Device for classifying extracellular vesicles, and in vitro method for classifying extracellular vesicles and detecting or analyzing proteins contained in extracellular vesicles

Abstract

The present disclosure relates to a device for classifying extracellular vesicles. The present disclosure also relates to an in vitro method for classifying extracellular vesicles and detecting or analyzing proteins contained in extracellular vesicles.

Description

Apparatus for classifying extracellular vesicles and in vitro methods for classifying extracellular vesicles and detecting or analyzing proteins contained in extracellular vesicles

[0001] The present disclosure relates to an apparatus for sorting extracellular vesicles (more specifically, a micro / nanofluidic device) and an in vitro method for sorting extracellular vesicles and detecting or analyzing proteins contained in the extracellular vesicles.

[0002] Cells release extracellular vesicles (EVVs) to the outside of the cell. The released EVVs contain contents that correspond to the physiological state of the releasing cell. Therefore, analyzing EVVs can infer the physiological state of the releasing cell. For example, analyzing exosomes is expected to enable inference of whether cancer cells are included in their source. Non-Patent Document 1 discloses a method for isolating exosomes released from cancer cells and analyzing their proteins.

[0003] Non-Patent Document 2 discloses a method for separating exosome membrane and luminal fractions and analyzing each. Furthermore, Patent Documents 1 and 2 disclose the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling method. In the thio-NAD cycling method, when a substrate having an androsterone skeleton, such as androsterone 3-phosphate, is produced in the presence of thio-NAD, NADH, and 3α-hydroxysteroid dehydrogenase (3α-HSD), thio-NADH is produced with high sensitivity in accordance with the amount of the substrate. This technique is also used in Non-Patent Documents 1 and 2. Thio-NADH can be quantified by absorbance at 405 nm.

[0004] JP5265816BJP5500985B

[0005] Iha et al. , Analytical Biochemistry, 654: 114831, 2022 Tsurusawa et al. , Cancers, 14(16): 3887, 2022

[0006] The present disclosure provides an apparatus for classifying extracellular vesicles and an in vitro method for classifying extracellular vesicles and detecting or analyzing proteins contained in the extracellular vesicles. In the present disclosure, the extracellular vesicles are preferably exosomes, the apparatus is an apparatus for obtaining an exosome fraction by size-based classification, and the method may be an in vitro method for classifying exosomes and detecting or analyzing proteins contained in the exosomes. The present disclosure also provides a method for quickly and easily preparing an exosome fraction from even a small sample and analyzing membrane or luminal proteins contained in the exosomes, for example, by combining polymer precipitation, classification using a microfluidic device, and highly sensitive protein detection technology. In the method of the present disclosure, the step of obtaining an exosome fraction preferably essentially involves only separation based on particle size and a step of purifying exosomes by precipitation using a polymer precipitation method.

[0007] According to the present disclosure, for example, the following inventions are provided: (1) An in vitro method for analyzing an analytical sample containing exosomes (e.g., an in vitro method for detecting or quantifying proteins contained in the lumen of exosomes), comprising: preparing a biological sample obtained from a subject; subjecting extracellular vesicles in the biological sample to size classification in a micro / nanofluidic device in a classification device, thereby obtaining at least one analytical sample containing exosomes; and detecting or quantifying proteins in the lumen of exosomes contained in each of the obtained analytical samples. (2) The method according to (1) above, wherein the detection or quantification is the detection or quantification of a product from a substrate by an enzyme-labeled antibody bound to a protein present in the analytical sample and the enzyme, and the detection or quantification of the product is carried out by generating thioNADH and / or thioNADPH by an enzymatic cycling reaction in the presence of NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH), and detecting the presence or amount of the generated thioNADH and / or thioNADPH. Preferably, the detection or quantification of a product from a substrate by an enzyme-labeled antibody bound to a protein present in the analytical sample and the enzyme is carried out by an enzyme-linked immunosorbent assay (ELISA) and an enzymatic cycling method or an improved enzymatic cycling method. More preferably, the ELISA comprises a dephosphorylation reaction of a substrate containing a phosphorylated 3α-hydroxysteroid with an alkaline phosphatase-labeled antibody, and the enzymatic cycling method comprises reacting the substrate containing a 3α-hydroxysteroid with 3α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of thio-nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide (NADH) to produce thio-NADH. (3) The method according to (1) or (2), wherein the micro / nanochannel device comprises: a receiver for receiving a sample (first sample); a first channel through which the first sample flows; and a first inlet through which the first sample flows from the first channel;a first inlet connected to a second flow path, the first inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size; a second flow path for allowing the passage of extracellular vesicles having a particle size equal to or smaller than the first reference particle size; a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size; the second flow path being connected to a first reservoir 11, in which a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated; and a second sample containing extracellular vesicles that do not pass through the first inlet (i.e., the inlet to the second flow path) flows into the first flow path, the first flow path being connected to a third flow path having a diameter that allows the second sample to pass through without clogging; and the micro / nano channel device having a reservoir into which the third sample flows from the third flow path, and a second analysis sample containing extracellular vesicles having a particle size exceeding the first reference particle size is accumulated in the reservoir. (4) The method according to (1) or (2), wherein the micro / nano channel device comprises a first channel through which a first sample flows, and a first inlet through which the first sample flows from the first channel; the first inlet is connected to a second channel, and the first inlet is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, and the second channel is a channel through which extracellular vesicles having a particle size equal to or smaller than the first reference particle size pass; the second channel is connected to a first reservoir, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the first reservoir; the first channel is connected to a third channel through which a second sample containing extracellular vesicles that do not pass through the inlet of the second channel flows, and which has a diameter that allows the second sample to pass without clogging; and the micro / nano channel device further comprises a second inlet through which the second sample flows from the third channel; The second inlet is connected to a fourth flow path, the second inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a second reference particle size, and the fourth flow path being a flow path for passing extracellular vesicles having a particle size equal to or smaller than the second reference particle size. The fourth flow path is connected to a second reservoir, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size is accumulated in the second reservoir. The third flow path is connected to a fifth flow path into which a third sample containing extracellular vesicles that do not pass through the second inlet flows, and which has a diameter that allows the third sample to pass without clogging.the micro / nano channel device further comprises a reservoir (e.g., a waste liquid container) into which the third sample flows from the fifth channel, or a third inlet into which the third sample flows from the fifth channel; When the fifth flow path has a third inlet, the third inlet is connected to a sixth flow path, and the third inlet is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a third reference particle size, and the sixth flow path is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the third reference particle size, and the sixth flow path is connected to a third reservoir, and a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than the third reference particle size is accumulated in the third reservoir, and the fifth flow path is connected to a seventh flow path into which a fourth sample containing extracellular vesicles that do not pass through the inlet of the sixth flow path flows, and which has a diameter that allows the fourth sample to pass without clogging, and the seventh flow path may be connected to a reservoir (e.g., a waste liquid container) into which the fourth sample flows or to a further flow path, and the first reference particle size < the second reference particle size < the third reference particle size is satisfied, A method in which any one or more of a first analytical sample, a second analytical sample, and a third analytical sample are analyzed (in particular, proteins contained in the lumen of exosomes contained in any one or more of the analytical samples are detected or quantified). (5) The method according to (3) or (4) above, in which the micro / nano channel device is formed in or on a disk-shaped substrate. (6) The method according to any of (3) to (5) above, in which the micro / nano channel device rotates, and centrifugal force caused by the rotation drives the sample in the channel, causing the sample to flow through the channel and classifying extracellular vesicles in the sample. (7) The method according to any of (3) to (6) above, in which the micro / nano channel device vibrates, thereby preventing or eliminating clogging of the inlet. (8) The method according to any one of (4) to (7) above, wherein the first flow path has a bend at a portion where an inlet to the second flow path is arranged, and the first sample flowing through the first flow path flows toward the inlet to the second flow path, thereby accumulating a first analysis sample from the first sample, the first analysis sample containing extracellular vesicles having a particle size equal to or smaller than a first reference particle size, in a first reservoir;(9) The method according to any one of (1) to (8), wherein obtaining a sample containing a population of extracellular vesicles from an isolated biological sample comprises precipitating the extracellular vesicles by a polymer precipitation method. (10) The method according to (9) above, wherein obtaining a sample containing a population of extracellular vesicles from the isolated biological sample further comprises subjecting the sample to centrifugation and / or filtering prior to the polymer precipitation method, thereby removing cells and cell debris. (11) A device for classifying extracellular vesicles, comprising a micro / nano channel device, the micro / nano channel device comprising: a receiver for receiving a sample (first sample); a first channel into which the first sample from the receiver flows; and a first inlet into which the first sample flows from the first channel, the first inlet being connected to a second channel, the first inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size; the second channel being a channel for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size; the second channel being connected to a first reservoir, in which a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated; the first channel being connected to a third channel into which a second sample containing extracellular vesicles that do not pass through the inlet of the second channel flows, the third channel having a diameter that allows the second sample to pass without clogging; the micro / nano channel device a second inlet through which the second sample flows from the third flow path;the second inlet is connected to a fourth flow channel, the second inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a second reference particle size, and the fourth flow channel being a flow channel for passing extracellular vesicles having a particle size equal to or smaller than the second reference particle size; the fourth flow channel being connected to a second reservoir, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size being accumulated in the second reservoir; the third flow channel being connected to a fifth flow channel into which a third sample containing extracellular vesicles that do not pass through the second inlet flows, and having a diameter that allows the third sample to pass without clogging; and the micro / nano channel device further comprises a reservoir (e.g., a waste liquid container) into which the third sample flows from the fifth flow channel, or a third inlet into which the third sample flows from the fifth flow channel; A classification device in which, when the fifth flow path is equipped with a third inlet, the third inlet is connected to a sixth flow path, the third inlet is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a third reference particle size, and the sixth flow path is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the third reference particle size, the sixth flow path is connected to a third reservoir, and a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than the third reference particle size is accumulated in the third reservoir, the fifth flow path is connected to a seventh flow path into which a fourth sample containing extracellular vesicles that do not pass through the inlet of the sixth flow path flows, and which has a diameter that allows the fourth sample to pass without clogging, and the seventh flow path may be connected to a reservoir (e.g., a waste liquid container) into which the fourth sample flows or to a further flow path, and the first reference particle size < the second reference particle size < the third reference particle size. (12) The classification device according to (11) above, wherein the micro / nano channel device is formed in or on a disk-shaped substrate. (13) The classification device according to (12) above, comprising a rotating unit that rotates the micro / nano channel device, and the micro / nano channel device rotates, and centrifugal force caused by the rotation drives samples in the channels, thereby causing each sample to flow through the channel and classifying extracellular vesicles in each sample. (14) The classification device according to any of (11) to (13) above, comprising a vibration generating unit that vibrates the micro / nano channel device,(15) A classification device according to any one of (11) to (14), wherein the first flow path has a bend at a portion where an inlet to a second flow path is arranged, and a first sample flowing through the first flow path flows toward the inlet to the second flow path, thereby accumulating a first analysis sample from the first sample in the first reservoir, the first analysis sample containing extracellular vesicles having a particle size equal to or smaller than a first reference particle size, and the third flow path has a bend at a portion where an inlet to a fourth flow path is arranged, and a third sample flowing through the third flow path flows toward the inlet to the fourth flow path, thereby accumulating a second analysis sample from the third sample in the second reservoir, the second analysis sample containing extracellular vesicles having a particle size equal to or smaller than a second reference particle size, and A classification device, wherein the fifth flow path has a bend at the portion where the inlet to the sixth flow path is located, and the fifth sample flowing through the fifth flow path flows toward the inlet to the sixth flow path, thereby accumulating a third analysis sample from the fifth sample, the third analysis sample containing extracellular vesicles having a particle size equal to or smaller than a third reference particle size, in a third reservoir.

