A hydrogel and uses thereof

Abstract

The present application provides a hydrogel embedded with biomaterials, which comprises a core gel material embedded with biomaterials, and the biomaterials are permeabilized biomaterials. The present application encapsulates biomolecules in a hydrogel, and the permeabilization in the hydrogel can increase the permeabilization strength of the biomolecules and the biocompatibility of various biological reactions.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a hydrogel and its applications. 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 hydrogel embedded with biomaterial, comprising a core gel material embedded with biomaterial, wherein the biomaterial is a permeable biomaterial.

[0007] 2. The hydrogel according to claim 1, further comprising an outer shell layer capable of encapsulating a core gel material embedded with biomaterial, the outer shell layer having a thickness of 1-2 μm.

[0008] 3. The hydrogel according to item 2, wherein,

[0009] The outer shell has a porous structure, and the pore size of the porous structure is smaller than the average size of the biomaterial.

[0010] 4. The hydrogel according to item 1, wherein,

[0011] The biomaterial is selected from proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles, and one or more of them in cells, as well as complexes formed therefrom, with cells being the preferred biomaterial.

[0012] 5. The hydrogel according to claim 1, wherein the permeable biomaterial is a slightly permeable biomaterial or a strongly permeable biomaterial;

[0013] Preferably,

[0014] 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.

[0015] The highly permeable biomaterial is a biomaterial that releases cell contents by disrupting the cell membrane.

[0016] 6. The hydrogel according to item 5, wherein,

[0017] The slight permeation treatment refers to a low-temperature treatment in a solution containing a nonionic surfactant, with the solution having a pH of 7-8.

[0018] 7. The hydrogel according to item 6, wherein,

[0019] Solutions containing nonionic surfactants also include one or more of the following: salts, buffer solutions, and bovine serum albumin.

[0020] 8. The hydrogel according to item 6, wherein,

[0021] The temperature for low-temperature treatment is (-10℃ to 10℃).

[0022] 9. The hydrogel according to item 6, wherein,

[0023] The nonionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPAL CA-630, and Octyl Glucoside.

[0024] 10. A hydrogel that can be embedded in biomaterials, comprising a core gel material and a shell layer having a thickness of 1-2 μm.

[0025] 11. The hydrogel according to any one of items 1-9 or the hydrogel according to item 10, wherein the core gel material has a porous structure;

[0026] Preferably,

[0027] Biomaterials can be embedded within the porous structure of the core gel material;

[0028] More preferably,

[0029] 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.

[0030] 12. The hydrogel according to any one of items 1-11, wherein,

[0031] The core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose.

[0032] Preferably,

[0033] The molecular weight of the core gel material is 0.18kDa-800kDa.

[0034] More preferably,

[0035] 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.

[0036] More preferably,

[0037] 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.

[0038] 13. The hydrogel according to any one of items 1-11, wherein,

[0039] In the hydrogel, the mass ratio of the core gel material to the outer shell layer is (2-25):

[0040] 1, preferably (5-20):1.

[0041] 14. A method for preparing the hydrogel according to any one of items 2-13, comprising the following steps:

[0042] Biomaterials are encapsulated within a core gel material phase;

[0043] 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:

[0044] The hydrogel is obtained by permeation treatment of biomaterial hydrogels;

[0045] 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.

[0046] 15. The method according to item 14, wherein,

[0047] 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.

[0048] 16. The method according to item 14 or 15, wherein,

[0049] The concentration range of the core gel material is 2%-50%.

[0050] 17. The method according to item 14 or 15, wherein,

[0051] The concentration of high molecular weight hydrophilic polymer in the outer shell phase ranges from 3% to 50%.

[0052] 18. The application of the hydrogels prepared by any one of items 1-13 and any one of items 14-17 in single-cell multi-omics library construction.

[0053] Compared with the prior art, the beneficial effects of this application are as follows:

[0054] 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.

[0055] 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.

[0056] This application utilizes a hydrogel platform with different molecular pore sizes inside and out, breaking through the current limitations of international oil-in-water microdroplet systems and enabling high-throughput single-cell multi-omics library construction; this application is applicable to any particles containing biofilms. Attached Figure Description

[0057] Figure 1 The following image shows a bright-field microscope image of the hydrogel in the examples and comparative examples.

[0058] Figure 2 The structural diagram of the hydrogel of this application is shown;

[0059] 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.

[0060] Figure 4 Images of cells before and after rupture are shown.

