WO2025103022A1 - Hydrogel, preparation method therefor, and use thereof - Google Patents

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

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 biological materials, a preparation method of a hydrogel embedded with biological materials, and an application of a hydrogel embedded with biological materials, such as a method for constructing a single-cell library for biological materials in a hydrogel embedded with biological materials, and an application of a hydrogel embedded with biological materials in constructing a single-cell library.

Background Art

Cells are the most basic units that make up life. At present, in the fields of tumors, 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, and deconstruct life with big data. At the level of tissues, organs, and individuals, the study of life processes from "breaking the whole into pieces" to "breaking the pieces into the whole" is realized, and profound insights are extracted from complex biological samples.

Single-cell sample processing in the single-cell multi-omics sequencing process refers to the process of preparing the whole cell mixture or the extracted cell nucleus mixture for downstream flow cytometric sorting into microplates or forming microdroplets through droplet microfluidics to prepare single-cell multi-omics libraries through biochemical reactions such as cell lysis. The extracted cell nucleus mixture naturally loses the biological molecules in the cytoplasm, resulting in the loss of natural single-cell multi-omics library information. The whole cell mixture is prone to rupture due to the characteristics of the cell membrane, resulting in a large amount of cross-contamination between cells in the single-cell multi-omics library.

Summary of the invention

This application is inspired by the compartmentalization phenomenon that is widely present in nature. For example, cells rely on permeability-regulatable 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. A hydrogel with different molecular pore sizes inside and outside (i.e., a hydrogel with uneven inside and outside) and its application are designed:

The technical solution of a biomaterial-embedded hydrogel and its application in this application is as follows:

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 item 1, further comprising an outer shell layer capable of covering the inner core gel material embedded with the biological material, wherein the thickness of the outer shell layer is 1-2 μm.

3. The hydrogel according to item 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 item 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 item 1, wherein the permeabilized biomaterial is a slightly permeabilized biomaterial or a strongly permeabilized biomaterial;

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 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.

7. The hydrogel according to item 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 item 6, wherein

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

9. The hydrogel according to item 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 items 1 to 9 or the hydrogel according to item 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 items 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 0.18 kDa-800 kDa;

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 items 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. A method for preparing a hydrogel according to any one of items 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 to obtain separated inner core gel material phase and outer shell phase.

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

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

17. The method according to item 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 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.

The technical solution for the application of hydrogel embedded with biomaterials in this application is as follows:

1. A method for constructing a single cell library for 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.

2. The method according to claim 1, wherein:

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

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.

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:

(i) constructing a mitochondrial DNA library;

(ii) constructing a library for the open chromatin interval;

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

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.

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.

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.

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

9. The method according to item 5, 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.

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.

11. The method according to item 10, wherein the solution containing the non-ionic surfactant further comprises one or more of salt, buffer, and bovine serum albumin.

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

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.

14. The method according to item 5, 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.

15. The method according to item 5, 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.

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.

17. The method according to item 5, 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; and the outer shell layer phase is a solution of the outer shell layer material.

18. The method according to item 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 a liquid-liquid separation treatment to obtain separated inner core gel material phase and outer shell phase.

19. The method according to claim 17, wherein:

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

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

21. Use of hydrogels embedded with biological materials in the construction of single cell libraries.

22. The use according to item 21, wherein the single cell library construction comprises 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. Use of hydrogels embedded with biomaterials in single-cell copy number variation sequencing.

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

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

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

27. The use according to any one of items 21 to 26, wherein the hydrogel embedded with biomaterial is the hydrogel embedded with biomaterial involved in any one of the methods of items 1 to 20.

Compared with the prior art, the beneficial effects of this application are:

The outer shell layer 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.

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 relatively 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 the 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.

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.

The hydrogel embedded with biomaterials in the present 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 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.

The hydrogel embedded with biomaterials in the present 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; potential regulatory elements can also be identified: helping to identify potential regulatory elements related to cell specificity, developmental processes or diseases.

The hydrogel embedded with biomaterials in this application constructs a library for the 3'-end transcriptome (RNA), which can be used to study gene expression: 3'-end transcriptome library construction is used to deeply study the gene expression pattern in cells, especially focusing on the 3'-end RNA; it can be used for single-cell analysis: suitable for single-cell RNA sequencing, which can reveal changes in gene expression at the single-cell level. It can also study transcription endpoint regulation: it helps to understand the regulation of RNA processing, splicing and stability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows bright field microscopic images of hydrogels in the examples and comparative examples.

FIG. 2 shows a structural diagram of the hydrogel of the present application.

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

FIG. 4 shows pictures of cells before and after emulsion breaking.

