Method for transferring biochemical sample

Abstract

The present invention relates to a method for transferring a biochemical sample, which enables easy movement of the biochemical sample for additional analysis or use by employing a sacrificial material. The present invention provides a method for transferring a biochemical sample, the method comprising the steps of: forming a sacrificial layer by supplying a sacrificial material to a sample at a first position; preparing a transfer composite by simultaneously separating a part or all of the sample and the sacrificial layer at the first position; moving the transfer composite to a second position; and removing the sacrificial layer of the transfer composite moved to the second position.

Description

Methods for transferring biochemical samples

[0001] The present invention relates to a method for transferring a biochemical sample, and more particularly, to a method for transferring a biochemical sample using a sacrificial material, which can easily transfer the biochemical sample for further analysis or use.

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

[0003] The analysis and processing of biochemical samples is essential in diverse fields, including medicine, biotechnology, and environmental science. Recent technological advancements have led to the development of various platforms capable of analyzing and utilizing biochemical samples from various perspectives. However, the lack of compatibility between these platforms complicates the simultaneous analysis and utilization of biochemical samples across multiple platforms. Current analysis and utilization systems rely on individual, unique analytical devices tailored to each analysis, resulting in limited interoperability and hindering the efficient utilization of biochemical samples across multiple platforms.

[0004] Furthermore, over time, biochemical samples become increasingly difficult to obtain, or their scarcity makes it difficult to extract diverse information from them. For example, while hospital biobanks store various types of biochemical samples, long-stored samples are difficult to re-acquire, limiting subsequent analysis and utilization. Furthermore, when samples are scarce, it can be difficult to obtain samples with the same biochemical information, potentially creating technical challenges for subsequent analysis and application. This can lead to the loss of clinically important information and potentially serious consequences, such as sample loss. Therefore, researchers urgently need methods to minimize sample loss during analysis and processing of biochemical samples and to utilize them efficiently.

[0005] The present invention aims to address these issues by providing a method and system for safely and efficiently transferring biochemical samples to another location using sacrificial materials. This maximizes the efficiency of subsequent analysis and utilization of biochemical samples, enhances compatibility between various biochemical sample analysis platforms, and technically addresses issues related to sample scarcity and the passage of time.

[0006] In order to solve the above-mentioned problem, the present invention provides a method for transferring a biochemical sample using a sacrificial material, which can easily transfer the biochemical sample for further analysis or use.

[0007] In order to solve the above-described problem, the present invention provides a method for transferring a biochemical sample, comprising the steps of: supplying a sacrificial material to a sample at a first location to form a sacrificial layer; simultaneously separating part or all of the sample and the sacrificial layer at the first location to prepare a transfer complex; and moving the transfer complex to a second location.

[0008] 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.

[0009] In one embodiment, the first location and the second location may include at least one selected from the group consisting of a slide, a substrate, a chip, a cover glass, a tube, a cap, and a well.

[0010] In one embodiment, the sacrificial material may comprise a polymer, a monomer, a unit, or an inorganic material.

[0011] In one embodiment, the sacrificial material may comprise a physically or chemically crosslinkable material.

[0012] In one embodiment, the step of removing the sacrificial layer of the transport complex may include a step of chemically or physically removing the sacrificial layer.

[0013] In one embodiment, the step of chemically or physically removing the sacrificial layer includes solvent dissolution, chemical etching, plasma treatment, acid treatment, alkaline treatment, mechanical polishing, grinding, scraping, sandblasting, thermal decomposition, plasma treatment, flame treatment, laser ablation, UV photochemical removal, plasma etching, ion beam etching, reactive ion etching, ultrasonic cleaning, liquid nitrogen expansion, hydrophobic coating followed by removal, electrochemical removal, ozone treatment, and It may include one or more methods selected from the group consisting of cryogenic blasting.

[0014] In one embodiment, the step of forming the sacrificial layer may further include a step of positioning a three-dimensional structure on the surface of the sample.

[0015] In one embodiment, the three-dimensional structure may spatially isolate the sacrificial material from a portion of the sample.

[0016] In one embodiment, the separating step may be a step in which only the sample at the area where the sacrificial material came into contact is selectively separated.

[0017] In one embodiment, the step of removing the sacrificial layer of the transport complex moved to the second position may be further included after the step of moving the transport complex to the second position.

[0018] The method for transferring a biochemical sample according to the present invention can enable further analysis and use of a biochemical sample utilized in a specific biochemical assay by transferring the biochemical sample using a sacrificial material.

[0019] In addition, the method for transferring biochemical samples according to the present invention can solve complex problems that are difficult to solve with a single data type, and when a problem occurs with one data type or there is a lot of noise, another data type can compensate for it, thereby increasing the reliability and robustness of the analysis.

[0020] In addition, the method for transferring biochemical samples according to the present invention can enable analysis and use using the latest tools by transferring numerous archived biochemical samples using sacrificial materials.

