Method for separating target substance contained in target liquid

Abstract

One aspect of the present disclosure provides a method for preserving a target substance in a target liquid. The method includes: allowing a fiber sheet to absorb the target liquid by bringing the fiber sheet into contact with the target liquid; and drying the fiber sheet and washing the fiber sheet. This makes it possible to separate the target substance easily or efficiently from foreign matter, for example. This makes it possible for the target substance to be separated easily, for example.

Description

Method for separating target substances contained in target liquid

[0001] The present disclosure relates to a method for separating a target substance contained in a target liquid.

[0002] BACKGROUND ART Selective capture or separation of substances such as biomolecules present in a liquid is important for investigating, adjusting, or processing the properties of the target substances.

[0003] For example, extracellular vesicles (exosomes; hereinafter, sometimes referred to as "EVs"), which are a type of biomolecule, have been found to have the ability to induce cancer metastasis as one of their functions in the body, and have attracted attention.

[0004] Extracellular vesicles are membrane vesicles approximately 40 to 1000 nm in size that are secreted from cells in the body and are present in bodily fluids such as blood, urine, saliva, and semen. Their surface contains membrane proteins, adhesion molecules, enzymes, etc. derived from the secretory cell, and their interior contains nucleic acids such as mRNA and miRNA. Therefore, it has been discovered that they can be transmitted to other cells and taken up, thereby affecting the recipient cells.

[0005] To date, various methods have been developed to selectively obtain target substances, including physical methods such as ultracentrifugation, chemical methods such as agglutination reagent methods, and capture methods using ZnO nanowire devices, etc. However, in order to capture, separate, or analyze biomolecules present in the body, for example, it has been necessary to obtain a certain volume of tumor, body fluid, etc.

[0006] As another example, capturing extracellular vesicles (EVs) using cellulose nanofibers is known (Patent Document 1). However, this document discloses a device for adsorbing EVs, and although it discloses that the gaps (nanopores) in the cellulose nanofibers should have a certain size distribution, it does not mention a method for physically preventing the re-release of EVs once captured.

[0007] In either case, there was no method for recovering biomolecules such as EVs from a minute amount of body fluid, for example, enough to wet the surface of an organ, skin, or the like.

[0008] WO2020 / 090859A1

[0009] According to one aspect of the present disclosure, there is provided a method for preserving a target substance in a target liquid. The method includes contacting a fiber sheet with the target liquid and allowing the target liquid to be absorbed by the fiber sheet; drying the fiber sheet; and washing the fiber sheet. This allows, for example, to simply or efficiently separate the target substance from impurities. This allows, for example, to simply separate the target substance.

[0010] According to another aspect of the present disclosure, there is provided a method for separating a target substance in a target liquid. The method includes contacting a fibrous sheet with the target liquid to absorb the target liquid into the fibrous sheet, drying the fibrous sheet, washing the fibrous sheet, and adding an aqueous solution to the dried fibrous sheet to release the captured target substance from the fibrous sheet. This allows, for example, the target substance to be separated from impurities in a simple or efficient manner.

[0011]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0012] FIG. 1 shows a flowchart of a method for separating a target substance in a target liquid according to an embodiment of the present disclosure. FIG. 2 shows a flowchart of a method for separating a target substance in a target liquid according to an embodiment of the present disclosure. FIG. 3 shows a series of CG images created for each step of a method for separating a target substance in a target liquid according to an embodiment of the present disclosure. FIG. 4 shows a series of SEM images taken for each step of a method for separating a target substance in a target liquid according to an embodiment of the present disclosure. FIG. 5 shows a graph of the porosity of a fiber sheet measured for each step of a method for separating a target substance in a target liquid according to an embodiment of the present disclosure. FIG. 6 shows a flowchart of a method for preserving a target substance in a target liquid according to an embodiment of the present disclosure. FIG. 7 shows CG images created for some of the steps of a method for separating a target substance in a target liquid according to an embodiment of the present disclosure. As an example of the present disclosure, the correlation of miRNA obtained in two experiments using a method for separating a target substance in a target liquid is shown. The annotation rate of synthetic miRNA intentionally introduced as an impurity to miRNA obtained using a method for separating a target substance in a target liquid is shown. The relationship between the drying time of a fiber sheet and the annotation rate of miRNA from separated EVs is shown in an example. (a) As an example of the present disclosure, the size distribution of EVs obtained by separation from human serum using a method for separating a target substance in a target liquid; and (b) the total amount thereof. As an example of the present disclosure, the expression level of membrane proteins in EVs obtained by separation from human serum using a method for separating a target substance in a target liquid is shown. (a) As an example of the present disclosure, the size distribution of EVs obtained by separation from mouse ascites using a method for separating a target substance in a target liquid; and (b) the total amount thereof. As an example of the present disclosure, the expression level of membrane proteins in EVs obtained by separation from mouse ascites using a method for separating a target substance in a target liquid is shown. As an example of the present disclosure, a heat map of miRNAs obtained from the pelvic peritoneum and liver surface of a mouse using a method for separating a target substance in a target liquid is shown. As an example of the present disclosure, a heat map of miRNAs obtained from the surface of a mouse tumor and the surface near the tumor using a method for separating a target substance in a target liquid, and, for comparison, a heat map of miRNAs obtained from mouse tumor tissue and tissue near the tumor.