[0008] The present disclosure also provides a method for obtaining an exosome fraction, including precipitating exosomes by polymer precipitation and separating exosomes from extracellular vesicles based on size, and a method for detecting or quantifying proteins using the same. (21) An in vitro method for detecting or quantifying proteins contained in exosomes in a biological sample, comprising: precipitating extracellular vesicles in the biological sample by polymer precipitation and dispersing them in a solution to obtain a first solution containing the extracellular vesicles; applying the first solution to a micro / nanofluidic device and classifying the extracellular vesicles by size in the micro / nanofluidic device to obtain a second solution (analysis sample) containing exosomes. (22) The method described in (21) above, further comprising detecting or quantifying exosomal proteins in the second solution (analysis sample). (23) The method described in (22) above, wherein the detection or quantification is performed by enzyme-linked immunosorbent assay (ELISA) and enzyme cycling or an improved enzyme cycling method. (24) The method according to (23) above, wherein the ELISA comprises a dephosphorylation reaction of a substrate containing a phosphorylated 3α-hydroxysteroid with an alkaline phosphatase-labeled antibody, and the enzymatic cycling method comprises reacting the substrate containing a 3α-hydroxysteroid with 3α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of thio-nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide (NADH) to produce thio-NADH. (25) The method according to (21) to (24) above, wherein the micro / nanochannel device comprises: a receiver for receiving a sample (first sample), a first channel into which the first sample from the receiver flows, and a first inlet into which the first sample flows from the first channel;the first inlet is connected to a second flow path, the first inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, and the second flow path being a flow path for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size; the second flow path being connected to a first reservoir, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size being accumulated in the first reservoir; the first flow path being connected to a third flow path into which a second sample containing extracellular vesicles that do not pass through the inlet of the second flow path flows, the third flow path having a diameter that allows the second sample to pass without clogging; and the micro / nano channel device further comprises a second inlet into which the second sample flows from the third flow path; the second inlet is connected to a fourth flow channel, the second inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a second reference particle size, and the fourth flow channel being a flow channel for passing extracellular vesicles having a particle size equal to or smaller than the second reference particle size; the fourth flow channel being connected to a second reservoir, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size being accumulated in the second reservoir; the third flow channel being connected to a fifth flow channel into which a third sample containing extracellular vesicles that do not pass through the second inlet flows, and having a diameter that allows the third sample to pass without clogging; and the micro / nano channel device further comprises a reservoir (e.g., a waste liquid container) into which the third sample flows from the fifth flow channel, or a third inlet into which the third sample flows from the fifth flow channel; When the fifth flow path has a third inlet, the third inlet is connected to a sixth flow path, and the third inlet is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a third reference particle size, and the sixth flow path is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the third reference particle size, and the sixth flow path is connected to a third reservoir, and a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than the third reference particle size is accumulated in the third reservoir, and the fifth flow path is connected to a seventh flow path into which a fourth sample containing extracellular vesicles that do not pass through the inlet of the sixth flow path flows, and which has a diameter that allows the fourth sample to pass without clogging, and the seventh flow path may be connected to a reservoir (e.g., a waste liquid container) into which the fourth sample flows or to a further flow path, and the first reference particle size < the second reference particle size < the third reference particle size.(26) The method according to any one of (21) to (25) above, wherein the steps up to obtaining each analytical sample do not include ultracentrifugation and affinity purification. (27) The method according to any one of (21) to (25) above, wherein the steps up to obtaining each analytical sample include only size-based fractionation of extracellular vesicles (e.g., filtering with a filter such as a 0.22 μm pore size filter, ultrafiltration with an ultrafiltration filter such as an ultrafiltration filter having a molecular weight cutoff of 100 kDa, and size-based classification with a micro / nano channel device), centrifugation, solution exchange, and separation of extracellular vesicles by polymer precipitation. (28) The method according to any one of (21) to (25) above, wherein the steps up to obtaining the first solution (pretreatment steps) only include fractionation of extracellular vesicles based on size (e.g., filtering with a filter such as a 0.22 μm pore size filter, ultrafiltration with an ultrafiltration filter such as an ultrafiltration filter having a molecular weight cutoff of 100 kDa), centrifugation, solution exchange, and separation of extracellular vesicles by polymer precipitation.

[0009] (31) A classification device comprising a micro / nano channel device 600, the micro / nano channel device 600 comprising: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21. The first inlet 31 is connected to the second flow path 22, and the first inlet 31 is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, and the second flow path 22 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size. The second flow path 22 is connected to a first reservoir 11, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the reservoir 11. The first flow path 21 is connected to a third flow path 23, into which a second sample containing extracellular vesicles that do not pass through the first inlet 31 (i.e., the inlet 31 to the second flow path 22) flows, and which has a diameter that allows the second sample to pass through without clogging. The micro / nano channel device is a classification device that has a reservoir 12 into which a third sample flows from a third channel 23, and a second analysis sample containing extracellular vesicles having a particle size greater than a first reference particle size accumulates in the reservoir 12.(32) The method according to any one of (21) to (24) above, wherein the micro / nano channel device 600 comprises: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21; a micro / nano channel device including a reservoir 12 into which the third sample flows from the third channel 23, and the reservoir 12 receives the second sample, the micro / nano channel device including the third sample, the micro / nano channel device including the third sample, and ...(33) The method according to any one of (1) to (9) above, wherein the micro / nano channel device 600 comprises: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21; a micro / nano channel device including a reservoir 12 into which the third sample flows from the third channel 23, and the reservoir 12 receives the second sample, the micro / nano channel device including the third sample, the micro / nano channel device including the third sample, and ...

[0010] 1 shows an example of a micro / nano channel device according to the present disclosure. 2 shows a further example of a micro / nano channel device according to the present disclosure. 3 shows examples of first to third inlets in a micro / nano channel device according to the present disclosure. Arrows in the figure indicate the direction of sample flow. 4 shows an example of a disk-shaped substrate equipped with a plurality of micro / nano channel devices according to the present disclosure. 5 shows an example of a disk-shaped substrate equipped with a plurality of micro / nano channel devices according to the present disclosure. 6 shows an example of a classification device including a disk-shaped substrate equipped with a micro / nano channel device according to the present disclosure. 7 shows an example of a scheme for separating and analyzing extracellular vesicles according to the present disclosure. 8 shows a further example of a micro / nano channel device according to the present disclosure. 9 shows an example of a classification device including a plurality of stacked micro / nano channel devices according to the present disclosure.

[0011] <Definitions> In this specification, the singular does not exclude the plural. In this specification, the term "comprises" includes the term "consisting of." The term "comprises" allows for the inclusion of a third, unrecited component, but the term "consisting of" does not allow for the inclusion of a third, unrecited component.

