Hydrogel, preparation method therefor, and use thereof

Abstract

Provided is a hydrogel with a biological material embedded therein, which comprises a core gel material with a biological material embedded therein, the biological material being a biological material subjected to permeabilization treatment. Enveloping biomolecules in a hydrogel and performing permeabilization treatment therein can improve the intensity of permeabilization and compatibility to various bioreactions of the biomolecules. Also provided is a method for constructing a single cell library for the biomaterial in the hydrogel with the biological material embedded therein. The method can be used for library construction for mitochondrial DNAs and / or library construction for open chromatin regions and / or library construction for 3' end transcriptome (RNAs).

Description

A hydrogel, preparation method and application thereof Technical Field

[0001] The present application relates to the field of biotechnology, and specifically to a hydrogel, a preparation method and application thereof, specifically, a hydrogel embedded with biomaterials, a preparation method of a hydrogel embedded with biomaterials, and an application of a hydrogel embedded with biomaterials, such as a method for constructing a single-cell library of biomaterials in a hydrogel embedded with biomaterials, and the use of a hydrogel embedded with biomaterials in constructing a single-cell library. Background Art

[0002] Cells are the most basic units of life. Currently, in the fields of oncology, immunity, development, and neurology, 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, which can fully restore cell characteristics and differences between populations, and can more accurately analyze the heterogeneity of cell populations. At the same time, single-cell sequencing can analyze life processes at single-cell resolution through the two dimensions of time and space, deconstructing life with big data. At the level of tissues, organs, and individuals, it realizes the study of life processes from "breaking the whole into pieces" to "breaking the pieces into the whole", and extracting profound insights from complex biological samples.

[0003] Single-cell sample processing during single-cell multi-omics sequencing involves the processing of intact cell mixtures or extracted nuclei mixtures through biochemical reactions such as cell lysis in preparation for downstream flow cytometric sorting into microplates or droplet formation via droplet microfluidics to prepare single-cell multi-omics libraries. Extracted nuclei mixtures inherently lack biomolecules from the cytoplasm, resulting in a loss of information in the native single-cell multi-omics library. Intact cell mixtures, due to the susceptibility of cell membranes to rupture, result in significant cell-to-cell cross-contamination in single-cell multi-omics libraries.

[0004] Summary of the Invention

[0005] This application is inspired by the widespread compartmentalization phenomenon in nature. For example, cells rely on permeability-controlled cell membranes and various organelles to compartmentalize biomacromolecules and regulate their diffusion, thereby enabling complex life activities while ensuring the exchange of substances with the outside world. This paper designs hydrogels with different molecular pore sizes inside and outside (i.e., heterogeneous hydrogels inside and outside) and their applications:

[0006] The technical solutions of the present application regarding a biomaterial-embedded hydrogel and its application are as follows:

[0007] 1. A hydrogel embedded with a biomaterial, comprising a core gel material embedded with the biomaterial, wherein the biomaterial is a permeabilized biomaterial.

[0008] 2. The hydrogel according to item 1, further comprising an outer shell layer capable of covering the inner core gel material embedded with the biological material, wherein the outer shell layer has a thickness of 1-2 μm.

[0009] 3. The hydrogel according to item 2, wherein

[0010] 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 biological material.

[0011] 4. The hydrogel according to item 1, wherein

[0012] The biological material is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed therefrom. Preferably, the biological material is a cell.

[0013] 5. The hydrogel according to item 1, wherein the permeabilized biomaterial is a slightly permeabilized biomaterial or a strongly permeabilized biomaterial;

[0014] Preferably,

[0015] The slightly permeabilized biological material is a biological material that allows small molecules and some larger molecules to freely enter and exit without lysing the cells or destroying the internal organic structure of the cells;

[0016] The strongly permeabilized biological material is a biological material whose cell membrane is destroyed and the cell contents are released.

[0017] 6. The hydrogel according to item 5, wherein

[0018] The slight permeabilization treatment refers to low-temperature treatment in a solution containing a non-ionic surfactant, and the pH of the solution containing the non-ionic surfactant is 7-8.

[0019] 7. The hydrogel according to item 6, wherein

[0020] The solution containing the nonionic surfactant further comprises one or more of salt, buffer solution and bovine serum albumin.

[0021] 8. The hydrogel according to item 6, wherein

[0022] The temperature of the low temperature treatment is (-10°C to 10°C).

[0023] 9. The hydrogel according to item 6, wherein

[0024] The nonionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPAL CA-630, and Octyl Glucoside.

[0025] 10. A hydrogel capable of embedding biomaterials, comprising an inner core gel material and an outer shell layer, wherein the outer shell layer has a thickness of 1-2 μm.

[0026] 11. The hydrogel according to any one of items 1 to 9 or the hydrogel according to item 10, wherein the inner core gel material has a porous structure;

[0027] Preferably,

[0028] Biomaterials can be embedded in the porous structure of the core gel material;

[0029] More preferably,

[0030] The pore size of the porous structure of the core gel material is 2-5 μm, and the pore size of the porous structure of the outer shell layer is 24 nm-86 nm.

[0031] 12. The hydrogel according to any one of items 1 to 11, wherein

[0032] The core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose;

[0033] Preferably,

[0034] The molecular weight of the inner core gel material is 0.18kDa-800kDa;

[0035] More preferably,

[0036] The outer shell layer comprises a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, and the high molecular weight hydrophilic polymer and / or the low molecular weight hydrophilic polymer are used to make the outer shell layer have a porous structure;

[0037] More preferably,

[0038] The hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate (PEGDA), polypropylene glycol, and ethylene oxide and propylene oxide.

[0039] 13. The hydrogel according to any one of items 1 to 11, wherein

[0040] In the hydrogel, the mass ratio of the inner core gel material to the outer shell layer is (2-25):1, preferably (5-20):1.

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

[0042] The biomaterial is encapsulated in the inner core gel material phase;

[0043] Microfluidic manipulation is used to generate hydrogels by controlling the solidification or semi-solidification of the core gel material phase, the shell phase, and the oil phase:

[0044] Performing permeabilization treatment in the biomaterial hydrogel to obtain the hydrogel;

[0045] The inner core gel material phase is a solution of the inner core gel material; and the outer shell phase is a solution of the outer shell material.

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

[0047] Before the biological material is wrapped in the inner core gel material phase, the inner core gel material phase and the outer shell phase are pre-mixed and then subjected to liquid-liquid separation treatment to obtain separated inner core gel material phase and outer shell phase.

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

[0049] The concentration of the inner core gel material ranges from 2% to 50%.

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

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

[0052] 18. Use of the hydrogel described in any one of items 1-13 or the hydrogel prepared by the method described in any one of items 14-17 in single-cell multi-omics library construction.

[0053] The technical solutions for the application of hydrogels embedded with biomaterials in this application are as follows:

[0054] 1. A method for constructing a single-cell library of a biomaterial embedded in a hydrogel containing the biomaterial, comprising:

[0055] Treating hydrogels embedded with biomaterials with transposases;

[0056] Flow sorting is used to sort the biomaterial-embedded hydrogels treated with transposase;

[0057] labeling the sorted biomaterial-embedded hydrogel;

[0058] The library is constructed based on the biological materials after tagging.

[0059] 2. The method according to claim 1, wherein

[0060] The transposase is selected from any one of Tn5, Mu, and Vibrio.

[0061] 3. The method according to item 1, wherein the microspheres used for labeling treatment are selected from any one of polystyrene PS microspheres, polymethyl methacrylate PMMA microspheres, polyethylene microspheres, and agarose soft gel microspheres.

[0062] 4. The method according to any one of items 1 to 3, wherein the library construction comprises any one, two or three of the following:

[0063] (i) constructing a mitochondrial DNA library;

[0064] (ii) library construction for the open chromatin interval;

[0065] (iii) Library construction of 3' end transcriptome (RNA).

[0066] 5. The method according to any one of items 1 to 4, wherein the hydrogel embedded with biomaterial comprises an inner core gel material embedded with biomaterial, and the biomaterial is a biomaterial that has been permeabilized.

[0067] 6. The method according to item 5, wherein the hydrogel embedded with biomaterials further comprises an outer shell layer capable of covering the inner core gel material embedded with biomaterials, and the thickness of the outer shell layer is 1-2 μm.

[0068] 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 biological material.

[0069] 8. The method according to item 5, wherein the biological material is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed therefrom, and preferably the biological material is a cell.

[0070] 9. The method according to item 5, wherein the permeabilized biomaterial is a slightly permeabilized biomaterial or a strongly permeabilized biomaterial;

[0071] Preferably,

[0072] The slightly permeabilized biological material is a biological material that allows small molecules and some larger molecules to freely enter and exit without lysing the cells or destroying the internal organic structure of the cells;

[0073] The strongly permeabilized biological material is a biological material whose cell membrane is destroyed and the cell contents are released.

[0074] 10. The method according to item 5, wherein the slight permeabilization treatment refers to low-temperature treatment in a solution containing a non-ionic surfactant, and the pH of the solution containing the non-ionic surfactant is 7-8.

[0075] 11. The method according to item 10, wherein the solution containing the nonionic surfactant further comprises one or more of salt, buffer, and bovine serum albumin.

[0076] 12. The method according to item 10, wherein the temperature of the low temperature treatment is (-10°C to 10°C).

[0077] 13. The method according to item 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.

[0078] 14. The method according to item 5, wherein the inner core gel material has a porous structure;

[0079] Preferably,

[0080] Biomaterials can be embedded in the porous structure of the core gel material;

[0081] More preferably,

[0082] The pore size of the porous structure of the core gel material is 2-5 μm, and the pore size of the porous structure of the outer shell layer is 24 nm-86 nm.

[0083] 15. The method according to item 5, wherein the core gel material is one or more selected from the group consisting of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose;

[0084] Preferably,

[0085] The molecular weight of the inner core gel material is 0.18kDa-800kDa;

[0086] More preferably,

[0087] The outer shell layer comprises a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, and the high molecular weight hydrophilic polymer and / or the low molecular weight hydrophilic polymer are used to make the outer shell layer have a porous structure;

[0088] More preferably,

[0089] The hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate (PEGDA), polypropylene glycol, and ethylene oxide and propylene oxide.