[0033] Figure 1 shows the results of PCA analysis of mouse miRNAs, as shown in the heat map above. As an example of the present disclosure, a heat map of miRNA expression encapsulated in EVs obtained from the pelvic peritoneum and liver surface using a fiber sheet on day 0 and day 4 is shown. As an example of the present disclosure, a PCA map of miRNA expression in EVs and tissues obtained using a fiber sheet is shown. The annotation rate of miRNAs obtained from the tumor surface and surfaces near the tumor in a human ovarian cancer patient, as shown in the heat map above, is shown. As an example of the present disclosure, a heat map of miRNA expression obtained by RNA sequencing for miRNAs from EVs and tumors obtained using a fiber sheet is shown. As an example of the present disclosure, a PCA map of miRNAs obtained from each organ or body fluid using a fiber sheet is shown. As an example of the present disclosure, the results of PCA analysis of various data are shown.

[0013] 1 shows a flowchart 100 of a method for separating a target substance in a target liquid according to one embodiment of the present disclosure. A fiber sheet is provided (S101). A target liquid containing a target substance is contacted with the fiber sheet, and the target liquid is absorbed by the fiber sheet (S102). The fiber sheet is then dried (S103). In some embodiments, the fiber sheet may be transported or stored in a dried state. The fiber sheet may then be washed.

[0014] 2 shows a flowchart 200 of a method for separating a target substance in a target liquid according to one embodiment of the present disclosure. A fiber sheet is provided (S201). A target liquid containing a target substance is brought into contact with the fiber sheet, and the target liquid is absorbed into the fiber sheet (S202). The fiber sheet is then dried (S203). The dried fiber sheet is washed to remove impurities while retaining the target substance in the fiber sheet (S204). The captured target substance is released from the fiber sheet (S205).

[0015] <Fiber Sheet> As used herein, the term "fiber sheet" generally refers to a flat member having pores formed by mechanically and / or chemically processing fibers. Fiber sheets include woven fabrics, nonwoven fabrics, textiles, paper, etc. The fiber sheet of the present disclosure preferably has hydrophilicity to the extent that an aqueous solution can penetrate into the interior by capillary blood action or higher.

[0016] In some embodiments, the fibers may be synthetic fibers, natural fibers, or a blend of multiple fiber types.

[0017] In some embodiments, the natural fibers may be plant fibers such as cellulose, cotton, hemp, linen, etc. In some embodiments, the natural fibers may be animal fibers such as wool, silk, cashmere, etc.

[0018] As used herein, the term "cellulose fiber sheet" generally refers to a fibrous sheet that consists primarily of or includes cellulose fibers. As used herein, "cellulose fiber" refers to fibers that consist primarily of cellulose. The cellulose fibers may be nanocellulose fibers (CNFs). Nanocellulose fibers generally have diameters of a few nanometers (nm) to tens of nm. Often, by way of non-limiting example, cellulose nanofibers have widths of about 3 nm to 100 nm.

[0019] Cellulose fibers can be obtained, for example, chemically and / or mechanically from wood pulp or other raw materials. A typical method for producing cellulose nanofibers is as follows: First, wood fibers (cellulose fibers) are extracted from wood chips and pulped. These cellulose fibers are composed of countless bundles of cellulose nanofibers. Next, these cellulose fibers are subjected to high-pressure collisions in a solvent in the presence of a TEMPO catalyst, thereby loosening the bundles of cellulose fibers. This allows the production of cellulose nanofibers. The above-mentioned methods for producing cellulose fibers and cellulose fiber sheets are non-limiting examples, and other production methods may also be used.

[0020] The resulting solvent containing the cellulose nanofibers is subjected to suction filtration, causing the cellulose nanofibers to aggregate or form a film due to surface tension. The cellulose nanofiber solvent may be water or the like. In one embodiment, the resulting film may be a nonwoven fabric.

[0021] Nanopores are formed by adding a liquid with low surface tension (hereinafter sometimes referred to as a "low surface tension solvent") to wet cellulose nanofibers that have been aggregated by suction filtration. This is then suctioned, and the solvent contained in the aggregated cellulose nanofiber mass is replaced with the low surface tension solvent, or the mass is dried. This forms nanopores inside the aggregate of cellulose nanofibers.

[0022] The size of the nanopore can be adjusted, for example, by adding a low surface tension solvent. The surface tension of the low surface tension solvent may be lower than the surface tension of water (72.75 mN / m at 20°C) and within a range in which nanopores can be formed. For example, the surface tension of the solvent at 20°C may be equal to or lower than 35 mN / m, 30 mN / m, 25 mN / m, 20 mN / m, etc. Low surface tension solvents include, but are not limited to, tertiary butyl alcohol (20.7 mN / m), ethanol (22.55 mN / m), isopropanol (20.8 mN / m), etc.

[0023] The above-mentioned methods for forming nanopores and adjusting their size are merely examples, and other methods may be used. For example, high-pressure treatment conditions that loosen cellulose fibers may be employed. For example, the type of pulp may be changed. For example, cellulose derived from other sources (e.g., microorganisms such as acetic acid bacteria (Acetobacter genus, etc.); animals such as sea squirts) may be used. By using these methods, the width of the cellulose nanofibers may be changed, and the size of the nanopores may be adjusted.

[0024] In some embodiments, the fibrous sheet may be formed from cellulose fibers (pulp). By dispersing the cellulose fibers (pulp) in a solvent, a fibrous sheet can be produced in much the same way as in the case of cellulose nanofibers.

[0025] The gaps (pore size) of cellulose fibers can be adjusted in much the same way as in the case of cellulose nanofibers. The width (diameter) of cellulose fibers is often about 20 μm to 40 μm. Therefore, the gap size may be a few nm to a few μm, about 10 nm to about 1000 nm, and about 1 μm to 100 μm.

