Sample analysis method through biochemical sample expansion and system therefor

Abstract

The present invention relates to a sample analysis method through biochemical sample expansion and a system therefor, which can perform analysis with high resolution and accuracy compared to conventional analysis methods by expanding a part or all of a biochemical sample. The present invention provides a sample analysis method through biochemical sample expansion, the method comprising the steps of: forming a conjugate precursor by binding an anchoring agent to a sample; forming a sample–hydrogel composite by reacting the conjugate precursor with a hydrogel precursor solution; expanding the sample-hydrogel composite by supplying a solvent to the sample-hydrogel composite; and isolating a desired analysis target from the expanded sample-hydrogel composite.

Description

Method for analyzing samples through biochemical sample expansion and system therefor

[0001] The present invention relates to a method for analyzing a sample through biochemical sample expansion and a system therefor, and more particularly, to a method for analyzing a sample through biochemical sample expansion and a system therefor, which enables analysis with higher resolution and accuracy than existing analysis methods by expanding part or all of a biochemical sample.

[0002] This invention was conducted with the support of Seoul National University (Industry-Academic Cooperation Foundation) (Project No. 0534-20230045).

[0003] Understanding the mechanisms of disease development is a crucial task in modern medicine and life science research. Accurately analyzing intracellular microstructures and uncovering how these structures relate to disease is crucial. Cells are intricately interwoven with diverse structures within them, and these structures play essential roles in maintaining cellular function, responding to external signals, and producing energy. However, these structures are often extremely small, making them difficult to observe accurately using conventional microscopy techniques. Therefore, technologies that allow for the observation of changes in intracellular microstructures through sample enlargement or high-resolution microscopy have emerged.

[0004] However, visualizing only the microstructures and biomolecules within biochemical samples has limitations in fully understanding disease mechanisms. Changes in cellular structure ultimately lead to alterations in genes, proteins, metabolites, and other substances, which are crucial factors in determining the onset and progression of disease. Therefore, analyzing changes in specific cellular structures at multiple levels is a crucial task in disease research.

[0005] To address the aforementioned issues, the present invention utilizes existing sample expansion and separation and analysis technologies to expand and visualize intracellular structures of interest, isolate the structures, and then perform high-resolution analysis using the isolated structures.

[0006] This invention could bring about groundbreaking changes in disease research. For example, by visualizing structural changes in mitochondria in cancer cells and analyzing how these changes regulate the expression of specific genes, we can gain a deeper understanding of the mechanisms of cancer development. Furthermore, by elucidating how specific structures in nerve cells are altered in neurodegenerative diseases and the resulting changes in biomolecular patterns, we could provide clues for developing new treatments.

[0007] In order to solve the above-mentioned problem, the present invention provides a method for analyzing a sample through biochemical sample expansion, which enables analysis with higher resolution and accuracy than existing analysis methods by expanding part or all of a biochemical sample, and a system therefor.

[0008] To solve the above-described problem, the present invention provides a method for analyzing a sample through biochemical sample expansion, comprising the steps of: forming a binding precursor by binding an anchoring agent to a sample; reacting the binding precursor with a hydrogel precursor solution to form a sample-hydrogel complex; expanding the sample-hydrogel complex by supplying a solvent to the sample-hydrogel complex; and separating a desired analyte from the expanded sample-hydrogel complex.

[0009] In one embodiment, the sample may include at least one selected from the group consisting of biological samples, microparticles, microstructures, nanostructures, DNA, RNA, proteins, peptides, microorganisms, viruses, protozoa, biofilms, microbiomes, extracellular vesicles, exosomes, small molecules, and chemicals.

[0010] In one embodiment, the anchoring agent may comprise a first functional group that binds to a biochemical molecule of the sample and a second functional group that binds to the hydrogel.

[0011] In one embodiment, the sample-hydrogel complex can be expanded in volume by 8 to 1,000,000 times by the solvent.

[0012] In one embodiment, the hydrogel precursor solution may comprise hydrogel monomers and water.

[0013] In one embodiment, the step of expanding the sample-hydrogel complex may further include a step of bonding the expanded sample-hydrogel complex to a substrate.

[0014] In one embodiment, the step of binding the expanded sample-hydrogel complex to the substrate may further include a pretreatment step for analysis.

[0015] In one embodiment, the step of isolating the desired analyte may be a step of isolating part or all of the expanded sample-hydrogel complex.

[0016] The method for analyzing a sample through biochemical sample expansion according to the present invention can enable analysis and utilization of a microscopic area of ​​a biochemical sample size ranging from 1 nm to 100 um, which was difficult to analyze using existing technologies, by expanding the biochemical sample and then separating and analyzing it.

[0017] In addition, the sample analysis method through biochemical sample expansion according to the present invention can analyze the signal transmission pathway occurring in each cell by expanding and then separating the contact area between these cells when studying the interaction between immune cells and cancer cells.

[0018] In addition, the sample analysis method through biochemical sample expansion according to the present invention expands the scope of application of existing technologies, enabling application to various biochemical analysis techniques, thereby providing high resolution and analysis precision for microscopic areas that are difficult to achieve with existing technologies, thereby opening up new possibilities for life science and medical research.

[0019] FIG. 1 is a schematic diagram illustrating a method and system for expanding a biochemical sample and then separating and analyzing the same according to one embodiment of the present invention.

[0020] Figure 2 schematically illustrates a biochemical sample expansion step according to one embodiment of the present invention.