FIG5 shows a graph of the average diameter of the hydrogels.

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.

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

FIG. 8 shows a picture of the hydrogel under slightly permeabilized conditions.

FIG. 9 shows a picture of the hydrogel under strong permeabilization conditions.

FIG. 10 shows a picture of cell nuclei encapsulated in hydrogel.

FIG. 11 shows a picture of cells encapsulated in hydrogel.

FIG. 12 shows a picture of cells encapsulated in hydrogel.

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 molecular pore sizes inside and outside.

FIG. 14 shows a picture of the hydrogel in Comparative Example 1. FIG.

FIG. 15 shows a picture of the hydrogel in Comparative Example 2.

FIG. 16 shows a picture of the hydrogel in Comparative Example 3.

FIG. 17 shows a picture of the thickness of the hydrogel shell layer of the present application.

FIG. 18 shows the fragment distribution after Tn5 tagmentation reaction.

Figure 19 shows the results of self-assembled Tn5 and enzyme activity verification in our laboratory. In Figure 19, A: Tn5 S5/S7 performs Tn5 labeling reaction on 50ng HEK293T genomic DNA, followed by PCR amplification; B: agarose electrophoresis is used to detect the amplified product.

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 positive (cell-containing hydrogel) hydrogels are sorted out as shown in Figure 20B.

FIG21 shows the average sequencing depth of mtDNA using the example of single-cell mtDNA library construction in three hydrogels.

FIG. 22 shows the results of human 293T cells and mouse 3T3 cells encapsulated in hydrogels for simultaneous library construction of mitochondrial DNA and chromatin accessibility at the cell level.

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

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

Figure 25 shows the results of the microfluidic platform co-encapsulated with hydrogel droplets and single-cell labeled microspheres containing Nextera capture sequence.

FIG26 shows the result 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

The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

In the present application, a hydrogel with different molecular pore sizes inside and outside also refers to a hydrogel with heterogeneous inside and outside.

The present application provides a hydrogel embedded with a biomaterial, wherein the hydrogel comprises an inner core gel material embedded with the biomaterial, and the biomaterial is a biomaterial that has been subjected to a permeabilization treatment.

In some embodiments, the hydrogel embedded with biomaterials also includes an outer shell layer capable of covering an inner core gel material embedded with biomaterials, 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.

The hydrogel of the present application includes: an artificial membrane with small pores 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; the inside 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.

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 biological material.

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

In some embodiments, 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.

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, 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, encapsulating the protein can improve its stability during the purification process, thereby helping to prepare more stable protein drugs and biological products.

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.

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, as follows: encapsulating sugar molecules helps to study the interaction of sugar molecules on the cell surface, which is very important for studying cell adhesion, immune response, recognition of infectious disease pathogens, etc.

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, 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.

When the biomaterial of the present application is a metabolite, when the metabolite is fixed in the gel material after the permeabilization treatment of the present application, it can be used for drug screening and toxicity assessment, as follows: The metabolite is fixed in the hydrogel, which can be used for drug screening and toxicity assessment. It 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.

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, 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 therapeutic research.

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.

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 openness measurement at the single cell level can be performed, thereby providing deep insights into epigenetics.

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

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 cell lysis or destruction of the internal organic structure of the cell; wherein the strongly permeabilized biomaterial is a biomaterial whose cell membrane is destroyed to release the cell contents.

In some embodiments, 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.

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

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

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

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.

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.

In some embodiments, the strong permeabilization treatment refers to treating the cells in a solution containing protease, or treating the cells in a solution containing SDS, or using a sonicator to disrupt the cells by using sonic energy.

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 MgCL ₂ , 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 NaCL ₂ , 1mM CaCL ₂ , and 0.05μg/μL Proteinase K, and incubating at 55°C for 30 minutes, and then incubating at 95°C for 10 minutes.

By slightly permeabilizing the hydrogel, the biological material can be fixed in the hydrogel in the present application, which can be used in the following applications:

(1) Bioseparation and dialysis:

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

(2) Cell sorting and enrichment:

Different types of cells can be fixed in hydrogels and, after slight permeabilization, can be used for 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.

(3) Biosensors:

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

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 simultaneous strong permeabilization of cells 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.

In the present application, the thickness of the outer shell layer of the hydrogel that can embed biomaterials can be detected by methods known to those skilled in the art. The thickness of the outer shell layer is 1-2 μm, which can be understood as the average thickness of the outer shell layer is 1-2 μm. Those skilled in the art can arbitrarily select a site in the outer shell layer to detect and determine its thickness, or arbitrarily select several sites, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 sites to detect and calculate the average value, that is, the average value is 1-2 μm. For example, the outer shell layer of the hydrogel can be observed with a microscope, and the thickness of the outer shell layer can be measured using the detection module of the microscope.