[0026] In some embodiments, the fibers may be chemical fibers. In some embodiments, the chemical fibers may be polymer fibers or synthetic fibers such as polyvinyl alcohol (PVA). The chemical fibers may be polyester fibers such as polyethylene terephthalate, polynaphthalene terephthalate, polyethylene naphthalate, and polytrimethylene terephthalate; polyamide fibers such as nylon; acrylic fibers such as acrylonitrile; polyolefin fibers such as polyethylene and polypropylene; regenerated cellulose fibers such as rayon, cupra, and polynosic; cellulose-based semi-synthetic fibers such as acetate; protein-based semi-synthetic fibers such as promix; polyurethane fibers; vinylon fibers; glass fibers; carbon fibers (including carbon nanotubes); and the like.

[0027] As used herein, "pore size" or "characteristic size" refers to the size of the pores in a fibrous sheet. The pore size may be 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 30 nm, or greater. The pore size may be 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, or smaller. The pore size may range from about 4 nm to about 200 nm, from about 40 nm to about 200 nm, from about 200 nm to about 500 nm, from about 4 nm to about 100 nm, from about 5 nm to about 90 nm, from about 10 nm to about 80 nm, or the like.

[0028] For example, a fibrous sheet having a pore size of about 40 nm to about 200 nm is suitable for capturing small EVs of a similar size. For example, a fibrous sheet having a pore size of about 200 nm to about 500 nm is suitable for capturing large EVs of a similar size. These are examples, and fibrous sheets having other pore sizes may be used to capture EVs of a similar size.

[0029] The fibrous sheet of the present disclosure preferably shrinks upon drying. In other words, the pore size of the fibrous sheet decreases as the absorbed solvent (aqueous or non-aqueous) evaporates. The fibrous sheet is preferably manufactured so that the pore size is equal to or larger than the size of the target object when wet, and smaller than the size of the target object after drying.

[0030] Pore ​​size can be measured before use, in a dry state, after washing, or after desorption of the capture substance. Pore size can be measured using mercury injection, electron microscopy, or a combination of these. Unlike porous solid materials, the interfiber spacing can be difficult to define unambiguously. While mercury injection is considered to reflect the pore size or interfiber distance of a fiber sheet, it does not necessarily provide the same value. Therefore, the results of mercury injection may be combined with other techniques (e.g., microscopy) or may be considered as an indicator of pore size.

[0031] Pore ​​size may be defined or determined based on measurements of the substance of interest (e.g., EVs) or a substance related thereto (e.g., nucleic acids encapsulated in EVs) or its properties. The properties of the fibrous sheet or its manufacturing conditions that produce the best measurement results or a predetermined result may be defined as properties that can be exchanged for pore size.

[0032] The pore size may alternatively be defined by, for example, the amount of EVs separated by the fiber sheet, the pore size of the EVs that give rise to the peak, the distribution or range of EV sizes, or the amount or profile of nucleic acids contained in the EVs separated by the fiber sheet, and may be expressed using these as indicators.

[0033] In some embodiments, the fibrous sheet may be configured such that, during manufacturing or when absorbing a target liquid, the target object can substantially enter or pass through the gaps in the fibrous sheet, and during cleaning (after drying or before cleaning), the target object is substantially retained in the fibrous sheet and / or does not flow out.

[0034] <Target Liquid> "Target liquid" includes a liquid that contains the target substance, a liquid that is thought to contain the target substance, or a liquid that does not contain the target substance but is used for the purpose of separating the target substance or for purposes related thereto.

[0035] The target liquid (also referred to as the target solution) may be a body fluid or a body fluid-derived liquid (diluted solution, treatment solution, etc.). The solution may be a non-body fluid (non-body fluid-derived) solution, an artificially prepared liquid, or a mixture of a body fluid or a body fluid-derived solution and a non-body fluid-derived solution. The solution may be a solution used for sample measurement or a solution used for calibration measurement. The solution may be used as is, or may be a liquid obtained by diluting or concentrating the stock solution. The solution may be a standard solution or a calibration solution. The sample to be measured may be a specimen. The solution may contain a physiological buffer solution such as phosphate-buffered saline (PBS) or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES) containing the substance to be recovered. The body fluid may contain an additive. The additive may include, for example, a stabilizer or a pH adjuster.

[0036] The body fluid may be, but is not limited to, blood, serum, plasma, lymphatic fluid, tissue fluid such as interstitial fluid, intercellular fluid, or interstitial fluid, body cavity fluid, serous cavity fluid, pleural fluid, peritoneal fluid, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid (synovial fluid), or aqueous humor (aqueous humor). The body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, or intestinal juice, or may be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk.

[0037] The bodily fluid may be a human bodily fluid. The bodily fluid may be an animal bodily fluid. The animal may be a reptile, a mammal, or an amphibian. The mammal may be a dog, a cat, a cow, a horse, a sheep, a pig, a hamster, a rat, a squirrel, or a primate such as a monkey, a gorilla, a chimpanzee, a bonobo, or a human.

[0038] <Target Substance> The target substance may be a biomolecule. The biomolecule may be an organelle or a vesicle. The vesicle may be, but is not limited to, a vacuole, a lysosome, a transport vesicle, a secretory vesicle, a gas vesicle, an extracellular matrix vesicle, an extracellular vesicle, or the like, or may include a plurality of these. The extracellular vesicle (EV) may be, but is not limited to, an exosome, an exosome complex, an exotome, a shedding microvesicle, a microvesicle, a membrane particle, a plasma membrane, an apoptotic vesicle, or the like.

[0039] The biomolecule may be or include, but is not limited to, a cell. The cell may be a red blood cell, a white blood cell, an immune cell, etc. The biomolecule may be a virus, a bacterium, etc.

[0040] The substance of interest may include nucleic acid.