[0021] FIG. 1 is a schematic diagram illustrating a method for transferring a biochemical sample using a sacrificial material according to one embodiment of the present invention.

[0022] Figure 2 is a schematic diagram of transferring a biochemical sample to a target substrate through a sacrificial layer formation step, a sacrificial layer movement step, and a sacrificial layer removal step according to one embodiment of the present invention.

[0023] Figure 3 is an experimental result confirming whether spatial information is maintained after a sample according to one embodiment of the present invention moves through each slide.

[0024] Figure 4 compares images before and after movement of a bone marrow aspiration smear sample according to one embodiment of the present invention.

[0025] FIG. 5 shows images of an H&E-stained FFPE sample before and after movement through a sacrificial layer according to one embodiment of the present invention.

[0026] Figure 6 is a result of an experiment to demonstrate that movement is possible on various types of substrates according to one embodiment of the present invention.

[0027] FIG. 7 is an example of use in which target cells are separated using a cell separation device, Spatially-resolved Laser Activated Cell Sorter (SLACS), after the sacrificial layer formation step, sacrificial layer movement step, and sacrificial layer removal step of a biochemical sample according to one embodiment of the present invention.

[0028] Figure 8 is a photograph of the concentration and electrophoresis results obtained after moving a sample from a human-simulating MCF7 cell line according to one embodiment of the present invention and performing RNAseq prep.

[0029] FIG. 9 is a photograph showing the expression levels of genes after RNAseq prep, after moving stained and unstained samples from a cell line according to one embodiment of the present invention.

[0030] 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.

[0031] 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 includes the meaning that the element is positioned directly above the other element or that additional elements may be interposed between them. 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.

[0032] 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 includes 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.

[0033] 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.

[0034] 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.

[0035] As used herein, the term "and / or" includes a combination of multiple listed items or any one of multiple listed items. As used herein, "A or B" includes "A," "B," or "both A and B."

[0036] The present invention relates to a method for transferring a biochemical sample, comprising the steps of: supplying a sacrificial material to a sample at a first location to form a sacrificial layer; simultaneously separating part or all of the sample and the sacrificial layer at the first location to prepare a transfer complex; and moving the transfer complex to a second location.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The above nanostructure (origami, nanoparticle, exosome) is a structure having a size from 1 nm to 1000 nm, and may include various structures as described below. In the case of the present invention, using the method described above, it is possible to transfer nanostructures on a specific substrate or surface. 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 nano-size as described above, but may also mean a micro-size structure containing these nanostructures.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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, protists, biofilms, microbiomes, small molecules, and chemicals, a complex of two or more compounds, or a complex of two or more compounds.

[0045] The sample used in the present invention is a sample to be biochemically analyzed, and in the case of the present invention, the biochemical sample utilized in a specific biochemical assay is moved using a sacrificial material, thereby enabling additional analysis and use. In conventional biochemical assays, the sample is often physically or chemically damaged when moved, and in particular, the spatial location information of the sample is often not preserved when moved. However, in the case of the present invention, by using a sacrificial material described below, not only can damage to the sample be minimized, but also the spatial location information of the sample can be maintained. In other words, the present invention has the advantage of enabling the reuse of a biochemical sample that has been used once through movement.

[0046] Specifically, for a single biochemical sample of interest, various types of analysis and utilization can provide richer information. Different types of data can complement each other to improve accuracy, and various types of data can provide a broader understanding of the context. For example, using sacrificial material to move specific regions containing important information on a pathology slide or biopsy slide enables not only pathological analysis but also molecular biological analysis, thereby obtaining deeper information and enhancing data accuracy.

[0047] Furthermore, since the present invention can move samples without damaging them, as described above, it can address complex problems that are difficult to solve with a single data type. Specifically, when one data type encounters problems or is noisy, another data type can compensate, increasing the reliability and robustness of the analysis. Finally, it provides new insights difficult to obtain with a single data type, enabling its application in a variety of fields.

[0048] Furthermore, the present invention utilizes sacrificial materials to transport numerous archived biochemical samples, enabling analysis and utilization using cutting-edge tools. This allows the transfer of previously generated biochemical samples to enable the application of cutting-edge analysis techniques.

[0049] Specifically, the latest technology can provide significantly more precise and accurate results than previously used analytical techniques. This allows for the analysis of changes and characteristics previously unidentifiable using current technology, thereby yielding new information from the samples at that time. Furthermore, as technology will continue to evolve, the present invention provides a technology that can be moved to analyze current samples using future, cutting-edge technologies, keeping pace with these advancements, thereby ensuring long-term utility.

[0050] Utilizing this technology can contribute to research that addresses previously unsolved problems, such as identifying the causes of disease or evolutionary research. Furthermore, by comprehensively analyzing past and present data, it can contribute to more integrated and comprehensive results, saving time and money on collecting new samples. In particular, some archive samples may come from rare organisms or environments that are currently difficult to collect again. Using the technology of the present invention to utilize cutting-edge technology on these rare samples can maximize information and enable future re-study. Finally, analyzing past samples with cutting-edge technology allows comparison of contemporary data with current data, enabling the identification of long-term changes and trends, and a deeper understanding of the causes of biological or environmental changes.