[0021] Figure 3 schematically illustrates a separation step after expansion of a biochemical sample according to one embodiment of the present invention.

[0022] FIG. 4 illustrates an example of utilizing a cell separation device in a sample separation step after a biochemical sample expansion step according to one embodiment of the present invention.

[0023] FIG. 5 illustrates an example of utilizing a Laser Microdissection system in a sample separation step after a biochemical sample expansion step according to one embodiment of the present invention.

[0024] Figure 6 is a photograph showing the nucleus and cytoplasm separated after expanding a sample from a human-simulating skbr3 cell line according to one embodiment of the present invention.

[0025] FIG. 7 illustrates an example of genetic analysis performed on the nucleus and cytoplasm separated after sample expansion in the skbr3 cell line according to one embodiment of the present invention.

[0026] Figure 8 is a photograph of the concentration and electrophoresis results after RNA-seq prep of the separated nucleus and cytoplasm according to one embodiment of the present invention.

[0027] FIG. 9 illustrates an example of analysis and the results thereof after separation of an expanded biochemical sample according to one embodiment of the present invention.

[0028] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention.

[0029] The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the technical spirit of the present technology can be sufficiently conveyed to those skilled in the art. In the drawings, the dimensions of each device component, such as width and thickness, are somewhat enlarged to clearly represent the components. The drawings are described from the perspective of an observer, and when an element is mentioned as being positioned above another element, this can all mean that the element is positioned directly above the other element or that additional elements may be interposed between the elements. Furthermore, those skilled in the art will be able to implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple drawings indicate substantially the same elements.

[0030] The terminology used in the present invention is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression may include the plural expression unless the context clearly indicates otherwise. In the present invention, it should be understood that the terms "comprises" or "has" are intended to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0031] Meanwhile, the meanings of terms described in this specification should be understood as follows. Terms such as "first" or "second" are intended to distinguish one component from another, and the scope of rights should not be limited by these terms. For example, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component.

[0032] In addition, singular expressions should be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as “include” or “have” should be understood to specify the presence of a described feature, number, step, operation, component, part, or combination thereof, but not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. In addition, in performing a method or a manufacturing method, each step constituting the method may occur in a different order from the stated order unless the context clearly indicates a specific order. That is, each step may occur in the same order as the stated order, may be performed substantially simultaneously, or may be performed in the opposite order.

[0033] In this specification, the term 'and / or' can include a combination of multiple listed items or any one of multiple listed items. In this specification, 'A or B' can include 'A', 'B', or 'both A and B'.

[0034] The present invention relates to a method for analyzing a sample through biochemical sample expansion, comprising the steps of: forming a binding precursor by binding an anchoring agent to a sample; reacting the binding precursor with a hydrogel precursor solution to form a sample-hydrogel complex; expanding the sample-hydrogel complex by supplying a solvent to the sample-hydrogel complex; and isolating a desired analyte from the expanded sample-hydrogel complex.

[0035] The above sample refers to a biochemical sample, and specifically may include at least one selected from the group consisting of biological samples, microparticles, microstructures, nanostructures, DNA, RNA, proteins, peptides, microorganisms, viruses, protozoa, biofilms, microbiomes, extracellular vesicles, exosomes, small molecules, and chemicals.

[0036] Looking at this more specifically, the above biological sample is a sample obtained from a living organism, and the biological sample may include all living organisms composed of single cells, including animals and plants, as well as cells cultured in vitro. The biological sample may include the living organism itself, as well as organs, tissues, and cells obtained from the living organism, and even various biochemical substances extracted from the cells.

[0037] The above microparticles and microstructures (microparticles, microbeads, microcontainers, droplets, nano / micro-structures / patterns) may be microparticles arranged in a monolayer or multilayer array form, and may also include microbeads, microcontainers, droplets, nano / micro-structures, or nano / micro-patterns. The microparticles and microstructures may be solids including polymers, metals, glass, silicon, etc., solutions such as aqueous solutions or oils, droplets, liquid-solid complexes including core-shell structures, etc. In addition, the above microparticles and structures may be manufactured by methods including self-assembly, pick-and-place assembly, random assembly, and transfer from another surface or substrate.

[0038] The above nanostructure (origami, nanoparticle, exosome) is a structure having a size from 1 nm to 1000 nm, and may include various structures as follows. The nanostructure may include various structures such as an origami structure containing DNA, RNA, protein, or other organic or inorganic substances, a DNA / RNA beam, a nanorobot, a nanopore, or a composite structure thereof. In addition, it may include not only a synthetic nanostructure having the above structure, but also a nanostructure derived from a living organism, such as an exosome, mitochondria, chromosome, or nucleus. In addition, the nanostructure may be a structure having a nanosize as described above, but may also mean a microsize structure including these nanostructures.

[0039] The biochemical samples, including DNA, RNA, proteins, and peptides, may include not only molecules extracted from living organisms but also all substances that have been artificially synthesized or modified. DNA comprises a sequence of deoxyribonucleic acid, RNA comprises ribonucleic acid, and proteins comprise a sequence of amino acids. In addition, various functional groups and bead nanostructures may be added through physical and chemical bonds.

[0040] The above microorganisms may include bacteria, microbiome, viruses, fungi, algae, and protozoa, and may include prokaryotic microorganisms, eukaryotic microorganisms, and non-cellular microorganisms. In addition, the above biofilm may include a biofilm or microbiome formed singly or in combination by the above microorganisms.