[0061] Figure 5 An image showing the average diameter of the hydrogel is displayed.

[0062] 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 layer has the following pore sizes: Figure 6 As shown in B.

[0063] Figure 7 The study demonstrated that the hydrogel could significantly retain DNA molecules larger than 968 bp.

[0064] Figure 8 An image of the hydrogel under slightly permeable conditions is shown.

[0065] Figure 9 An image of a hydrogel under highly permeable conditions is shown.

[0066] Figure 10An image showing a cell nucleus encapsulated in a hydrogel is shown.

[0067] Figure 11 The image shows cells encapsulated in a hydrogel.

[0068] Figure 12 The image shows cells encapsulated in a hydrogel.

[0069] 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.

[0070] Figure 14 An image of the hydrogel in Comparative Example 1 is shown.

[0071] Figure 15 An image of the hydrogel in Comparative Example 2 is shown.

[0072] Figure 16 An image of the hydrogel in Comparative Example 3 is shown.

[0073] Figure 17 An image showing the thickness of the hydrogel shell layer of this application is displayed.

[0074] Figure 18 The fragment distribution after the Tn5-tagged reaction is shown.

[0075] 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.

[0076] 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;

[0077] Figure 21 The figure shows the average sequencing depth of mtDNA, using a single-cell mtDNA library construction in three hydrogels as an example.

[0078] Figure 22The 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.

[0079] Figure 23 The experiment demonstrates the construction of mtDNA and chromatin accessibility libraries in hydrogels (based on the Tn5 S5 / S7 library construction assay).

[0080] 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)).

[0081] Figure 25 The results show the microfluidic platform co-encapsulated with hydrogel droplets and single-cell-tagged microspheres containing the Nextera capture sequence.

[0082] 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

[0083] 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.

[0084] 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.

[0085] 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.

[0086] This application provides a hydrogel embedded with biomaterial, which includes a core gel material embedded with biomaterial, wherein the biomaterial is a biomaterial that has undergone permeation treatment.

[0087] In some embodiments of this application, 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.

[0088] 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.

[0089] In some embodiments of this application, 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.

[0090] In this application, the average size of the biological material refers to the average diameter of the biological material.

[0091] In some embodiments of this application, 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, with cells being the preferred biological material.

[0092] When the biomaterial used in this application is a protein, after permeation treatment, the biomaterial is immobilized in a gel material and can be used for protein purification, specifically as follows: The hydrogel in this application can be used as a medium for protein separation and purification, improving protein purity through selective protein adsorption and elution. Simultaneously, encapsulating the protein can improve its stability during purification, thereby contributing to the preparation of more stable protein drugs and biological products.

[0093] When the biological material in this application is nucleic acid, after permeation treatment, when the biological material is fixed in a gel material, it can be used for molecular diagnostics, as follows: the nucleic acid fixed in the hydrogel in this application can be used for molecular diagnostics, such as detecting specific genes or pathogens by PCR amplification.

[0094] When the biomaterial of this application is sugar, after permeation treatment, when the biomaterial is fixed in a 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.

[0095] When the biomaterial of this application is lipid, after permeation treatment, when the biomaterial is fixed in the gel material, it can be used for drug delivery, specifically as follows: lipids encapsulated in hydrogels can be used to improve drug delivery, and by preparing lipid nanoparticles, the solubility and bioavailability of drugs can be improved.

[0096] When the biomaterial in this application is a metabolite, after permeation treatment, the biomaterial immobilized in a gel material 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 helps to evaluate the impact of new drugs on metabolic pathways, as well as the potential toxicity of the drug. This is very useful for drug development and toxicity studies.

[0097] When the biomaterial of this application is a polypeptide, after permeation treatment, the biomaterial is fixed in a gel material and can be used for drug development, specifically as follows: encapsulating polypeptides in a hydrogel can improve the stability of the drug and enhance its targeting, which is helpful for drug development and therapeutic research.

[0098] When the biomaterials used in this application are bacteria and viruses, after permeation treatment, the biomaterials are fixed in the gel material. The encapsulation of bacteria or viruses in the hydrogel is beneficial for the analysis of cell genomes or viral genetic material at the single-cell level. This is helpful for vaccine development and can also be used for the study of pathogens and understanding their behavior.

[0099] When the biomaterial used in this application is a cell nucleus, after permeation treatment, when the biomaterial is fixed in a 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.

