KR20210068812A - Apparatus and method for preparing a liquid biopsy sample with an exosome subpopulation or the lower - Google Patents

Abstract

The present invention relates to an apparatus and a method for preparing a liquid biopsy sample with an exosome subpopulation. The apparatus for preparing a liquid biopsy sample with an exosome subpopulation according to an embodiment of the present invention comprises: an immobilization member (10) in a solid state; a first capture material (20) which specifically binds to a first isolation marker (m1) of a predetermined disease-associated exosome (1a) among exosomes (1) secreted from a plurality of cells, so as to capture the disease-associated exosome (1a); a first reversible linker (30) which detachably couples the immobilization member (10) and the first capture material (20); and a second capture material (40) which binds to the immobilization member (10) that is not coupled with the first capture material (20) and specifically binds to a second separation marker (m2) of a target exosome (1b) in an exosome subpopulation (S), which is a population of the disease-associated exosome (1a), so as to capture the target exosome (1b).

Description

Exosome subpopulation liquid biopsy sample manufacturing apparatus and manufacturing method {APPARATUS AND METHOD FOR PREPARING A LIQUID BIOPSY SAMPLE WITH AN EXOSOME SUBPOPULATION OR THE LOWER}

The present invention relates to an apparatus and method for preparing a liquid biopsy sample composed of a subpopulation of exosomes related to a specific disease, such as cancer.

Recently, the concept of 'liquid biopsy' to minimize the pain of screening for specific diseases such as cancer and degenerative diseases and to implement early diagnosis was introduced. Liquid biopsy is a non-invasive method that extracts bodily fluids such as blood, saliva, and urine relatively easily and analyzes specific disease-related cells or components (peptides, proteins, nucleic acids, organelles, specific substances, etc.) way. Since it is possible to confirm the occurrence of a specific disease at an early stage through this, it is attracting attention as an alternative to the existing test methods such as imaging, biopsy, and blood test.

When a liquid biopsy is used to diagnose a specific disease, for example, a cancer disease, compared to a tissue biopsy, there are generally advantages as follows. First, the use of body fluids in liquid biopsy is relatively easy and the risk of complications is reduced, compared to the usual invasive surgical tissue harvesting, which is potentially risky and expensive. Second, while tissue biopsy only provides a snapshot of the disease at a single site when necessary and cannot monitor disease progression and treatment every moment, liquid biopsy enables real-time continuous monitoring of cancer disease progression. Do. Third, through tissue biopsy, it is difficult to fully understand the composition of tumors due to the diversity of tumors. However, in liquid biopsy, biomaterials generated from tumor cells are collected, enabling a more comprehensive analysis of the composition of tumors at the molecular level. Do. Fourth, in tissue biopsy, the sample size is limited and it is insufficient when divided for various diagnostic purposes. However, in liquid biopsy, multiple samples can be collected from various body fluids as needed. Fifth, compared to the limited clinical usefulness of test results due to low accuracy in tissue biopsy, liquid biopsy provides relatively higher accuracy and sensitivity, so it can provide reliable results for clinical decision-making.

Exosomes are attracting attention as liquid biopsy biomarkers. Exosomes (30 ~ 120 nm in size) that are secreted in the form of microvesicles from cells in the human body and reflect the characteristics of the derived cells as they are are called 'avatars' of cells, and can be extracted from various body fluids such as blood, saliva, and urine. Do. Because exosomes are endoplasmic reticulum with a lipid bilayer, they are not only stable in the body, but also free from attack by protease, RNase, and DNase. Therefore, research to use exosomes as new biomarkers for diagnosing specific diseases such as cancer and degenerative diseases is increasing, and in fact, early diagnosis and companion diagnosis of cancer using components such as proteins and RNA contained in exosomes as markers. Purpose-built products are being developed.

Exosome-containing protein and RNA biomarkers that can be used for diagnosis during liquid biopsy for specific diseases, particularly cancer diseases, are as follows. First of all, looking at exosome biosynthesis and processes, first, micro-vesicles (MVEs) such as exosomes flip peaks inside the cell, at which time intracytoplasmic proteins, mRNAs, and microRNAs (miRNAs) containing Forms small intracellular vesicles. Second, these internal vesicles are released to the outside as exosomes as MVEs fuse with the cell membrane. Third, the released exosomes begin migrating toward a destination determined by the attachment of specific ligands on their surface. Fourth, exosomes reaching the target cell are introduced through the endocytosis pathway or are fused with the target cell membrane to release the exosome components directly into the cytoplasm.