[0090] 16. The method according to item 5, wherein in the hydrogel, the mass ratio of the inner core gel material to the outer shell layer is (2-25):1, preferably (5-20):1.

[0091] 17. The method according to claim 5, wherein the method for preparing the hydrogel comprises the following steps:

[0092] The biomaterial is encapsulated in the inner core gel material phase;

[0093] Microfluidic manipulation is used to generate hydrogels by controlling the solidification or semi-solidification of the core gel material phase, the shell phase, and the oil phase:

[0094] Performing permeabilization treatment in the biomaterial hydrogel to obtain the hydrogel;

[0095] The inner core gel material phase is a solution of the inner core gel material; and the outer shell phase is a solution of the outer shell material.

[0096] 18. The method according to claim 17, wherein, before the biological material is encapsulated in the inner core gel material phase, the inner core gel material phase and the outer shell phase are pre-mixed and then subjected to liquid-liquid separation treatment to obtain separated inner core gel material phase and outer shell phase.

[0097] 19. The method according to claim 17, wherein

[0098] The concentration of the inner core gel material ranges from 2% to 50%.

[0099] 20. The method of claim 18, wherein the concentration of the high molecular weight hydrophilic polymer in the shell phase is in the range of 3% to 50%.

[0100] 21. Use of hydrogels embedded with biomaterials in the construction of single-cell libraries.

[0101] 22. The method according to claim 21, wherein the single cell library construction comprises any one, two or three of the following:

[0102] (i) constructing a mitochondrial DNA library;

[0103] (ii) library construction for the open chromatin interval;

[0104] (iii) Library construction of 3' end transcriptome (RNA).

[0105] 23. Use of hydrogels embedded with biomaterials in single-cell copy number variation sequencing.

[0106] 24. Use of a hydrogel embedded with biological material for transposase treatment.

[0107] 25. Use of hydrogels embedded with biological materials in flow cytometry.

[0108] 26. Use of hydrogels embedded with biomaterials for single cell labeling.

[0109] 27. The use according to any one of items 21 to 26, wherein the hydrogel embedded with a biomaterial is the hydrogel embedded with a biomaterial according to the method according to any one of items 1 to 20.

[0110] Compared with the prior art, the present invention has the following advantages:

[0111] The outer shell has an artificial membrane with small pores: it can realize controllable material exchange (enzymes, primers and PCR amplification products, etc.); the inner core gel material is a non-hollow large-pore matrix: it can support the biomolecular membrane system and reduce the diffusion efficiency of biomolecules; the inner core gel material contains biomolecules that can be permeabilized: the cell permeabilization step will remove the biomolecular membrane liposomes, thereby allowing larger molecules such as antibodies to enter the interior of the biomolecules, while better preserving the physiological properties of the biomolecules.

[0112] The present application encapsulates biological molecules in a hydrogel, and performs a permeabilization treatment in the hydrogel, which can increase the strength of the permeabilization of the biological molecules and the compatibility with various biological reactions. When the permeabilization conditions are relatively mild (i.e., weak permeabilization treatment or slight permeabilization treatment), the internal hydrogel can enhance the support for the biomembrane molecules and maximize the preservation of the biomembrane structure. When the permeabilization conditions are strong, the internal hydrogel plays a role in reducing the diffusion efficiency of the biological molecules; at the same time, the external hydrogel membrane can play a role in selective permeability of the substance. In summary, hydrogels with different molecular pore sizes inside and outside can perform high-throughput reagent addition or reduction and prevent cross-contamination of biological molecules.

[0113] The hydrogel platform with different molecular pore sizes inside and outside the present application breaks through the limitations of the current international oil-in-water microdroplet system and enables the construction of high-throughput single-cell multi-omics libraries; the present application is applicable to any particles with biofilms.

[0114] The hydrogel embedded with biomaterials in this application is used to construct a mitochondrial DNA library. The mitochondrial DNA library construction can be used to study mitochondrial genetic variation in cells and understand the structure and function of mitochondrial DNA; it can be used to explore mitochondrial-related diseases. Mitochondrial DNA variation is related to 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. Mitochondrial DNA plays an important role in evolutionary research. Through library construction, the differences in mitochondrial DNA between species can be understood.

[0115] The hydrogel embedded with biomaterials in this application constructs a library of chromatin open intervals, which can be used to study gene regulation. The construction of the chromatin open interval library helps to identify and study gene regulatory regions, including promoters and enhancers; it can be used for functional annotation: by analyzing the open intervals, the function and regulatory mechanism of the gene can be predicted; it can also identify potential regulatory elements: help identify potential regulatory elements related to cell specificity, developmental processes or diseases.

[0116] The biomaterial-embedded hydrogels described in this application construct libraries of 3'-end transcriptomes (RNAs), which can be used to study gene expression. 3'-end transcriptome libraries are used to delve deeper into gene expression patterns in cells, particularly focusing on the 3'-end of RNA. They can also be used for single-cell analysis, such as single-cell RNA sequencing, which can reveal changes in gene expression at the single-cell level. They can also be used to study transcriptional endpoint regulation, helping to understand the regulation of RNA processing, splicing, and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIG1 shows bright field microscopy images of the hydrogels in the examples and comparative examples.

[0118] FIG2 shows a structural diagram of the hydrogel of the present application.

[0119] Figure 3 shows a schematic diagram of the droplet microfluidic chip for generating hydrogels in the present application; wherein 1 represents the shell layer material phase inlet, 2 represents the core gel material phase inlet, 3 represents the oil phase inlet, and 4 represents the hydrogel collection outlet.

[0120] FIG4 shows pictures of cells before and after emulsion breaking.

[0121] FIG5 shows a graph of the average diameter of the hydrogels.

[0122] FIG6 shows a scanning electron micrograph of the interior of the hydrogel of Example 1; the porous structure of the inner core gel material is shown in FIG6A, and the pore size of the porous structure of the outer shell layer is shown in FIG6B.

[0123] FIG. 7 shows that the hydrogel can significantly retain DNA molecules larger than 968 bp.

[0124] FIG8 shows a picture of the hydrogel under slightly permeabilized conditions.

[0125] FIG9 shows images of hydrogels under strong permeabilization conditions.

[0126] FIG10 shows a picture of cell nuclei encapsulated in hydrogel.

[0127] FIG11 shows a picture of cells encapsulated in hydrogel.

[0128] FIG12 shows a picture of cells encapsulated in hydrogel.

[0129] Figure 13 shows the cross-contamination rate of human-mouse mixtures in single-cell scATAC-seq data: Figure 13 A is a group of intact cells (human 293T cells and mouse 3T3 cells mixed in equal proportions), and Figure 13 B is a group of intact cells (human 293T cells and mouse 3T3 cells mixed in equal proportions) encapsulated in hydrogels with different inner and outer molecular pore sizes.

[0130] FIG14 shows a photograph of the hydrogel in Comparative Example 1.

[0131] FIG15 shows a photograph of the hydrogel in Comparative Example 2.

[0132] FIG16 shows a photograph of the hydrogel in Comparative Example 3.

[0133] FIG. 17 shows a graph of the thickness of the hydrogel shell layer of the present application.

[0134] FIG18 shows the fragment distribution after Tn5 tagmentation reaction.

[0135] Figure 19 shows the results of our laboratory's self-assembled Tn5 and enzyme activity verification. In Figure 19 (A), Tn5 S5 / S7 was used to perform Tn5 tagmentation on 50 ng of HEK293T genomic DNA, followed by PCR amplification; in Figure 19 (B), the amplified product was detected by agarose gel electrophoresis.

[0136] Figure 20 shows the results of human 293T cells encapsulated in hydrogels, which can be flow-sorted after Tn5 labeling reaction and nucleic acid dye, wherein the sorting strategy is as shown in Figure 20A, and the hydrogels that are positive (cell-containing hydrogels) are sorted out as shown in Figure 20B.

[0137] FIG21 shows the average sequencing depth of mtDNA using the example of constructing single-cell mtDNA libraries in three hydrogels.

[0138] Figure 22 shows the results of simultaneous library construction of mitochondrial DNA and chromatin accessibility at the cell level by encapsulating human 293T cells and mouse 3T3 cells in hydrogels.

[0139] Figure 23 shows the mtDNA and chromatin accessibility library construction test in hydrogel (based on Tn5 S5 / S7 library construction test.)

[0140] Figure 24 shows the results of 3-terminal transcriptome sequencing in permselective membrane droplets (library construction based on Tn5 S5 / S7 followed by in situ reverse transcription, and library construction using Tn5 S7 / S7 (tagmentation) after reverse transcription).

[0141] FIG25 shows the results of a microfluidic platform in which hydrogel droplets and single-cell labeling microspheres containing Nextera capture sequences are co-encapsulated.

[0142] Figure 26 shows the results of high-throughput deep sequencing of mitochondrial DNA, chromatin accessibility and 3' end transcriptome at the single-cell level using a self-developed hydrogel droplet microfluidic platform with different inner and outer molecular pore sizes. DETAILED DESCRIPTION

[0143] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0144] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0145] In this application, a hydrogel with different molecular pore sizes inside and outside also refers to a hydrogel with heterogeneous inside and outside.

[0146] The present application provides a hydrogel embedded with a biomaterial, wherein the hydrogel comprises a core gel material embedded with the biomaterial, and the biomaterial is a biomaterial that has undergone a permeabilization treatment.

[0147] In some embodiments, the hydrogel embedded with biomaterial further comprises an outer shell layer capable of encapsulating the inner core gel material embedded with biomaterial, and the thickness of the outer shell layer is 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 therebetween.

[0148] The hydrogel of the present application includes: an artificial membrane with a small pore size on the outside: to achieve controllable material exchange (enzymes, primers and PCR amplification products, etc.); an internal non-hollow large-pore matrix: to support the biomolecular membrane system and reduce the diffusion efficiency of biomolecules; and the interior contains biomolecules that can be permeabilized: the cell permeabilization step will remove the biomolecular membrane liposomes, thereby allowing larger molecules such as antibodies to enter the interior of the biomolecules, while better preserving the physiological properties of the biomolecules.