[0100] When the biomaterials of this application are two or more types of cells or their complexes, and are immobilized in a gel material after permeation treatment, they help to understand the interactions between cells, thereby providing profound insights into cell biology.

[0101] In some embodiments of this application, the permeation-treated biomaterial is a slightly permeable biomaterial or a strongly permeable biomaterial; preferably, 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 strongly permeable biomaterial is a biomaterial that releases cell contents by disrupting the cell membrane.

[0102] In some embodiments of this application, 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.

[0103] In some embodiments of this application, the solution containing the nonionic surfactant further includes one or more of the following: salt, buffer solution, and bovine serum albumin.

[0104] In some embodiments of this application, the salt is selected from one or more of sodium chloride, magnesium chloride, sodium sulfate, and magnesium sulfate.

[0105] In some embodiments of this application, the buffer solution is selected from one or more of Tris-HCl, phosphate buffer, acetate buffer, and HEPES buffer.

[0106] In some embodiments of this application, the nonionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPAL CA-630, and Octyl Glucoside.

[0107] In some embodiments of this application, the temperature of the low-temperature treatment is (-10℃ to 10℃). For example, the temperature of the low-temperature treatment can be -10℃, -8℃, -6℃, -4℃, -2℃, 0℃, 2℃, 4℃, 6℃, 8℃, 10℃ or any range thereof.

[0108] In some embodiments of this application, the enhanced permeability treatment refers to treatment in a solution containing protease, treatment in a solution containing SDS, or treatment using an acoustic disruptor to rupture cells using acoustic energy.

[0109] In some embodiments of this application, 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-HClph7.4, 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.

[0110] Cells can be immobilized in the hydrogel described in this application by using a slightly permeable hydrogel, which can have the following applications:

[0111] (1) Bioseparation and dialysis:

[0112] It can be used to separate biomolecules and particles, such as proteins, DNA, and RNA. This facilitates the better application of bioseparation techniques, such as electrophoresis, dialysis, and filtration, to purify and analyze biological samples.

[0113] (2) Cell sorting and enrichment:

[0114] By immobilizing different types of cells in hydrogels and then 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.

[0115] (3) Biosensors:

[0116] 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.

[0117] 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.

[0118] In this application, the thickness of the outer shell layer of the biomaterial-embedd hydrogel can be detected using methods known to those skilled in the art. The thickness of the outer shell layer is 1-2 μm, which can be understood as the average thickness of the outer shell layer being 1-2 μm. Those skilled in the art can arbitrarily select one site in the outer shell layer to detect and determine its thickness, or arbitrarily select several sites, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites, to detect and calculate their average value, i.e., the average value is 1-2 μm. For example, the outer shell layer of the hydrogel can be observed using a microscope, and the thickness of the outer shell layer can be measured using the microscope's detection module.

[0119] This application also provides a hydrogel that can be embedded in biomaterials, comprising a core gel material and a shell layer, wherein the thickness of the shell layer is 1-2 μm; for example, the thickness of the 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.

[0120] In this application, the thickness of the outer shell layer can be controlled by controlling the mass ratio between the material phase forming the outer shell layer and the core gel material phase containing biomolecules. Those skilled in the art will understand that as long as the mass ratio of the two can be effectively controlled, the required outer shell layer thickness can be achieved.

[0121] In one specific embodiment, the thickness of the outer shell layer is 1-2 μm, which is obtained by setting the flow rate ratio during the microfluidic generation of the hydrogel from the droplets. 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 of the outer shell material phase and the biomolecule-containing core gel material phase are merely an example, and those skilled in the art will understand that appropriate adjustments can be made when operating with different equipment.

[0122] 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.

[0123] 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.

[0124] In some embodiments of this application, in the two types of hydrogels described above, the core gel material has a porous structure; the biomaterial can be embedded inside the porous structure of the core gel material.

[0125] In some embodiments of this application, 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 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 any range therefrom.

[0126] In some embodiments of this application, in the two types of hydrogels described above, the core gel material is a hydrophilic polymer; the core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose.

[0127] In some embodiments of this application, the molecular weight of the core gel material is 0.18 kDa-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, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, or 160 kDa. 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa, 470kDa, 480kDa, 490kDa, 500kDa, 600kDa, 700kDa, 800kDa, or any range between them.

[0128] In some embodiments of this application, 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 makes the outer shell layer have a porous structure.

[0129] 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).

[0130] In some embodiments of this application, 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.