Protein components involved in exosome biosynthesis in the first step include hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), signal transducing adapter melecule (STAM1), tumor susceptability gene (TSG101), and CD81. As proteins involved in the exosome release step of the second step, RAB2B, RAB5A, RAB7, and RAB9A are known. Such protein molecules are not only potential therapeutic candidates in the development of therapeutic agents, but also can be used as main targets in the development of detection and isolation technologies for exosomes.

In addition, exosomes released into body fluids contain various cellular components such as double-helical DNAs, RNAs, proteins, and lipid components. According to recent studies, exosome-loaded DNA molecules represent the entire genome and may also reflect the mutational status of tumor cells that have released exosomes. Therefore, analysis of double-helix DNA molecules in exosomes can be particularly useful for early detection of cancer diseases or metastases. In the case of exosome-containing proteins, the composition changes depending on the cell or tissue released, but most exosomes contain a general aggregate of evolutionarily conserved protein molecules. Protein content in exosomes is important for the development of techniques for isolation and detection of exosomes and analysis methods. Furthermore, exosome protein molecules can be used as biomarkers in information gathering and diagnosis of other pathological conditions. In the case of exosome-containing RNA, the difference in the contents of cell-derived and exosome-derived mRNA and miRNA, and the functionality of exosome-containing mRNAs were reported, and the results of studies on several miRNAs published simultaneously were sparked an explosion of research. In fact, miRNAs are attracting attention as special targets in the development of therapeutics as well as diagnostics. In many reports using the new detection technology, miRNAs are present in almost all body fluids and thus are emerging as potential biomarkers for diagnostic analysis. Indeed, according to the results of analysis of various body fluid samples, a large number of different miRNAs have been found depending on the source of the sample.

In particular, as a key issue related to cancer, exosomes are known to play a role in tumorigenesis, cancer metastasis, and drug resistance. In the microenvironment where tumors are generated, tumor cells, like other cells, also secrete exosomes and other microvesicles, which contribute to tumor cell migration during tumor progression and metastasis by promoting angiogenesis. Tumor cell-derived ERs also contain molecules involved in immunosuppression, which may inactivate T lymphocytes or natural killer cells, or promote differentiation of regulatory T lymphocytes or myeloid cells to suppress the immune response. can In addition, metastasis of tumor activity by exosomes secreted from tumor cells has also been reported.

However, since exosomes derived from all cells exist in a mixed form in the body fluid, there is a limitation in accurately diagnosing specific diseases such as cancer with exosome samples in the form of a bulk population simply isolated from body fluid. . Moreover, in the early stage of disease onset, the concentration of exosomes derived from specific disease-related cells in body fluids is relatively very low, so for early diagnosis, separation and concentration of target exosomes from body fluids must be preceded. Among the existing exosome separation technologies, the density difference-based ultracentrifugation method is adopted as the gold standard separation process due to its high reproducibility and relatively stable process. However, there are disadvantages in that expensive equipment is absolutely necessary and the separation time is long. In order to compensate for these drawbacks, a method for separating exosomes by precipitating them, using size exclusion chromatography, or using microfluidics as disclosed in the patent literature of the following prior literature, has been developed.

However, since these existing exosome isolation techniques provide all the exosomes present in the body fluid in the form of a single population, there is a limitation in selectively detecting trace amounts of exosomes derived from specific cells such as cancer. In fact, biochemical components (membrane protein, internally mounted protein, RNA, etc.) are variously included in the exosome membrane or the inside of the exosomes released from the cell, so they are very heterogeneous in composition. In particular, it has been reported that certain exosomes among exosomes derived from cancer cells contain mRNA or miRNA involved in the creation of a local environment at the outpost stage of angiogenesis and cancer metastasis. Moreover, as described above, during cancer metastasis, the specific exosomes are known to contain biochemical components involved in inducing the death of immune cells or transforming normal cells into cancer cells.

When diagnosing a specific disease such as cancer, it is very difficult to obtain high diagnostic accuracy when a bulk population including all exosomes present in body fluid is used as a liquid biopsy sample. This is because it causes negative results on assay specificity and sensitivity due to sample matrix effects such as non-specific adhesion caused by non-specific exosomes. In order to overcome this analytical problem, multiple biomarkers (eg, a plurality of different miRNA species) that have low specificity but are known to be increased in body fluids of patients with specific diseases can be simultaneously measured and used to determine whether or not a disease occurs. However, the synergistic effect by the use of multiple biomarkers is bound to be limited, because the disease specificity of most exosome markers not specifically related to disease is fundamentally low.

Accordingly, there is an urgent need for a method to solve the problem of not being able to selectively isolate exosomes associated with the occurrence of a specific disease, such as cancer, and use it for diagnosis of a specific disease.