[0149] 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.

[0150] In this application, the average size of a biological material refers to the average diameter of the biological material.

[0151] In some embodiments, the biomaterial is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed therefrom. Preferably, the biomaterial is a cell.

[0152] When the biological material of the present application is a protein, when the protein is fixed in the gel material after the permeabilization treatment of the present application, it can be used for protein purification, specifically as follows: the hydrogel in the present 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, the encapsulation of the protein can improve its stability during the purification process, thereby helping to prepare more stable protein drugs and biological products.

[0153] When the biological material of the present application is nucleic acid, when the nucleic acid is fixed in the gel material after the permeabilization treatment of the present application, it can be used for molecular diagnosis, as follows: the nucleic acid fixed in the hydrogel in the present application can be used for molecular diagnosis, and specific genes or pathogens can be detected by PCR amplification.

[0154] When the biological material of the present application is sugar, when the sugar is fixed in the gel material after the permeabilization treatment of the present application, it can be used for glycobiology research, specifically as follows: the encapsulation of sugar molecules helps to study the interaction of sugar molecules on the cell surface, which is very important for studying cell adhesion, immune response, recognition of infectious disease pathogens, etc.

[0155] When the biomaterial of the present application is lipid, when the lipid is fixed in the gel material after the permeabilization treatment of the present application, it can be used for drug delivery, specifically as follows: lipid encapsulated in hydrogel can be used to improve drug delivery, and by preparing lipid nanoparticles, the solubility and bioavailability of the drug can be improved.

[0156] When the biomaterial of the present application is a metabolite, after the permeabilization treatment of the present application, the metabolite is fixed in the gel material, which can be used for drug screening and toxicity assessment. Specifically, the metabolite is fixed in the hydrogel, which can be used for drug screening and toxicity assessment. This provides a new way to evaluate the impact of new drugs on metabolic pathways and study the potential toxicity of drugs, which is beneficial to drug development and toxicity research.

[0157] When the biomaterial of the present application is a polypeptide, when the polypeptide is fixed in the gel material after the permeabilization treatment of the present application, it can be used for drug development, specifically as follows: encapsulating the polypeptide in the hydrogel can enhance and improve the stability of the drug and enhance its targeting, which is beneficial to drug development and treatment research.

[0158] When the biological material of the present application is bacteria and / or viruses, when the bacteria and / or viruses are fixed in the gel material after the permeabilization treatment of the present application, the bacteria or viruses encapsulated in the hydrogel are conducive to the analysis of the cell genome or viral genetic material at the single-cell level, which is conducive to the development of vaccines and can also be used for the study of pathogens.

[0159] When the biological material of the present application is a cell nucleus, when the cell nucleus is fixed in the gel material after the permeabilization treatment of the present application, the cell nucleus is encapsulated in the hydrogel and after the permeabilization treatment, chromatin accessibility measurement at the single-cell level can be performed, thereby providing profound insights into epigenetics.

[0160] When the biomaterial of the present application is two or more cells or their complexes, and after the permeabilization treatment of the present application, the two or more cells or their complexes are fixed in the gel material, it helps to study and understand the interaction between cells and opens up profound insights for the study of cell biology.

[0161] In some embodiments, the permeabilized biomaterial is a slightly permeabilized biomaterial or a strongly permeabilized biomaterial; wherein the slightly permeabilized biomaterial is a biomaterial that allows small molecules and some larger molecules to freely enter and exit without lysing the cells or destroying the organic structure inside the cells; wherein the strongly permeabilized biomaterial is a biomaterial in which the cell membrane is destroyed to release the cell contents.

[0162] In some embodiments, the mild permeabilization treatment refers to low-temperature treatment in a solution containing a non-ionic surfactant, wherein the pH of the solution containing the non-ionic surfactant is 7-8.

[0163] In some embodiments, the solution containing a nonionic surfactant further comprises one or more of salt, buffer, and bovine serum albumin.

[0164] In some embodiments, the salt is selected from one or more of sodium chloride, magnesium chloride, sodium sulfate, and magnesium sulfate.

[0165] In some embodiments, the buffer is selected from one or more of Tris-HCl, phosphate buffer, acetate buffer, and HEPES buffer.

[0166] In some embodiments, the nonionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPAL CA-630, and Octyl Glucoside.

[0167] In some embodiments, the temperature of the low temperature treatment is (-10°C to 10°C), for example, the temperature of the low temperature treatment can be -10°C, -8°C, -6°C, -4°C, -2°C, 0°C, 2°C, 4°C, 6°C, 8°C, 10°C or any range therebetween.

[0168] In some embodiments, the strong permeabilization treatment refers to treatment in a solution containing protease, or treatment in a solution containing SDS, or cell disruption treatment using a sonicator using sonic energy.

[0169] In one embodiment, the strong permeabilization treatment refers to treatment in a solution containing a protease, wherein the protease is selected from one or more of Proteinase K, trypsin, Chymotrypsin, Elastase, and Pepsin. The slight permeabilization treatment (or weak permeabilization treatment) refers to treating the hydrogel containing the biomaterial with a slight permeabilization reagent composed of a final concentration of 10mM Tris-HCL ph7.4, 10mM NaCL, 3mM MgCL2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40, and incubating on ice for 3-5 minutes. The strong permeabilization treatment refers to treating the hydrogel containing the biomaterial with a strong permeabilization reagent composed of a final concentration of 0.1M NaCL2, 1mM CaCL2, and 0.05μg / μL Proteinase K, and incubating at 55°C for 30 minutes, and then incubating at 95°C for 10 minutes.

[0170] By slightly permeabilizing the hydrogel, biological materials can be fixed in the hydrogel of this application, which can be used in the following applications:

[0171] (1) Bioseparation and dialysis:

[0172] It can be used to separate biomolecules and particles such as proteins, DNA, RNA, etc. This facilitates the application of bioseparation techniques such as electrophoresis, dialysis, and filtration to purify and analyze biological samples.

[0173] (2) Cell sorting and enrichment:

[0174] Different types of cells can be fixed in hydrogels and slightly permeabilized to allow labeling of cell-specific molecules, which can be used for cell sorting and enrichment, helping to isolate specific cell subpopulations or single cells for single-cell research or cell therapy.

[0175] (3) Biosensors:

[0176] Immobilizing cells in hydrogels can be used to create biosensors that detect specific biomolecules, cytokines, or light-sensitive signals. This is very useful for medical diagnostics, environmental monitoring, and biosensing applications.

[0177] Single-cell whole-genome analysis has always been a challenge because genes are nested within chromosomes. Traditional methods require the use of strong permeabilization lysis buffers, but this limits the ability to simultaneously perform strong cell permeabilization in a microfluidic system. The hydrogel technology proposed in this application can strongly permeabilize cells and encapsulate them together with single-cell barcoded microspheres, thereby simplifying single-cell whole-genome sequencing and overcoming the limitations of traditional methods. This innovative method is expected to bring efficient single-cell genome analysis.

[0178] In the present application, the thickness of the outer shell of the biomaterial-embeddable hydrogel can be measured by methods known to those skilled in the art. The thickness of the outer shell is 1-2 μm, which can be understood as an average thickness of the outer shell of 1-2 μm. Those skilled in the art can select any one site in the outer shell for testing and determining its thickness, or can select any number of sites, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites, for testing and calculating the average value, i.e., the average value is 1-2 μm. For example, the outer shell of the hydrogel can be observed using a microscope, and the thickness of the outer shell can be measured using the detection module of the microscope.

[0179] The present application also provides a hydrogel that can be embedded in biomaterials, which comprises an inner core gel material and an outer shell layer, and the thickness of the outer shell layer is 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 therebetween.

[0180] In the present 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 material phase forming the inner core gel containing biological molecules. Those skilled in the art will understand that as long as the mass ratio of the two can be effectively controlled, the outer shell layer thickness required by the present application can be achieved.

[0181] In one specific embodiment, the shell layer has a thickness of 1-2 μm, achieved by setting a flow rate ratio during droplet microfluidics hydrogel formation, wherein the flow rate setting ratio (in μL / min) is as follows: the flow rate of the shell layer material phase and the biomolecule-containing core gel material phase is set at a ratio of 1:(1-2). For example, the shell layer material phase is set at 1.11 μL / min; the biomolecule-containing core gel material phase is set at 1.55 μL / min to control hydrogel formation. The above flow rates of the shell layer material phase and the biomolecule-containing core gel material phase are merely examples, and those skilled in the art will appreciate that appropriate adjustments can be made when operating with different equipment.

[0182] In one embodiment, the thickness of the outer shell of the hydrogel after formation is observed under a bright field microscope and is 1-2 μm. As shown in FIG17 , the thickness of the outer shell of the hydrogel is within the range of 1-2 μm.

[0183] In this application, Figure 2 shows a schematic diagram of biomolecules encapsulated in a hydrogel with different pore sizes inside and outside the hydrogel. The resulting hydrogel can be placed in large quantities in a 1.5 mL centrifuge tube for permeabilization and the addition and removal of solutions. The pore size of the hydrogel shell is smaller than the average size (diameter) of the internal biomaterial, which plays a role in selective permeability. The hydrogel core gel material is a non-hollow, large-pore matrix that can support the biomolecular membrane system and reduce the diffusion efficiency of biomolecules.

[0184] In some embodiments, in the above hydrogel, the inner core gel material has a porous structure; and the biomaterial can be embedded in the porous structure of the inner core gel material.

[0185] In some embodiments, the pore size of the porous structure of the inner core gel material is 2-5 μm; for example, the pore size of the porous structure of the inner 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 therebetween.

[0186] In some embodiments, in the above-mentioned hydrogel, the inner core gel material is a hydrophilic polymer; the inner core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches, and glucose.

[0187] In some embodiments, the molecular weight of the inner core gel material is 0.18kDa-800kDa; for example, the molecular weight of the inner core gel material can be 0.18kDa, 0.2kDa, 0.3kDa, 0.4kDa, 0.5kDa, 0.6kDa, 0.7kDa, 0.8kDa, 0.9kDa, 1kDa, 5kDa, 10kDa, 20kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, 450kDa, 500kDa, 600kDa, 700kDa, 800kDa or any range therebetween.