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, wherein 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.

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 can 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.

In a specific embodiment, the thickness of the outer shell layer is 1-2 μm, which is obtained by setting the flow rate ratio when generating the hydrogel by droplet microfluidics, wherein the flow rate setting ratio (in μL/min) is as follows: the flow rate setting ratio of the outer shell material phase and the inner core gel material phase containing the biological molecules is 1: (1-2). For example: the outer shell material phase is set at 1.11 μL/min; the inner core gel material phase containing the biological molecules is set at 1.55 μL/min to control the generation of the hydrogel. The flow rates of the outer shell material phase and the inner core gel material phase containing the biological molecules are merely examples, and those skilled in the art will appreciate that appropriate adjustments can be made when changing different equipment for operation.

In one embodiment, the thickness of the outer shell layer of the generated hydrogel is 1-2 μm as observed by bright field microscopy. As shown in FIG17 , the thickness of the outer shell layer of the hydrogel is within the range of 1-2 μm.

In the present application, FIG2 shows a schematic diagram of biomolecules encapsulated in a hydrogel with different pore sizes of the inside and outside molecules. The generated hydrogel can be placed in a 1.5 mL centrifuge tube in large quantities for permeabilization treatment and 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, which can support the biomolecular membrane system and reduce the diffusion efficiency of biomolecules.

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

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

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.

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.

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 are used to make the outer shell layer have a porous structure.

In the present 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).

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 (Ethylene oxide propylene oxide).

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

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

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

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

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.

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

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.

The present application provides a method for preparing the above-mentioned hydrogel, which comprises the following steps: encapsulating 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 biological material 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.

In some embodiments, 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 to obtain separated inner core gel material phase and outer shell phase.

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.

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

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

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.

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

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.

To prepare biomolecules, resuspend HEK293T cells in 1 mL of DPBS containing 0.04% BSA, 300 g, 3 min, 4 ° C. Add 1.0 mL of DPBS containing 0.04% BSA to resuspend the biomolecules, and pipette 10 μL onto a counting plate, taking about 1 million biomolecules and resuspending them with 300 μL of Dextran-rich solution.

Use a 3mL BD syringe to hold 2% FS10 in HFE-7500; a 1mL BD syringe to hold PEGDA-rich; and a 1mL BD syringe to hold dextran-rich biomolecule suspension.

Place the syringe filled with reagents in the syringe pump and collect the droplets with a 1.5mL EP tube. Set the microfluidic flow rate to 6.67μL/min for the oil phase, 1.11μL/min for the PEGDA-rich phase, and 1.55μL/min for the Dextran-rich phase.

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.

Pipette 10 μL of droplet onto the Countess slide to observe the formation of reaction compartments, whether the size of the reaction compartments is uniform, and whether there is agglomeration;

Add 500 μL of HFE-7500 containing 20% (vol/vol) perfluorooctanol to the droplets and centrifuge for 5 seconds. Remove the oil at the bottom of the centrifuge tube, add 500 μL of DPBS buffer containing 0.1% (vol/vol) Pluronic F-68, mix with a pipette, and centrifuge for 5 seconds. Discard the supernatant in the centrifuge tube, and collect the hydrogel in a 1.5 mL centrifuge tube.

Slight permeabilization of biomolecules can be performed in hydrogels.

The specific conditions of slight permeabilization are as follows: the hydrogel containing biological materials was treated with a slight permeabilization reagent consisting of a final concentration of 10 mM Tris-HCL pH 7.4, 10 mM NaCL, 3 mM MgCL ₂ , 1% (vol/vol) BSA, and 0.1% (vol/vol) NP40 and incubated on ice for 3 minutes.

The present 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.

The present application provides a method for constructing a single-cell library for the biomaterial in the above-mentioned hydrogel embedded with biomaterial, 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 biomaterial with a transposase; sorting the hydrogel embedded with biomaterial treated with the transposase by flow sorting; labeling the sorted hydrogel embedded with biomaterial; and constructing a library for the labeled biomaterial.

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

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

In the present application, tagging refers to the step of cutting on the 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 fragments by PCR, ligation or any other suitable method known to those skilled in the art.