[0051] The first position and the second position may include at least one selected from the group consisting of a slide, a substrate, a chip, a cover glass, a tube, a cap, and a well. As described above, the present invention is a method for moving a sample for biochemical analysis. The most commonly used support for samples in such biochemical analysis are slides, substrates, chips, cover glasses, tubes, caps, and wells, and the present invention can facilitate the movement of each sample between these slides, substrates, chips, cover glasses, tubes, caps, and wells.

[0052] The sacrificial material may be a material whose phase or viscosity changes due to external stimuli. The sacrificial material may be supplied to the sample to form a sacrificial layer, and at this time, a transport complex may be formed that simultaneously fixes the sample and allows the sample to move.

[0053] Looking into this in detail, the sacrificial material can be applied on the sample at the first location. Thereafter, the sacrificial material is hardened through an external stimulus to form a sacrificial layer. At this time, if a sacrificial material that does not chemically interact with the sample is used, the sample can be physically fixed, and the surface of the sample can be coated with the sacrificial material. After the formation of the sacrificial layer is completed, when the sacrificial layer is separated from the first location, part or all of the sample is separated simultaneously with the sacrificial layer, and a transfer complex can be formed. To facilitate this separation, the bonding strength between the sample and the surface at the first location is preferably lower than the bonding strength between the sacrificial material and the sample surface. Thereafter, as described below, when the transfer complex is moved to a second location and the sacrificial material is removed, damage to the sample is minimized, and the movement to the second location can be completed while maintaining the location information of the sample.

[0054] Additionally, the surface of the first location may be pretreated to facilitate such movement. For example, the surface of the first location may be surface-treated to have hydrophilic or hydrophobic properties. Additionally, physical or chemical surface treatments may be performed to adjust the bonding strength with the sample. Furthermore, it is also possible to adjust the bonding strength with the sample by forming a microstructure on the surface of the first location.

[0055] Examples of such pretreatment methods may include plasma surface treatment, chemical / physical surface treatment, electrochemical surface treatment, coating / painting treatment, thermal surface treatment, hydrophilic / hydrophobic surface treatment, and solvent coating / application.

[0056] Examples of the chemical surface treatment may include acid treatment, alkaline cleaning, oxidation, chemical etching, anodizing, oxidation treatment of aluminum and other metals, chromate conversion coating, phosphating, passivation, carburizing, nitriding, sulfurizing or fluorination, and examples of the physical surface treatment may include sandblasting, shot peening, mechanical polishing, grinding, buffing, laser treatment, ultrasonic cleaning, vacuum deposition, physical vapor deposition (PVD), sputtering, vapor deposition, It may include cavitation peening or rolling.

[0057] As an example of the electrochemical surface treatment, electroplating, electropolishing, electrolytic anodizing, electroetching, electroforming, electrocoloring, anodizing, case hardening or electrospinning can be used, and as an example of the coating / painting treatment, painting, powder coating, powder spray coating, liquid coating, epoxy coating, polyurethane coating, titanium coating, enameling, chromium plating, zinc plating, nickel plating, tin plating, catapult (E-coating), organic silicon coating (Silicone) Coating, polymer coating, ceramic coating, or Teflon coating can be used. In addition, as examples of the thermal surface treatment, heat treatment, carburizing, nitriding, plasma treatment, laser heat treatment, flame spraying, thermal spray coating, or thermal diffusion coating can be used.

[0058] In addition to the surface treatment methods described above, treatments such as plasma treatment, ion implantation, cold spray, nanocoating, non-conductive coating, waterproof coating, anti-fouling coating, anti-corrosion coating, superhydrophobic coating, organic coating, antimicrobial coating, UV protection coating, heat resistant coating, or water-repellent coating may also be performed.

[0059] In addition, in the case of the above preprocessing method, it can be performed in the first position as described above, and preprocessing can also be performed in the case of the second position.

[0060] In addition to the pretreatment of the first location as described above, a pretreatment process may also be performed on the surface of the sample. This pretreatment of the sample may be performed to increase the storage and mobility of the sample, and may be performed using a biomolecule coating including DNA, RNA, protein, cells, exosomes, or organelles, a small molecule coating, a nanostructure coating, a polymer coating, a metal oxide coating, or a hydrogel coating.

[0061] The sacrificial material is a material that is supplied to the surface of the sample to form a sacrificial layer, and may include a polymer, a monomer, a unit, or an inorganic material. As described above, the sacrificial material of the present invention may use a material that changes phase or viscosity due to an external stimulus, and may include a material that is physically or chemically crosslinkable. That is, the sacrificial material can form a solidified sacrificial layer by the above-described stimulus.