[0131] In some embodiments of this application, 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.

[0132] In some embodiments of this application, 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, 1.1 kDa, 1.2 kDa, 1.3 kDa, 1.4 kDa, 1.5 kDa, 1.6 kDa, 1.7 kDa, 1.8 kDa, 1.9 kDa, 2.0 kDa, 2.1 kDa, 2.2 kDa, 2... .3kDa, 2.4kDa, 2.5kDa, 2.6kDa, 2.7kDa, 2.8kDa, 2.9kDa, 3.0kDa, 3.1kDa, 3.2kDa, 3.3kDa, 3.4kDa, 3.5kDa, 3.6kDa, 3.7kDa, 3. 8kDa, 3.9kDa, 4.0kDa, 4.1kDa, 4.2kDa, 4.3kDa, 4.4kDa, 4.5kDa, 4.6kDa, 4.7KDa, 4.8kDa, 4.9kDa, 5.0kDa, 5.1kDa, 5.2kDa, 5.3k Da, 5.4kDa, 5.5kDa, 5.6kDa, 5.7kDa, 5.8kDa, 5.9kDa, 6.0kDa or any range therebetween.

[0133] In some embodiments of this application, 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, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 3... 6nm, 37nm, 38nm, 39nm, 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm, 60nm, 61nm, 62nm, 63nm, 64nm, 65nm, 66nm, 67nm, 68nm, 69nm, 70nm, 71nm, 72nm, 73nm, 74nm, 75nm, 76nm, 77nm, 78nm, 79nm, 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, or any range thereof.

[0134] In some embodiments of this application, the porosity of the porous structure of the outer shell layer can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or any range thereof.

[0135] In some embodiments of this application, 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.

[0136] In some embodiments of this application, the mass ratio of the core gel material to the outer shell layer in the hydrogel is (5-20):1.

[0137] In some embodiments of this application, 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.

[0138] 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.

[0139] In some embodiments of this application, 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.

[0140] In some embodiments of this application, the concentration range of the core gel material is 2%-50%; for example, the concentration range of the core gel material can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any range between them.

[0141] The concentration here refers to the concentration obtained by dividing mass (g) by volume (mL), and the concentration includes biological materials.

[0142] In some embodiments of this application, 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%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any range between them.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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 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.

[0152] Slight permeability of biomolecules can be achieved in hydrogels.

[0153] 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.

[0154] 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).

[0155] Example 1

[0156] 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).

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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, the hydrogel with different molecular pore sizes on the inside and outside, should be collected in a 1.5 mL centrifuge tube.

[0164] 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.

[0165] 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.

[0166] The thickness of the outer shell layer is 1.5-2 μm.

[0167] Example 2

[0168] 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.

[0169] Example 3

[0170] 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.

[0171] Example 4

[0172] 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.

[0173] Example 5

[0174] 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.

[0175] Example 6

[0176] 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.

[0177] Example 7

[0178] 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.

[0179] Example 8

[0180] 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.

[0181] Example 9

[0182] The only difference between Example 9 and Example 1 is that Dextran is replaced with polyvinyl alcohol; otherwise, they are the same.

[0183] Example 10

[0184] The only difference between Example 10 and Example 1 is that PEGDA (MW8kDa) is replaced with polypropylene glycol (MW8kDa), otherwise they are the same.

[0185] Example 11

[0186] 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.

[0187] Comparative Example 1

[0188] The only difference between Comparative Example 1 and Example 1 is that no slight permeation treatment was performed.

[0189] Comparative Example 2

[0190] 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.

[0191] Comparative Example 3

[0192] 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.

[0193] Table 1

[0194]

[0195]

[0196] 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.

[0197] 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.

[0198] Examples 12-19

[0199] The preparation method is the same as in Example 1, except for the slight permeation treatment conditions in Table 2.

[0200] Table 2

[0201]

[0202]

[0203] 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.

[0204] Experimental Example

[0205] Experiment Example 1: Hydrogel Cell Loss Test

[0206] 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.)

[0207] 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.

[0208] 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.)

[0209] 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).

[0210] Experiment Example 2: Diameter Testing of Hydrogels with Different Inner and Outer Molecular Pore Sizes

[0211] 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).

[0212] 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).

[0213] Experimental Example 3

[0214] The collected hydrogels were analyzed by cryo-scanning electron microscopy (Cryo-SEM) to observe their cross-sectional morphology.