[0012] As used herein, the term "subject" refers to mammals, rodents such as mice and rats, livestock animals such as pigs and cows, pets such as dogs and cats, and primates such as humans and monkeys. The subject is preferably a human. The subject may be a healthy individual, a subject at risk of developing a disease or pathological condition, or a subject with a disease or pathological condition.

[0013] As used herein, "extracellular vesicles" refer to vesicles secreted by cells. The membranes of extracellular vesicles are derived from the membranes of cells and therefore contain at least one or more components of the cell membrane (e.g., lipids and membrane proteins that make up the cell membrane). Examples of extracellular vesicles include apoptotic vesicles (1 μm to 5 μm in diameter), microvesicles (100 nm to 1000 nm in diameter), exosomes (50 nm to 150 nm in diameter), and exomeres (approximately 35 nm or less in diameter). In vivo, cells secrete extracellular vesicles extracellularly (particularly into body fluids), and under culture conditions, cells secrete extracellular vesicles extracellularly (particularly into culture medium). Extracellular vesicles are typically isolated from cell culture supernatants or tissue supernatants by affinity chromatography, size exclusion chromatography, polymer precipitation, ultracentrifugation, ultrafiltration, or a combination thereof. The properties of the isolated extracellular vesicles vary significantly depending on the isolation method. Furthermore, because cells secrete a variety of extracellular vesicles, it is not always easy to identify, concentrate, enrich, isolate, or purify useful extracellular vesicles with specific properties. Depending on the cell of origin, exosomes express one or more, typically all, proteins selected from the group consisting of Alix, Tsg101, tetraspanins (CD81, CD63, and CD9), heat shock proteins (HSP60, HSP70, HSC70, HSP90, etc.), and flotillin. Exosomes or their quantity can be confirmed, for example, by the expression of one or more tetraspanins. Exosomes or their quantity can also be confirmed by particle size. Microvesicles (MVs) are formed by outward budding of the cell membrane and have diameters of approximately 100 to 1,000 nm. MVs are known to express selectins, CD40, and the like. Apoptotic vesicles are vesicles produced by cells undergoing programmed cell death (apoptosis) and have diameters of 50 to 5,000 nm. Apoptotic vesicles are known to express annexin V, phosphatidylserine, and the like.

[0014] As used herein, "CD9" refers to a member of a protein family called tetraspanins, which has a transmembrane domain and is present on the cell membrane or exosome membrane. The standard sequence of human CD9 is registered with the National Center for Biotechnology Information (NCBI) as NCBI Reference Sequence: NP_001760. CD9 includes proteins that have a sequence corresponding to the sequence registered as NCBI Reference Sequence: NP_001760 and have the function of CD9. Examples of antibodies that bind to CD9 include, but are not limited to, clones HI9a, MZ3, M2 / 57, SN4 C3-3A2, Bu16, MEM-61, and MF1. Antibodies that bind to CD9 and have the heavy chain CDRs 1-3 and light chain CDRs 1-3 of any of these clones may also be useful anti-CD9 antibodies.

[0015] As used herein, "CD63" refers to a member of the tetraspanin protein family, which has a transmembrane domain and is present on the cell membrane or exosome membrane. The standard sequence of human CD63 is registered with the National Center for Biotechnology Information (NCBI) under GenBank Accession No. AAH13017.1. CD63 includes proteins that have a sequence corresponding to the sequence registered under GenBank Accession No. AAH13017.1 and have the function of CD63. Antibodies that bind to CD63 include, but are not limited to, clones MEM-259, Ts63, TEA3 / 18, CC25, REA1055, RFAC4, ME491, MX-49.129.5, H5C6, 3H6, and AD1. Antibodies that bind to CD63 and have the heavy chain CDRs 1-3 and light chain CDRs 1-3 of any of these clones may also be useful anti-CD63 antibodies.

[0016] As used herein, "CD81" refers to a member of the tetraspanin protein family, which has a transmembrane domain and is present on the cell membrane or exosome membrane. The standard sequence of human CD81 is registered with the National Center for Biotechnology Information (NCBI) under GenBank Accession No. AAH93047.1. CD81 includes proteins that have a sequence corresponding to the sequence registered under GenBank Accession No. AAH93047.1 and have the function of CD81. Antibodies that bind to CD81 include, but are not limited to, clones 1D6, 5A6, 2F7, Eat2, REA513, JS-81, M38, and A1504. Antibodies that bind to CD81 and have the heavy chain CDRs 1-3 and light chain CDRs 1-3 of any of these clones may also be useful anti-CD81 antibodies.

[0017] As used herein, "isolation" refers to the removal of at least one other component from the environment in which it was produced. Thus, isolation does not require purification to the point of containing only a single component, but allows other components to be present in the same composition. Isolation of extracellular vesicles only requires that they are separated from other components to a degree sufficient for analysis.

[0018] As used herein, the term "biological sample" refers to a sample (e.g., extracellular fluid and bodily fluid) obtained from a subject. Biological samples include blood, saliva, tears, urine, ascites, pleural fluid, and secretions (e.g., endocrine and exocrine fluids, e.g., digestive fluids such as pancreatic juice). As used herein, a biological sample is a liquid sample (also referred to as a "biological liquid sample") suitable for application to a micro / nanofluidic device. As used herein, a "blood sample" refers to blood (e.g., peripheral blood) or a blood-derived sample (e.g., serum and plasma) obtained from a subject.

[0019] As used herein, "antibody" refers to an immunoglobulin. Antibodies may be of various isotypes, such as IgG, IgE, IgM, IgA, or IgD. Antibodies may be polyclonal or monoclonal. Antibodies have a signal peptide when produced intracellularly, but the signal peptide is removed when secreted extracellularly. Antibodies include full-length antibodies and antigen-binding fragments thereof. When testing samples from a certain animal species, antibodies that bind to proteins of that animal species should be used. For example, when testing human samples, antibodies that bind to human proteins are used. Antibodies bind to antigens via complementarity-determining regions (CDRs). Antibodies comprise a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH1-CH3), and a light chain comprising a light chain variable region (VL) and a light chain constant region (CL). An antibody has three CDRs in its heavy chain variable region (i.e., heavy chain CDRs 1 to 3) and three CDRs in its light chain variable region (i.e., light chain CDRs 1 to 3). The portions of the variable region other than the CDRs are less variable between antibodies and are called framework regions.

[0020] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment of an antibody that maintains its ability to bind to an antigen. Examples of antigen-binding fragments include Fab, Fab', and F(ab'). ２ , Fv, scFv (single-chain Fv), diabody, sc(Fv) ２ (Single chain (Fv) ２ For example, when an antibody is digested with papain, Fab can be obtained. Alternatively, when an antibody is digested with pepsin, F(ab') can be obtained. ２ can be obtained, which can be further reduced to obtain Fab'. Antigen-binding fragments of other antibodies can also be produced by methods well known to those skilled in the art. Such antibody antigen-binding fragments can be used in the present invention.

[0021] As used herein, an "enzyme-labeled antibody" refers to an antibody used in an enzyme labeling method, which is labeled with a specific enzyme. The enzyme-labeled antibody binds to an immobilized antigen, and after the sample and unbound antibody are washed away, a substrate is reacted with the enzyme-labeled antibody remaining on the solid phase, and the presence or amount of the reaction product is used to estimate the amount of antigen in the sample.

[0022] As used herein, a "microchannel device" refers to a device having a channel through which a sample flows. In a microchannel device, the channel has a height and width on the order of micrometers and / or submicrometers (nanometers), allowing a sample containing extracellular vesicles having a particle size appropriate for the purpose of the channel to flow smoothly. As used herein, the term "microchannel device" encompasses a device (nanochannel device) having only a channel with a height and width on the order of submicrometers (nanometers), and may also be referred to as a micro / nanochannel device. In any aspect of the present disclosure, the micro / nanochannel device is preferably a nanochannel device.

[0023] As used herein, an "analytical sample" is a sample that can be subjected to analysis. If there are multiple analytical samples, not all of them need actually be analyzed, but at least one of them will be analyzed.

[0024] <Method of the present disclosure> The present disclosure provides an in vitro method for detecting or quantifying proteins contained in the lumen of extracellular vesicles. In a preferred embodiment, the extracellular vesicles comprise exosomes. Accordingly, the present disclosure also provides an in vitro method for detecting or quantifying proteins contained in the lumen of exosomes.

[0025] The method of the present disclosure includes obtaining an isolated biological sample. Extracellular vesicles are contained in the biological sample obtained from a subject. The isolated biological sample is pretreated to make it suitable for application to micro / nanofluidic devices. For example, solids that clog the channels are removed by centrifugation, filtering, ultrafiltration, or polymer precipitation. Centrifugation can be performed at a strength sufficient to prevent the precipitation of extracellular vesicles (e.g., 2000 × g for 30 minutes). Filtering can be performed, for example, using commercially available filters with pore sizes of 0.45 μm or 0.22 μm. Ultrafiltration can be performed, for example, using an ultrafiltration filter with a molecular weight cutoff of 100 kDa. Polymer precipitation is a method in which a hydrophilic polymer is added to a sample to remove water molecules from the surface of the extracellular vesicles, thereby reducing the solubility of the extracellular vesicles and thereby precipitating the extracellular vesicles. The hydrophilic polymer is not particularly limited, and various hydrophilic polymers, such as polyethylene glycol, can be used. The pretreatment may include one or more, two, three, or all of centrifugation, filtering, ultrafiltration, and polymer precipitation. The population containing extracellular vesicles obtained after pretreatment contains at least exosomes and may additionally contain apoptotic vesicles and microvesicles (MVs). After pretreatment, exosomes are not isolated from other extracellular vesicles. Extracellular vesicles have a particle size range of 50 nm to 1 μm, and methods for obtaining a sample containing such a population of extracellular vesicles can be appropriately performed by those skilled in the art.