[0062] More specifically, the sacrificial material may include natural polymers such as gelatin, alginate, chitosan, collagen, elastin, fibrin, glycosaminoglycan, hyaluronic acid or latex, and poly-2-hydroxyethyl methacrylate, poly-dimethyl siloxane, poly-ethylene, poly-ethylene glycol, poly-ethylene terepthalate, poly-e-caprolactone, poly-lactic-co-glycolic acid, poly-methyl-methacrylate, It may include synthetic polymers such as poly-tetrafluoroethylene, poly-isoprene, or poly-propylene. Combinations of these may also be used.

[0063] Additionally, the sacrificial material may include a hydrophilic polymer, and examples of the hydrophilic polymer include gelatin, collagen, chitosan, hyaluronic acid, alginic acid, oxidized alginate, pectin, dextran, polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyhydroxy ethyl methacrylate, polyethylene glycol, poly vinyl pyrrolidone, poly styrene sulfonate, casein, or albumin.

[0064] In addition, the sacrificial material may include a biodegradable polymer, and examples of the biodegradable polymer include cellulose, starch, alginate, chitin, chitosan, hyaluronate, collagen, gelatin, albumin, poly-3-hydroxyalkanoate, poly-ethylene succinate, poly-butylene terephthalate, polyglycolide, polylactic acid, poly-ε-caprolactone, polyvinyl alcohol, poly-ester carbonate, polyanhydride, It may include poly-α-cyanoacrylate, polyphosphazene or poly-orthoester.

[0065] In addition, the sacrificial material may include a monomer, and examples of the monomer include ethylene, propylene, styrene, vinyl chloride, terephthalic acid, ethylene glycol, butadiene, styrene-butadiene rubber (SBR), acrylonitrile, methyl methacrylate, caprolactam, hexamethylenediamine, adipic acid, isoprene, lactic acid, glycolic acid, methylenediphenyl diisocyanate (MDI), polyol, ethylene oxide, It may contain glyoxal, urea, phenol, formaldehyde, melamine, formaldehyde, ethylenediamine, alkylene oxides or styrene.

[0066] Additionally, the sacrificial material may include a monomer, and examples of the monomer may include amino acids, nucleotides, glucose, N-acetylglucosamine, glycerol, fatty acids, sugars, isoprene, acetyl-CoA, pyruvate, or N-acetylmuramic acid.

[0067] Additionally, the sacrificial material may include an inorganic material. The inorganic material that may be used may be a metal or ceramic, and may be formed by a method such as sputtering, deposition, or plating.

[0068] In addition, the sacrificial material is preferably supplied to the surface of the sample and has a low viscosity to ensure smooth application before the phase or viscosity changes due to external stimuli. To this end, the sacrificial material may include a solvent, and the solvent may include a polar solvent, a non-polar solvent, an organic solvent, or an inorganic solvent.

[0069] 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).

[0070] The nonpolar solvent may include benzene, hexane, chloroform, diethyl ether, diisopropyl ether or 1,4-dioxane.

[0071] The organic solvent is 1,2-dichloroethane (dichloroethylene), 1,2-dichloroethylene (dichloroacetylene), carbon tetrachloride, carbon disulfide, 1,1,2,2-tetrachloroethane (dichloroacetylene), chloroform, trichloroethylene, normal hexane, 1,4-dioxane, dichloromethane (dichloromethylene), 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, ortho-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 and mineral tarpen), petroleum naphtha, petroleum benzene, petroleum ether, coal tar naphtha or turpentine.

[0072] The above inorganic solvent may include water, ammonia, carbon dioxide, carbon tetrachloride, phosphorus, sulfur, salt, amine, sulfuric acid, nitric acid, mercury or gallium.

[0073] The sacrificial material manufactured as described above can be supplied to the sample using various methods. For example, it can be applied to the sample using drop casting, spin coating, dip coating, roll coating, slot die coating, spray coating, spin casting, flow coating, nozzle printing, or ink jet, and two or more of the above methods can be applied simultaneously or sequentially.

[0074] After the sacrificial material is supplied as described above, the phase or viscosity may change due to external stimuli as described above, thereby forming a sacrificial layer. At this time, since the sacrificial material contains a polymer, monomer, unit, or inorganic substance as described above, the phase may be changed or the viscosity may be increased by crosslinking these substances. In other words, the sacrificial material may be solidified or gelled by external stimuli to form the sacrificial layer.

[0075] The step of forming the sacrificial layer may include a crosslinking process. During the crosslinking process, physical crosslinking, chemical crosslinking, etc. may occur in the applied sacrificial material. Physical crosslinking may include ionic crosslinking, hydrogen bonding, or block copolymer bonding, while chemical crosslinking may include covalent bonding.

[0076] In addition, for the cross-linking process, the sacrificial material may include a cross-linking agent, and may be cross-linked without the cross-linking agent through a method such as temperature change, light (wavelength: ultraviolet, infrared, visible light, gamma ray) irradiation, pH change, enzyme addition, or ion addition.