[0188] In some embodiments, in the above two hydrogels, the outer shell layer includes a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, and the high molecular weight hydrophilic polymer and / or the low molecular weight hydrophilic polymer give the outer shell layer a porous structure.

[0189] In this application, a high molecular weight hydrophilic polymer refers to a polymer having a weight average molecular weight greater than about 6 kilodaltons (kDa), and a low molecular weight hydrophilic polymer refers to a polymer having a weight average molecular weight less than about 6 kilodaltons (kDa).

[0190] 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), and ethylene oxide propylene oxide.

[0191] In some embodiments, the molecular weight of the high molecular weight hydrophilic polymer is 6 kDa-20 kDa; for example, the molecular weight of the high molecular weight hydrophilic polymer can be 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa or any range therebetween.

[0192] 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 therebetween.

[0193] In some embodiments, the pore size of the porous structure of the outer shell layer is 24 nm-86 nm; for example, the pore size of the porous structure of the outer shell layer is 24 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 86 nm or any range therebetween.

[0194] In some embodiments, the porous structure of the outer shell layer may have a porosity of 80%, 81%, 83%, 85%, 87%, 89%, 90%, or any range therebetween.

[0195] In some embodiments, in the hydrogel, the mass ratio of the inner core gel material to the outer shell layer is (2-25):1; for example, the mass ratio of the inner 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 therebetween.

[0196] In some embodiments, in the hydrogel, the mass ratio of the inner core gel material to the outer shell layer is (5-20):1.

[0197] In some embodiments, in the above two hydrogels, in the hydrogel, the mass of the low molecular weight hydrophilic polymer is not higher than the mass of the high molecular weight hydrophilic polymer; preferably, the ratio of the mass of the high molecular weight hydrophilic polymer to the mass of the low molecular weight hydrophilic polymer is (1-2):1; for example, the ratio of the mass of the high molecular weight hydrophilic polymer to the mass of 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.

[0198] The present application provides a method for preparing the above-mentioned hydrogel, which includes the following steps: encapsulating a biological material in an inner core gel material phase; generating a hydrogel by controlling the solidification or semi-solidification of the inner core gel material phase, the outer shell phase, and the oil phase using microfluidic operation; performing a permeabilization treatment in the biomaterial hydrogel to obtain a hydrogel; the inner core gel material phase is a solution of the inner core gel material; and the outer shell phase is a solution of the outer shell material.

[0199] In some embodiments, before the biomaterial is encapsulated in the inner core gel material phase, the inner core gel material phase and the outer shell phase are pre-mixed and then subjected to liquid-liquid separation to obtain separated inner core gel material phase and outer shell phase.

[0200] In some embodiments, the concentration range of the inner core gel material is 2%-50%; for example, the concentration range of the inner core gel material can be 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or any range therebetween.

[0201] The concentration here refers to the concentration obtained by dividing the mass g by the volume mL, where the concentration includes biological materials.

[0202] In some embodiments, the concentration of the high molecular weight hydrophilic polymer in the shell phase is in the range of 3%-50%; for example, the concentration of the high molecular weight hydrophilic polymer can be in the range of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or any range therebetween.

[0203] The present application provides the use of the above-mentioned hydrogel or the hydrogel prepared by the above-mentioned method in the construction of a single-cell multi-omics library.

[0204] In one embodiment of the present application, a certain amount of PEGDA (MW 8 kDa), a certain amount of PEGDA (MW 575 Da) and a certain amount of Dextran (MW 500 kDa) were taken to prepare a hydrogel mixture, the volume was adjusted to 1 mL, and after mixing evenly, the mixture was centrifuged at 16000 g in a desktop centrifuge for 30 minutes to induce liquid-liquid phase separation.

[0205] After centrifugation, a clear separation boundary was observed between the upper PEGDA-rich phase and the lower dextran-rich phase, and the upper and lower phase droplets were respectively aspirated into corresponding centrifuge tubes.

[0206] To prepare biomolecules, resuspend HEK293T cells in 1 mL of DPBS containing 0.04% BSA and centrifuge at 300g 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. Resuspend approximately 1 million biomolecules in 300 μL of Dextran-rich solution.

[0207] 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 biomolecule suspension.

[0208] Place the loaded syringe into the syringe pump and collect the droplets using a 1.5 mL EP tubing. Set the microfluidic flow rate to 6.67 μL / min for the oil phase, 1.11 μL / min for the PEGDA-rich phase, and 1.55 μL / min for the dextran-rich phase.

[0209] About 200 μL of droplets collected in a 1.5 mL centrifuge tube were placed under a UV lamp and irradiated with UV light for 2 minutes to solidify the PEGDA in the droplets into a gel.

[0210] Pipette a 10 μL droplet onto a Countess slide to observe the formation of reaction compartments. Observe whether the reaction compartments are uniform in size and whether there are any agglomerates.

[0211] Add 500 μL of HFE-7500 containing 20% ​​(vol / vol) perfluorooctanol to the droplet and centrifuge briefly for 5 seconds. Aspirate the oil at the bottom of the tube, add 500 μL of DPBS buffer containing 0.1% (vol / vol) Pluronic F-68, pipette to mix, and centrifuge briefly for 5 seconds. Discard the supernatant from the tube, and the lower layer, containing the hydrogel with different molecular pore sizes, is collected in a 1.5 mL centrifuge tube.

[0212] Slight permeabilization of biomolecules can be achieved in hydrogels.

[0213] The specific conditions for slight permeabilization are as follows: the hydrogel containing the biomaterial is treated with a slight permeabilization reagent consisting of a final concentration 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.

[0214] This application utilizes the complementary compatibility of natural high-molecular-weight dextran (Dextran) and polyethylene glycol diacrylate (PEGDA), and through droplet microfluidics, 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) is prepared to encapsulate biomolecules with good hydrophilicity and biocompatibility in a hydrogel sample processing system.

[0215] The present application provides a method for constructing a single-cell library of biomaterials embedded in the above-mentioned hydrogel with embedded biomaterials, that is, a method for constructing a single-cell multi-omics library based on the above-mentioned hydrogel with different molecular pore sizes inside and outside, which comprises: treating the hydrogel embedded with biomaterials with a transposase; sorting the hydrogel embedded with biomaterials treated with the transposase with a flow cytometry method; labeling the sorted hydrogel embedded with biomaterials; and constructing a library of the labeled biomaterials.

[0216] Those skilled in the art will appreciate that transposase treatment, flow cytometry sorting, ligation, and library construction are common methods in the art, as long as the purpose is met.

[0217] The hydrogel embedded with the biomaterial can be either a slightly permeabilized hydrogel or a strongly permeabilized hydrogel.

[0218] In this application, tagging refers to the step of cutting a DNA molecule by a transposome complex including a transposase and inserting a specific DNA fragment at a specific position. After a purification step to remove the transposase, additional sequences are added to the ends of the inserted fragment by PCR, ligation, or any other suitable method known to those skilled in the art.

[0219] In some embodiments, the transposase is selected from any one of Tn5, Mu, and Vibrio.

[0220] Second-generation sequencing library construction is to fragment or sieve the DNA sample into a target sequence of a specified length, and then add oligonucleotide sequencing adapters P5 and P7 for subsequent sequencing. Traditional library construction methods need to go through steps such as DNA fragmentation, end repair, adapter connection, library amplification, multiple purification and sorting, which is time-consuming. When transposase such as Tn5 is used for sequencing library construction in this application, the multi-step reactions such as DNA fragmentation, end repair, adapter connection can be converted into 1-step reaction, greatly shortening the library construction time and improving work efficiency. The construction of the second-generation sequencing library in this application is all based on the library construction method of transposase.

[0221] In some embodiments, the microspheres used for labeling treatment are selected from any one of polystyrene PS microspheres, polymethyl methacrylate PMMA microspheres, polyethylene microspheres, and agarose soft gel microspheres.

[0222] The labeled microspheres carry a unique sequence identifier. This application allows each cell to be uniquely labeled by co-encapsulating different cells with uniquely identified labeled 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 contributing to a more comprehensive understanding of the heterogeneity of cell populations, such as for studying differences between cell subtypes, identifying rare cell types, and exploring cell responses under different physiological conditions.

[0223] Flow sorting is a highly accurate biological analysis technique used to analyze and classify mixtures of single cells or particles. Single cell separation can be performed by sorting cell-containing hydrogels into 96-well plates (independent reaction chambers), and single cell analysis can be achieved in combination with the subsequent reaction steps of this application. The beneficial effects are as follows: Most scientific research institutes are equipped with flow sorters, and most laboratories can use this method for single-cell analysis. Cells can be stained with a certain biomarker in advance, and then specific cells can be sorted out by flow sorting for subsequent single-cell analysis experiments.

[0224] In some embodiments, library construction includes any one, two, or three of the following: (i) library construction of mitochondrial DNA; (ii) library construction of chromatin open intervals; (iii) library construction of 3' end transcriptome (RNA).

[0225] The hydrogel embedded with biomaterials in this application is used to construct a mitochondrial DNA library: it has the following beneficial effects:

[0226] 1) Research on mitochondrial inheritance: Construction of mitochondrial DNA library can be used to study mitochondrial genetic variation in cells and understand the structure and function of mitochondrial DNA.

[0227] 2) Exploring mitochondrial-related diseases: Mitochondrial DNA variations are associated with a variety of diseases (such as mitochondrial diseases, neurodegenerative diseases, etc.). Library construction helps to study the mechanisms of these diseases.

[0228] 3) Evolutionary research: Mitochondrial DNA plays an important role in evolutionary research. Through library construction, we can understand the differences in mitochondrial DNA between species.

[0229] The biomaterial-embedded hydrogel in this application constructs a library in the open chromatin interval, which has the following effects:

[0230] 1) Study of gene regulation: Construction of chromatin open region libraries helps to identify and study gene regulatory regions, including promoters and enhancers.

[0231] 2) Functional annotation: By analyzing open regions, gene functions and regulatory mechanisms can be predicted. Identification of potential regulatory elements: This helps identify potential regulatory elements related to cell specificity, developmental processes, or diseases.