US 10-1807256 B1

The present invention is to solve the problems of the prior art described above, and one aspect of the present invention is to identify a specific disease-related target exosome subpopulation in body fluids when diagnosing specific diseases such as cancer, degenerative diseases, heart diseases, and infectious diseases. An object of the present invention is to provide a sequential multiplex immunoaffinity separation technique using mild conditions so that it can be isolated and recovered in yield and in its original state and provided as a liquid biopsy sample.

Exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention is a solid state fixing member; a first capture material that specifically binds to a first separation marker of a predetermined disease-related exosome among exosomes secreted from a plurality of cells to capture the disease-related exosome; a first reversible linker separably coupling the fixing member and the first capture material; And the first capture material is coupled to the non-bonded fixing member, and specifically binds to a second separation marker of the target exosome in the exosome subpopulation, which is a group of disease-related exosomes, to form the target exosome. and a second capture material to capture.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, a second reversible linker for separably coupling the fixing member to which the first capture material is not bound and the second capture material; may further include.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, at least one of the first reversible linker and the second reversible linker is a ligand; a binder that is polymerized with the capture material to form a polymer, the ligand is detachably attached, and the structure is changed by an attachment reaction with the ligand; and a recognition material that is fixed to the surface of the fixing member and specifically binds to the binder whose structure is changed, and is separated from the binder when the ligand is detached from the binder.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, at least one of the first reversible linker and the second reversible linker is a ligand; a binder fixed to the surface of the fixing member, to which the ligand is detachably attached, and whose structure is changed by an attachment reaction with the ligand; and a recognition material that is polymerized with the capture material to form a polymer and specifically binds to the binder whose structure is changed, but is separated from the binder when the ligand is desorbed from the binder.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, the ligand is any selected from the group consisting of sugar molecules, ions, substrates, antigens, peptides, vitamins, growth factors, and hormones. It may include more than one.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, the binder is selected from the group consisting of a sugar-binding protein, an ion-binding protein, an enzyme, an antibody, an aptamer, a cell receptor, and a nanostructure. It may include any one or more selected.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, the recognition material is an antibody, a protein receptor, a cell receptor, an aptamer, an enzyme, a nanoparticle, a nanostructure, and a heavy metal chelator ( chelator) may include any one or more selected from the group consisting of.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, at least one of the first reversible linker and the second reversible linker is a ligand that polymerizes with the capture material to form a polymer ; and a binder fixed to the surface of the fixing member, coupled to the ligand, and separated from the ligand by a competition reaction with the competing ligand.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, at least one of the first reversible linker and the second reversible linker may include a ligand fixed to the surface of the fixing member; It may include; a binder that is polymerized with the capture material to form a polymer, is bound to the ligand, and is separated from the ligand by a competitive reaction with the competing ligand.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, the binder is a divalent metal ion, avidin, sugar-binding protein, ion-binding protein, enzyme, antibody, aptamer, protein receptor , cell receptors, and any one or more selected from the group consisting of nanostructures, wherein the ligand is, His-tag (polyhistidine-tag), vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides , nucleic acids, growth factors, and any one or more selected from the group consisting of hormones, wherein the competitive ligand is imidazole, vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, It may include any one or more selected from the group consisting of growth factors, and hormones.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, the fixing member is a hydrogel, a magnetic bead, a latex bead, a glass bead, a nanometal structure, a porous membrane, and a non-porous membrane It may include any one or more selected from the group consisting of.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, a reactor for accommodating the fixing member therein; may further include.

In addition, in the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention, the fixing member coupled to the disease-related exosome or the target exosome may be at least one of magnetic force, gravity, and centrifugal force. can be concentrated by

On the other hand, the exosome subpopulation liquid biopsy sample preparation method according to an embodiment of the present invention is (a) a first separation marker specifically binding to a predetermined disease-related exosome among exosomes secreted from a plurality of cells. 1 binding the capture material to a fixing member in a solid state using a first reversible linker; (b) reacting the sample solution containing the population of exosomes with the first capture material bound to the fixing member to capture the disease-related exosomes, thereby forming the disease-related exosome population separating the population; (c) recovering the exosome subpopulation by dissociating the first reversible linker so that the captured disease-related exosomes are separated from the fixing member; (d) binding a second capture material that specifically binds to a second separation marker of the target exosome in the exosome subpopulation to the fixing member; And (e) by reacting the exosome subpopulation solution containing the recovered exosome subpopulation and the second capture material bound to the fixing member to capture the target exosome, the target exosome population Including; isolating the exosome subpopulation.

In addition, in the exosome subpopulation liquid biopsy sample preparation method according to an embodiment of the present invention, in step (d), the second capture material may be coupled to the fixing member by a second reversible linker.