[0077] The above cross-linking agent can be activated by a method such as temperature change, light (wavelength: ultraviolet, infrared, visible light, gamma ray) irradiation, pH change, enzyme addition, or ion addition.

[0078] 이때 사용될 수 있는 상기 가교제는 glutaraldehyde, maleic acid, fumaric acid, malic rmsacid, sulfosuccinic acid, phthalic acid, iso-phthalic acid, terephthalic acid, aconitic acid (cis and trans), citric acid, hexamethylene diisocyanate, boronic acid, Butylated melamine, Organic acid, Melamine, Polyisocyanate, Polyisocyanate, Polyisocyanate, Metal-Chelate, Epoxy, Organic agent, Organic agent, PolyAziridine, Ethylene glycol diacrylate, Di(ethylene glycol) diacrylate, Tetra(ethylene glycol) diacrylate, Ethylene glycol dimethacrylate, Di(ethylene glycol) dimethacrylate, Tri(ethylene glycol) dimethacrylate, N,N’-Methylenebisacrylamide, N,N’-(1,2-Dihydroxyethylene)bisacrylamide, N-(1-Hydroxy-2,2-dimethoxyethyl)acrylamide, Divinylbenzene, epichlorohydrin, trimethylpropane triglycidyl ether, ethylene glycol diglycidyl ether, sodium citrate, sodium tripolyphosphate, sulfosuccinic acid, oxalic acid, 칼슘이온 또는 철이온을 포함할 수 있다.

[0079] In addition, the crosslinking agent may include a photoinitiator, and examples of the photoinitiator include Azobisisobutyronitrile (AIBN), Benzoyl peroxide, Camphorquinone, Irgacure 2959, Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), BAPO-OLi, VA-086, Eosin-Y, Camphorquinone, Riboflavin, WSPI, BDEA, or P2CK.

[0080] 상기 가교제는 열 개시제를 포함할 수 있으며, 상기 열 개시재의 일예로서 tert-Amyl peroxybenzoate, 4,4-Azobis(4-cyanovaleric acid), 1,1'-Azobis(cyclohexanecarbonitrile), 2,2'-Azobisisobutyronitrile (AIBN), Benzoyl peroxide, 2,2-Bis(tert-butylperoxy)butane, 1,1-Bis(tert-butylperoxy)cyclohexane, 2,5-Bis(tert-butylperoxy)-2,5-dimethylhexane, 2,5-Bis(tert-Butylperoxy)-2,5-dimethyl-3-hexyne, Bis(1-(tert-butylperoxy)-1-methylethyl)benzene, 1,1-Bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane, tert-Butyl hydroperoxide, tert-Butyl peracetate, tert-Butyl peroxide, tert-Butyl peroxybenzoate, tert-Butylperoxy isopropyl carbonate, Cumene hydroperoxide, Cyclohexanone peroxide, Dicumyl peroxide, Lauroyl peroxide, 2,4- Pentanedione peroxide, Peracetic acid 또는 Potassium persulfate를 사용할 수 있다.

[0081] In addition, the crosslinking agent may include a chemical initiator, and examples of the chemical initiator may include Carbodiimide, NHS ester, Imidoester, Pentafluorophenyl ester, Hydroxymethyl phosphine, maleimide, Haloacetyl (Bromo- or Iodo-), pyridyldisulfide, Thiosulfonate, Vinylsulfone, Hydrazide, Alkoxyamine, Diazirine, Aryl Azide, or Isocyanate.

[0082] In addition, after the sacrificial material is applied as described above, the sacrificial layer may be formed by simply drying the material to evaporate the solvent mixed with the sacrificial material. In this case, examples of the drying method include natural drying, hot air drying, cold air drying, vacuum drying, spray drying, freeze drying, drum drying, or refrigerated drying. In addition, the drying method may be used to form the sacrificial layer by evaporating the solvent mixed with the sacrificial material, but may also be used to dry the sacrificial layer after the sacrificial layer is formed.

[0083] Before the step of forming the sacrificial layer, a step of positioning a three-dimensional structure on the surface of the sample may be further included.

[0084] The above three-dimensional structure serves to form the outer surface of the sacrificial material, and refers to a structure that allows the sacrificial material to form a sacrificial layer within a certain range.

[0085] A closer look reveals that, as described above, the sacrificial material may be supplied mixed with a solvent for smooth application. In this case, simple application may result in increased flowability due to the low viscosity of the sacrificial material, which may result in the sacrificial material being applied to undesirable areas or failure to form a sacrificial layer of the desired height.

[0086] Therefore, by positioning the three-dimensional structure on the surface of the sample as described above, the sacrificial material can be applied only within a single point range, and the sacrificial material can be applied to a desired height so that the sacrificial layer has a sufficient thickness.