[0026] The micro / nano channel device is suitable for analyzing minute amounts of samples. The volume of the biological sample applied to the micro / nano channel device is not particularly limited, but may be, for example, 1 μL to 500 μL, 10 μL to 100 μL, 5 μL to 50 μL, 1 μL to 10 μL, or 1 μL to 5 μL. Analysis of minute amounts of biological samples may be possible.

[0027] The method of the present disclosure further includes subjecting the extracellular vesicles in the pretreated biological sample to size classification in a micro / nanofluidic device, thereby obtaining at least one or more analytical samples containing exosomes.３ pcs / μL or more, 10 ４ pcs / μL or more, 10 ５ pcs / μL or more, 10 ６ 10 or more per μL ７ The sample for analysis may contain extracellular vesicles at a concentration of, for example, 10 ３ pieces / μL ~ 10 ６ pieces / μL, 10 ６ pieces / μL ~ 10 １０ pieces / μL, 10 ７ pieces / μL ~ 10 ９ pieces / μL, 10 ８ pieces / μL ~ 10 １０ pcs / μL, or 10 ９ pieces / μL ~ 10 １１ The amount of extracellular vesicles obtained can be adjusted appropriately depending on the protein to be measured.

[0028] Extracellular vesicles isolated by polymer precipitation generally contain many contaminants. However, for application to micro / nanofluidic devices, substances that cause clogging of the micro / nanofluidic devices are removed by pretreatment. Furthermore, extracellular vesicles in the sample are sorted by size in the micro / nanofluidic device. Therefore, the influence of contaminants on the application of biological samples to micro / nanofluidic devices can be minimized.

[0029] In a micro / nanofluidic device, size classification includes classification into one or more, two or more, three or more, or four or more groups, and one or more, two or more, three or more, or four or more analytical samples are obtained, respectively. According to the present disclosure, at least one or more, two or more, three or more, four or more, or all of the obtained analytical samples can be subjected to analysis.

[0030] Analysis can be performed on whole exosomes, or on the membrane and luminal fractions of exosomes after separating them. Separation can be achieved by disrupting exosomes to extract the luminal fraction and then solubilizing the membrane fraction after extraction. Exosome disruption can be achieved by mechanical treatment (e.g., freeze-thawing or sonication) or chemical treatment (e.g., detergent treatment). Furthermore, after extraction of the luminal fraction, the membrane fraction can be solubilized by detergent treatment under conditions suitable for membrane fraction solubilization. Separation of exosomes into membrane and luminal fractions can also be performed using, for example, a commercially available kit (e.g., Mem-PER Plus Membrane Protein Extraction kit (89842; Thermo Fisher Scientific)) (see Iha et al., Analytical Biochemistry, 654: 114831, 2022).

[0031] Analysis of proteins contained in exosomes, or analysis of proteins contained in the membrane fraction or lumen fraction of exosomes can be performed, for example, in the case of measuring 100 μL of sample per assay. －１５ moles / assay or greater, 10 －１６ moles / assay or greater, 10 －１７ moles / assay or more or 10 －１８ moles / assay or more (e.g., 10 －１５ moles / assay ∼10 －２１ moles / assay, e.g., 10 －１５ moles / assay ∼10 －２０ moles / assay, e.g., 10 －１５ moles / assay ∼10 －１９ moles / assay, also e.g., 10 －１５ moles / assay ∼10 －１８This can be carried out by a protein detection or quantification technique having a detection sensitivity of at least 1000 mol / assay. Such detection or quantification techniques include enzyme cycling methods, particularly modified enzyme cycling methods, combined with enzyme-linked immunosorbent assays (ELISAs) (see, for example, JP5265816B and JP5500985B). Analysis of proteins contained in exosomes or analysis of proteins contained in the membrane or lumen fractions of exosomes can be carried out particularly by the modified enzyme cycling method.

[0032] ELISA can be, for example, a sandwich ELISA. In sandwich ELISA, an immobilized antibody and an enzyme-labeled antibody can be used. A component such as a protein to be analyzed is contacted with a surface (suitably blocked) on which the antibody is immobilized, allowing it to bind to the surface. Unbound proteins are then washed away, and the protein on the surface is detected with the enzyme-labeled antibody. In the enzyme-labeled antibody method, an enzyme (e.g., alkaline phosphatase) in the enzyme-labeled antibody dephosphorylates a substrate to obtain a reaction product.

[0033] The problem with purifying extracellular vesicles using micro / nanofluidic devices is that they cannot process large amounts of samples. More specifically, this ELISA does not have sufficient detection sensitivity. However, the enzyme cycling method can detect 10 －１５ A detection sensitivity of 10 moles / assay can be obtained, and according to the improved enzyme cycling method (see, for example, JP5265816B and JP5500985B), －１８ A detection sensitivity of 10 moles / assay can be achieved (10 －２１ (Examples of detecting substances at the mole / assay level have also been found.) Therefore, in the present disclosure, the improved enzyme cycling method combined with enzyme-linked immunosorbent assay (ELISA) is preferably used for analyzing analytical samples.

[0034] An example of an improved enzyme cycling method is the thio-nicotinamide adenine dinucleotide (thio-NAD) cycling method. In the thio-NAD cycling method, 3α-hydroxysteroid dehydrogenase (3α-HSD) is used. In the presence of NNADH and thio-NAD, 3α-HSD catalyzes substrate cycling between 3α-hydroxysteroids and their corresponding 3-ketosteroids. Thio-NAD is reduced by the above reaction to form thio-NADH. Quantifying thio-NADH enables detection and quantification of 3α-hydroxysteroids. Thio-NADH has an absorbance at 405 nm. Therefore, the amount of thio-NADH can be measured by measuring the absorbance at 405 nm. 3α-hydroxysteroids can be produced from their phosphorylated forms using alkaline phosphatase. Using alkaline phosphatase as the enzyme and phosphorylated 3α-hydroxysteroids as the substrate in an enzyme-labeled antibody assay allows for the production of thio-NADH in response to the amount of antigen. In a preferred embodiment, the alkaline phosphatase substrate may be a substrate having an androsterone skeleton (wherein the androsterone skeleton has a hydroxy group at the 3α position and a carbonyl group at the 17 position), such as androsterone 3-phosphate, e.g., 17β-methoxy-5β-androstan-3α-ol 3-phosphate. Although not particularly limited, alkaline phosphatase can be attached with, for example, a maleimide group or an N-hydroxysuccinimide group (NHS group), and then linked to an amino group or a sulfhydryl group of an antibody via these groups. Other well-known and commonly used techniques, such as enzyme labeling utilizing binding between biotin and avidin, can be used to prepare enzyme-labeled antibodies.

[0035] The proteins to be detected or quantified may preferably include proteins of the tetraspanin family (e.g., one, two, or all selected from the group consisting of CD9, CD63, and CD81). Measuring these proteins can verify that the obtained extracellular vesicles contain exosomes. Other proteins to be detected or quantified include membrane proteins or luminal proteins contained in extracellular vesicles such as exosomes (see, for example, Iha et al., 2022, supra). The membrane proteins or luminal proteins may be disease markers. Disease markers include, but are not limited to, tumor markers and cranial nerve disease markers. The proteins to be detected or quantified may include one or more, preferably two or more, proteins other than exosome markers such as tetraspanin family proteins.

[0036] Proteins to be detected or quantified include, for example, amyloid beta. Amyloid beta (Aβ) is the main component of amyloid plaques found in the brains of Alzheimer's disease patients. Aβ is derived from the amyloid precursor protein and is produced by cleavage by beta-secretase and gamma-secretase. Aβ(1-40), Aβ(1-42), and the Aβ(1-42) / Aβ(1-40) ratio can be detected or quantified. Aβ(1-40) can be detected, for example, by antibodies that bind to Aβ(1-40), including, but not limited to, clone 2294, 5C3, BAM-10, BDI350, and Ab40.1, as well as antibodies having the heavy chain CDRs 1-3 and light chain CDRs 1-3 of these antibodies. Aβ(1-42) can be detected, for example, by an antibody that binds to Aβ(1-42), including, but not limited to, clones EPR9296, mOC64, 2C2G5, 12F4, and NR221P, as well as antibodies having the heavy chain CDRs 1-3 and light chain CDRs 1-3 of these antibodies. For example, these peptides may be quantified by an ELISA assay using a standard product. As markers for cranial nerve diseases, in addition to amyloid beta, tau protein and neurofilament light chain (NfL) are known as markers for Alzheimer's disease; α-synuclein, dopamine transporter, and NfL are known as markers for Parkinson's disease; and huntingtin and NfL are known as markers for Huntington's disease.Tumor markers are not particularly limited, but examples thereof include blood cancers such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, and gallbladder cancer. Examples of tumor markers include markers for solid cancers such as ovarian cancer, bile duct cancer, kidney cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, bladder cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, as well as markers for sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma. Tumor markers include, but are not limited to, CEA, AFP, hCG, CA-125, CA19-9, PSA, HER2 / neu, MUC1, MAGE, NY-ESO-1, WT1, PSMA, CTA, EGFR, BCL-2, mutant KRAS, cyclin D1, TERT, CD20, mutant BRAF, TAG-72, glial cell tumor tissue factor, CX3CR1, and mutant MET. Proteins that may be measured include MHC class I and / or MHC class II.