[0041] The nucleic acid may be or may contain ribonucleic acid (RNA). The RNA may be, but is not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), non-coding RNA (ncRNA), microRNA (miRNA), ribozyme, double-stranded RNA (dsRNA), or the like, or may contain a plurality of these. The RNA may be modified. The RNA or miRNA may be involved in the onset or progression of cancer, cardiovascular disease, neurodegenerative disease, psychiatric disorder, chronic inflammatory disease, or the like. The miRNA may be a type of RNA that promotes or positively regulates carcinogenesis (onco miRNA (oncogenic miRNA)), or a type of RNA that suppresses or negatively regulates carcinogenesis (tumor suppressor miRNA).

[0042] The nucleic acid may be or include deoxyribonucleic acid (DNA).

[0043] <Absorption> The target liquid can be absorbed into the fibrous sheet. The target liquid is absorbed into the fibrous sheet by contacting at least a portion of the fibrous sheet. The target liquid may be absorbed into the fibrous sheet by surface tension. The target liquid may be absorbed into the fibrous sheet by pressure.

[0044] In some embodiments, the fibrous sheet may be brought into contact with the surface of a target liquid that exists macroscopically in a liquid state. In some embodiments, the fibrous sheet may be immersed in the target liquid. For example, the fibrous sheet may be brought into contact with or immersed in collected urine to absorb the urine.

[0045] In some embodiments, the fiber sheet may be brought into contact with a target liquid present on a solid surface (the surface of a target portion on which a target substance is present; hereinafter also referred to as "target surface"). The target surface may be in a wet environment. In this case, the fiber sheet may be attached to the solid surface. For example, the fiber sheet may be brought into contact with the surface of a tissue or organ (hereinafter simply referred to as an organ) of an organism. The organism may be a plant or an animal. The organ of the organism may be a biological organ such as skin, mucous membrane, or internal organ. The surface of the organ of the organism may be accessible non-invasively, invasively by surgical techniques, or minimally invasively, such as endoscopic surgery. The surface may be the surface of an organ, or an exposed internal surface that has become accessible for the first time by incision, etc.

[0046] The organs of an animal include the circulatory system, digestive system, endocrine system, urinary system, lymphatic system, integumentary system, nervous system, reproductive system, respiratory system, and locomotor system (including skeleton and muscles). However, the use of these terms and classification methods are merely examples, and other methods may be adopted.

[0047] For example, the fibrous sheet can be applied to the surface of an internal organ such as a liver that is exposed during surgery or that has been removed from the body through surgical or endoscopic procedures, etc., to absorb body fluids on the surface.

[0048] In some embodiments, a fiber sheet may be applied to or brought into contact with the skin or mucosa to absorb body fluids present on the surface of the skin or mucosa. If the amount of body fluid present on the body surface is insufficient, a solution may be dripped onto the body surface to promote absorption into the fiber sheet.

[0049] In some embodiments, the fiber sheet may be applied to or brought into contact with areas that are not normally visible from the outside, such as the armpits (or underarms), oral cavity (including the tongue), nasal cavity, esophagus, stomach, intestines, or vagina, to absorb bodily fluids present or secreted on the surface of the skin or mucous membrane.

[0050] In some embodiments, bodily fluids may be collected and then contacted with a fiber sheet at a location remote from the subject to absorb the bodily fluid, such as urine, tears, saliva, gastric fluid, vaginal fluid, semen, blood, ascites, pleural fluid, etc.

[0051] <Drying> In some embodiments, the fiber sheet is dried after absorbing the target liquid. Drying may be performed at room temperature, at a temperature elevated above room temperature or by heating, or at a temperature lower than room temperature. The temperature may be set so that the subsequent measurement of the target substance is performed stably or so as not to substantially adversely affect the measurement. Drying may be performed at the humidity of the local and prevailing climatic conditions, or at a low, controlled humidity. If the drying time is too short, the drying may not be complete. If the drying time is too long, the process may be inefficient. Therefore, an appropriate drying time may be determined.

[0052] <Pore size control> Figure 3 shows, using a cellulose nanofiber (CNF) sheet as a fiber sheet, each step of capturing extracellular vesicles as target substances, separating them from contaminants, and then releasing them from the fiber sheet, using computer graphics (CG).

[0053] As shown in Figure 3(a), extracellular vesicles enter the interior of a CNF sheet upon contact with a target liquid. Substances larger than the pore size of the CNF sheet cannot enter the interior of the sheet. Next, the pore size of the CNF sheet shrinks upon drying (Figure 3(b)). As a result, the extracellular vesicles inside are trapped by the CNFs and cannot escape again. When washed, the pore size of the CNF sheet increases slightly due to contact with the aqueous solution. However, by performing the washing under appropriate conditions, the pore size of the CNF sheet does not increase too much, and the extracellular vesicles remain trapped inside the CNF sheet and cannot escape to the outside (Figure 3(c)). Substances smaller than the pore size (e.g., impurities) can be flushed out of the CNF sheet. By sufficiently increasing the pore size of the CNF sheet under different solution conditions, the extracellular vesicles can be extracted from the CNF sheet (Figure 3(d)).

[0054] In this manner, by using the method according to some embodiments described herein, a substance (target substance) that is smaller than the initial pore size but larger than the pore size after washing can be separated and obtained from substances (contaminants) with other pore sizes. In other words, by selecting a fiber sheet having one or both of these two pore sizes corresponding to the size of the target substance, the target substance can be selectively separated and / or obtained.

[0055] Figure 4(a) to (d) show a series of SEM images of the surface of a CNF sheet corresponding to each step in Figure 3(a) to (d). Here, human saliva was used as the target liquid, and the CNF sheet was fabricated with pores of a size that could selectively capture extracellular vesicles.

[0056] Figure 4(a) shows the surface structure of the initial CNF sheet. As shown in Figure 4(b), numerous extracellular vesicles were captured by the CNF sheet after contact with the target liquid. After washing (Figure 4(c)), the pore size of the CNF sheet became smaller, making the internal structure difficult to see, but subsequent experiments revealed that extracellular vesicles were captured within the sheet. When the pore size of the CNF sheet was increased sufficiently under different solution conditions, the extracellular vesicles were removed from the CNF sheet. As a result, almost no extracellular vesicles were observed (Figure 4(d)).