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

The second generation sequencing library construction is to fragment or screen the DNA sample into a target sequence of a specified length, and then add oligonucleotide sequencing adapters P5 and P7 for subsequent sequencing. The traditional library construction method requires steps such as DNA fragmentation, end repair, adapter connection, library amplification, multiple purification and sorting, which is time-consuming. When the present application uses transposase such as Tn5 for sequencing library construction, multi-step reactions such as DNA fragmentation, end repair, and adapter connection can be converted into a single-step reaction, greatly shortening the library construction time and improving work efficiency. The construction of the second generation sequencing library in the present application is a library construction method based on transposase.

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.

The labeled microspheres carry a unique sequence identifier. This application can uniquely identify each cell by co-encapsulating different cells with uniquely identified labeled microspheres. The advantage of this method is that it allows a large number of single cells to be analyzed simultaneously, providing high-throughput single-cell data, which helps to more fully understand the heterogeneity of cell populations, such as studying differences between cell subtypes, identifying rare cell types, and exploring the response of cells under different physiological conditions.

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 certain biomarkers in advance, and then specific cells can be sorted out by flow sorting for subsequent single-cell analysis experiments.

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).

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

1) Study of 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.

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

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.

The hydrogel embedded with biological materials in this application constructs a library for the open interval of chromatin, which has the following effects:

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

2) Functional annotation: By analyzing the open interval, the function and regulatory mechanism of the gene can be predicted. Identification of potential regulatory elements: Helps identify potential regulatory elements related to cell specificity, developmental processes or diseases.

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

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

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

3) Study the regulation of transcription endpoints: It helps to understand the regulation of RNA processing, splicing and stability.

The present application provides the use of the above-mentioned hydrogel embedded with biomaterials 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; (iii) library construction of 3' end transcriptome (RNA).

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

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

The present application provides use of the above-mentioned hydrogel embedded with biological materials in flow sorting.

The present application provides use of the above-mentioned hydrogel embedded with biological materials in single cell labeling.

Example 1

0.036 g PEGDA (MW 8 kDa), 0.024 g PEGDA (MW 575 Da) and 0.6 g Dextran (MW 500 kDa) were taken to prepare a hydrogel mixture, which was fixed to 1 mL. After mixing evenly, the mixture was centrifuged at 16000 g for 30 minutes in a desktop centrifuge to induce liquid-liquid phase separation. Among them, the mass ratio of high molecular weight hydrophilic polymer to low molecular weight hydrophilic polymer was 3:2, the mass ratio of inner core gel material to outer shell layer was 10:1, the inner core gel material was Dextran, and the outer shell layer was PEGDA (MW 8 kDa) and PEGDA (MW 575 Da).

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.

Prepare biomolecules, resuspend HEK293T cells in 1 mL of DPBS containing 0.04% BSA, 1800 rpm, 3 min, 4°C. Add 1.0 mL of DPBS containing 0.04% BSA to resuspend biomolecules, and pipette 10 μL onto a counting plate, take about 1 million biomolecules and resuspend with 300 μL of Dextran-rich solution.

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.

Place the syringe filled with reagents in the syringe pump and collect the droplets with a 1.5mL EP tube. Set the microfluidic flow rate to 6.67μL/min for the oil phase, 1.11μL/min for the PEGDA-rich phase, and 1.55μL/min for the Dextran-rich phase.

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.

Pipette 10 μL of droplet onto the Countess slide to observe the formation of reaction compartments, whether the size of the reaction compartments is uniform, and whether there is agglomeration;

Add 500 μL of HFE-7500 containing 20% (v/v) perfluorooctanol to the droplets and centrifuge for 5 seconds. Remove the oil at the bottom of the centrifuge tube, add 500 μL of DPBS buffer containing 0.1% (vol/vol) Pluronic F-68, mix with a pipette, and centrifuge for 5 seconds. Discard the supernatant in the centrifuge tube, and collect the hydrogel in a 1.5 mL centrifuge tube.

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 composed of a final concentration of 10 mM Tris-HCL pH 7.4, 10 mM NaCL, 3 mM MgCL ₂ , 1% (vol/vol) BSA, and 0.1% (vol/vol) NP40 and incubated on ice for 3 minutes.

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

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

Example 2

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

Example 3

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 layer is 15:1, and the rest is the same.

Example 4

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

Example 5

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 layer is 25:1, and the rest is the same.

Example 6

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

Example 7

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

Example 8

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

Example 9

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

Example 10

The only difference between Example 10 and Example 1 is that PEGDA (MW 8 kDa) is replaced by polypropylene glycol (MW 8 kDa), and the rest is the same.

Embodiment 11

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

Comparative Example 1

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

Comparative Example 2

The only difference between Comparative Example 2 and Example 1 is that it does not contain Dextran (MW 500kDa), and the rest is the same.

Comparative Example 3

The only difference between Comparative Example 3 and Example 1 is that it does not contain PEGDA (MW8 kDa) and PEGDA (MW575 Da), and the rest is the same.