[0041] The above small molecules and reagents include compounds, chemicals, drugs, etc., and can be patterned in an array form using methods such as sputtering, inkjet printing, ultrasonic patterning, and CVD. This array includes a library for screening, such as a chemical library, a drug library, and a small molecule library, and the location information can be an identifier of each substance.

[0042] In addition, the sample may be a single type of sample, but may be a mixture of two or more of the biological samples, microparticles, microstructures, nanostructures, DNA, RNA, proteins, peptides, microorganisms, viruses, protozoa, biofilms, microbiomes, extracellular vesicles, exosomes, small molecules, and chemicals, a complex of two or more compounds, or a complex of two or more compounds.

[0043] An anchoring agent can be combined with the sample to form a bonding precursor. In the present invention, as described below, a hydrogel is bonded to the sample, and then the hydrogel is expanded to expand the sample. At this time, since the sample and the hydrogel are not directly bonded, the anchoring agent can be first bonded to the sample, as described above, and then the sample and the hydrogel can be bonded via the anchoring agent.

[0044] At this time, the sample can be combined with the anchoring agent by contacting it with a chemical anchoring agent solution or by perfusing it with an anchoring agent solution and then reacting it under sufficient time and temperature conditions to allow a chemical reaction to occur.

[0045] The anchoring agent may include a first functional group that binds to a biochemical molecule of the sample and a second functional group that binds to the hydrogel. That is, the anchoring agent may bind to the sample through the first functional group and to the hydrogel through the second functional group.

[0046] The first functional group may include at least one selected from the group consisting of N-hydroxysuccinimide (NHS), an ester group, an epoxy group, an aldehyde group, or a carboxamide group, and the first functional group may specifically bind to a carboxylic group or an amino group of proteins, peptides, nucleic acids, lipids, proteoglycans, lipopolysaccharides, carbohydrates, metabolites, vitamins, hormones, cofactors, and coenzymes contained in the sample.

[0047] The second functional group may include at least one selected from the group consisting of a vinyl group, an aryl group, an acrylate group, a methacrylate group, an acrylonitrile group, and an acrylamide group. In addition, as described above, the second functional group may be combined with a hydrogel.

[0048] As described above, by combining an anchoring agent and a sample, a binding precursor can be formed, and then the binding precursor can be reacted with a hydrogel precursor solution to form a sample-hydrogel complex.

[0049] The hydrogel precursor solution used at this time may include a hydrogel monomer and water.

[0050] The hydrogel monomer may include, but is not limited to, acrylate-based monomers, vinyl-based monomers, or polysaccharide-based monomers.

[0051] Looking at this specifically, the acrylic monomers may include Acrylamide (AAm), Sodium acrylate (SA), N,N-Dimethylacrylamide (DMAA), 2-Hydroxyethyl methacrylate (HEMA), Polyethylene glycol diacrylate (PEGDA), Poly(ethylene glycol) methacrylate (PEGMA), N-Isopropylacrylamide (NIPAM), Acrylic acid (AA), Methacrylic acid (MAA), Glycidyl methacrylate (GMA), or Vinyl acetate.

[0052] The above vinyl-based monomers may include vinylpyrrolidone (VP), vinyl alcohol (VOH), vinyl chloride (VCl), or vinylidene fluoride (VDF).

[0053] The above polysaccharide-based monomers may include Chitosan, Alginate, Hyaluronic acid, Carboxymethylcellulose (CMC) or Dextran.

[0054] In addition to the above monomers, Pluronic F127, Gelatin methacryloyl (GelMA), Chondroitin sulfate, Sodium methacrylate (SMA), Itaconic acid (IA), Trans-aconitic acid (TAA), Ethyl-2-(hydroxymethyl)-acrylate (EHA), Pentaerythritol tetraacrylate (PT), Propoxylated trimethylol propane triacrylate (TPT), Pentaerythritol triacrylate (PA), Dipentaerythritol pentaacrylate or dipentaerythritol hexaacrylate (DPHA), Trimethylolpropane triacrylate (TTA), Bis(trimethylol) Propane)-tetraacrylate (DiTA), Trimethylolpropane trimethacrylate (TTMA), Glycerol propoxylated (1PO / OH) triacrylate (GPT), Ethoxylated trimethylolpropane Triacrylate (TET), It may also contain pentaerythritol allyl ether (PAE), sodium 4-hydroxy-2-methylenebutyrate (SHMB), or N,N-dimethylaminopropylacrylamide (DMPAA).

[0055] The above water refers to H2O and may include deionized water (DI water), ionized water, distilled water, triple distilled water, or phosphate buffered saline (PBS).

[0056] The above hydrogel precursor solution may contain, in addition to the above hydrogel monomer and water, an initiator, a crosslinking agent, an accelerator, a preservative, an antioxidant, or a protein denaturant.

[0057] The above initiator refers to a compound that activates a crosslinking agent that binds the hydrogel and the anchoring agent, and may include thermal initiators, photoinitiators, or redox initiators.

[0058] At this time, the thermal initiator may be Ammonium persulfate (APS), Potassium persulfate (KPS), 2,2'-Azobis(2-methylpropionitrile) (AIBN), or Benzoyl peroxide (BPO), the photoinitiator may be Irgacure 2959, Irgacure 651, Camphorquinone, Riboflavin, Eosin Y, or Rose Bengal, and the redox initiator may be Tetramethylethylenediamine (TEMED) + Ammonium persulfate (APS), Sodium bisulfite (SBS), Ascorbic acid + Hydrogen peroxide, or Sodium thiosulfate. In addition, it is also possible to use an initiator such as Ceric ammonium nitrate (CAN) or Ferrous ammonium sulfate.