[0057] The pore size of the CNF sheet at each step was measured using the mercury injection method. Figure 5 shows the porosity (%) determined by the mercury injection method. A to E represent the following states: A: unused state after production; B: state after saliva was dropped and dried; C: state after 10 seconds of washing with PBS; D: state after 10 seconds of washing with PBS and 5 minutes of treatment with lysis buffer; and E: state after 5 minutes of washing with PBS to fully open the pores and extract the captured substance.

[0058] Here, in steps B to E, tBuOH treatment was performed before mercury injection measurement. That is, after each step, the sheet was immediately immersed in tBuOH. After one hour, the sheet was removed, dried at room temperature, and analyzed. The CNF sheet must be dried to perform the mercury injection method. If no treatment is performed, there is a risk that the pores will shrink again due to drying. The tBuOH treatment suppresses the subsequent shrinkage of pore size that may occur due to drying, allowing for more accurate pore size measurement.

[0059] Embodiment 3: In-situ Crushing of Target Objects Figure 6 shows a flowchart 300 of a method for separating a target substance in a target liquid according to one embodiment of the present disclosure. A fiber sheet is provided (S301). A target liquid containing the target substance is brought into contact with the fiber sheet, and the target liquid is absorbed into the fiber sheet (S302). The fiber sheet is then dried (S303). The dried fiber sheet is washed to remove impurities while retaining the target substance in the fiber sheet (S304). The target substance captured in the fiber sheet is crushed to obtain the internal substance of the target substance (S305).

[0060] In some embodiments, the target substance may not necessarily be crushed within the fiber sheet to obtain the internal substance. In some embodiments, the method for separating a target substance in a target liquid may comprise crushing the target substance captured in the fiber sheet instead of S305.

[0061] Figure 7 shows, using computer graphics (CG), the steps of using a cellulose nanofiber (CNF) sheet as a fiber sheet to disrupt extracellular vesicles captured as target substances and extracting the miRNA contained therein.

[0062] The extracellular vesicles (not shown) are captured by the CNF sheet and washed under appropriate conditions. This allows contaminants, such as free miRNA (EV-free miRNA), to be flushed out of the CNF sheet (Figure 7(a)). Next, the CNF sheet is immersed in a disruption solution (e.g., lysis buffer), allowing the CNF sheet to be saturated with the disruption solution. The disruption solution disrupts the extracellular vesicles, allowing the miRNA contained within them to be extracted from the CNF sheet (Figure 7(b)).

[0063] It was confirmed that the miRNA profile in EVs obtained using this method was relatively stable. Figure 8 shows the correlation of miRNA between two miRNA extractions using the above method. Each point represents one miRNA. The horizontal axis represents the read count in measurement #1, and the vertical axis represents the read count in measurement #2. The read count was determined using an RNA sequencer (MiSEQ, Illumina). This confirmed the reproducibility of these two extractions and measurements.

[0064] Because there was a suspicion that free miRNA was being read, measurements were performed using synthetic miRNA. Specifically, a 50 pM solution of synthetic miR-21-3p was prepared. This solution was analyzed directly using an RNA sequencer (a). Furthermore, the above solution was absorbed and captured on a manufactured CNF sheet, treated with lysis buffer for 5 minutes, and purified using a column. This solution was analyzed using an RNA sequencer (b). Meanwhile, a CNF sheet was allowed to absorb water, and the resulting solution was obtained by introducing lysis buffer into it, and this solution was analyzed using an RNA sequencer (c). Figure 9 shows a comparison of these annotation rates.

[0065] When only water was used, miR-21-3p was not detected (c). When a solution of synthetic miR-21-3p was absorbed into the CNF sheet (b), the amount of miR-21-3p detected (annotation rate) was extremely small compared to when the same solution was measured directly (a). Therefore, it was revealed that free miRNA adsorbed to the CNF sheet has almost no effect on the measurement results of EV-derived miRNA.

[0066] Example: Drying In this example, 10 μL of the same sample as above was dropped onto a 1 cm x 1 cm CNF sheet (hereinafter referred to as the fiber sheet) using a pipette. This fiber sheet was kept at room temperature and in the laboratory's ambient air environment for 3 and 7 days. This allowed the fiber sheet to dry. It was then washed by immersion in PBS. The fiber sheet was then immersed in 1.5 mL of lysis buffer (Norgen) and shaken using a vortex mixer. 1 mL of this lysis buffer was removed and purified using the Norgen kit to obtain 50 μL of miRNA solution. The RNA in the resulting solution was analyzed using an RNA sequencer (MiSEQ, Illumina). The number of reads was 1.5 million to 2.3 million or more.

[0067] Figure 10 shows the average annotation rates obtained from multiple samples after 3 and 7 days of drying. The annotation rates were approximately 0.03 to 0.07% (average approximately 0.05%) after 3 days of drying, and approximately 0.07 to 0.14% (average approximately 0.105%) after 7 days of drying. In this experiment, the annotation rate was higher after 7 days than after 3 days, suggesting that drying was more advanced after 7 days.

[0068] It was observed that the annotation rate did not change even when the fiber sheet was stored for 5-6 days or more under standard atmospheric conditions in Japan. From this, it is believed that the fiber sheet will be completely dry in 5-6 days. This degree of drying may be confirmed by other methods. The speed and degree of drying can be further improved depending on conditions such as drying time and humidity.

[0069] Example: EVs in human serum Using a method according to one embodiment of the present disclosure, human serum was absorbed into a fiber sheet and EVs contained therein were separated.