Table 1

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 usually 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.

Note: The mass of the outer shell refers to the sum of the mass of the high molecular weight hydrophilic polymer and the low molecular weight hydrophilic polymer. The mass in the above table refers to the mass required when the inner core gel material, high molecular weight hydrophilic polymer and low molecular weight hydrophilic polymer are fixed to 1 mL.

Example 12-Example 19

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

Table 2

According to the configurations in Tables 1 and 2 above, a hydrogel was formed, and the collected hydrogel was placed on a glass slide and placed under a microscope to observe the hydrogel structure. The results are shown in FIG1 , and it can be seen that Examples 1-7, Examples 9-11, Examples 12-19, and Comparative Example 1 can all form hydrogels with complete structures (circular without gaps when observed in a plane), while the hydrogel formed in Example 8 has gaps, and Comparative Examples 2 and 3 cannot form a hydrogel structure. The inventor selected the hydrogel in Example 1 or the hydrogel in Reference Example 1 for subsequent experiments.

Experimental example

Experimental Example 1 Hydrogel Cell Loss Test

1. Collect the generated water-in-oil droplets into a 1.5mL centrifuge tube through microfluidic operation, and take 10uL droplets six times onto a glass slide to observe the proportion of cell-containing droplets, and finally calculate the average proportion of cell-containing droplets to the total droplets in the six measurements. (The proportion of cell-containing droplets to the total droplets in the six measurements are: 0.1157, 0.1114, 0.1037, 0.1051, 0.1012, 0.125)

2. Place the approximately 200 μL droplets collected in a 1.5 mL centrifuge tube under a UV lamp and irradiate with UV light for 2 minutes to solidify the shell material in the droplets into gel. After gelation, add 500 μL of HFE-7500 containing 20% (v/v) perfluorooctanol and centrifuge for 5 seconds in a centrifuge. Aspirate the oil at the bottom of the centrifuge tube, add 500 μL of DPBS buffer containing 0.1% (w/v) PluronicF-68, mix well with a pipette, and centrifuge for 5 seconds in a centrifuge. Discard the supernatant in the centrifuge tube, and collect the hydrogel in a 1.5 mL centrifuge tube.

3. Take 10uL of the collected hydrogel of Example 1 six times and place it on a glass slide to observe the proportion of hydrogel containing cells, and finally calculate the average proportion of hydrogel containing cells to the total hydrogel in the six measurements. (The proportion of the number of hydrogel containing cells to the total number of hydrogels in the six measurements is: 0.1034, 0.1096, 0.1143, 0.1, 0.0952, 0.1105.)

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

Experimental Example 2 Diameter test of hydrogels with different inner and outer molecular pore sizes

The hydrogel of Example 1 was adsorbed onto a glass slide and the diameter of the hydrogel was measured under a microscope (a total of 64 hydrogel diameters were measured, and the diameters were: 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, 56.579, 55.447 ... .447,55.447,55.447,55.447,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,54.316,53.184,53.184,53.184,5 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).

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 64 hydrogel diameters (average value and standard deviation)

Experimental Example 3

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

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

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 hydrogel structure normally, as shown in Figure 16.

Experimental Example 4

The preparation method of Example 1 is referred to, except that HEK293T cells are replaced with pFB25 plasmids, and the average mass concentration of pFB25 plasmids encapsulated in each hydrogel is 7 nM. The plasmids are encapsulated in hydrogels, and the prepared hydrogels are placed in primers containing a specific amplification length of the plasmid, There are 4 primers with specific amplification lengths in the nucleic acid dye and PCR reaction solution, and the corresponding specific amplicon lengths are 150 bp, 547 bp, 968 bp, and 1187 bp. After PCR, the fluorescence intensity of the nucleic acid dye in the hydrogel of each amplicon length is counted by fluorescence microscopy.

As shown in Figure 7, the hydrogel prepared in Example 1 was tested and DNA molecules larger than 968 bp were obviously retained in the hydrogel: Figure 7 A is an example of the fluorescence intensity of specific amplicons in the four hydrogels (Scale bar: 50 um), and Figure 7 B is a statistical analysis of the relative fluorescence intensity of specific amplicons in the hydrogel.