[0059] The crosslinking agent is a compound that crosslinks the hydrogel and the anchoring agent, and may include an acrylate-based crosslinker, a vinyl-based crosslinker, or a carbodiimide-based crosslinker.

[0060] The acrylic crosslinking agent may be N,N'-Methylenebisacrylamide (MBA), Polyethylene glycol diacrylate (PEGDA), Ethylene glycol dimethacrylate (EGDMA), Diacrylate polyethylene glycol (PEGDA), Tetraethylene glycol diacrylate (TEGDA), or Trimethylolpropane triacrylate (TMPTA). The vinyl crosslinking agent may be Divinylbenzene (DVB) or N,N'-Ethylenebisacrylamide. The carbodiimide crosslinking agent may be 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or N-Hydroxysuccinimide (NHS). In addition, Glutaraldehyde, Genipin, Tannic acid, Epichlorohydrin, or Formaldehyde may be used as a crosslinking agent.

[0061] The above accelerator may be an acrylamide-based accelerator such as Tetramethylethylenediamine (TEMED) or Sodium bisulfite (SBS), and in addition, accelerators such as N,N,N',N'-Tetramethyl-ethylenediamine (TEMED), L-Ascorbic acid, Sodium sulfite, Thiourea, or Thioglycolic acid may be used.

[0062] The Preservation Agents include RNase Inhibitors, Poly(A) polymerase, Cap-binding proteins, DNase Inhibitors, EDTA, DNA Ligase, Topoisomerase, DNA Methyltransferase, Protease Inhibitors, PMSF (Phenylmethylsulfonyl fluoride), Leupeptin, Aprotinin, Protein Disulfide Isomerase (PDI), Chaperones (eg, Hsp70, GroEL), Ubiquitin-activating enzymes (E1, E2, E3), Peptidase Inhibitors, Pepstatin, Bestatin, Prolyl oligopeptidase or Carboxypeptidase inhibitors.

[0063] The above antioxidant may be a vitamin-based antioxidant, a polyphenol-based antioxidant, a flavonoid-based antioxidant, a carotenoid-based antioxidant, a phenolic acid antioxidant, a selenium and mineral-based antioxidant, a thiol-based antioxidant, an organic acid antioxidant, a synthetic antioxidant, or a plant-based antioxidant.

[0064] Specifically, the vitamin series antioxidant may include Ascorbic Acid, Tocopherol, Retinol, Beta-carotene, Lycopene, Lutein, Zeaxanthin or Coenzyme Q10 (Ubiquinone), the polyphenol series antioxidant may include Quercetin, Rutin, Epicatechin, Catechin, Resveratrol, Polydatin, Ellagic Acid, Chlorogenic Acid or Gallic Acid, the flavonoid series antioxidant may include Anthocyanins, Flavones, Flavonols, Flavanones or Isoflavones, the carotenoid series antioxidant may include Alpha-carotene, Astaxanthin, Canthaxanthin or Cryptoxanthin, the phenolic acid antioxidant may include Caffeic Acid, Ferulic Acid, Cinnamic Acid or p-Coumaric Acid, and the selenium and mineral series antioxidant may include Selenium, Zinc, Copper or Manganese, and the The thiol series antioxidants may include Glutathione, Alpha Lipoic Acid or N-Acetylcysteine, the organic acid antioxidants may include Melatonin, Ubiquinol, Taurine, Ergothioneine or Proanthocyanidins, the synthetic antioxidants may include Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), Propyl Gallate, 4-Hydroxy-TEMPO, or Tert-Butylhydroquinone (TBHQ), and the plant-based antioxidants may include Gingerol, Curcumin,May contain Silymarin or Oleuropein.

[0065] The above protein denaturing agent may include an acidic solution, a basic solution, an organic solvent, a denaturant, a surfactant, an oxidizing agent, a reducing agent, a salt, a dehydrating agent, or a heavy metal ion.

[0066] The acidic solution may be protein denaturation Hcl or Acetic acid, the basic solution may be NaOH or NH3, the organic solvent may be Ethanol, Acetone or Methanol, the denaturant may be Urea, Ammonium persulfate or Guanidine hydrochloride, the surfactant may be Sodium Dodecyl Sulfate (SDS) or Triton X-100, the oxidizing agent or reducing agent may be Dithiothreitol (DTT), β-Mercaptoethanol or Hydrogen peroxide, the salt may be NaCl or Ammonium sulfate, the dehydrating agent may be Glycerol, and the heavy metal ion may be Hg 2+ , Pb 2+ or Cu 2+ It could be.

[0067] The hydrogel precursor solution prepared as described above can be supplied to the sample through various methods, and specifically, can be supplied through drop casting, spin coating, dip coating, roll coating, slot die coating, spray coating, spin casting, flow coating, nozzle printing, or ink jet to form a sample-hydrogel complex.