[0087] The above three-dimensional structure may be spatially isolated so that the sacrificial material is in contact with a portion of the sample. When the three-dimensional structure is positioned on a portion of the sample, the sacrificial layer may be formed only on a portion of the sample, thereby allowing only a portion of the sample to be moved. That is, when the three-dimensional structure is used as described above, only the portion of the sample in which the sacrificial material is in contact can be selectively separated. In this case, not only can it be used for the purpose of selectively analyzing only a desired portion of the sample, but since it is possible to move only a portion of the sample, the usability of the sample can be greatly increased when performing other analyses in the future.

[0088] After the manufacture of the sacrificial layer is completed as described above, the sacrificial layer is separated from the first position. At this time, as described above, the bonding strength between the first position and the sample is lower than the bonding strength between the sample and the sacrificial layer, so that during the separation process of the sacrificial layer, the sample can also be separated to form a transfer complex.

[0089] In the case of the transport complex separated as described above, it moves to the second location and is then combined in the same form as the first location.

[0090] The step of removing the sacrificial layer of the transport complex moved to the second position may be further included after the step of moving the transport complex to the second position.

[0091] The transport complex moved to the second position may have its sacrificial layer removed. As described above, if the sacrificial layer is removed after the transport is completed, the sample can be moved while maintaining the same positional information as the first position.

[0092] To this end, the step of removing the sacrificial layer is a process of removing the cross-linked polymer, monomer, unit, or inorganic material used in the sacrificial material so that only a part or all of the sample remains at the second location. The step of removing the cross-linked sacrificial material can be performed through a method including chemical removal, thermal removal, laser removal, photochemical removal, plasma removal, ion beam removal, or ultrasonic removal.

[0093] Examples of the chemical removal include solvent dissolution, chemical etching, plasma treatment, acid treatment, or alkaline treatment. Examples of the thermal removal include thermal decomposition, plasma treatment, or flame treatment. Examples of the plasma and ion removal include plasma etching, ion beam etching, or reactive ion etching. Examples of the ultrasonic removal include ultrasonic cleaning or liquid nitrogen expansion. In addition to the above methods, other methods such as hydrophobic coating followed by removal, electrochemical removal, ozone treatment, and cryogenic blasting can be used.

[0094] When the above-mentioned dissolution is used, a polar solvent, a non-polar solvent, an organic solvent, or an inorganic solvent may be used as the solvent. Preferably, the solvent used is the same as the solvent used to dilute the sacrificial material, but a different solvent may also be used. The group of compounds that can be used in the solvent is the same as the solvent used in combination with the sacrificial material, and therefore, a detailed description thereof will be omitted.

[0095] In addition, when the above dissolution is utilized, the solubility can be controlled by adjusting the temperature of the solvent used, and the removal process can be sequentially performed at multiple temperature levels. In addition, the solubility can be controlled by adjusting the pH of the solvent used, and the removal process can be sequentially performed at multiple pH levels.

[0096] In addition, when dissolution is used as described above, the three-dimensional structure can serve to fix the sample to the second position. Since the solvent used for the dissolution is fluid, if a large amount of solvent is supplied during the dissolution process, the sample may shift from its position. Therefore, by fixing the sample's position using the three-dimensional structure, the sample's displacement can be prevented.

[0097] Additionally, the three-dimensional structure may serve to position the solvent used for the dissolution above the sacrificial layer. This may also prevent the solvent from coming into contact with any area other than the upper portion of the sacrificial layer.

[0098] When using the above dissolution, simple dissolution-washing, ultrasonic washing, heating-solvent removal, mechanical auxiliary solvent removal, solvent stripping, flushing-removal using a circulation system, solvent diffusion-evaporation, electrochemical dissolution, electrochemical dissolution, encapsulation dissolution or other auxiliary methods can be used as a solvent supply method.

[0099] Specifically, as a simple dissolution-washing method, immersion in solvent is a method of immersing a substance in a solvent to allow it to dissolve naturally, solvent washing is a method of dissolving and removing a substance by directly spraying or pouring a solvent on the substance, and wiping with solvent is a method of wiping a substance using a cloth or pad soaked in solvent.

[0100] Among the above ultrasonic cleaning methods, ultrasonic bath cleaning is a method of effectively dissolving and removing a substance by placing a substrate in a solvent bath where ultrasonic waves are generated, and ultrasonic spray cleaning is a method of removing a substance from a surface using a solvent sprayed using ultrasonic waves.

[0101] As mechanical auxiliary solvent removal, Brushing with Solvent is a method of removing materials by rubbing the surface with a brush dipped in solvent, Spraying and Scraping is a method of spraying solvent and physically removing materials with a scraper, Pulsed Jet Cleaning is a method of removing materials by spraying solvent with a high-pressure pulse jet, and Vibratory Cleaning is a method of dissolving and removing materials by applying vibration while using a solvent.

[0102] As a solvent stripping method, paste and gel solvent stripping are methods of applying a gel-type solvent to dissolve and remove a substance in a fixed state, and solvent stripping film is a method of attaching a film containing a solvent on a substance and removing it together with the film after dissolving it.