Experimental Example 5 Slight Permeabilization Conditions

For comparison, the hydrogels encapsulating HEK293T cells were placed in DPBS buffer and slightly permeabilized buffer (buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 0.1% (vol/vol) NP-40, 1% (vol/vol) BSA), wherein the conditions of the slightly permeabilized buffer (buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 0.1% (vol/vol) NP-40, 1% (vol/vol) BSA) were the conditions of the hydrogel prepared in Example 1 (the results are shown in B of FIG8 ), and the DPBS buffer conditions were the preparation method of reference Example 1, except that the biomolecules were not slightly permeabilized but only treated with DPBS buffer (the results are shown in A of FIG8 ). After incubation on ice for 5 minutes, the hydrogels were placed in DPBS containing nucleic acid dyes for staining, and after staining, they were analyzed under a fluorescence microscope.

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.

Experimental Example 6: Strong permeabilization conditions

The hydrogels encapsulating HEK293T cells were placed in DPBS buffer and strong permeabilization buffer (buffer containing 0.1% Triton X-100, 10 mg/ml protease K), respectively. The conditions of the strong permeabilization buffer (buffer containing 0.1% Triton X-100, 10 mg/ml protease K) were the hydrogel prepared in Example 11 (the results are shown in B in Figure 9), and the DPBS buffer conditions were the preparation method of reference Example 11, except that the biomolecules were not subjected to strong permeabilization treatment but 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.

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.

Experimental Example 7:

After the extracted 293T cell nucleus and the inner core gel material are mixed, the generated 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 droplet into a gel. After gelation, 500μL of HFE-7500 containing 20% (v/v) perfluorooctanol is added, and the mixture is centrifuged in a centrifuge for 5s. The oil at the bottom of the centrifuge tube is sucked off, and 500μL of DPBS buffer containing 0.1% (w/v) Pluronic F-68 is added, and the mixture is mixed by pipetting, and the mixture is centrifuged in a centrifuge for 5s. 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 is that HEK293T cells are replaced with 293T cell nuclei). The collected hydrogel is placed on a glass slide for observation.

As shown in FIG. 10 , the hydrogel prepared in Experimental Example 7 can be used to encapsulate the nucleus of human 293T cells.

Experimental Example 8:

Refer to the method of Example 1, wherein 293T cells are replaced with human peripheral blood mononuclear cells (PBMC), and the rest is the same. After the extracted human peripheral blood mononuclear cells (PBMC) and the inner core gel material are mixed, the generated droplets are collected into a 1.5mL centrifuge tube by microfluidic operation, and placed under a UV lamp, UV irradiation for 2min, so that the shell material in the droplet solidifies into gel. After gelation, 500μL of HFE-7500 containing 20% (v/v) perfluorooctanol is added, and centrifuged in a flash centrifuge for 5s. The oil at the bottom of the centrifuge tube is sucked off, 500μL of DPBS buffer containing 0.1% (w/v) Pluronic F-68 is added, the pipette is blown and mixed, and the instant centrifuge is centrifuged for 5s. The supernatant in the centrifuge tube is discarded, and the lower layer, that is, 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 slide for observation.

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.

Experimental Example 9:

The hydrogel prepared in Example 1 was divided into 4 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 structure of each group was observed under a microscope after being stored at room temperature (22°C) and low temperature (-80°C) for 48 hours.

As shown in FIG12 , the hydrogel can be exposed to 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 remain intact under microscopic observation.

Experimental Example 10

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

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.

Experimental Example 11 Tn5 labeling reaction of hydrogel

Tn5 labeling reaction is a key step in high-throughput sequencing library preparation. Ideally, after Tn5 labeling reaction using hydrogel, DNA molecules will be divided into short fragments of 100bp to 600bp, while DNA fragments greater than or equal to 968bp are retained in the hydrogel system. This means that the DNA fragments after Tn5 labeling can diffuse freely inside and outside the hydrogel network and can be amplified efficiently. Therefore, in theory, the fragment distribution of the amplified product should be between 100bp and 600bp.

By performing Tn5 labeling reaction and subsequent insert sequence amplification experiments on hydrogels encapsulating 293T cell genomic DNA (each hydrogel contains an average of 1.66 pg of genomic DNA), the inventors found that the hydrogels can successfully perform Tn5 labeling reactions and generate a 100 bp to 600 bp fragment distribution that meets the second-generation sequencing standards. The results are shown in Figure 18.

Experimental Example 12. Library construction of mitochondrial DNA at the single cell level:

The hydrogel of Example 1 was placed in a total volume of 50 μL Tn5 (S5/S7) tagmentation reaction system containing 0.2 μg/μL Tn5 (S5/S7), 1 x tagmentation buffer, and 5 mM MgCL ₂ , and incubated at 37°C for 30 min.

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

A total volume of 25 μL of 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).