[0070] Figure 11(a) shows the size distribution of EVs extracted from the fiber sheet. EV sizes were measured using a nanoparticle analysis system (NanoSight, Malvern PANalytical). The horizontal axis represents particle size (nm), and the vertical axis represents particle concentration (x10 ９ As shown in Figure 11(a), it was found that particles were obtained with a peak at about 105 nm and a distribution of about 60 nm to about 260 nm. From this, it was estimated that the measured particles were EVs.

[0071] Furthermore, a new fiber sheet was immersed in PBS for 10 seconds, and the resulting solution was subjected to the same measurement. ８ / mL). Figure 11(b) shows a comparison between the case of an unused fiber sheet (PBS, left) and the case of Figure 11(a). From this, it was found that these particles are almost EV, and the influence of dust and other substances can be ignored.

[0072] As shown in Figure 12, specific proteins expressed in the obtained particles were detected using an exosome identification and quantification device (Exoview Human Tetraspanin Kit, NanoView Bioscience). Anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody were used as primary antibodies. Detection of CD9, CD63, and CD81 confirmed that the obtained particles were EVs. Detection of CD63 and CD9 and suppression of nonspecific adsorption to IgG confirmed that the obtained particles were EVs.

[0073] Example: EVs in ascites on the surface of mouse ovarian cancer tumors Using a method according to an embodiment of the present disclosure, a fiber (CNF) sheet was attached to the surface of the tumor in a tumor-bearing mouse transplanted with ovarian cancer, and the ascites was absorbed, and EVs contained therein were isolated.

[0074] Female athymic (NCr-nude) mice were used. The mouse ovarian cancer cell line ID8 was injected intraperitoneally (IP). 14-day-old mice underwent laparotomy, and a fiber sheet was attached to the tumor surface, and the body fluid (ascites) on the surface was absorbed and collected. In the case of the ID8 cell line, ascites usually accumulates within 30-40 days, but not within 14 days.

[0075] Figure 13(a) shows the size distribution of EVs extracted from the fiber sheet. EV sizes were measured using a nanoparticle analysis system (NanoSight, Malvern PANalytical). The horizontal axis represents particle size (nm), and the vertical axis represents particle concentration (x10 ７ As shown in Figure 11(a), it was found that particles were obtained with a maximum peak at approximately 130 nm and multiple sub-peaks at other sizes, with a distribution of approximately 60 nm to approximately 400 nm. From this, it was estimated that the measured particles were EV.

[0076] Furthermore, a new fiber sheet was immersed in PBS for 10 seconds, and the resulting solution was subjected to the same measurement. Figure 13(b) shows a comparison between the case of a new fiber sheet (PBS, left) and the case of Figure 13(a). From this, it was found that these particles were almost entirely EV, and the influence of dust and other substances could be ignored.

[0077] As shown in Figure 14, specific proteins expressed in the obtained particles were detected using an exosome identification and quantification device (Exoview Human Tetraspanin Kit, NanoView Bioscience). Anti-CD63, anti-CD9, and anti-CD81 antibodies were used as primary antibodies. The detection of CD9, CD63, and CD81 confirmed that the obtained particles were EVs. The detection of CD63 and CD9 and the suppression of nonspecific adsorption to IgG confirmed that the obtained particles were EVs.

[0078] Figure 15 shows a heat map of miRNA expression obtained by RNA sequencing for 17 miRNAs encapsulated in EVs extracted from the fiber sheet (liver surface (n = 5), peritoneal surface (n = 5)). Five miRNAs (mmu-miR-615-3p, mmu-miR-196b-5p, mmu-miR-196a-5p, mmu-miR-10b-3p, and mmu-miR-10b-5p) were highly expressed in EVs from the pelvic peritoneum compared to the liver surface. On the other hand, 12 miRNAs (mmu-miR-335-5p, mmu-miR-214-3p, mmu-miR-199a-5p, mmu-miR-106b-3p, mmu-miR-31-5p, mmu-miR-93-5p, mmu-miR-126a-5p, mmu-miR-126b-3p, mmu-miR-126a-3p, mmu-miR-126b-5p, mmu-miR-122-3p, and mmu-miR-802-5p) were highly expressed in EVs from the liver surface. Thus, it was found that the miRNAs contained in EVs on the surface differ depending on the organ or body part.

[0079] Samples were obtained from a fiber sheet attached to the surface of a tumor (Sheet-Tumor, Evsheet T1-T3), a fiber sheet attached to normal tissue (Sheet-Normal, Evsheet N1-N3), tumor tissue itself (Tissue-TumorTumor, Tissue T1-T3), and normal tissue itself (Tissue-Normal, Tissue N1-N3, obtained by absorbing a small amount of ascites from a normal mouse with a tissue sheet).

[0080] For body fluids captured with the fiber sheet, the fiber sheet was washed with PBS while the EVs were trapped, and then the EVs were disrupted. The RNA in the resulting solution was analyzed using an RNA sequencer (MiSEQ, Illumina).

[0081] Figure 16 shows a heat map of the 254 miRNAs obtained by the RNA sequencing. As can be seen, the miRNAs in EVs obtained using the EV sheet had a different miRNA profile from that in tissues. Furthermore, the ascites from tumor-bearing mice and those from non-tumor-bearing normal mice showed completely different miRNA profiles.

[0082] Principal component analysis (PCA) was performed on the detected miRNAs. As shown in Figure 17, the first principal component (PC1) is a variable representing "sheet" (-) and "tissue" (+), the second principal component (PC2) is a variable representing "cancer" (-) and "normal" (+), and the third principal component (PC3) is a variable representing "cancer" (-) and "normal" (+). Based on the miRNA obtained from the tissue, cancer tissue and normal tissue could be clearly distinguished (PC2). On the other hand, based on the miRNA obtained using the fiber sheet, cancer tissue and normal tissue could be even more clearly distinguished. As can be seen, the miRNA in EVs obtained using the EV sheet had a different miRNA profile from that of the tissue. Furthermore, the ascites from cancer-bearing mice and non-tumor-bearing normal mice exhibited completely different miRNA profiles.