[0103] Solvent flushing is a method of removing substances by washing the inside of a pipe or system with a solvent, and a solvent circulation system is a method of continuously dissolving and removing substances by circulating a solvent.

[0104] Solvent Penetration and Diffusion, a solvent diffusion-evaporation method, is a method in which a solvent penetrates into the interior of a substance to gradually dissolve and remove the substance, while Evaporation and Residue Removal is a method in which a substance is dissolved using a solvent and then the substance is removed as the solvent evaporates.

[0105] Electrolytic cleaning, an electrochemical dissolution method, is a method of dissolving and removing substances through an electrochemical reaction using a solvent as an electrolyte, and electrosolvent cleaning is a method of using an electric field to make a solvent remove substances more effectively.

[0106] Microencapsulated Solvent Cleaning, as an encapsulation dissolution method, is a method of dissolving and removing a substance at a specific location by encapsulating a solvent, and Solvent Pack Application: is a method of attaching a pack containing a solvent to a substance and removing it together with the pack after dissolution.

[0107] In addition to the above-mentioned removal methods, other removal methods may be used, and as such removal methods, high-pressure solvent jetting is a method of removing a substance by spraying a solvent at high pressure, sponge and pad solvent treatment is a method of physically removing a substance by using a sponge or pad soaked in solvent, and capsule break and solvent application is a method of removing a substance by breaking the encapsulated solvent and then dissolving it.

[0108] When the above laser removal method is used, the wavelength of the laser may include ultraviolet light, visible light, infrared light or gamma rays, and a material, dye or pigment having a high laser absorption rate may be added to the sacrificial material to control the degree to which the sacrificial layer is removed.

[0109] Additionally, when a laser ablation method is used, cryogenic ablation, vacuum ablation, or gas flushing may be used to minimize damage to the sample caused by the laser.

[0110] After the movement as described above is completed, the sample may be preprocessed. As described above, the sample may be moved to a second location for further analysis, and thus a preprocessing step for the analysis may be performed. The preprocessing method used in this case may be any preprocessing method required for the analysis, and the specific method is the same as the preprocessing method prior to the movement, so it is omitted.

[0111]

[0112] 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.

[0113]

[0114] Example 1

[0115] Experiments were conducted using bone marrow aspiration smear samples. As shown in Figure 2, after the sacrificial layer formation step, sacrificial layer transfer step, and sacrificial layer removal step, cells present on each slide were transferred to another slide.

[0116] The sacrificial layer used at this time was manufactured with the following composition:

[0117] Gelatin 10-30% (w / w)

[0118] Solvent: distilled water

[0119] The above compositions were mixed to prepare a homogeneous sacrificial layer composition. The composition was applied to a slide containing a bone marrow aspiration smear sample stained with Wright & Giemsa for 1 minute at room temperature. The mixture was then dried at 4°C to form a rigid sacrificial layer capable of supporting the section.

[0120] The dried sacrificial layer and sample were separated from the original slide, positioned so that the sample was facing the substrate to which it was to be transferred, and placed on a CosmoSlide pre-coated with 20 μL of distilled water. Adhesion between the sacrificial layer / sample and the CosmoSlide was induced for approximately 20 minutes at room temperature. Subsequently, the sacrificial layer / sample, along with the CosmoSlide, were immersed in distilled water at 37 °C for 30 minutes to completely dissolve and remove the sacrificial layer.

[0121] As shown in Fig. 3, the sample was stably transferred without tearing or deformation through this method, and it was confirmed that transfer to other types of substrates was possible.

[0122] In addition, changes in the cell microstructure and spatial arrangement were confirmed by comparing the enlarged images of the cell sample before and after movement. The images in Fig. 4 were acquired using an optical microscope and were photographed at magnifications of 2x, 4x, 20x, and 40x. As shown in Fig. 4, when observing the cell sample before and after movement, it was confirmed that the cell microstructure and spatial position information were maintained.

[0123] In this example, when the H&E-stained FFPE sample was observed with a 40x optical microscope for images before and after migration through the sacrificial layer, it was confirmed that the cell morphology and histological structure were maintained the same before and after migration, as shown in Fig. 5.

[0124]

[0125] Example 2

[0126] Experiments were conducted using tissue section samples. As shown in Figure 2, after the sacrificial layer formation step, sacrificial layer transfer step, and sacrificial layer removal step, cells present on each slide were transferred to another slide.

[0127] The sacrificial layer used at this time was manufactured with the following composition:

[0128] Polyvinyl alcohol 10.24% (w / w)

[0129] Polyethylene glycol 4.26% (w / w)

[0130] Solvent: distilled water

[0131]

[0132] The above components were mixed to prepare a homogeneous sacrificial layer composition. The prepared composition was applied to a slide containing a tissue section or cell culture at 4 °C for 30 seconds, and then cooled in a -80 °C environment for 5 minutes to solidify.