[0037] In one aspect, the series of steps from ELISA to the Thio-NAD cycling method may also be automated using CLOCK technology (see JP 2017-75807 A and Abe et al., Analytical Methods, 12: 4858-4866, 2020). CLOCK technology uses a micro / nanofluidic device (particularly a disk-shaped device) having a reservoir for introducing reagents, a reaction chamber, and a channel connecting the reservoir and the reaction chamber. The centrifugal force generated by rotating the micro / nanofluidic device is utilized to deliver the reagents to the reaction chamber in the appropriate order, allowing the reaction to occur. More specifically, the device has multiple channels with channels extending from the reservoir containing the reagents toward the reaction chambers, away from the center of rotation (starting point) during centrifugation. Each channel is configured to have a channel of the same or different length. If the channels have different lengths, the order in which the reagents are introduced into the reaction chambers is adjusted accordingly. The flow channel leading to the reaction chamber may include two U-shaped flow channels and a channel sandwiched between them (i.e., a channel extending in a direction counter to the centrifugal force), which can prevent leakage of reagents into the reaction chamber. This micro / nano channel device does not require drive members such as valves or pumps and can be easily manufactured by injection molding.

[0038] According to the present disclosure, there is provided an in vitro method for detecting or quantifying proteins contained in exosomes in a biological sample, the method comprising: precipitating extracellular vesicles in the biological sample by polymer precipitation and dispersing them in a solution to obtain a first solution containing the extracellular vesicles; applying the first solution to a micro / nanofluidic device and classifying the extracellular vesicles by size in the micro / nanofluidic device to obtain a second solution (analysis sample) containing exosomes. This method may further comprise detecting or quantifying exosomal proteins in the second solution (analysis sample). Detection or quantification can be performed by ELISA and enzyme cycling or modified enzyme cycling, as described above. This enables exosome analysis without the need for cumbersome and time-consuming steps such as ultracentrifugation during exosome preparation. Although the polymer precipitation method has been identified as having a problem of high levels of impurities, these can be removed by applying filtering (e.g., filtering using a 0.22 μm pore size filter and ultrafiltration using a filter with a 100 kDa molecular weight cutoff) and micro / nanofluidic devices. Micro / nanofluidic devices cannot process large volumes of sample, for example, only microliter-order (less than 1 mL) samples at best. However, enzyme cycling, particularly improved enzyme cycling, enables protein detection from minute samples. Thus, the present disclosure provides a simple and rapid method for obtaining and analyzing exosome fractions. The method of the present disclosure allows sample pretreatment and classification to be completed in a short period of time. The method of the present disclosure can complete all steps from a prepared biological sample to the detection and quantification of proteins in exosomes contained therein, for example, within one week, six days, five days, four days, three days, or two days, e.g., within two to four days, or within two, three, or four days. In the method of the present disclosure, for example, sample pretreatment and classification can be completed within one week, six days, five days, four days, three days, or two days, for example, within two to four days, or within one to three days, or within one, two, three, or four days.In the method of the present disclosure, the acquisition of a biological sample containing extracellular vesicles and polymer precipitation requires, for example, about 2 to 3 hours, and the classification using a micro / nanofluidic device requires, for example, about 20 to 30 minutes. Therefore, the time required to obtain an exosome fraction by classification is, for example, about 2 hours and 20 minutes to about 3.5 hours. The time required to further fractionate the exosome fraction into membrane and luminal components is, for example, about 2 to 3 hours. The enzyme cycling method requires, for example, about 3 to 5 hours. Therefore, the time required to detect proteins from a biological sample can be within 1 day, 18 hours, 15 hours, 12 hours, or 10 hours, for example, about 7 to 11 hours, for example, about 8 to 9 hours.

[0039] <Micro / nanochannel device of the present disclosure> A micro / nanochannel device can be used in the above-described method. The present disclosure provides a micro / nanochannel device suitable for use in the above-described method, and a device for sorting extracellular vesicles (particularly exosomes) that includes the micro / nanochannel device.

[0040] According to the present disclosure, a device for classifying extracellular vesicles (particularly exosomes) comprises a micro / nano channel device.

[0041] According to the present disclosure, the micro / nano channel device includes: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21; the first inlet 31 is connected to the second flow channel 22, the first inlet 31 being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, the second flow channel 22 being a flow channel for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size, the second flow channel 22 being connected to a first reservoir 11, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size being accumulated in the first reservoir 11, the first flow channel 21 being connected to a third flow channel 23 into which a second sample containing extracellular vesicles that do not pass through the first inlet 31 (i.e., the inlet 31 to the second flow channel 22) flows and which has a diameter that allows the second sample to pass through without clogging; the sample further includes a reservoir (e.g., a waste liquid container) 41 into which the third sample flows from the third flow path 23, or a second inlet 32 ​​into which the second sample flows from the third flow path 23; When the third flow path 23 is provided with the second inlet 32, the second inlet 32 ​​is connected to the fourth flow path 24, the second inlet 32 ​​is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding the second reference particle size, and the fourth flow path 24 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the second reference particle size, the fourth flow path 24 is connected to the second reservoir 12, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size is accumulated in the second reservoir 12, and the third flow path 23 is connected to a fifth flow path 25 into which a third sample containing extracellular vesicles that do not pass through the second inlet 32 ​​(i.e., the inlet 32 ​​to the fourth flow path 24) flows and which has a diameter that allows the third sample to pass without clogging, and the micro / nano flow path device comprises: the sample further includes a reservoir (e.g., a waste liquid container) 41 into which the third sample flows from the fifth flow path 25, or a third inlet 33 into which the third sample flows from the fifth flow path 25;When the fifth flow path 25 is equipped with a third inlet 33, the third inlet 33 is connected to the sixth flow path 26, and the third inlet 33 is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a third reference particle size, and the sixth flow path 26 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the third reference particle size. The sixth flow path 26 is connected to a third reservoir 13, and a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than the third reference particle size is accumulated in the third reservoir 13. The fifth flow path 25 is connected to a seventh flow path 27, into which a fourth sample containing extracellular vesicles that do not pass through the third inlet (i.e., the inlet 33 to the sixth flow path 26) flows, and which has a diameter that allows the fourth sample to pass without clogging. The seventh flow path may be connected to a reservoir (e.g., a waste liquid container) 41 into which the fourth sample flows or to a further flow path.

[0042] In one aspect, as shown in FIG. 8 , the micro / nano channel device 600 includes: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21. The first inlet 31 is connected to the second flow path 22, and the first inlet 31 is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size. The second flow path 22 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size. The second flow path 22 is connected to a first reservoir 11, and a first analysis sample (or waste liquid) containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the reservoir 11 (or waste liquid container 11). The first flow path 21 is connected to a third flow path 23, into which a second sample containing extracellular vesicles that do not pass through the first inlet 31 (i.e., the inlet 31 to the second flow path 22) flows and which has a diameter that allows the second sample to pass through without clogging. The micro / nano channel device includes a reservoir 12 into which a third sample flows from a third channel 23. A second analysis sample containing extracellular vesicles having a particle size exceeding a first reference particle size accumulates in the reservoir 12. In this case, the first reference particle size may be the lower limit of the exosome particle size, for example, about 30 nm to about 65 nm, about 40 nm to about 60 nm, about 45 nm to about 55 nm, or about 50 nm. The first sample is filtered, for example, with a 0.22 μm pore size filter. In this example, the reservoir 11 contains extracellular vesicles equal to or smaller than the first reference value, and the reservoir 12 contains an exosome fraction. Therefore, the second analysis sample (a sample containing extracellular vesicles equal to or larger than the first reference particle size) contained in the reservoir 12 can be subjected to analysis. The first analysis sample contained in the reservoir 11 may also be analyzed to monitor extracellular vesicles. The first analytical sample may not be analyzed if not necessary, and may, for example, be discarded.