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;

Perform the following PCR reaction

Step 1: 72°C, 5 min

Step 2: 98°C, 30 seconds

Step 3: 98°C, 10 s

Step 4: 63°C, 30 seconds

Step 5: 72°C, 1 min

(Repeat step 5, a total of 8 times)

Step 6: Keep at 12℃.

After PCR, 1.2x volume of Vazyme DNA clean beads (N411-01) was used for purification, and then 30 μL of nuclease-free water was used for elution. The eluted solution is the final library.

Related data:

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

Purchase Tn5 naked enzyme (ABclonal, RM21303) and perform insert sequence assembly. After establishing this system, Tn5 naked enzyme can be assembled with DNA fragments of any sequence. Take the assembly of Nextera S5/S7 as an example. After assembly, human 293T cell genomic DNA is used to verify the enzyme activity. The results are shown in Figure 19.

FIG. 19A shows the verification of the enzyme activity of Tn5 assembled with 293T cell genomic DNA. Tn5 containing NexteraS5/S7 sequence can perform labeling reaction on genomic DNA. After gap filling and PCR amplification of sequencing adapter primers, the fragments after the reaction are subjected to agarose gel electrophoresis and the standard 100bp-700bp diffuse bands can be seen. FIG. 19B shows that compared with the group without Tn5, the Tn5 enzyme labeling reaction can produce obvious diffuse bands. This proves that the Tn5 naked enzyme and insertion sequence assembly system of the present application is successful.

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

(3) Positive hydrogels can be sorted into 96-well plates for mitochondrial DNA sequencing, and 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 (deep sequencing depth) for each site of the mitochondrial genome at the single-cell level, which 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.

Example 13. Simultaneous construction of mitochondrial DNA and chromatin accessibility libraries at the single cell level:

Take the hydrogel of Example 10, use the DNBelab C (BGI contains Tn5) series high-throughput single-cell ATAC library preparation kit of BGI to perform single-cell labeling reaction, droplet PCR, purify the droplet PCR product, and use it for sequencing adapter PCR amplification reaction;

After PCR, 1.2x volume of Vazyme DNA clean beads (N411-01) was used for purification, and then 30 μL of nuclease-free water was used for elution. The eluted solution is the final library.

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

Human 293T cells and mouse 3T3 cells were encapsulated in hydrogels with different molecular pore sizes inside and outside to simultaneously construct libraries for 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 nuclear base group reads showed a high enrichment of transcription start sites (TSS) (Figures 22B and C), and the average mitochondrial genome sequencing depth of each cell was about 96×, and the coverage was uniform (Figures 22D and E).

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

The hydrogel in Example 1 was slightly permeabilized and subjected to the first labeling reaction for labeling mitochondrial DNA and chromatin open regions. The labeling reaction system was a total volume of 50 μL labeling reaction system, which contained Tn5 (S5/S7) with a final concentration of 0.2 μg/μL, a final concentration of 1 x tagmentation buffer, and a final concentration of 5 mM MgCL _{2 .} The reaction was incubated at 30° C. for 30 min.

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

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

Step 1: 10 min, 50 °C;

Step 2: 8°C, 12 seconds;

Step 3: 15℃, 45s

Step 4: 20℃, 45s

Step 5: 30°C, 30 seconds

Step 6: 42°C, 2 min

Step 7: 50°C, 3 min

Repeat steps 2-7 a total of 3 times

Step 8: 50°C, 5 min

The second labeling reaction was performed in the hydrogel to label RNA and cDNA heteroduplexes. The labeling reaction system was a total volume of 50 μL labeling reaction system, which contained Tn5 (S7/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 MgCL ₂ , and was incubated at 37°C for 30 minutes.

Single-stranded nucleotide chain removal and Tn5 transposase labeling reaction gap filling reaction were carried out in the hydrogel. The reaction system had a total volume of 50 μL, which contained a final concentration of 0.5 mM dNTP, a final concentration of 8 U/μL Maxima H minus Reverse Transcriptase, a final concentration of 2 U/μL Exo1, and a final concentration of 1x NaCL RT buffer, and was incubated at 37°C for 15 min.

The hydrogel and Nextera capture sequence single-cell labeling microspheres (RAN biotech.050.065.2.ATAC) were co-encapsulated in the droplets; the microfluidic flow rate was 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 labeling microsphere phase.

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.

After PCR, 1.2x volume of Vazyme DNA clean beads (N411-01) were used for purification, and 30 μL of nuclease-free water was used for elution. The eluted solution is the final library.

Related data:

(1) The hydrogel can be used to perform mtDNA and chromatin open region tagging reaction (first tagging reaction) with 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 tagging reaction in selectively permeable membrane droplets can retain 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).