[0232] The hydrogel embedded with biomaterials in this application constructs a library for the 3' end transcriptome (RNA), which has the following effects:

[0233] 1) Study gene expression: 3' end transcriptome library construction is used to deeply study gene expression patterns in cells, especially focusing on the 3' end of RNA.

[0234] 2) Single-cell analysis: Applicable to single-cell RNA sequencing, which can reveal changes in gene expression at the single-cell level.

[0235] 3) Studying transcriptional endpoint regulation: helps to understand the regulation of RNA processing, splicing and stability.

[0236] The present application provides the use of the above-mentioned biomaterial-embedded hydrogel in the construction of a single-cell library. The single-cell library construction includes any one, two, or three of the following: (i) library construction of mitochondrial DNA; (ii) library construction of chromatin open intervals; and (iii) library construction of the 3' transcriptome (RNA).

[0237] The present application provides the use of the above-mentioned hydrogel embedded with biomaterials in single-cell copy number variation sequencing.

[0238] The present application provides use of the above-mentioned hydrogel embedded with biomaterials for transposase treatment.

[0239] The present application provides use of the above-mentioned hydrogel embedded with biomaterials in flow sorting.

[0240] The present application provides use of the above-mentioned biomaterial-embedded hydrogel in single cell labeling.

[0241] Example 1

[0242] A hydrogel mixture was prepared by taking 0.036g PEGDA (MW 8kDa), 0.024g PEGDA (MW 575Da), and 0.6g Dextran (MW 500kDa). The volume was adjusted to 1mL, mixed thoroughly, and centrifuged at 16,000g 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 was Dextran, and the outer shell was PEGDA (MW 8kDa) and PEGDA (MW 575 Da).

[0243] After centrifugation, a clear separation boundary was observed between the upper PEGDA-rich phase and the lower dextran-rich phase, and the upper and lower phase droplets were respectively aspirated into corresponding centrifuge tubes.

[0244] To prepare biomolecules, resuspend 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. Resuspend approximately 1 million biomolecules in 300 μL of Dextran-rich solution.

[0245] 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 biomolecule suspension.

[0246] Place the loaded syringe into the syringe pump and collect the droplets using a 1.5 mL EP tubing. Set the microfluidic flow rate to 6.67 μL / min for the oil phase, 1.11 μL / min for the PEGDA-rich phase, and 1.55 μL / min for the dextran-rich phase.

[0247] About 200 μL of droplets collected in a 1.5 mL centrifuge tube were placed under a UV lamp and irradiated with UV light for 2 minutes to solidify the PEGDA in the droplets into a gel.

[0248] Pipette a 10 μL droplet onto a Countess slide to observe the formation of reaction compartments. Observe whether the reaction compartments are uniform in size and whether there are any agglomerates.

[0249] Add 500 μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanol to the droplet and centrifuge briefly for 5 seconds. Aspirate the oil at the bottom of the tube, add 500 μL of DPBS buffer containing 0.1% (vol / vol) Pluronic F-68, pipette to mix, and centrifuge briefly for 5 seconds. Discard the supernatant from the tube, and collect the hydrogel layer (with different molecular pore sizes inside and outside) in a 1.5 mL centrifuge tube.

[0250] The biomolecules can be slightly permeabilized in the hydrogel. The specific conditions for slight permeabilization are as follows: the hydrogel containing the biomaterial is treated with a slight permeabilization reagent consisting of a final concentration of 10mM Tris-HCl pH 7.4, 10mM NaCL, 3mM MgCL2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40 and incubated on ice for 3 minutes.

[0251] The pore size of the porous structure of the core gel material was analyzed by cryo-scanning electron microscopy, and as shown in Figure 6A, the pore size is 2-5 μm. The pore size of the porous structure of the outer shell was analyzed by transmission electron microscopy, and as shown in Figure 6B, the pore size is 24 nm to 86 nm.

[0252] The thickness of the outer shell layer is 1.5-2 μm.

[0253] Example 2

[0254] The only difference between Example 2 and Example 1 is that the amount of Dextran is 0.3 g, and the mass ratio of the inner core gel material to the outer shell is 5:1. The rest of the contents are the same.

[0255] Example 3

[0256] The only difference between Example 3 and Example 1 is that the amount of Dextran is 0.9 g, the mass ratio of the inner core gel material to the outer shell is 15:1, and the rest are the same.

[0257] Example 4

[0258] The only difference between Example 4 and Example 1 is that the amount of Dextran is 1.2 g, and the mass ratio of the inner core gel material to the outer shell is 20:1. The rest of the process is the same.

[0259] Example 5

[0260] The only difference between Example 5 and Example 1 is that the amount of Dextran is 1.5 g, the mass ratio of the inner core gel material to the outer shell is 25:1, and the rest are the same.

[0261] Example 6

[0262] The only difference between Example 6 and Example 1 is that the amount of Dextran is 1.8 g, and the mass ratio of the inner core gel material to the outer shell is 30:1. The rest of the process is the same.

[0263] Example 7

[0264] The only difference between Example 7 and Example 1 is that the amount of Dextran is 0.12 g, and the mass ratio of the inner core gel material to the outer shell is 2:1. The rest of the contents are the same.

[0265] Example 8

[0266] The only difference between Example 8 and Example 1 is that the amount of Dextran is 0.06 g, the mass ratio of the inner core gel material to the outer shell is 1:1, and the rest are the same.

[0267] Example 9

[0268] The only difference between Example 9 and Example 1 is that Dextran is replaced by polyvinyl alcohol, and the rest is the same as Example 1.

[0269] Example 10

[0270] The only difference between Example 10 and Example 1 is that PEGDA (MW8kDa) is replaced by polypropylene glycol (MW8kDa), and the rest are the same.

[0271] Example 11

[0272] The only difference between Example 11 and Example 2 is that the mild permeabilization conditions are replaced by strong permeabilization conditions, wherein the strong permeabilization conditions are as follows: the hydrogel containing the biomaterial is treated with a strong permeabilization reagent consisting of a final concentration of 0.1 M NaCL2, 1 mM CaCL2, and 0.05 μg / μL Proteinase K, incubated at 55°C for 30 minutes, and then incubated at 95°C for 10 minutes.

[0273] Comparative Example 1

[0274] The only difference between Comparative Example 1 and Example 1 is that the slight permeabilization treatment is not performed.

[0275] Comparative Example 2

[0276] The only difference between Comparative Example 2 and Example 1 is that Dextran (MW 500 kDa) is not contained, and the rest are the same.

[0277] Comparative Example 3

[0278] The only difference between Comparative Example 3 and Example 1 is that PEGDA (MW 8 kDa) and PEGDA (MW 575 Da) are not contained, and the rest are the same.

[0279] Table 1

[0280] Note: The shell layer thickness shown in Table 1 refers to the result of measuring the thickness of the shell layer using the detection module of the microscope. Due to the existence of errors, the shell layer thickness data in Examples 1-11 are generally within the range of ±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 thickness of the shell layer, and in actual detection, the thickness of the shell layer can be 1.53 μm-1.87 μm.

[0281] 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 mass in the table above refers to the mass required to make the inner core gel material, high molecular weight hydrophilic polymer, and low molecular weight hydrophilic polymer to a constant volume of 1 mL.

[0282] Example 12-Example 19

[0283] The preparation method is similar to that of Example 1, except for the slight permeabilization treatment conditions in Table 2.

[0284] Table 2

[0285] Hydrogels were formed according to the configurations in Tables 1 and 2 above. The collected hydrogels were placed on glass slides and observed under a microscope. The results are shown in Figure 1. Examples 1-7, Examples 9-11, Examples 12-19, and Comparative Example 1 all formed structurally intact hydrogels (circular and without notches when viewed from the top), while the hydrogel formed in Example 8 had notches, and Comparative Examples 2 and 3 failed to form hydrogel structures. The inventors selected the hydrogels from Example 1 or those referring to Example 1 for subsequent experiments.

[0286] Experimental example

[0287] Experimental Example 1 Hydrogel Cell Loss Test

[0288] 1. The generated water-in-oil droplets were collected into a 1.5mL centrifuge tube using microfluidics. Six 10µL droplets were placed on a glass slide to observe the percentage of cell-containing droplets. The average percentage of cell-containing droplets to the total droplets was calculated. (The percentages of cell-containing droplets to the total droplets in the six measurements were: 0.1157, 0.1114, 0.1037, 0.1051, 0.1012, and 0.125, respectively.)

[0289] 2. Place the approximately 200 μL droplet collected in a 1.5 mL centrifuge tube under a UV lamp and irradiate for 2 minutes to solidify the shell material in the droplet into a gel. After gelation, add 500 μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanol and centrifuge for 5 seconds. Aspirate the oil at the bottom of the centrifuge tube and add 500 μL of DPBS buffer containing 0.1% (w / v) Pluronic F-68. Mix by pipetting and centrifuge for 5 seconds. Discard the supernatant in the centrifuge tube. The lower layer, i.e., the hydrogel with different molecular pore sizes inside and outside, is collected in a 1.5 mL centrifuge tube.

[0290] 3. Six 10 μL aliquots of the collected hydrogel from Example 1 were placed on a glass slide to measure the percentage of cell-containing hydrogel. The average percentage of cell-containing hydrogels to the total hydrogels was calculated. (The percentages of cell-containing hydrogels to the total hydrogels in the six measurements were: 0.1034, 0.1096, 0.1143, 0.1, 0.0952, and 0.1105, respectively.)

[0291] The results are shown in Figure 4. As shown in Figure 4, the hydrogel prepared in Example 1 was tested and showed no obvious cell loss before demulsification (water-in-oil droplet state) and after demulsification (hydrogel state) (no statistical difference in cell percentage, independent sample t-test P value was 0.3254).

[0292] Experimental Example 2 Diameter Test of Hydrogels with Different Internal and External Molecular Pore Sizes

[0293] The hydrogel of Example 1 was absorbed onto a glass slide and the diameter of the hydrogel was measured under a microscope (a total of 64 hydrogels were measured, and the diameters were: 61.105, 61.105, 61.105, 61.105, 59.974, 59.974, 58.842, 58.842, 58.842, 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, 55.447 ... .447,55.447,55.447,55.447,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,53.184,53.184,53.184,5 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).