[0043] In one aspect, as shown in FIG. 1 , the micro / nano channel device 100 includes: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21. The first inlet 31 is connected to the second flow path 22, and the first inlet 31 is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, and the second flow path 22 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size, and the second flow path 22 is connected to a first reservoir 11, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the first reservoir 11, and the first flow path 21 is connected to a third flow path 23 into which a second sample containing extracellular vesicles that do not pass through the first inlet 31 (i.e., the inlet 31 to the second flow path 22) flows, and which has a diameter that allows the second sample to pass without clogging, and the micro / nano channel device further comprises a second inlet 32 ​​into which the second sample flows from the third flow path 23, When the third flow path 23 is provided with the second inlet 32, the second inlet 32 ​​is connected to the fourth flow path 24, the second inlet 32 ​​being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding the second reference particle size, and the fourth flow path 24 being a flow path for passing extracellular vesicles having a particle size equal to or smaller than the second reference particle size, the fourth flow path 24 being connected to the second reservoir 12, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size is accumulated in the second reservoir 12, the third flow path 23 being connected to a fifth flow path 25 into which a third sample containing extracellular vesicles that do not pass through the second inlet 32 ​​(i.e., the inlet 32 ​​to the fourth flow path 24) flows and which has a diameter that allows the third sample to pass without clogging, and the micro / nano flow path device is provided with a reservoir (e.g., a waste liquid container) 41 into which the third sample flows from the fifth flow path 25.

[0044] In a preferred embodiment, the first standard particle size < the second standard particle size < the third standard particle size are satisfied.

[0045] With this configuration, in the micro / nano channel device (e.g., the micro / nano channel device 100) of the present disclosure, a first sample flows through the first channel 21 and flows toward the first inlet 31. Particles having a particle size equal to or smaller than a first reference particle size pass through the first inlet 31 and the second channel 22 to accumulate in the first reservoir 11 and become a first analysis sample. On the other hand, particles having a particle size exceeding the first reference particle size cannot pass through the first inlet 31, flow into the third channel 23, and flow toward the second inlet 32. Particles having a particle size equal to or smaller than a second reference particle size pass through the second inlet 32 ​​and the fourth channel 24 to accumulate in the second reservoir 12 and become a second analysis sample. Particles having a particle size exceeding the second reference value cannot pass through the second inlet 32, and flow into the fifth channel 25. In this way, the micro / nanofluidic device of the present disclosure classifies extracellular vesicles according to (or based on) size, and provides an analytical sample containing extracellular vesicles of different particle sizes. The fourth sample contained in reservoir 41 may be used for analysis or may be discarded if not needed.

[0046] In one aspect, as shown in FIG. 2 , the micro / nano channel device 200 includes: a receiver 10 for receiving a sample (first sample); a first channel 21 through which the first sample flows; and a first inlet 31 through which the first sample flows from the first channel 21. The first inlet 31 is connected to the second flow path 22, and the first inlet 31 is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, and the second flow path 22 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size, and the second flow path 22 is connected to a first reservoir 11, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the first reservoir 11, and the first flow path 21 is connected to a third flow path 23 into which a second sample containing extracellular vesicles that do not pass through the first inlet 31 (i.e., the inlet 31 to the second flow path 22) flows, and which has a diameter that allows the second sample to pass without clogging, and the micro / nano channel device further comprises a second inlet 32 ​​into which the second sample flows from the third flow path 23, When the third flow path 23 is provided with the second inlet 32, the second inlet 32 ​​is connected to the fourth flow path 24, the second inlet 32 ​​being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding the second reference particle size, and the fourth flow path 24 being a flow path for passing extracellular vesicles having a particle size equal to or smaller than the second reference particle size, the fourth flow path 24 being connected to the second reservoir 12, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size is accumulated in the second reservoir 12, and the third flow path 23 is connected to a fifth flow path 25 into which a third sample containing extracellular vesicles that do not pass through the second inlet 32 ​​(i.e., the inlet 32 ​​to the fourth flow path 24) flows, and which has a diameter that allows the third sample to pass without clogging; and the micro / nano channel device further comprises a third inlet 33 into which the third sample flows from the fifth flow path 25,The third inlet 33 is connected to the sixth flow path 26, and the third inlet 33 is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a third reference particle size. The sixth flow path 26 is a flow path for passing extracellular vesicles having a particle size equal to or smaller than the third reference particle size. The sixth flow path 26 is connected to a third reservoir 13, and a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than the third reference particle size is accumulated in the third reservoir 13. The fifth flow path 25 is connected to a seventh flow path 27, which receives a fourth sample containing extracellular vesicles that do not pass through the third inlet (i.e., the inlet 33 to the sixth flow path 26) and has a diameter that allows the fourth sample to pass without clogging. The seventh flow path has a reservoir (e.g., a waste liquid container) 41 into which the fourth sample flows.

[0047] In a preferred embodiment, the first standard particle size < the second standard particle size < the third standard particle size are satisfied.

[0048] With this configuration, in the micro / nano channel device (e.g., the micro / nano channel device 200) of the present disclosure, a first sample flows through the first channel 21 and flows toward the first inlet 31. Particles having a particle size equal to or smaller than a first reference particle size pass through the first inlet 31 and the second channel 22 to accumulate in the first reservoir 11 and become a first analysis sample. On the other hand, particles having a particle size exceeding the first reference particle size cannot pass through the first inlet 31, flow into the third channel 23, and flow toward the second inlet 32. Particles having a particle size equal to or smaller than a second reference particle size pass through the second inlet 32 ​​and the fourth channel 24 to accumulate in the second reservoir 12 and become a second analysis sample. Particles having a particle size exceeding the second reference particle size cannot pass through the second inlet 32, and flow into the fifth channel 25. Particles having a particle size equal to or smaller than the third reference particle size pass through the third inlet 33 and the sixth channel 26 and accumulate in the third reservoir 13, forming a third analysis sample. Particles having a particle size exceeding the third reference particle size cannot pass through the third inlet 33, flow into the seventh channel 27, and are introduced into a reservoir (e.g., a waste container) 41 into which the fourth sample flows. In this way, the micro / nanochannel device of the present disclosure classifies extracellular vesicles according to (or based on) size, providing an analysis sample containing extracellular vesicles of different particle sizes. The fourth sample contained in the reservoir 41 may be used for analysis or may be discarded if not needed.

[0049] In a preferred embodiment of the micro / nanofluidic device 100, the first standard particle size may be about 30 nm to about 65 nm, about 35 nm to about 60 nm, about 40 nm to about 60 nm, about 45 nm to about 55 nm, or about 50 nm. In a preferred embodiment, the second standard particle size may be about 120 nm to about 180 nm, about 130 nm to about 170 nm, about 140 nm to about 160 nm, or about 150 nm. This allows, for example, separation of exosomes and non-exosomes from extracellular vesicles. Samples containing extracellular vesicles or debris with a size equal to or smaller than the first standard particle size may be discarded or collected for analysis. Analysis may include, for example, analysis of cells, extracellular vesicles other than exosomes, or their debris. For example, the presence of cells, extracellular vesicles other than exosomes, or their debris may be utilized for quality control of exosomes and their isolation conditions. For example, if the amount of exosome fragments is below a standard value, the exosomes are evaluated as having been well isolated, and the exosome-containing fraction is collected. However, if the amount of exosome fragments is above or exceeds the standard value, the exosomes are evaluated as not having been well isolated, and the exosome-containing fraction is not analyzed, and the fraction can be discarded.

[0050] In a preferred embodiment of the micro / nanofluidic device 200, the first reference particle size is about 30 nm to about 65 nm, about 40 nm to about 60 nm, about 45 nm to about 55 nm, or about 50 nm. In a preferred embodiment, the third reference particle size may be about 120 nm to about 180 nm, about 130 nm to about 170 nm, about 140 nm to about 160 nm, or about 150 nm. This allows, for example, exosomes and non-exosomes to be separated from extracellular vesicles. The second reference particle size is between the first and third reference particle sizes and is suitable for further size classification and analysis of exosomes. The second reference particle size may be set arbitrarily and is not particularly limited, but may be about 70 nm to about 120 nm, about 80 nm to about 120 nm, about 90 nm to about 110 nm, or about 100 nm. Samples containing extracellular vesicles or debris with a particle size equal to or greater than the third standard particle size can be discarded or collected for analysis. Analysis can include, for example, analysis of cells, extracellular vesicles other than exosomes, or their fragments. For example, the presence of cells, extracellular vesicles, or their fragments can be used for quality control of exosomes and their isolation conditions. For example, if the amount of cellular debris is below a standard value, the exosomes can be considered to have been well isolated, and a fraction containing exosomes can be collected. However, if the amount of exosome fragments is equal to or greater than the standard value, the exosomes can be considered to have been poorly isolated, and the exosome-containing fraction can be discarded without analysis.

[0051] In one preferred embodiment, the first particle size reference is about 50 nm, the second particle size reference is about 100 nm, and the third particle size reference is about 150 nm.

[0052] Those skilled in the art will understand that if it is desired to further classify exosomes by size, a classification unit can be added to the flow channel, including an inlet and reservoir for this purpose and a flow channel connecting the inlet and reservoir. For example, the classification unit may have an nth inlet, a 2nth flow channel, and an nth reservoir, where n is a natural number, and the nth inlet and nth reservoir are connected by the 2nth flow channel. The nth inlet can be designed to satisfy the nth reference particle size < the nth + 1th reference particle size, and the 2nth flow channel can be designed to allow extracellular vesicles that have passed through the nth inlet to pass through. The nth reservoir can be connected to the 2nth flow channel, and the nth analysis sample can be accumulated in the nth reservoir. Adding such a classification unit to the flow channel enables even more detailed classification of extracellular vesicles. The method of the present invention can also be applied to extracellular vesicles other than exosomes. Therefore, according to the present invention, there is provided a method for separating extracellular vesicles, in which the first, second, and third standard particle sizes can be determined according to the particle size of the vesicles to be obtained, and can be appropriately determined by those skilled in the art. Examples of extracellular vesicles include apoptotic vesicles (1 μm to 5 μm in diameter), microvesicles (100 nm to 1000 nm in diameter), exosomes (50 nm to 150 nm in diameter), and exomeres (approximately 35 nm or less in diameter). By setting the upper and lower limits of each standard particle size, or by setting the upper and lower limits, it is possible to separate all of the extracellular vesicles, and by setting the standard particle size at a value intermediate between the lower limits, it is possible to separate vesicles within a specific particle size range.