(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'-end transcriptome sequencing in selective permeable membrane droplets (based on Tn5 S5/S7 for library construction and then in situ reverse transcription, and reverse transcription and library construction using Tn5 S7/S7 (labeling), where 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 rate of the intragenic region shows high coverage at the 3' end.

(3) A hydrogel droplet with different molecular pore sizes inside and outside and a single-cell labeled microsphere containing Nextera capture sequence co-encapsulated microfluidic platform, and 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 microsphere (the microsphere is 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.

(4) The hydrogel can integrate the co-encapsulated chip of the single-cell encoded microspheres of Nextera capture sequence to simultaneously construct the mitochondrial DNA and chromatin openness library and the 3'-end transcriptome library, and 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 using the independently developed hydrogel droplet microfluidic platform with different inner and outer molecular pore sizes: the mitochondrial DNA sequencing reads account 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; 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 interruption and labeling efficiency of nuclear chromatin open areas (C); the exon region accounts for 61% of the transcriptome reads (E) and the coverage rate of the intragenic region shows a high coverage of the 3' end, indicating the 3' end transcriptome sequencing quality of this platform.

This application is based on a hydrogel system to construct a single-cell multi-omics library based on mitochondrial DNA, which can minimize the 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.

Although the present invention has been disclosed as above by way of embodiments, it is not intended to limit the present invention. Any person having ordinary knowledge in the technical field may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope of the patent application attached hereto.

Claims (45)

A hydrogel embedded with biomaterials comprises an inner core gel material embedded with the biomaterials, wherein the biomaterials are permeabilized biomaterials. 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 thickness of the outer shell layer is 1-2 μm. 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. 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. The hydrogel according to claim 1, wherein the permeabilized biomaterial is a slightly permeabilized biomaterial or a strongly permeabilized biomaterial; 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. 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. 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. The hydrogel according to claim 6, wherein The temperature of the low temperature treatment is (-10°C to 10°C). 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. A hydrogel capable of embedding biological materials comprises an inner core gel material and an outer shell layer, wherein the thickness of the outer shell layer is 1-2 μm. 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. 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. 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. 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. 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 to obtain separated inner core gel material phase and outer shell phase. The method according to claim 14 or 15, wherein The concentration of the inner core gel material ranges from 2% to 50%. 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%. Use of the hydrogel described in any one of claims 1 to 13 or the hydrogel prepared by the method described in any one of claims 14 to 17 in single-cell multi-omics library construction. A method for constructing a single cell library for 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. The method according to claim 19, wherein The transposase is selected from any one of Tn5, Mu, and Vibrio. 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. The method according to any one of claims 19 to 21, wherein the library construction comprises 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). The method according to any one of claims 19-22, 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. The method according to claim 23, wherein the hydrogel embedded with biomaterial further comprises an outer shell layer capable of covering the inner core gel material embedded with biomaterial, and the thickness of the outer shell layer is 1-2 μm. The method according to claim 24, wherein the shell layer has a porous structure, and the pore size of the porous structure of the shell layer is smaller than the average size of the biological material. The method according to claim 23, wherein the biological material is selected from one or more of proteins, nucleic acids, sugars, lipids, metabolites, peptides, bacteria, viruses, organelles and cells, and complexes formed therefrom, and preferably the biological material is a cell. 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. The method according to 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. The method according to claim 28, wherein the solution containing the non-ionic surfactant further comprises one or more of salt, buffer, and bovine serum albumin. The method according to claim 28, wherein the temperature of the low temperature treatment is (-10°C to 10°C). The method according to claim 28, wherein the non-ionic surfactant is selected from one or more of NP40, Triton X-100, Brij-35, Tween-20, IGEPAL CA-630, and Octyl Glucoside. The method according to 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. The method according to 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. The method according to 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. The method according to 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; and the outer shell layer phase is a solution of the outer shell layer material. The method according to claim 35, 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. The method according to claim 35, wherein The concentration of the inner core gel material ranges from 2% to 50%. The method according to claim 35, wherein the concentration of the high molecular weight hydrophilic polymer in the shell phase is in the range of 3% to 50%. Use of hydrogels embedded with biomaterials for single-cell library construction. The use according to claim 39, wherein the single cell library construction comprises 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). Use of biomaterial-embedded hydrogels for single-cell copy number variation sequencing. Use of hydrogels embedded with biomaterials for transposase treatment. Use of hydrogels embedded with biomaterials in flow cytometry. Use of hydrogels embedded with biomaterials for single-cell labeling. The use according to any one of claims 39 to 44, wherein the hydrogel embedded with biomaterial is the hydrogel embedded with biomaterial involved in the method according to any one of claims 19 to 38.