[0068] In addition, the polymerization and cross-linking reactions forming the sample-hydrogel complex may include polymerization reactions such as radical polymerization, anionic polymerization, cationic polymerization, copolymerization, step-growth polymerization, ring-opening polymerization, metathesis polymerization, and ionic polymerization, and cross-linking reactions such as chemical cross-linking, radical cross-linking, photocrosslinking, ionic cross-linking, thermal cross-linking, and enzymatic cross-linking, and in addition, supercritical fluid polymerization, electrospinning polymerization, and self-healing cross-linking. It may include reactions such as (Self-Healing Cross-Linking), shape-memory polymerization and cross-linking.

[0069] More specifically, the radical polymerization may include free radical polymerization, living radical polymerization, atom transfer radical polymerization (ATRP), or reversible addition-fragmentation chain transfer polymerization (RAFT polymerization), the anionic polymerization may include living anionic polymerization, the cationic polymerization may include living cationic polymerization, the copolymerization may include random copolymerization, block copolymerization, graft copolymerization, and alternating copolymerization, and the monomer polymerization (step-growth polymerization) may include It may include condensation polymerization, addition polymerization, and the ring-opening polymerization may include cationic ring-opening polymerization, anionic ring-opening polymerization, and radical ring-opening polymerization.The above metathesis polymerization may include ROMP (Ring-Opening Metathesis Polymerization) and ADMET (Acyclic Diene Metathesis), and the above disproportionation polymerization may include anionic disproportionation polymerization and cationic disproportionation polymerization.

[0070] In addition, the chemical cross-linking may include cyclization, condensation reaction, epoxy-amine cross-linking, isocyanate-alcohol cross-linking (polyurethane formation), and silane cross-linking, the radical cross-linking may include peroxide cross-linking and electron beam cross-linking, the photocross-linking may include UV cross-linking and photoinitiated radical cross-linking, and the ionic cross-linking may include cross-linking using multivalent cations and anionic cross-linking. And, the thermal cross-linking may include thermally-induced cross-linking and thermosetting cross-linking, and the enzymatic cross-linking may include transglutaminase cross-linking and oxidative enzyme cross-linking.

[0071] After the sample-hydrogel complex is formed as described above, a solvent may be supplied to the sample-hydrogel complex to expand the sample-hydrogel complex. Generally, a hydrogel increases in volume when in contact with a solvent, and in the case of the present invention, since the sample forms a sample-hydrogel complex with the hydrogel, supplying a solvent as described above can expand the sample-hydrogel complex.

[0072] The solvent used at this time may include a polar solvent, a non-polar solvent, an organic solvent, or an inorganic solvent.

[0073] The polar solvent may include water, ethanol, methanol, IPA (isopropyl alcohol), DMSO (dimethylsulfoxide), DMF (dimethylformamide), MeCN (acetonitrile), MC (dichloromethane), THF (tetrahydrofuran), or EA (ethyl acetate).

[0074] The nonpolar solvent may include benzene, hexane, chloroform, diethyl ether, diisopropyl ether or 1,4-dioxane.

[0075] The above organic solvents are 1. 2-dichloroethane (ethylene dichloride), 1. 2-dichloroethylene (acetylene dichloride), carbon tetrachloride, carbon disulfide, 1. 1. 2. 2-tetrachloroethane (acetylene tetrachloride), chloroform, trichloroethylene, normal hexane, 1. 4-dioxane, dichloromethane (methylene dichloride), methanol, methylcyclohexanone, methylcyclohexanol, methyl butyl ketone, methyl ethyl ketone, methyl isobutyl ketone, 1-butanol, 2-butanol, cyclohexanone, styrene, acetone, ethylene glycol monomethyl ether (methyl cellosolve), ethylene glycol monoethyl ether (cellosolve), ethylene glycol monoethyl ether acetate (cellosolve acetate), May contain ethylene glycol monobutyl ether (butyl cellosolve), ethyl ether, N. N-dimethylformamide, o-dichlorobenzene, isobutyl alcohol, isopentyl alcohol (isoamyl alcohol), isopropyl alcohol, methyl acetate, butyl acetate, ethyl acetate, isobutyl acetate, isopentyl acetate (isoamyl acetate), isopropyl acetate, pentyl acetate (amyl acetate), propyl acetate, cresol, chlorobenzene, xylene, tetrachloroethylene (para-chloroethylene), tetrahydrofuran, toluene, 1. 1. 1-trichloroethane, gasoline, mineral spirits (mineral thinner, petroleum spirit, white spirit or mineral tarpen), petroleum naphtha, petroleum benzene, petroleum ether, coal tar naphtha or turpentine.

[0076] The above inorganic solvent may include water, ammonia, carbon dioxide, carbon tetrachloride, phosphorus, sulfur, salt, amine, sulfuric acid, nitric acid, mercury or gallium.

[0077] The sample-hydrogel complex can be expanded by 8-1,000,000 times its volume by the solvent. If the sample-hydrogel complex is expanded by less than 2 times its volume, it is difficult to expect a confirmation effect. If the sample-hydrogel complex is expanded by more than 30 times, the tissue of the sample may be destroyed, making it difficult to perform an accurate analysis in the step described below.

[0078] After the step of expanding the sample-hydrogel complex, a step of bonding the expanded sample-hydrogel complex to a substrate may be further included. The above-described expansion step may be performed after the sample is supplied onto the substrate, but in this case, the lower portion of the sample comes into contact with the substrate, causing friction during expansion. If such friction occurs, the sample may be damaged during the expansion process, and since the expansion on the substrate is a two-dimensional expansion, it may be difficult for the sample to be uniformly expanded. Therefore, it is preferable that the sample be expanded in a solution or at a location other than the substrate, and then bonded to the substrate.