[0053] Each reservoir is designed to allow for the removal of a sample for analysis. For example, the reservoir may have an opening that allows access to the sample for analysis with a microchip or needle. Alternatively, the opening may have a lid that can be opened or closed. The lid may be physically destroyed (e.g., the lid is a rupturable membrane) to allow the sample for analysis to be removed from the reservoir, or the lid may be opened and the sample for analysis to be removed from the reservoir.

[0054] In a preferred embodiment, the inlets 31, 32, and 33 have holes that act as structural obstacles to prevent the passage of extracellular vesicles having diameters exceeding the first, second, and third reference diameters, respectively. The second, third, and fourth channels have the same cross-sectional shape as the inlets 31, 32, and 33, or have larger cross-sectional shapes, respectively, thereby allowing extracellular vesicles having diameters equal to or smaller than the first, second, and third reference diameters, respectively, to accumulate in the reservoirs 11, 12, and 13, respectively.

[0055] In a preferred embodiment, the inlets 31, 32, and 33 are equipped with filtration filters that can prevent the passage of extracellular vesicles having particle sizes exceeding the first, second, and third reference particle sizes, respectively. A filtration filter having an appropriate pore size can be appropriately selected by a person skilled in the art. The second, third, and fourth flow paths have the same cross-sectional shape as the inlets 31, 32, and 33, respectively, or have a larger cross-sectional shape, thereby allowing extracellular vesicles having particle sizes equal to or smaller than the first, second, and third reference particle sizes, respectively, to accumulate in the reservoirs 11, 12, and 13, respectively.

[0056] In some embodiments, the holes can be rectangular or circular. In some embodiments, the cross section of the flow channel can also be rectangular or circular.

[0057] In the micro / nanochannel device of the present disclosure, as shown in Fig. 3, a sample flows through a channel 111 formed toward each inlet 112. Therefore, extracellular vesicles that pass through each inlet 112 pass through a channel 113 connected to a reservoir 114 and are accumulated in the reservoir 114. Furthermore, extracellular vesicles that do not pass through each inlet 112 flow without leakage into a channel 115. In the figure, the inlet 112 is formed as a channel that is bent at a substantially right angle, but this is not necessarily required, and it is sufficient that the channel and the inlet are formed so that the sample flowing through the channel 111 efficiently comes into contact with the inlet 112.

[0058] In one aspect, the biological sample loaded onto the receiver 10 moves through the flow channel by capillary action, and extracellular vesicles are separated. In another aspect, the biological sample loaded onto the receiver 10 moves through the flow channel by the pressure of the liquid supply, and extracellular vesicles are separated.

[0059] In some embodiments, the micro / nano channel device itself does not have any actuating parts such as pumps or valves. Therefore, the micro / nano channel device is easy to manufacture. For example, the micro / nano channel device can be fabricated by injection molding. The inner surfaces of the channels, filters, receivers, and reservoirs that come into contact with the liquid in the micro / nano channel device can be hydrophilic (e.g., can have a hydrophilic coating).

[0060] In one preferred embodiment, reservoirs 11, 12, and 13, and reservoir (e.g., waste container) 41 each have negative pressure, which draws the sample loaded into receiver 10 into the flow path, thereby achieving classification of extracellular vesicles.

[0061] In a preferred embodiment, the micro / nano channel device (e.g., micro / nano channel devices 100, 200, and 600) is provided on or within a disk-shaped substrate (see, for example, Figures 4 and 5), and the centrifugal force generated by rotation draws the sample loaded into the receiver 10 into the channel, thereby achieving classification of extracellular vesicles. In the figure, multiple micro / nano channel devices are mounted on one substrate, but this is not necessarily required; it is also acceptable to mount only one micro / nano channel device. However, from the perspective of improving processing capacity, it is preferable that multiple micro / nano channel devices be mounted on one substrate.

[0062] In a preferred embodiment, as shown in FIG. 9 , the substrates may be removably stacked. For example, multiple substrates may be stacked to allow classification of multiple analytes at once. In this case, the receivers of each micro / nanochannel device are configured to be accessible from the top layer of the substrate. For example, the receivers themselves may be located on or within the substrate, but it is preferable that the receivers or the inlets of the channels leading to the receivers are located on the top layer of the substrate. After classification, the stacked substrates are separated from each other, and the analytical samples contained in each reservoir are collected.

[0063] In a preferred embodiment, the micro / nano channel device is vibrated to prevent clogging. For example, the micro / nano channel device is configured so that vibration is applied to the inlet to prevent clogging of the inlet (e.g., the micro / nano channel device is placed on a vibration generator to apply vibration to the micro / nano channel device). Alternatively, the micro / nano channel device is configured so that vibration is applied to the channel to prevent clogging of the channel (e.g., the micro / nano channel device is placed on a vibration generator to apply vibration to the micro / nano channel device).

[0064] <Extracellular vesicle (particularly exosome) classification device according to the present disclosure> According to the present disclosure, a device for classifying extracellular vesicles (particularly exosomes) is provided. The device 500 for classifying extracellular vesicles (particularly exosomes) according to the present disclosure has a micro / nano channel device according to the present disclosure. Briefly, it is envisioned that the micro / nano channel device according to the present disclosure can be rotated and / or vibrated.

[0065] From the viewpoint of rotating the micro / nano channel device of the present disclosure, the micro / nano channel device of the present disclosure is preferably provided on or in a disk-shaped substrate 501. The substrate 501 may have any shape as long as it is rotatable. The substrate 501 may have a rotationally symmetric shape. The substrate may have a regular polygonal shape or a disk shape. The substrate 501 may rotate around its center of gravity as the rotation axis. The micro / nano channel device of the present disclosure may also be provided on the back surface of the substrate 501. In this case, the receiver 10 of the micro / nano channel device formed on the back surface is preferably configured to allow sample introduction from the front surface.

[0066] In order to process many biological samples simultaneously, a plurality of micro / nanochannel devices according to the present disclosure may be provided on a substrate (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more). For example, in the example of Figures 4 and 5, four micro / nanochannel devices according to the present disclosure are mounted on one substrate 501.

[0067] In a preferred embodiment, the extracellular vesicle (particularly exosome) classification device 500 has a vibration mechanism 504, which can apply vibration to the micro / nano channel device 501 of the present disclosure. Vibration may be applied to the micro / nano channel device of the present disclosure constantly, or may be applied at predetermined timings. Vibration of the micro / nano channel device of the present disclosure will be beneficial for preventing clogging of the sample in each channel and each inlet.

[0068] In a preferred embodiment, a device 500 for classifying extracellular vesicles (particularly exosomes) has a rotation mechanism 502, a support shaft 503, and a substrate 501, and the rotation mechanism 502 and the substrate 501 are connected (fixed) by the support shaft 503. This allows the substrate 501 to be rotated by driving the rotation mechanism 502, and therefore allows the micro / nano channel device formed on or within the substrate 501 to be rotated and centrifugal force to be applied thereto. The rotation can be performed at a speed appropriate for the sample to proceed through the channel.

[0069] In one preferred embodiment, the classification device 500 for extracellular vesicles (particularly exosomes) has a vibration mechanism 504, a rotation mechanism 502, a support shaft 503, and a substrate 501.

[0070] In this way, the extracellular vesicles can be classified by size, and the classified sample can then be subjected to analysis.

[0071] Explanation of symbols in the drawings 100: Example 1 of micro / nano channel device 200: Example 2 of micro / nano channel device 10: Sample inlet (receiver) 11: First reservoir 12: Second reservoir 13: Third reservoir 21: First channel 22: Second channel 23: Third channel 24: Fourth channel 25: Fifth channel 26: Sixth channel 27: Seventh channel 31: Inlet of second channel 32: Inlet of fourth channel 33: Inlet of sixth channel 41: Reservoir (e.g., waste liquid container) 111: nth channel 112: pth inlet 113: (n+1)th channel 114: pth reservoir 115: (n+2)th channel 300: Example of a disc-shaped substrate on which a plurality of Example 1 of micro / nano channel devices are mounted 400: Example of a disk-shaped substrate on which a plurality of micro / nano channel device example 2 is mounted 500: Example of a classification device 501: Disk-shaped substrate on which a micro / nano channel device is mounted 502: Motor 503: Support shaft connecting the disk-shaped substrate and the motor 504: Vibration generator 600: Example of a micro / nano channel device 3 700: Example of a classification device including stacked micro / nano channel devices

Claims

1. An in vitro method for detecting or quantifying proteins contained in the lumen of exosomes, comprising: preparing a biological sample obtained from a subject; subjecting extracellular vesicles in the biological sample to size-based classification within a micro / nanochannel device in a classification device, thereby obtaining at least one analysis sample containing exosomes; and detecting or quantifying the proteins in the lumen of exosomes contained in each obtained analysis sample.

