads accounted for approximately 60% (A); and the mitochondrial genome sequencing depth was high (8000x) with uniform coverage (D), indicating that this platform can effectively detect mitochondrial DNA mutations; the nuclear genome reads showed a high enrichment of transcription start sites (TSS) (B) and nucleosome size gradient fragments, characterizing the high fragmentation and tagging efficiency of open chromatin regions (C); the exon regions accounted for 61% of the transcriptome reads (E), and the intragene region coverage showed high 3' end coverage, characterizing the 3' end transcriptome sequencing quality of this platform.

[0322] This application utilizes a hydrogel system to construct single-cell multi-omics libraries primarily based on mitochondrial DNA. This approach minimizes cross-contamination of cytoplasmic contents between cells (such as mitochondrial DNA cross-contamination and RNA cross-contamination in the cytoplasm). It enables high-throughput single-cell mtDNA deep sequencing and simultaneous mapping of chromatin accessibility, or high-throughput single-cell mtDNA deep sequencing, simultaneous mapping of chromatin accessibility and transcriptome.

[0323] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Anyone skilled in the art may make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended patent application.

Claims

1. A method for constructing a single-cell library of biomaterials embedded in a hydrogel, comprising: Hydrogels containing embedded biomaterials were treated using transposases; Flow cytometry was used to sort hydrogels containing embedded biomaterials that had been treated with transposases. The sorted hydrogels embedded with biomaterials were then labeled. Library construction was performed on the labeled biological materials; Furthermore, the biomaterials are biomaterials that have undergone permeability treatment. The permeation-treated biomaterial is a slightly permeable biomaterial; The slightly permeable biomaterial is a biomaterial that allows small molecules and some larger molecules to freely enter and exit without cell lysis or destruction of the internal organic structure of the cell. The slight permeation treatment refers to a low-temperature treatment in a solution containing a nonionic surfactant, wherein the pH of the solution containing the nonionic surfactant is 7-8. Solutions containing nonionic surfactants also include salts, buffer solutions, and bovine serum albumin; The temperature for low-temperature treatment is -10℃ to 10℃; The nonionic surfactant is selected from NP40; The salt is selected from one or more of sodium chloride, magnesium chloride, sodium sulfate, and magnesium sulfate; The buffer solution is selected from one or more of Tris-HCl, phosphate buffer, acetate buffer, and HEPES buffer. The biomaterial-embedded hydrogel includes a core gel material embedded with biomaterial; The hydrogel embedded with biomaterials also includes an outer shell layer capable of encapsulating the core gel material embedded with biomaterials, the outer shell layer having a thickness of 1-2 µm; The mass ratio of the core gel material to the outer shell layer is (2-25):1; The core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starch, and glucose; The molecular weight of the core gel material is 10 kDa-800 kDa; The outer shell layer comprises a high molecular weight hydrophilic polymer and a low molecular weight hydrophilic polymer, which gives the outer shell layer a porous structure. The hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate, polypropylene glycol, ethylene oxide, and propylene oxide. The molecular weight of the high molecular weight hydrophilic polymer is 6 kDa-20 kDa; The low molecular weight hydrophilic polymer has a molecular weight of 0.2 kDa-6 kDa.

2. The method according to claim 1, wherein, The transposase is selected from any one of Tn5, Mu, and Vibrio.

3. The method of claim 1, wherein, The microspheres used for labeling are selected from any one of polystyrene (PS) microspheres, polymethyl methacrylate (PMMA) microspheres, polyethylene microspheres, and agarose soft microspheres.

4. The method according to any one of claims 1 to 3, wherein, Library construction includes any one, two, or three of the following: (i) Constructing a library of mitochondrial DNA; (ii) Constructing a library from open chromatin regions; (iii) Construct a library from the 3' end transcriptomic RNA.

5. The method of claim 1, wherein, The pore size of the porous structure of the outer shell is smaller than the average size of the biomaterial.

6. The method of claim 1, wherein, The biomaterial is a cell.

7. The method of claim 5, wherein, The core gel material has a porous structure.

8. The method of claim 7, wherein, Biomaterials can be embedded within the porous structure of the core gel material.

9. The method of claim 7, wherein, The porous structure of the core gel material has a pore size of 2-5 µm, and the porous structure of the outer shell layer has a pore size of 24 nm-86 nm.

10. The method of claim 1, wherein, In the hydrogel, the mass ratio of the core gel material to the outer shell layer is (5-20):

1.

11. The method of claim 3, wherein, The preparation method of hydrogel includes the following steps: Biomaterials are encapsulated within a core gel material phase; Hydrogels are generated by controlling the curing or semi-curing of the core gel material phase, the outer shell phase, and the oil phase using microfluidic manipulation: The hydrogel is obtained by permeation treatment of biomaterial hydrogels; The core gel material phase is a solution of the core gel material; The outer shell phase is a solution of the outer shell material.

12. The method of claim 11, wherein, Before the biomaterial is encapsulated in the core gel material phase, the core gel material phase and the outer shell phase are premixed and then subjected to liquid-liquid separation to obtain the separated core gel material phase and outer shell phase.

13. The method according to claim 11, wherein, The concentration range of the core gel material is 2% - 50%.

14. The method of claim 12, wherein, The concentration of high molecular weight hydrophilic polymer in the outer shell phase ranges from 3% to 50%.

15. Use of a hydrogel embedded with biomaterials in single cell library construction; wherein, The biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel involved in any one of claims 1-14.

16. The use according to claim 15, wherein, The single-cell library construction includes any one, two, or three of the following: (i) Constructing a library of mitochondrial DNA; (ii) Constructing a library from open chromatin regions; (iii) Construct a library from the 3' end transcriptomic RNA.

17. Use of a hydrogel embedded with a biomaterial in single-cell copy number variation sequencing; wherein, The biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel involved in any one of claims 1-14.

18. Use of a hydrogel embedded with a biomaterial for transposase treatment; wherein, The biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel involved in any one of claims 1-14.

19. Use of a hydrogel embedded with a biomaterial in flow sorting; wherein, The biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel involved in any one of claims 1-14.

20. Use of a hydrogel embedded with a biomaterial in single cell tagging; wherein, The biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel involved in any one of claims 1-14.

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