[0215] 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.

[0216] 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... Figure 16 As shown.

[0217] Experiment Example 4

[0218] 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.

[0219] 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.

[0220] Experimental Example 5: Slightly Permeable Conditions

[0221] 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.

[0222] 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.

[0223] Experimental Example 6: Strong Permeability Conditions

[0224] 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.

[0225] 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.

[0226] Experiment Example 7:

[0227] 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.

[0228] like Figure 10 As shown, the human 293T cell nuclei can be encapsulated in the hydrogel prepared in Example 7 of this experiment.

[0229] Experimental Example 8:

[0230] 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.

[0231] 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.

[0232] Experiment Example 9:

[0233] 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.

[0234] 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.

[0235] Experimental Example 10

[0236] 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.

[0237] 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 out 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.

[0238] Experimental Example 11: Tn5 Tagging Reaction of Hydrogels

[0239] 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.

[0240] 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.

[0241] Experiment Example 12. Library Construction of Mitochondrial DNA at the Single-Cell Level:

[0242] 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.

[0243] The hydrogel after incubation was stained with 1x EvaGreen (YEASEN, 10223ES76) nucleic acid dye.

[0244] 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).

[0245] 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.

[0246] Perform the following PCR reaction

[0247] Step 1: 72℃, 5 min

[0248] Step 2: 98℃, 30s

[0249] Step 3: 98℃, 10s

[0250] Step 4: 63℃, 30s

[0251] Step 5: 72℃, 1 min

[0252] (Repeat step 5, for a total of 8 times)

[0253] Step 6: Maintain at 12℃.

[0254] 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.

[0255] Relevant data:

[0256] (1) Assembly and activity verification of the Tn5(S5 / S7) transposase used in Experiment Example 12:

[0257] 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.

[0258] 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.

[0259] (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.

[0260] (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.

[0261] Example 13. Simultaneous library construction using mitochondrial DNA and chromatin accessibility at the single-cell level:

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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).

[0266] Example 14: Simultaneous library construction of mitochondrial DNA, chromatin openness, and 3' transcriptome at the single-cell level:

[0267] 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.

[0268] RNA reverse transcription is performed in a hydrogel to form heteroduplex RNA and DNA.

[0269] 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.

[0270] Step 1: 10 min, 50℃;

[0271] Step 2: 8℃, 12s;

[0272] Step 3: 15℃, 45s

[0273] Step 4: 20℃, 45s

[0274] Step 5: 30℃, 30s

[0275] Step 6: 42℃, 2min

[0276] Step 7: 50℃, 3 min

[0277] Repeat steps 2-7, for a total of 3 times.

[0278] Step 8: 50℃, 5min

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] After PCR, the DNA was purified using 1.2x volumes of Vazyme DNAclean beads (N411-01), followed by elution with 30 μL of nuclease-free water. The eluted solution was the final library.

[0284] Relevant data:

[0285] (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 ATn5 S5 / S7 (specific sequence) tagging reaction, standard library construction and indexing PCR were performed, and the average library size was 487bp, 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).

[0286] (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.

[0287] (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.

[0288] (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.

[0289] 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.

[0290] 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 hydrogel embedded with biomaterial, comprising a core gel material embedded with biomaterial, wherein the biomaterial is a permeable biomaterial. 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 hydrogel also includes an outer shell layer capable of encapsulating a core gel material embedded with biomaterials, the outer shell layer having a thickness of 1-2 µm; In the hydrogel, 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 hydrogel according to claim 1, wherein, The pore size of the porous structure of the outer shell is smaller than the average size of the biomaterial.

3. The hydrogel according to claim 1, wherein, The biomaterial is a cell.

4. The hydrogel according to any one of claims 1-3, wherein, The core gel material has a porous structure.

5. The hydrogel according to claim 4, wherein, Biomaterials can be embedded within the porous structure of the core gel material.

6. The hydrogel according to claim 4, 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.

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

1.

8. A method for preparing the hydrogel according to any one of claims 1-7, comprising 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.

9. The method according to claim 8, 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.

10. The method according to claim 8, wherein, The concentration range of the core gel material is 2% - 50%.

11. The method according to claim 8, wherein, The concentration of high molecular weight hydrophilic polymer in the outer shell phase ranges from 3% to 50%.

12. The application of the hydrogel according to any one of claims 1-7, or the hydrogel prepared by any one of claims 8-11, in single-cell multi-omics library construction.

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