2. The method according to claim 1, wherein the detection or quantification is a detection or quantification of a product from a substrate by an enzyme-labeled antibody bound to a protein present in the analysis sample, and the detection or quantification of the product is performed by generating thio-NADH and / or thio-NADPH by an enzyme cycling reaction in the presence of NADH and / or NADPH, thio-NAD and / or thio-NADP, and dehydrogenase (DH), and detecting the presence or measuring the amount of the generated thio-NADH and / or thio-NADPH.

3. The method according to claim 1 or 2, wherein the micro / nanochannel device comprises: a receiver for receiving a sample (first sample); a first channel through which the first sample flows; a first inlet through which the first sample flows into the first channel, the first inlet being an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size, the second channel being a channel for passing extracellular vesicles having a particle size equal to or smaller than the first reference particle size, the second channel being connected to a first reservoir 11, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the reservoir; the first channel is connected to a third channel having a diameter that allows a second sample containing extracellular vesicles that do not pass through the first inlet (i.e., the inlet to the second channel) to flow through without clogging; the micro / nanochannel device comprises a reservoir into which a third sample flows from the third channel, and a second analysis sample containing extracellular vesicles having a particle size exceeding the first reference particle size is accumulated in the reservoir.

4. The method according to claim 1 or 2, wherein the micro / nano-channel device comprises: a first channel through which a first sample flows; a first inlet through which the first sample flows into the first channel, the first inlet being connected to a second channel and being an inlet that prevents extracellular vesicles having a particle size exceeding a first reference particle size from passing through, the second channel being a channel for allowing extracellular vesicles having a particle size not exceeding the first reference particle size to pass through, the second channel being connected to a first reservoir, and a first analysis sample containing extracellular vesicles having a particle size not exceeding the first reference particle size being accumulated in the first reservoir; the first channel being connected to a third channel having a diameter that allows a second sample containing extracellular vesicles that do not pass through the inlet of the second channel to flow in and pass through without clogging; the micro / nano-channel device further comprising a second inlet through which the second sample flows from the third channel, the second inlet being connected to a fourth channel and being an inlet that prevents extracellular vesicles having a particle size exceeding a second reference particle size from passing through, the fourth channel being a channel for allowing extracellular vesicles having a particle size not exceeding the second reference particle size to pass through, the fourth channel being connected to a second reservoir, and a second analysis sample containing extracellular vesicles having a particle size not exceeding the second reference particle size being accumulated in the second reservoir; the third channel being connected to a fifth channel having a diameter that allows a third sample containing extracellular vesicles that do not pass through the second inlet to flow in and pass through without clogging; the micro / nano-channel device further comprising a reservoir into which the third sample flows from the fifth channel or a third inlet through which the third sample flows from the fifth channel.When the fifth flow path includes the third inlet, the third inlet is connected to the sixth flow path. The third inlet is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding the third reference particle size. The sixth flow path is a flow path for allowing extracellular vesicles having a particle size equal to or smaller than the third reference particle size to pass through. The sixth flow path is connected to the third reservoir. A third analysis sample containing extracellular vesicles having a particle size equal to or smaller than the third reference particle size is accumulated in the third reservoir. The fifth flow path is connected to a seventh flow path having a diameter that allows a fourth sample containing extracellular vesicles that do not pass through the inlet of the sixth flow path to flow through without clogging. The seventh flow path may be connected to a reservoir into which the fourth sample flows or a further flow path. A method, satisfying the condition that the first reference particle size < the second reference particle size < the third reference particle size, in which any one or more of the first analysis sample, the second analysis sample, and the third analysis sample are analyzed.

5. The method according to claim 3 or 4, wherein the micro / nanochannel device is formed within or on a disk-shaped substrate.

6. The method according to any one of claims 3 to 5, wherein the micro / nano channel device rotates, and the centrifugal force generated by the rotation drives the sample in the channel, whereby the sample flows through the channel and the extracellular vesicles in the sample are classified.

7. The method according to any one of claims 3 to 6, wherein the micro / nano channel device vibrates, thereby preventing or eliminating clogging at the inlet.

8. The method according to any one of claims 4 to 7, wherein the first channel has a bend at a portion where the inlet to the second channel is disposed, and the first sample flowing through the first channel flows toward the inlet to the second channel, whereby a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than a first reference particle size is accumulated in the first reservoir; the third channel has a bend at a portion where the inlet to the fourth channel is disposed, and the third sample flowing through the third channel flows toward the inlet to the fourth channel, whereby a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than a second reference particle size is accumulated in the second reservoir; the fifth channel has a bend at a portion where the inlet to the sixth channel is disposed, and the fifth sample flowing through the fifth channel flows toward the inlet to the sixth channel, whereby a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than a third reference particle size is accumulated in the third reservoir.

9. The method according to any one of claims 1 to 8, wherein obtaining a sample containing a population of extracellular vesicles from an isolated biological sample includes sedimenting the extracellular vesicles by a polymer precipitation method.

10. The method according to claim 9, wherein obtaining a sample containing a population of extracellular vesicles from an isolated biological sample further includes subjecting the sample to centrifugation and / or filtering before the polymer precipitation method, thereby removing cells and cell debris.

11. An extracellular vesicle classification device, comprising a micro / nano flow channel device, wherein the micro / nano flow channel device includes a receiver for receiving a sample (a first sample), a first flow channel into which the first sample from the receiver flows, and a first inlet through which the first sample flows from the first flow channel. The first inlet is connected to a second flow channel and is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a first reference particle size. The second flow channel is a flow channel for allowing extracellular vesicles having a particle size equal to or smaller than the first reference particle size to pass through. The second flow channel is connected to a first reservoir, and a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than the first reference particle size is accumulated in the first reservoir. The first flow channel is connected to a third flow channel having a diameter that allows a second sample containing extracellular vesicles that do not pass through the inlet of the second flow channel to flow in and pass through without clogging. The micro / nano flow channel device further includes a second inlet through which the second sample flows from the third flow channel. The second inlet is connected to a fourth flow channel and is an inlet that prevents the passage of extracellular vesicles having a particle size exceeding a second reference particle size. The fourth flow channel is a flow channel for allowing extracellular vesicles having a particle size equal to or smaller than the second reference particle size to pass through. The fourth flow channel is connected to a second reservoir, and a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than the second reference particle size is accumulated in the second reservoir. The third flow channel is connected to a fifth flow channel having a diameter that allows a third sample containing extracellular vesicles that do not pass through the second inlet to flow in and pass through without clogging. The micro / nano flow channel device further includes a reservoir into which the third sample flows from the fifth flow channel or a third inlet through which the third sample flows from the fifth flow channel.When the fifth flow path includes a third inlet, the third inlet is connected to a sixth flow path. The third inlet is an inlet that prevents extracellular vesicles having a particle size exceeding a third reference particle size from passing through. The sixth flow path is a flow path for allowing extracellular vesicles having a particle size equal to or less than the third reference particle size to pass through. The sixth flow path is connected to a third reservoir. A third sample for analysis containing extracellular vesicles having a particle size equal to or less than the third reference particle size is accumulated in the third reservoir. The fifth flow path is connected to a seventh flow path having a diameter that allows a fourth sample containing extracellular vesicles that do not pass through the inlet of the sixth flow path to flow through without clogging. The seventh flow path may be connected to a reservoir into which the fourth sample flows or a further flow path. A classification device satisfying the condition: first reference particle size < second reference particle size < third reference particle size.

12. The classification device according to claim 11, wherein the micro / nano channel device is formed within or on a disk-shaped substrate.

13. The classification device according to claim 12, comprising a rotating part for rotating the micro / nano channel device, wherein the micro / nano channel device rotates, and the centrifugal force generated by the rotation drives the sample in the channel, whereby each sample flows through the channel and the extracellular vesicles in each sample are classified.

14. The classification device according to any one of claims 11 to 13, comprising a vibration generating part for vibrating the micro / nano channel device, wherein the micro / nano channel device vibrates, thereby preventing blockage of the inlet.

15. The classification device according to any one of claims 11 to 14, wherein the first channel has a bend at a portion where the inlet to the second channel is arranged, and the first sample flowing through the first channel flows toward the inlet to the second channel, whereby a first analysis sample containing extracellular vesicles having a particle size equal to or smaller than a first reference particle size is accumulated in the first reservoir; the third channel has a bend at a portion where the inlet to the fourth channel is arranged, and the third sample flowing through the third channel flows toward the inlet to the fourth channel, whereby a second analysis sample containing extracellular vesicles having a particle size equal to or smaller than a second reference particle size is accumulated in the second reservoir; the fifth channel has a bend at a portion where the inlet to the sixth channel is arranged, and the fifth sample flowing through the fifth channel flows toward the inlet to the sixth channel, whereby a third analysis sample containing extracellular vesicles having a particle size equal to or smaller than a third reference particle size is accumulated in the third reservoir.

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