[0079] The substrates that can be used at this time are glass slides, silicon wafers, polymer substrates, aminosilane-coated substrates, nickel-coated substrates, poly-L-lysine-coated substrates, avidin-biotin-coated substrates, hydrogel-coated substrates, nanoparticle-coated substrates, substrates in which spatially encoded DNA exists in an array form, substrates in which spatially encoded RNA exists in an array form, substrates in which spatially encoded peptides exist in an array form, substrates in which spatially encoded proteins exist in an array form, calcium fluoride substrates, carbon nanotube substrates, transparent conductive oxide substrates, ITO coated substrates (Indium Tin Oxide-Coated Substrate), Printed Circuit Board (PCB), Ceramic Substrate, Flexible Substrate, Plastic Slide, Microfluidic Substrate, Porous Substrate, Metal Substrate, Paper Substrate, Sapphire Wafer, Silicon Carbide Wafer,A graphene substrate, a quartz substrate, a transparent conductive substrate, an alumina substrate, a zirconia substrate, a liquid substrate, or a ceramic-polymer composite substrate can be used.

[0080] Additionally, a pretreatment step for analysis may be further included after the step of binding the expanded sample-hydrogel complex to the substrate.

[0081] The above pretreatment step may include chemical pretreatment, physical pretreatment, mechanical pretreatment, thermal pretreatment, optical pretreatment, electrical pretreatment, biological pretreatment, plasma pretreatment, surface treatment, or hybrid pretreatment.

[0082] Specifically, the chemical pretreatment may include glutaraldehyde cross-linking, EDC / NHS chemical cross-linking, aminosilane functionalization, cross-linking of polyacrylamide hydrogels, oxidative treatment, premixing of nanoparticle additives, and pH-induced functionalization, and the physical pretreatment may include freeze-thaw cycling, drying, rehydration, ultrasonic treatment, compression molding, osmotic preconditioning, and surface smoothing, and the mechanical pretreatment may include The thermal pretreatment may include cutting and shaping, stretching treatment, grinding treatment, and rolling treatment, and the thermal pretreatment may include thermal cross-linking, annealing, high-temperature treatment, and hot pressing, and the optical pretreatment may include UV exposure,The electrical pretreatment may include electrical field exposure, electrochemical treatment, ion implantation, and electrolysis, the biological pretreatment may include enzymatic treatment, cell adhesion enhancement, bacterial culturing pretreatment, and antibody coating, and the plasma and other surface treatments may include plasma surface treatment, corona treatment, oxidative plasma treatment, and plasma amination, and the hybrid pretreatment may include thermo-chemical It may include thermal-chemical pretreatment, photo-electrical pretreatment, and chemical-mechanical pretreatment.

[0083] The expanded sample-hydrogel complex can isolate a desired analyte after being attached to the substrate. As described above, in the case of the present invention, the sample is formed into a sample-hydrogel complex and then expanded, so that observation can be made with higher magnification and resolution than with existing methods. Furthermore, in this case, the gap between each component of the sample increases due to the expansion, so that the desired analyte can be more easily isolated. In addition, the step of isolating the desired analyte may be a step of isolating part or all of the expanded sample-hydrogel complex.

[0084] In this case, the separation step can be performed by a method including physical separation methods, chemical separation methods, mechanical separation methods, electrical separation methods, optical separation methods, biological separation methods, thermal separation methods, gas and liquid separation methods, and hybrid separation methods.

[0085] Specifically, the physical separation methods may include centrifugation, filtration, precipitation, ultrasonication, dehydration, and extrusion.

[0086] The above chemical separation methods may include dissolution, solvent exchange, pH adjustment, oxidation / reduction treatment, and extraction.

[0087] The above mechanical separation methods may include cutting, mechanical stretching, scraping, and mechanical grinding.

[0088] The electrical separation methods may include electrophoresis, ion exchange, electrolysis, and dielectrophoretic focusing.

[0089] The above optical separation methods may include photo-activated separation, laser cutting, laser ablation, and optical trapping.

[0090] The above biological separation methods may include separation through enzymatic degradation and antibody-antigen binding.

[0091] The above thermal separation methods may include thermal decrosslinking, thermal dissolution, and thermal separation.

[0092] The above gas and liquid separation methods may include gas adsorption, liquid-liquid extraction, gas diffusion, and solvent switching.

[0093] The above hybrid separation methods may include thermal-electrical separation, chemical-physical separation, and optical-mechanical separation.

[0094] The diameter (circle basis) or side length (square basis) of the size of the separable area may include microscale (0.01 to 100 um) and milliscale (0.1 to 3 mm). In the case of the present invention, since the sample can be separated after being expanded as described above, even when using the same separation method and scale as the existing method, higher accuracy can be achieved.

[0095] Methods for analyzing the above separated samples may include Genomics, Transcriptomics, Proteomics, Metabolomics, Epigenomics, Microbiomics, Lipidomics, Single-Cell Omics, Spatial Omics, Proteomics - Protein Analysis Platforms, Multiplex Assay Platforms, Metabolomics, Protein-Protein Interaction Analysis, and Other Omics and Analysis Tools.

[0096] Specifically, the above genomics may include Next-Generation Sequencing (NGS), Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES), ChIP-Seq, and Bisulfite Sequencing.