[0133] The solidified sacrificial layer provided structural support for the tissue section, and both the sacrificial layer and the section were cleanly separated from the original slide. The separated section was then placed at the desired location on the substrate to be transferred and maintained at 4 °C for 20 minutes to melt the sacrificial layer. Subsequently, the sacrificial layer was completely removed by washing with distilled water at 4 °C for 15 minutes.

[0134] As shown in Fig. 6, it was confirmed that normal transfer was possible from a Cosmoslide to a Xenium slide and from an SPL Hybridwell glass slide to a PEN membrane slide. In other words, it was confirmed that tissue sections could be stably transferred without tearing or deformation through this method, and that transfer to various types of substrates was possible.

[0135]

[0136] Example 3

[0137] After moving the sample in the same manner as in Example 1, the target cells were separated using a cell separation device, Spatially-resolved Laser Activated Cell Sorter (SLACS), for analysis (Fig. 7).

[0138] When RNAseq prep was performed on three single cells (C1, C2, C3) before transfer and three single cells (T1, T2, T3) after transfer to use as a control, a significant amount of cDNA concentration was obtained as shown in Fig. 8, and when confirmed through the electrophoresis results, it was confirmed that the RNAseq was performed well as the gel images were good for all samples.

[0139] In addition, when stained and unstained cell samples were transferred to each substrate and analyzed after RNAseq, it was confirmed that high gene counts were observed in both cases, as shown in Fig. 9. That is, based on the results of Figs. 8 and 9, it was confirmed that the method of Example 1 of the present invention enables transfer without damaging cells.

[0140]

[0141] Examples 4-8

[0142] In addition to the gelatin of Example 1 and the polyvinyl alcohol and polyethylene glycol of Example 2, other materials that could be used in the sacrificial layer were tested. Each sacrificial material was selected as shown in Table 1.

[0143] Sacrificial substance solvent Example 4 Hyaluronic acid product Example 5 Alginate product Example 6 Polyacrylic acid product Example 7 Polyvinyl pyrrolidine water Example 8 Latex hexane

[0144] Experimental Example 1

[0145] The same experiment as Example 1 was conducted using the sacrificial materials of Examples 4 to 8 above. The experiment was conducted 20 times for each sample to check whether residue was generated after separation of the sacrificial layer and whether spatial information was preserved after movement. The number of defects is shown in Table 2 below.

[0146] After separation, residual material generation space information is not preserved. Example 110 Example 221 Example 422 Example 521 Example 611 Example 711 Example 843

[0147] As shown in Table 2, when water-soluble polymers were used (Examples 1-7), the generation of residue after separation was reduced, and spatial information was also well preserved. However, in Example 8, which used a non-water-soluble polymer, the generation of residue was also minimized, and spatial information was also largely preserved.

[0148]

[0149] 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 sacrificial layer by supplying a sacrificial material to a sample at a first location; A step of manufacturing a transfer composite by simultaneously separating part or all of the sample and the sacrificial layer at the first position; and A step of moving the above transport complex to a second position; A method for transferring a biochemical sample comprising:

2. In paragraph 1, A method for transferring a biochemical sample, 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 transferring a biochemical sample, characterized in that the first position and the second position include at least one selected from the group consisting of a slide, a substrate, a chip, a cover glass, a tube, a cap, and a well.

4. In paragraph 1, A method for transferring a biochemical sample, wherein the sacrificial material comprises a polymer, a monomer, a unit, or an inorganic substance.

5. In paragraph 4, A method for transferring a biochemical sample, wherein the sacrificial material comprises a physically or chemically cross-linkable material.

6. In paragraph 1, A method for transferring a biochemical sample, characterized in that the step of removing the sacrificial layer of the above transport complex comprises a step of chemically or physically removing the sacrificial layer.

7. In paragraph 6, The step of chemically or physically removing the sacrificial layer includes solvent dissolution, chemical etching, plasma treatment, acid treatment, alkaline treatment, mechanical polishing, grinding, scraping, sandblasting, thermal decomposition, plasma treatment, flame treatment, laser ablation, UV photochemical removal, plasma etching, ion beam etching, reactive ion etching, ultrasonic cleaning, liquid nitrogen expansion, hydrophobic coating followed by removal, electrochemical removal, ozone treatment, and A method for transferring a biochemical sample, characterized in that it comprises at least one method selected from the group consisting of cryogenic blasting.

8. In paragraph 1, Before the step of forming the above sacrificial layer, A step of positioning a three-dimensional structure on the surface of the sample; A method for transferring a biochemical sample, characterized in that it further comprises:

9. In paragraph 8, A method for transferring a biochemical sample, wherein the three-dimensional structure is spatially isolated such that the sacrificial material is in contact with a portion of the sample.

10. In paragraph 9, A method for transferring a biochemical sample, characterized in that the above-mentioned separating step is a step in which only the sample of the area in contact with the sacrificial material is selectively separated.

11. In paragraph 1, A method for transferring a biochemical sample, characterized in that it further comprises the step of removing the sacrificial layer of the transfer complex moved to the second position after the step of moving the transfer complex to the second position.

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