[0083] Figure 18 shows heat maps of miRNA expression encapsulated in EVs obtained from the pelvic peritoneum and liver surface using a fiber sheet on days 0 and 4 (n = 5 for each). Thus, early cancer progression can be detected. Figure 19 shows PCA maps of miRNA expression in EVs obtained using a fiber sheet and in tissues. A change from day 0 to day 4, i.e., early cancer progression, was also observed on the PCA map. This change on the PCA map is thought to indicate a shift in cancer progression toward ascites on day 28. Thus, by attaching a fiber sheet, physiological EVs can be obtained from trace amounts of body fluid, allowing for analysis of the very early stages of cancer progression.

[0084] Example: EVs in ascites on the surface of human ovarian cancer tumors Using a method according to an embodiment of the present disclosure, a fiber sheet was attached to the surface of a human ovarian cancer tumor, the ascites was absorbed, and the EVs contained therein were isolated.

[0085] A fiber sheet was attached to the surface of a tumor removed from an ovarian cancer patient by laparotomy and allowed to absorb ascites (Surface). The fiber sheet was then immersed in ascites that naturally flowed from the removed tumor and allowed to absorb the ascites (Near). The EVs captured within each fiber sheet were isolated. The fiber sheet with the EVs captured was washed with PBS, and then the EVs were crushed. The miRNAs contained in each were analyzed using an RNA sequencer.

[0086] The EVs contained in the ascites had significantly different gene profiles (not shown). This indicates that there is tumor-derived ascites fluid that is different from the large amount of ascites fluid that has been commonly analyzed in the past. Furthermore, it was revealed that the EVs contained in the ascites fluid have different gene profiles.

[0087] Figure 20 shows the annotation rates for the sequencing results obtained from each sample. The annotation rate for miRNA obtained from the tissue surface was extremely high, at approximately 75%. On the other hand, the annotation rate for miRNA obtained from ascites that naturally flowed from the tissue was lower than that from the fiber surface, but was also extremely high, at approximately 34%. This is interpreted as being due to differences in annotation rate between samples rich in tumor-derived EVs and body fluids affected by various humoral factors, due to factors such as the inclusion of various nucleic acids.

[0088] Figure 21 shows a heat map of miRNA expression obtained by RNA sequencing for miRNAs from EVs collected using the fiber sheet and tumor tissue (n = 3, tumor surface-fiber sheet, tumor tissue ascites-fiber sheet, and cancer tissue). Differences in miRNA expression were observed between these samples. Figure 22 shows PCA maps of miRNAs obtained using the tumor surface-fiber sheet, ascites-fiber sheet, tumor tissue, serum-fiber sheet, and urine-fiber sheet (n = 3). This clustering analysis also revealed that tumor surface EVs have a unique miRNA profile compared to tumor tissue and ascites. The miRNA profile of tumor surface EVs was also found to be closer to that of tumor tissue than that of ascites EVs (not shown). These results suggest that tumor surface EVs are EVs released from tumors and that the fiber sheet was used to collect them.

[0089] Figures 23(A) and (B) show PCA maps of various data sets. Ascites EVs showed a distinct profile. Tumor tissue and tumor surface profiles were similar. Interestingly, EVs obtained by attaching a fiber sheet to the interior of a metastatic tumor (the exposed surface after opening the tumor) had a profile similar to that of the primary tumor tissue. On the other hand, the primary tumor tissue and metastatic tumor tissue had different profiles. Therefore, using a fiber sheet, this new approach can reveal previously unknown profiles of a patient's cancer. For example, based on the type, quantity, and profile of material (RNA) in EVs obtained with the fiber sheet, it is possible to infer the nature of the tissue (e.g., metastatic, primary, etc.), the type of cancer, and the stage of the cancer.

[0090] In some embodiments, the fiber sheet may be attached to a non-invasively accessible site (e.g., skin, mucous membrane) to collect biomolecules such as EVs. In some embodiments, the fiber sheet may be attached to an internal site invasively to collect biomolecules such as EVs. For example, the internal site may be accessed by a surgical procedure. Examples of surgical procedures include, but are not limited to, laparotomy and laparoscopic surgery. In a single surgery, the fiber sheet may be attached to multiple organs or sites to collect biomolecules such as EVs from each. The amount, type, profile, etc. of biomolecules (e.g., EVs) or substances contained therein (e.g., nucleic acids such as DNA and RNA, proteins, etc.) collected by the fiber sheet may be determined. Information useful for diagnosis may also be generated. For example, the type / subtype of disease, the stage / progression of disease, and whether the disease is localized or diffuse may be estimated, and based on this, predictions or strategies such as diagnosis, prognosis, and prevention may be proposed.