[0097] The above transcriptomics may include Microarray, qPCR (Quantitative PCR), RNA-Seq, Single-cell RNA-Seq, NanoString Technologies, and Long-read RNA-Seq.

[0098] The above proteomics may include LC-MS / MS (Liquid Chromatography-Mass Spectrometry), TIMS (Trapped Ion Mobility Spectrometry), SDS-PAGE, Western Blotting, iTRAQ (Isobaric Tags for Relative and Absolute Quantitation), TMT (Tandem Mass Tags), 2D-Gel Electrophoresis, and Shotgun Proteomics.

[0099] The metabolomics may include GC-MS (Gas Chromatography-Mass Spectrometry), LC-MS (Liquid Chromatography-Mass Spectrometry), NMR (Nuclear Magnetic Resonance), CE-MS (Capillary Electrophoresis-Mass Spectrometry), Metabolite Profiling, and Fluxomics.

[0100] The above epigenomic analysis may include ATAC-Seq (Assay for Transposase-Accessible Chromatin using Sequencing), MeDIP-Seq (Methylated DNA Immunoprecipitation Sequencing), Hi-C, DNase-Seq, and Histone Modification Assays.

[0101] The above microbial community analysis (Microbiomics) may include 16S rRNA Sequencing, Metagenomics, Metatranscriptomics, Metaproteomics, and Metabolomics (Microbial).

[0102] The above Lipidomics may include Shotgun Lipidomics, LC-MS / MS (Lipidomics), Imaging Mass Spectrometry, and Lipid Profiling.

[0103] The above single-cell analysis (Single-Cell Omics) may include Single-cell RNA-Seq, Single-cell ATAC-Seq, Single-cell Proteomics, Single-cell Metabolomics, and Single-cell Epigenomics.

[0104] The spatial omics include Visium Spatial Gene Expression (10x Genomics), Spatial Transcriptomics, (CO-Detection by Indexing), NanoString GeoMx DSP (Digital Spatial Profiler), and Multiplexed Ion Beam Imaging (MIBI).

[0105] The above proteomics - protein analysis platforms may include SomaLogic (SOMAscan), Olink Proteomics, SWATH-MS (Sequential Windowed Acquisition of All Theoretical Mass Spectra), Meso Scale Discovery (MSD), and SILAC (Stable Isotope Labeling by Amino acids in Cell culture).

[0106] The above multiple assay platforms may include Luminex xMAP, Quanterix Simoa (Single Molecule Array), MagPix (Luminex), and ELISA (Enzyme-Linked Immunosorbent Assay).

[0107] Metabolomics may include MS Imaging (Mass Spectrometry Imaging), Orbitrap MS, Thermo Fisher Q Exactive, and Biocrates AbsoluteIDQ.

[0108] The above protein-protein interaction analysis may include Yeast Two-Hybrid System, Co-Immunoprecipitation (Co-IP), Proximity Ligation Assay (PLA), and Bimolecular Fluorescence Complementation (BiFC).

[0109] The above other Omics and Analysis Tools may include Phosphoproteomics, Glycomics, Interactomics, Multi-Omics Data Integration, and CRISPR Screening.

[0110]

[0111] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily implement them. Furthermore, when describing the present invention, detailed descriptions of related, known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention. Furthermore, certain features presented in the drawings may be enlarged, reduced, or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.

[0112]

[0113] Example 1

[0114] As shown in Fig. 4, sample expansion was performed. Specifically, SKBR3 cell lines were fixed and pretreated twice (15 min each) with 100 mM sodium bicarbonate buffer (pH 8.5, DNase / RNase-free). Then, acrylate groups were introduced to the protein by reacting with an anchoring agent containing glycidyl methacrylate (GMA, Sigma-Aldrich) (100 mM sodium bicarbonate, final concentration 0.04% w / v GMA) at room temperature for 3 h. Since GMA is soluble up to about 3% in aqueous solution, it was stirred vigorously after addition, and after the reaction, the cells were washed three times with PBS.

[0115] Next, a hydrogel precursor solution was prepared. The hydrogel precursor solution was composed of 8.6% (w / v) sodium acrylate, 2.5% (w / v) acrylamide, 0.15% (w / v) N,N′-methylenebisacrylamide, 2 M NaCl, and 1× PBS. Based on this hydrogel precursor, a hydrogel precursor solution was prepared by mixing a 10% (w / v) ammonium persulfate (APS) solution and a 10% (w / v) TEMED solution at a final ratio of 47:1:1:1. After diffusing the solution at 4°C for 30 min, a free radical polymerization reaction was performed at 37°C for 2 h in a gelling chamber using a coverslip as a spacer to form a sample-hydrogel complex (Fig. 2).

[0116] The formed sample-hydrogel complex was cut into an appropriate size and treated with digestion buffer (8 U / mL Proteinase K, 0.5% Triton X-100, 1 mM EDTA, 50 mM Tris-HCl, 2 M NaCl, pH 8) at 37°C for 4 hours to overnight. The complex after digestion was immersed in distilled water three times (each for 15 minutes) consecutively or cultured for 2 hours to expand to approximately 4-10 times its length (64-1000 times its volume) (see Fig. 4).

[0117] To mimic human samples, the SKBR3 cell line was used as an example of a biochemical sample. Mitochondria and nuclei were immunofluorescently stained, then separated using a laser after sample expansion (see Figure 6). Images of the expanded and separated mitochondria and nuclei demonstrated the successful isolation of the desired organelles. Furthermore, we confirmed that nuclear and mitochondrial gene markers were expressed separately in the regions where each organelle was separated (see Figure 7).