[0294] As shown in FIG5 , the entire hydrogel prepared in Example 1 was tested and the diameter of the hydrogel was 55 μm: FIG5 A is an example of a hydrogel under a microscope, and FIG5 B is a statistical analysis of the diameters of 64 hydrogels (mean and standard deviation).

[0295] Experimental Example 3

[0296] The collected hydrogel was subjected to cryo-scanning electron microscopy (Cryo-SEM) analysis to observe its cross-sectional morphology.

[0297] As shown in FIG6 , the hydrogel prepared in Example 1 was tested and found that the pore size of the inner core gel embedded with the biomaterial was a macroporous matrix of about 2 μm.

[0298] However, it was found that the hydrogel generated in Comparative Example 1 was eccentric and had a gap, as shown in Figure 14, and the hydrogel generated in Comparative Example 2 was eccentric and had a gap, as shown in Figure 15; the hydrogel generated in Comparative Example 3 was broken and could not form a normal hydrogel structure, as shown in Figure 16.

[0299] Experimental Example 4

[0300] The preparation method was similar to that of Example 1, except that the HEK293T cells were replaced with the pFB25 plasmid, and the average pFB25 plasmid concentration per hydrogel was 7 nM. The plasmids were then encapsulated in the hydrogels and placed in a PCR reaction solution containing primers specific for the plasmid's amplification length, a nucleic acid dye, and four different primers corresponding to specific 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 measured using a fluorescence microscope.

[0301] As shown in FIG7 , the hydrogel prepared in Example 1 was tested and DNA molecules larger than 968 bp were clearly retained in the hydrogel: FIG7 A is an example of the fluorescence intensity of specific amplicons in four hydrogels (Scale bar: 50 μm), and FIG7 B is a statistical analysis of the relative fluorescence intensity of specific amplicons in the hydrogel.

[0302] Experimental Example 5: Mild Permeabilization Conditions

[0303] For comparison, HEK293T cell-encapsulated hydrogels were placed in DPBS buffer and a slightly permeabilized 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 slightly permeabilized 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 the same as that used for the hydrogel prepared in Example 1 (results shown in FIG8B ). The DPBS buffer was the same as that used in Example 1, except that the biomolecules were not slightly permeabilized but were treated with DPBS buffer alone (results shown in FIG8A ). After incubation on ice for 5 minutes, the hydrogels were stained with DPBS containing a nucleic acid dye and analyzed under a fluorescence microscope.

[0304] As shown in FIG8 , FIG8A is a picture of a hydrogel without permeabilization treatment, and FIG8B is a picture of a hydrogel treated with slight permeabilization conditions: it can be seen that the biomolecules are well confined in the hydrogel.

[0305] Experimental Example 6: Strong Permeabilization Conditions

[0306] The comparison of the hydrogels encapsulating HEK293T cells was carried out by placing them in DPBS buffer and a strong permeabilization buffer (buffer containing 0.1% Triton X-100, 10mg / ml protease K), respectively. The conditions of the strong permeabilization buffer (buffer containing 0.1% Triton X-100, 10mg / ml protease K) were the hydrogels prepared in Example 11 (the results are shown in B in Figure 9 ), and the DPBS buffer conditions were the same as those in Example 11, except that the biomolecules were not subjected to a strong permeabilization treatment but were only treated with DPBS buffer (the results are shown in A in Figure 9 ). After the hydrogels placed in DPBS buffer were incubated on ice for 5 minutes, the hydrogels were placed in DPBS containing nucleic acid dyes for staining and analyzed under a fluorescence microscope. After the hydrogels placed in the strong permeabilization buffer were incubated at 55 degrees for 30 minutes, the hydrogels were placed in DPBS containing nucleic acid dyes for staining and analyzed under a fluorescence microscope.

[0307] As shown in FIG9 , FIG9A is a picture of a hydrogel without permeabilization treatment, and FIG9B is a picture of a hydrogel treated with strong permeabilization conditions: it can be seen that the biomolecules are well confined in the hydrogel.

[0308] Experimental Example 7:

[0309] After the extracted 293T cell nuclei and the inner core gel material are mixed, the resulting droplets are collected into a 1.5mL centrifuge tube using microfluidics and placed under a UV lamp for 2 minutes to solidify the shell material in the droplets into a gel. After gelation, 500μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanol is added and the mixture is centrifuged in a flash centrifuge for 5 seconds. The oil at the bottom of the centrifuge tube is aspirated, and 500μL of DPBS buffer containing 0.1% (w / v) Pluronic F-68 is added. The mixture is pipetted and mixed, and the mixture is centrifuged in a flash centrifuge for 5 seconds. The supernatant in the centrifuge tube is discarded, and the lower layer, i.e., the hydrogel with different molecular pore sizes inside and outside, is collected in a 1.5mL centrifuge tube (refer to the preparation method of Example 1, the only difference being that HEK293T cells are replaced with 293T cell nuclei). The collected hydrogel is placed on a glass slide for observation.

[0310] As shown in FIG10 , the hydrogel prepared in Experimental Example 7 can be used to encapsulate the nuclei of human 293T cells.

[0311] Experimental Example 8:

[0312] Refer to the method of Example 1, wherein 293T cells are replaced with human peripheral blood mononuclear cells (PBMC), and the rest are the same. After the extracted human peripheral blood mononuclear cells (PBMC) and the inner core gel material are mixed, the resulting droplets are collected into a 1.5mL centrifuge tube by microfluidic operation and placed under a UV lamp for 2 minutes to solidify the shell material in the droplets into a gel. After the gel is formed, 500μL of HFE-7500 containing 20% ​​(v / v) perfluorooctanol is added, and the mixture is centrifuged in a flash centrifuge for 5 seconds. The oil at the bottom of the centrifuge tube is aspirated, and 500μL of DPBS buffer containing 0.1% (w / v) Pluronic F-68 is added. The mixture is pipetted and mixed, and the mixture is centrifuged in a flash centrifuge for 5 seconds. The supernatant in the centrifuge tube is discarded, and the lower layer, i.e., the hydrogel with different molecular pore sizes inside and outside, is collected in a 1.5mL centrifuge tube. The collected hydrogel is placed on a glass slide for observation.

[0313] As shown in Figure 11, human peripheral blood mononuclear cells (PBMCs) can be encapsulated in hydrogels with different molecular pore sizes inside and outside to perform permeabilization and other multi-step biochemical reaction steps.

[0314] Experimental Example 9:

[0315] The hydrogels prepared in Example 1 were divided into four groups and placed in a buffer solution containing 5% dimethyl sulfoxide (Group 1), 25% glycerol (Group 2), 80% ethanol (Group 3), and 0.1% polypropylene glycol and ethylene oxide (Group 4). The hydrogel structures of each group were observed under a microscope after storage at room temperature (22°C) and at low temperature (-80°C) for 48 hours.

[0316] As shown in FIG12 , the hydrogel can be treated with organic reagents required for different common biological reactions (dimethyl sulfoxide, glycerol, ethanol, and polypropylene glycol and ethylene oxide) or stored at different temperatures for 48 hours, and the structure can be observed under a microscope to remain intact.

[0317] Experimental Example 10

[0318] Refer to the method of Example 1, in which 293T cells are replaced with human 293T cells and mouse 3T3 cells mixed in equal proportions, and half of the cells are encapsulated in a hydrogel. The generated hydrogel (encapsulated with human 293T cells and mouse 3T3 cells mixed in equal proportions) and the other half of the cell mixture (human 293T cells and mouse 3T3 cells mixed in equal proportions) are placed in a slightly permeabilized buffer (buffer containing 10mM Tris-HCL ph7.4, 10mM NaCL, 3mM MgCL2, 1% (vol / vol) BSA, 0.1% (vol / vol) NP40), incubated on ice for 5 minutes, and then subjected to Tn5 transposase reaction. After the reaction, the cells are co-encapsulated with single-cell encoded microspheres, and then demulsified and the library is constructed after encapsulation. The constructed library is subjected to second-generation sequencing and bioinformatics analysis. The results are shown in Figure 13.

[0319] A comparison of intact cells and intact cells encapsulated in hydrogels with different inner and outer molecular pore sizes under the same permeabilization conditions (buffer containing 10mM Tris-HCL pH 7.4, 10mM NaCL, 3mM MgCL2, 1% (vol / vol) BSA, and 0.1% (vol / vol) NP40) revealed that the cross-contamination rate of intact cells was 86.12% (Figure 13A) and the cross-contamination rate of intact cells encapsulated in hydrogels with different inner and outer molecular pore sizes was 10.01% (Figure 13B). This indicates that under the same permeabilization conditions, encapsulating intact cells in hydrogels with different inner and outer molecular pore sizes can significantly reduce the cross-contamination rate between cells.

[0320] Experimental Example 11 Tn5 labeling reaction of hydrogel

[0321] The Tn5 tagmentation reaction is a key step in high-throughput sequencing library preparation. Ideally, after Tn5 tagmentation using a hydrogel, DNA molecules are fragmented into short fragments ranging from 100 to 600 bp, while DNA fragments greater than or equal to 968 bp are retained within the hydrogel system. This means that the Tn5-tagged DNA fragments can diffuse freely within and outside the hydrogel network and can be amplified efficiently. Therefore, the fragment distribution of the amplified product should theoretically be between 100 and 600 bp.

[0322] By performing Tn5 tagmentation and subsequent insert amplification experiments on hydrogels encapsulating 293T cell genomic DNA (average 1.66 pg of genomic DNA per hydrogel), the inventors found that the hydrogels could successfully undergo Tn5 tagmentation and generate fragments ranging from 100 bp to 600 bp that meet next-generation sequencing standards. The results are shown in Figure 18.

[0323] Experimental Example 12. Library construction of mitochondrial DNA at the single-cell level:

[0324] The hydrogel of Example 1 was placed in a 50 μL Tn5 (S5 / S7) tagmentation reaction system containing a final concentration of 0.2 μg / μL Tn5 (S5 / S7), a final concentration of 1 x tagmentation buffer, and a final concentration of 5 mM MgCl2, and incubated at 37°C for 30 min.