[0091] The present disclosure also includes the following embodiments: A001 A method for preserving a target substance in a target liquid, comprising: providing a fiber sheet; contacting the fiber sheet with the target liquid to allow the target liquid to be absorbed by the fiber sheet; drying the fiber sheet; and washing the fiber sheet. A001b A method for preserving a target substance in a target liquid, comprising: providing a fiber sheet; contacting the fiber sheet with the target liquid to allow the target liquid to be absorbed by the fiber sheet; and drying the fiber sheet. A002 A method for separating a target substance in a target liquid, comprising: providing a fiber sheet; contacting the fiber sheet with the target liquid to allow the target liquid to be absorbed by the fiber sheet; drying the fiber sheet; washing the fiber sheet; and adding an aqueous solution to the dried fiber sheet to release the captured target substance from the fiber sheet. A003 A method for separating a target substance in a target liquid, comprising: providing a fibrous sheet; contacting the fibrous sheet with a target liquid to absorb the target liquid into the fibrous sheet; drying the fibrous sheet; washing the fibrous sheet; and crushing the target substance captured in the fibrous sheet to obtain the internal substance. A011 The method of any one of embodiments A001 to A003, or any embodiment, wherein the fibrous sheet is substantially configured such that, when absorbing the target liquid, the target substance can enter the interior of the fibrous sheet, and the target substance is retained within the fibrous sheet during drying and washing the fibrous sheet. A021 The method of any one of embodiments A001 to A011, or any embodiment, wherein the target substance is extracellular vesicles (EVs). A022. The method of embodiment A021 or any embodiment, wherein the fibrous sheet has a pore size substantially between 40 nm and 500 nm.A023 The method of embodiment A022, or any embodiment, wherein the fibrous sheet has a pore size substantially between 40 nm and 200 nm. A024 The method of embodiment A022, or any embodiment, wherein the fibrous sheet has a pore size substantially between 200 nm and 500 nm. A025 The method of any one of embodiments A001 to A024, or any embodiment, wherein the substance of interest is a virus. A026 The method of embodiment A025, or any embodiment, wherein the fibrous sheet has a pore size substantially between 20 nm and 500 nm. A031 The method of any one of embodiments A001 to A026, or any embodiment, wherein the liquid of interest is a body fluid. A041 The method according to any one of embodiments A001 to A026, or any embodiment, wherein contacting the fiber sheet with the target liquid comprises contacting at least one surface of the fiber sheet with a target object containing the target liquid. A042 The method according to embodiment A041, or any embodiment, wherein the target object containing the target liquid is at least one selected from the group consisting of skin, mucous membrane, organ, and tumor. A043 The method according to embodiment A041 or A042, or any embodiment, wherein contacting the fiber sheet with the target liquid comprises attaching the fiber sheet to the surface of the target object and allowing the fiber sheet to absorb body fluid present on the surface of the target object. A051 The method according to any one of embodiments A001 to A043, or any embodiment, wherein drying the fiber sheet is performed at room temperature. A052 The method of any one of embodiments A001 to A051 or any embodiment, wherein drying the fibrous sheet is carried out in an air atmosphere or a vacuum atmosphere.A053 The method of any one of embodiments A001 to A052, or any embodiment, wherein drying the fibrous sheet comprises reducing the pore size. A061 The method of any one of embodiments A001 to A053, or any embodiment, wherein washing the fibrous sheet comprises washing the fibrous sheet so that the target substance is retained in the fibrous sheet and / or does not flow out.

[0092] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the invention. It is therefore intended that the present invention cover all such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for preserving a target substance in a body fluid, To provide a fiber sheet; The fiber sheet is attached to the surface of the target object, and the fiber sheet is allowed to absorb the bodily fluids present on the surface of the target object; Drying the fiber sheet; and Washing the aforementioned fiber sheet; Equipped with, The aforementioned target substance is an extracellular vesicle (EV) or a virus. The aforementioned object is at least one selected from the group consisting of tumors, organs, and mucous membranes. method.

2. A method for separating a target substance from a bodily fluid, To provide a fiber sheet; The fiber sheet is attached to the surface of the target object, and the fiber sheet is allowed to absorb the bodily fluids present on the surface of the target object; To dry the fiber sheet; Washing the aforementioned fiber sheet; and The process includes adding an aqueous solution to the dried fiber sheet to release the captured target substance from the fiber sheet, The aforementioned target substance is an extracellular vesicle (EV) or a virus. The aforementioned object is at least one selected from the group consisting of tumors, organs, and mucous membranes. method.

3. A method for separating a target substance from a bodily fluid, To provide a fiber sheet; The fiber sheet is attached to the surface of the target object, and the fiber sheet is allowed to absorb the bodily fluids present on the surface of the target object; To dry the fiber sheet; Washing the aforementioned fiber sheet; and The process includes crushing the target substance captured by the fiber sheet and obtaining the internal substance, The aforementioned target substance is an extracellular vesicle (EV) or a virus. The aforementioned object is at least one selected from the group consisting of tumors, organs, and mucous membranes. method.

4. A method according to any one of claims 1 to 3, The aforementioned fiber sheet is When absorbing the bodily fluid, the target substance can enter the interior of the fiber sheet. During the drying and washing of the fiber sheet, the target substance is retained inside the fiber sheet. A method that is essentially structured in such a way.

5. The method according to claim 4, The method wherein the fiber sheet substantially has a pore size between 40 nm and 500 nm.

6. The method according to claim 4, The method wherein the fiber sheet substantially has a pore size between 40 nm and 200 nm.

7. The method according to claim 4, The method wherein the fiber sheet substantially has a pore size between 200 nm and 500 nm.

8. A method according to any one of claims 1 to 3, Bringing the aforementioned fiber sheet into contact with the aforementioned bodily fluids is, A method comprising bringing at least one surface of the fiber sheet into contact with a target object containing the bodily fluid.

9. A method according to any one of claims 1 to 3, The method for drying the aforementioned fiber sheet is carried out at room temperature.

10. A method according to any one of claims 1 to 3, The method for drying the aforementioned fiber sheet is carried out in an air atmosphere or a vacuum atmosphere.

11. A method according to any one of claims 1 to 3, A method comprising drying the fiber sheet, wherein the pore size of the fiber sheet is reduced.

12. A method according to any one of claims 1 to 3, A method for cleaning the fiber sheet, comprising cleaning the fiber sheet such that the target substance is retained on the fiber sheet and / or flows out to the outside.