[0118] When RNAseq prep was performed by isolating three nuclear regions (N1, N2, N3) of a single cell and three cytoplasm regions (C1, C2, C3) of a single cell, along with the negative control (NC) and positive control (PC), a significant amount of cDNA concentration was obtained as shown in Fig. 8. When the electrophoresis results were confirmed, the same gel image as the positive control (PC) was confirmed in N1, N2, N3, C1, C2, and C3, confirming that RNAseq was performed successfully.

[0119] Figure 9 is a diagram illustrating an example of how various analyses can be applied after separating an expanded biochemical sample. This technology is an example of how RNA analysis, DNA analysis, and microbiome analysis can be performed on cell lines and tissues. Figure 9 shows how, after expanding a cell line sample, gene expression levels were quantified through RNA-seq and the expression levels of cell line-related markers were confirmed. Secondly, an example of calculating copy number alterations through whole genome analysis and analyzing microbial composition using 16S rRNA or shotgun metagenomic sequencing to assess inter-individual diversity is illustrated. In other words, it was confirmed that the method of the present invention can physically expand tissues and cell microstructures to recover nucleic acids at a high yield from the same location, and that molecular analysis on various individuals is possible.

[0120]

[0121] Examples 2-4

[0122] An experiment was conducted to determine the effects of different types of hydrogel precursors used in Example 1. The experiment was conducted in the same manner as in Example 1, but using different hydrogel precursors as shown in Table 1 below. Examples 2 to 4 and Comparative Example 1 were conducted in the same manner as in Example 1, and the expansion ratios (by volume) after culture are shown in Table 1.

[0123] Precursor Expansion Ratio (times) Example 1 Sodium acrylate, acrylamide, N,N′-methylenebisacrylamide 3 25 Example 2 Vinylpyrrolidone 2 18 Example 3 Hyaluronic acid 1 24 Example 4 Poly(ethylene glycol) methacrylate 2 86 Comparative Example 1 Not used 1

[0124] As shown in Table 1, it was confirmed that appropriate expansion was performed even when various hydrogel monomers were used. However, in the case of Comparative Example 1, which did not use a hydrogel monomer, it was confirmed that the sample did not expand.

[0125]

[0126] Examples 5-7

[0127] The same experiment was conducted using the anchoring agent shown in Table 2 below instead of glycidyl methacrylate (GMA), which was used as the anchoring agent in Example 1. Examples 5 to 4 and Comparative Example 1 below were conducted in the same manner as Example 1, and the expansion ratio (by volume) after culture is shown in Table 2.

[0128] Anchoring agent Expansion ratio (fold) Example 1 Glycidyl methacrylate (GMA) 325 Example 5 Methacrylic acid-N-hydroxysuccinimide (MA-NHS) 320 Example 6 NHS-PEG-(meth)acrylate 319 Example 7 Acryloyl-X, SE (AcX) 308 Comparative example 2 Not used 1

[0129] As shown in Table 2, since the anchoring agent is a substance that mediates bonding with the hydrogel, it was confirmed that the same hydrogel (Example 1, sodium acrylate, acrylamide, N,N′-methylenebisacrylamide) could be expanded at a similar rate regardless of its type. In addition, in the case of Comparative Example 2, since no anchoring agent was used, it was confirmed that the bonding between the hydrogel and the sample did not occur, and therefore, although the hydrogel expanded, the sample did not expand.

[0130]

[0131] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A step of forming a binding precursor by binding an anchoring agent to a sample; A step of forming a sample-hydrogel complex by reacting the above-mentioned binding precursor and hydrogel precursor solution; A step of supplying a solvent to the sample-hydrogel complex to expand the sample-hydrogel complex; and A step of separating a desired analyte from the expanded sample-hydrogel complex; A method for analyzing samples through biochemical sample expansion including .

2. In paragraph 1, A method for analyzing a sample through biochemical sample expansion, characterized in that the sample comprises at least one selected from the group consisting of biological samples, microparticles, microstructures, nanostructures, DNA, RNA, proteins, peptides, microorganisms, viruses, protozoa, biofilms, microbiomes, extracellular vesicles, exosomes, small molecules, and chemicals.

3. In paragraph 1, A method for analyzing a sample through biochemical sample expansion, wherein the anchoring agent comprises a first functional group that binds to a biochemical molecule of the sample and a second functional group that binds to the hydrogel.

4. In paragraph 1, A method for analyzing a sample through biochemical sample expansion, characterized in that the sample-hydrogel complex is expanded in volume by 8-1,000,000 times by the solvent.

5. In paragraph 1, A method for analyzing a sample through biochemical sample expansion, wherein the hydrogel precursor solution comprises a hydrogel monomer and water.

6. In paragraph 1, A method for analyzing a sample through biochemical sample expansion, characterized in that it further comprises a step of binding the expanded sample-hydrogel complex to a substrate after the step of expanding the sample-hydrogel complex.

7. In paragraph 6, A method for analyzing a sample through biochemical sample expansion, characterized in that it further comprises a pretreatment step for analysis after the step of binding the expanded sample-hydrogel complex to a substrate.

8. In paragraph 1, A method for analyzing a sample through biochemical sample expansion, characterized in that the step of separating the desired analysis target is a step of separating part or all of the expanded sample-hydrogel complex.

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