[0325] After incubation, the hydrogel was stained with 1 x EvaGreen (YEASEN, 10223ES76) nucleic acid dye;

[0326] A total volume of 25 μL PCR reaction system was added to a 96-well plate, which contained i5-primer and i7-primer with a final concentration of 1 μM (i5-primer and i7-primer sequences were both from Nextera Index XT Kit v2, FC-131-2001, Illumina) and a final concentration of 1 x KAPA HiFi HotStart ReadyMix (KK2600).

[0327] The stained hydrogels were flow-sorted and the hydrogels with strong EvaGreen fluorescence signals were placed in a 96-well plate pre-added with the PCR reaction system;

[0328] Perform the following PCR reaction

[0329] Step 1: 72°C, 5 min

[0330] Step 2: 98°C, 30 seconds

[0331] Step 3: 98°C, 10 seconds

[0332] Step 4: 63°C, 30 seconds

[0333] Step 5: 72°C, 1 min

[0334] (Repeat step 5, a total of 8 times)

[0335] Step 6: Keep at 12°C.

[0336] After PCR, the DNA was purified using 1.2x the volume of Vazyme DNA clean beads (N411-01) and eluted with 30 μL of nuclease-free water. The eluted solution is the final library.

[0337] Related data:

[0338] (1) Assembly and enzyme activity verification of Tn5 (S5 / S7) transposase used in Experimental Example 12:

[0339] Purchase Tn5 naked enzyme (ABclonal, RM21303) and perform insert sequence assembly. Once this system is established, Tn5 naked enzyme can be assembled with DNA fragments of any sequence. Using Nextera S5 / S7 as an example, enzyme activity was verified using human 293T cell genomic DNA after assembly. The results are shown in Figure 19.

[0340] Figure 19A demonstrates the activity of assembled Tn5 using genomic DNA from 293T cells. Tn5 containing the Nextera S5 / S7 sequence can tag the genomic DNA. After gap filling and PCR amplification using sequencing adapter primers, agarose gel electrophoresis reveals the presence of a standard 100-700 bp smear. Figure 19B shows that compared to the group without Tn5, the Tn5 enzyme tagmentation reaction produces a distinct smear. This demonstrates the success of the present application's Tn5 naked enzyme and insert sequence assembly system.

[0341] (2) The hydrogel can be adapted to flow cytometry (flow cytometry gating strategy), and the results are shown in Figure 20. Human 293T cells were encapsulated in the hydrogel and subjected to Tn5 labeling reaction and nucleic acid dye and flow cytometry sorting. The sorting strategy is shown in Figure 20A, and the hydrogels that are positive (cell-containing hydrogels) are sorted out as shown in Figure 20B.

[0342] (3) The positive hydrogels can be sorted into 96-well plates for mitochondrial DNA sequencing. The final sequencing coverage is shown in Figure 21: The applicant randomly selected three single-cell libraries. Taking the three hydrogels involved in the three single-cell libraries as an example, the average sequencing depth of mtDNA was 38739.78x, 41798.13x, and 48956.15x, respectively. Among them, the average sequencing depth refers to how many times each site is measured. It can be seen that the single-cell library of this application has an average of tens of thousands of measurements per site in the mitochondrial genome at the single-cell level (deep sequencing depth). This shows that when the hydrogel of this application is used for DNA library construction, the sequencing technology is good, which is conducive to downstream mutation analysis.

[0343] Example 13. Simultaneous library construction of mitochondrial DNA and chromatin accessibility at the single-cell level:

[0344] The hydrogel of Example 10 was used to perform single-cell tagmentation reaction and droplet PCR using the DNBelab C (containing 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;

[0345] After PCR, the DNA was purified using 1.2x the volume of Vazyme DNA clean beads (N411-01) and eluted with 30 μL of nuclease-free water. The eluted solution is the final library.

[0346] The sequencing results after simultaneous sequencing of mitochondrial DNA and chromatin accessibility at the single-cell level are shown in Figure 22.

[0347] Human 293T cells and mouse 3T3 cells were encapsulated in hydrogels with different molecular pore sizes inside and outside to simultaneously construct libraries of mitochondrial DNA and chromatin accessibility at the cellular level. The size distribution of the library fragments after library construction is shown in Figure 22. The nucleobase reads showed a high enrichment of transcription start sites (TSS) (Figures 22B and C). The average mitochondrial genome sequencing depth of each cell was approximately 96×, and the coverage was uniform (Figures 22D and E).

[0348] Example 14 Simultaneous construction of libraries for mitochondrial DNA, chromatin accessibility, and 3'-end transcriptome at the single-cell level:

[0349] The hydrogel in Example 1 was slightly permeabilized and subjected to the first labeling reaction for labeling mitochondrial DNA and open chromatin regions. The labeling reaction system was a 50 μL labeling reaction system containing Tn5 (S5 / S7) at a final concentration of 0.2 μg / μL, a final concentration of 1 x tagmentation buffer, and a final concentration of 5 mM MgCL2. The cells were incubated at 30°C for 30 min.

[0350] RNA reverse transcription is performed in the hydrogel to form RNA and DNA heteroduplexes;

[0351] The total volume of the reverse transcription system is 100 μL, which contains TruseqR1_oligo_dT with a final concentration of 2 μM, dNTP with a final concentration of 0.5 mM, Maxima H minus Reverse Transcriptase with a final concentration of 10 U / μL, RiboLock RNase inhibitor with a final concentration of 2 U / μL, SUPERaseIn RNase inhibitor with a final concentration of 0.2 U / μL, RnaseOUT RNase Inhibitor with a final concentration of 0.4 U / μL, NaCL RT buffer with a final concentration of 1x, and PEG8000 with a final concentration of 12%.

[0352] Step 1: 10 min, 50°C;

[0353] Step 2: 8°C, 12 seconds;

[0354] Step 3: 15°C, 45 seconds

[0355] Step 4: 20°C, 45 seconds

[0356] Step 5: 30°C, 30 seconds

[0357] Step 6: 42°C, 2 min

[0358] Step 7: 50°C, 3 min

[0359] Repeat steps 2-7 a total of 3 times

[0360] Step 8: 50°C, 5 min

[0361] A second tagmentation reaction was performed in the hydrogel to label RNA and cDNA heteroduplexes. The tagmentation reaction consisted of a 50 μL volume containing a final concentration of 0.2 μg / μL Tn5 (S7 / S7), 1 x tagmentation buffer, and 5 mM MgCl2. The reaction was incubated at 37°C for 30 minutes.

[0362] Single-strand nucleotide chain removal and Tn5 transposase tagmentation gap filling reaction were performed in the hydrogel. The reaction system was prepared in a total volume of 50 μL, containing dNTPs at a final concentration of 0.5 mM, Maxima H minus reverse transcriptase at a final concentration of 8 U / μL, Exo1 at a final concentration of 2 U / μL, and NaCl RT buffer at a final concentration of 1x. The reaction was incubated at 37°C for 15 minutes.

[0363] The hydrogel and Nextera capture sequence-encapsulated single-cell tagging microspheres (RAN biotech.050.065.2.ATAC) were co-encapsulated in the droplets. The microfluidic flow rates were set at 6.67 μL / min for the oil phase, 8 μL / min for the PCR master mix phase, 8 μL / min for the hydrogel phase, and 4 μL / min for the single-cell tagging microsphere phase.

[0364] The droplet PCR products were purified and divided into two equal parts by volume, one for mitochondrial DNA and chromatin open sequencing adapter PCR amplification reaction, and the other for transcriptome sequencing adapter PCR amplification reaction.

[0365] After PCR, the DNA was purified using 1.2x the volume of Vazyme DNA clean beads (N411-01) and eluted with 30 μL of nuclease-free water. The eluted solution is the final library.

[0366] Related data:

[0367] (1) The hydrogel can be used to perform mtDNA and chromatin open region tagging reaction (first tagging reaction) using Tn5 S5 / S7 transposase. The results are shown in Figure 23. In Figure 23A, after the Tn5 S5 / S7 (specific sequence) tagging reaction, standard library construction indexing PCR was performed, and the average library size was 487 bp, which was consistent with the theoretical value. Figure 23B shows the analysis of cell nucleus openness after library construction, showing that the tagging reaction in the permselective membrane droplet can retain the chromatin openness information. Figure 23C shows the analysis of mtDNA sequencing depth and coverage after library construction, showing that both coverage and sequencing depth were high (8000x).

[0368] (2) In situ reverse transcription was performed in the hydrogel and RNA / DNA hybrid chain labeling reaction was performed using Tn5 S7 / S7, and the results are shown in Figure 24. Figure 24 shows the results of 3' transcriptome sequencing in selective permeability membrane droplets (library construction based on Tn5 S5 / S7 was followed by in situ reverse transcription, and library construction was performed using Tn5 S7 / S7 (labeling) after reverse transcription, wherein Figure 24 A shows that the average library size is 350 bp, which is consistent with the theoretical value; Figure 24 B shows that the exon region accounts for 61% of the transcriptome reads; Figure 24 C shows that the coverage of the intragenic region shows high coverage at the 3' end.

[0369] (3) A microfluidic platform was co-encapsulated with hydrogel droplets having different molecular pore sizes inside and outside and single-cell labeled microspheres containing Nextera capture sequence. The results are shown in Figure 25. Figure 25 shows a microfluidic platform co-encapsulated with hydrogel droplets and single-cell labeled microspheres containing Nextera capture sequence, wherein Figure 25 A shows the design of the co-encapsulated droplet microfluidic chip; Figure 25 B shows a real image of the single-cell labeled microspheres (the microspheres are soluble in the presence of DTT); Figure 25 C shows a real image of the co-encapsulated chip; Figure 25 D shows the droplet morphology before droplet PCR; Figure 25 E shows the droplet morphology after droplet PCR.

[0370] (4) The hydrogel can be integrated with the co-encapsulated chip of the Nextera capture sequence single-cell encoding microspheres to simultaneously construct the mitochondrial DNA and chromatin openness libraries and 3' end transcriptome libraries. The results are shown in Figure 26. Figure 26 shows the results of high-throughput deep sequencing of mitochondrial DNA, chromatin openness and 3' end transcriptome at the single cell level by the independently developed hydrogel droplet microfluidic platform with different inner and outer molecular pore sizes: the mitochondrial DNA sequencing read length (reads) accounts for about 60% (A); and the mitochondrial genome sequencing depth is high (8000x) and the coverage is uniform (D), indicating that this platform can effectively detect mitochondrial DNA mutations; the nuclear base group reads show a high enrichment of transcription start sites (TSS) (B) and the nucleosome size is a gradient fragment, indicating the high fragmentation and labeling efficiency of the nuclear chromatin open region (C); the exon region accounts for 61% of the transcriptome reads (E) and the coverage rate of the gene region shows a high coverage of the 3' end, indicating the 3' end transcriptome sequencing quality of this platform.

[0371] This application is based on a hydrogel system to construct a single-cell multi-omics library based on mitochondrial DNA, which can maximize the reduction of cross-contamination of cytoplasmic contents between cells (such as mitochondrial DNA cross-contamination in the cytoplasm, RNA cross-contamination in the cytoplasm), and can perform high-throughput single-cell mtDNA deep sequencing and simultaneous mapping of chromatin accessibility or high-throughput single-cell mtDNA deep sequencing and simultaneous mapping of chromatin accessibility and transcriptome.

[0372] Although the present invention has been disclosed above with reference to the embodiments, they are not intended to limit the present invention. Anyone with ordinary knowledge in the art may make slight changes and modifications 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 patent application attached hereto.

Claims

1. A hydrogel embedded with biomaterials, comprising an inner core gel material embedded with biomaterials, wherein the biomaterials are permeabilized biomaterials. 2 . The hydrogel according to claim 1 , further comprising an outer shell layer capable of covering the inner core gel material embedded with the biological material, wherein the outer shell layer has a thickness of 1-2 μm.

3. The hydrogel according to claim 2, 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 biological material.

4. The hydrogel according to claim 1, wherein The biological material is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed therefrom. Preferably, the biological material is a cell.

5. The hydrogel according to claim 1, wherein The permeabilized biological material is a slightly permeabilized biological material or a strongly permeabilized biological material; Preferably, The slightly permeabilized biological material is a biological material 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 permeabilized biological material is a biological material whose cell membrane is destroyed and the cell contents are released.

6. The hydrogel according to claim 5, wherein The slight permeabilization treatment refers to low-temperature treatment in a solution containing a non-ionic surfactant, and the pH of the solution containing the non-ionic surfactant is 7-8.

7. The hydrogel according to claim 6, wherein The solution containing the nonionic surfactant may further include one or more of salt, buffer solution and bovine serum albumin.

8. The hydrogel according to claim 6, wherein The temperature of the low temperature treatment is (-10°C to 10°C).

9. The hydrogel according to claim 6, 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.

10. A hydrogel embeddable in biomaterials, comprising an inner core gel material and an outer shell layer, wherein the thickness of the outer shell layer is 1-2 μm.

11. The hydrogel according to any one of claims 1 to 9 or the hydrogel according to claim 10, wherein: The inner core gel material has a porous structure; Preferably, The biological material can be embedded in the porous structure of the inner core gel material; More preferably, The pore size of the porous structure of the inner core gel material is 2-5 μm, and the pore size of the porous structure of the outer shell layer is 24 nm-86 nm.

12. The hydrogel according to any one of claims 1 to 11, wherein The inner core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches and glucose; Preferably, The molecular weight of the inner core gel material is 0.18kDa-800kDa; More preferably, The outer shell layer comprises a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, and the high molecular weight hydrophilic polymer and / or the low molecular weight hydrophilic polymer are used to make the outer shell layer have a porous structure; More preferably, The hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate (PEGDA), polypropylene glycol, and ethylene oxide and propylene oxide.

13. The hydrogel according to any one of claims 1 to 11, wherein In the hydrogel, the mass ratio of the inner core gel material to the outer shell layer is (2-25):1, preferably (5-20):

1.

14. The method for preparing the hydrogel according to any one of claims 2 to 13, comprising the following steps: The biomaterial is encapsulated in the inner core gel material phase; Microfluidic manipulation is used to generate hydrogels by controlling the solidification or semi-solidification of the inner core gel material phase, the outer shell phase, and the oil phase: The biomaterial hydrogel is permeabilized to obtain the hydrogel; The inner core gel material phase is a solution of the inner core gel material; and the outer shell layer phase is a solution of the outer shell layer material.

15. The method according to claim 14, wherein: Before the biological material is wrapped in the inner core gel material phase, the inner core gel material phase and the outer shell phase are pre-mixed and then subjected to liquid-liquid separation treatment to obtain separated inner core gel material phase and outer shell phase.

16. The method according to claim 14 or 15, wherein: The concentration of the inner core gel material ranges from 2% to 50%.

17. The method according to claim 14 or 15, wherein: In the shell phase, the concentration of the high molecular weight hydrophilic polymer is in the range of 3% to 50%.

18. Use of the hydrogel according to any one of claims 1 to 13 or the hydrogel prepared by the method according to any one of claims 14 to 17 in single cell multi-omics library construction.

19. A method for constructing a single cell library of a biomaterial embedded in a hydrogel containing the biomaterial, comprising: Treating hydrogels embedded with biomaterials with transposases; Using flow sorting to sort the biomaterial-embedded hydrogels treated with transposase; labeling the sorted hydrogel embedded with biomaterials; The library is constructed for the biological materials after tagging.

20. The method according to claim 19, wherein: The transposase is selected from any one of Tn5, Mu, and Vibrio.

21. The method according to claim 19, wherein: The microspheres used for labeling treatment are selected from any one of polystyrene PS microspheres, polymethyl methacrylate PMMA microspheres, polyethylene microspheres, and agarose soft gel microspheres.

22. The method according to any one of claims 19 to 21, wherein: Library construction includes any one, two or three of the following: (i) constructing a mitochondrial DNA library; (ii) constructing a library for the open chromatin interval; (iii) Library construction of 3' end transcriptome (RNA).

23. The method according to any one of claims 19 to 22, wherein: The biomaterial-embedded hydrogel comprises an inner core gel material embedded with the biomaterial, and the biomaterial is a biomaterial that has been permeabilized.

24. The method according to claim 23, wherein: The hydrogel embedded with biomaterials further comprises an outer shell layer capable of covering the inner core gel material embedded with biomaterials, and the thickness of the outer shell layer is 1-2 μm.

25. The method according to claim 24, 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 biological material.

26. The method of claim 23, wherein: The biological material is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, polypeptides, bacteria, viruses, organelles and cells, and complexes formed therefrom. Preferably, the biological material is a cell.

27. The method according to claim 23, wherein: The permeabilized biological material is a slightly permeabilized biological material or a strongly permeabilized biological material; Preferably, The slightly permeabilized biological material is a biological material 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 permeabilized biological material is a biological material whose cell membrane is destroyed and the cell contents are released.

28. The method of claim 23, wherein: The slight permeabilization treatment refers to low-temperature treatment in a solution containing a non-ionic surfactant, and the pH of the solution containing the non-ionic surfactant is 7-8.

29. The method according to claim 28, wherein: The solution containing the nonionic surfactant may further include one or more of salt, buffer solution and bovine serum albumin.

30. The method of claim 28, wherein: The temperature of the low temperature treatment is (-10°C to 10°C).

31. The method of claim 28, 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.

32. The method of claim 23, wherein: The inner core gel material has a porous structure; Preferably, The biological material can be embedded in the porous structure of the inner core gel material; More preferably, The pore size of the porous structure of the inner core gel material is 2-5 μm, and the pore size of the porous structure of the outer shell layer is 24 nm-86 nm.

33. The method of claim 23, wherein: The inner core gel material is selected from one or more of dextran, polyvinyl alcohol, hydroxypropyl starches and glucose; Preferably, The molecular weight of the inner core gel material is 0.18kDa-800kDa; More preferably, The outer shell layer comprises a high molecular weight hydrophilic polymer and / or a low molecular weight hydrophilic polymer, and the high molecular weight hydrophilic polymer and / or the low molecular weight hydrophilic polymer are used to make the outer shell layer have a porous structure; More preferably, The hydrophilic polymer of the outer shell layer is selected from one or more of polyethylene glycol diacrylate (PEGDA), polypropylene glycol, and ethylene oxide and propylene oxide.

34. The method of claim 23, wherein: In the hydrogel, the mass ratio of the inner core gel material to the outer shell layer is (2-25):1, preferably (5-20):

1.

35. The method of claim 23, wherein: The method for preparing the hydrogel comprises the following steps: The biomaterial is encapsulated in the inner core gel material phase; Microfluidic manipulation is used to generate hydrogels by controlling the solidification or semi-solidification of the inner core gel material phase, the outer shell phase, and the oil phase: The biomaterial hydrogel is permeabilized to obtain the hydrogel; The inner core gel material phase is a solution of the inner core gel material; The shell phase is a solution of the shell material.

36. The method of claim 35, wherein: Before the biological material is wrapped in the inner core gel material phase, the inner core gel material phase and the outer shell phase are pre-mixed and then subjected to liquid-liquid separation treatment to obtain separated inner core gel material phase and outer shell phase.

37. The method of claim 35, wherein: The concentration of the inner core gel material ranges from 2% to 50%.

38. The method of claim 35, wherein: In the shell phase, the concentration of the high molecular weight hydrophilic polymer is in the range of 3% to 50%.

39. Use of hydrogels embedded with biomaterials in the construction of single cell libraries.

40. The use according to claim 39, wherein The single cell library construction includes any one, two or three of the following: (i) constructing a mitochondrial DNA library; (ii) constructing a library for the open chromatin interval; (iii) Library construction of 3' end transcriptome (RNA).

41. Use of hydrogels embedded with biomaterials in single-cell copy number variation sequencing.

42. Use of a hydrogel embedded with biological material for transposase treatment.

43. Use of hydrogels embedded with biological materials in flow cytometry.

44. Use of hydrogels embedded with biomaterials for single cell labeling.

45. The use according to any one of claims 39 to 44, wherein The biomaterial-embedded hydrogel is the biomaterial-embedded hydrogel according to any one of claims 19 to 38.

Images