In addition, in the exosome subpopulation liquid biopsy sample preparation method according to an embodiment of the present invention, the second reversible linker is dissociated so that the captured target exosome is separated from the fixing member to form the exosome subpopulation. The step of recovering; may further include.

In addition, in the exosome subpopulation liquid biopsy sample preparation method according to an embodiment of the present invention, between the step (b) and the step (c), using any one or more of magnetic force, gravity, and centrifugal force, the Concentrating the fixing member in which the disease-related exosomes are captured; may further include.

In addition, in the exosome subpopulation liquid biopsy sample preparation method according to an embodiment of the present invention, after step (e), using any one or more of magnetic force, gravity, and centrifugal force, the target exosome is captured Concentrating the fixing member; may further include.

The features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

Prior to this, the terms or words used in the present specification and claims should not be construed in a conventional and dictionary meaning, and the inventor may properly define the concept of the term to describe his invention in the best way. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

According to the present invention, although the target disease association is relatively low, the specificity with high association with the target disease is high through the separation of the exosome subgroup for the exosome marker (primary segregation marker) of a comprehensive upper group including disease-related exosomes. Exosomes can be concentrated and recovered to the extent that they can be separated later, and biomarkers involved in the spread of target diseases are contained through the separation of exosome subpopulations for exosome-specific markers (secondary separation markers) in the primary separation solution. The exosomes can be provided as a liquid biopsy sample in a concentrated form.

Through analysis of biomarkers eluted by dissolving exosomes in liquid biopsy samples separated and concentrated in the form of subgroups, early diagnosis and diagnosis of specific diseases such as cancer, degenerative disease, heart disease, and infectious disease It can significantly increase the accuracy.

1 is a schematic diagram comparing a biomarker analysis process using a conventional liquid biopsy sample (left) and a biomarker analysis process using a liquid biopsy sample prepared according to the present invention (right).
Figure 2 is a configuration diagram schematically showing an exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention.
3 to 6 are schematic diagrams illustrating the first reversible linker and the second reversible linker shown in FIG. 2 according to various embodiments.
7 to 11 are flowcharts of a method for preparing a subpopulation exosome liquid biopsy sample according to an embodiment of the present invention.
12 is a flowchart of a method for preparing a liquid biopsy sample of exosome subpopulation according to another embodiment of the present invention.
13 is a flowchart of a method for preparing an exosome subpopulation liquid biopsy sample according to another embodiment of the present invention.
14 to 17 are experimental results of simultaneous measurement of CD9 and Cav1 markers for each of the exosome standard sample and the sample obtained after immunoaffinity separation using an unlabeled sensor in order to verify the effect of separating the exosome subgroup according to the present invention. As , FIG. 12 is a CD9 expression concentration in a normal exosome sample, FIG. 13 is a Cav1 expression concentration in a normal exosome sample, FIG. 14 is a CD9 expression concentration in a cancer cell exosome sample, FIG. 15 is Cav1 in a cancer cell exosome sample Expression concentrations are respectively indicated.
18 is a schematic diagram showing the targeting synergistic effect according to the concentration of the target exosome and the reduction of the analysis window when sequential CD63+/Cav1+ exosome subpopulation is measured (blue curve in FIG. 14) using the CD63+ exosome subgroup as an analysis sample. .

The objects, specific advantages and novel features of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings and preferred embodiments. In the present specification, in adding reference numbers to the components of each drawing, it should be noted that only the same components are given the same number as possible even though they are indicated on different drawings. Also, terms such as “first” and “second” are used to distinguish one component from another, and the component is not limited by the terms. Hereinafter, in describing the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the gist of the present invention will be omitted.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic diagram comparing a biomarker analysis process using a conventional liquid biopsy sample (left) and a biomarker analysis process using a liquid biopsy sample prepared according to the present invention (right).

The present invention relates to a technique for preparing a liquid biopsy sample by separating exosomes in body fluid into subgroups and sequentially extending beyond them into subgroups.

As a diagnostic method for early confirmation of the occurrence of a specific disease, 'liquid biopsy', which extracts body fluids such as blood, saliva, and urine, and analyzes exosomes released from specific disease-related cells have. A technique for using exosomes extracted from bodily fluids as new biomarkers for diagnosing specific diseases such as cancer, degenerative diseases, infectious diseases, and heart diseases has been recently studied. However, since exosomes derived from all cells exist in a mixed form in the body fluid, as in the prior art shown on the left of FIG. 1, exosome samples in the form of a bulk population simply separated from body fluids are There is a limit in accurately diagnosing a specific disease, such as It is quite difficult to find specific biomarker molecules contained in the target exosomes in the whole body fluid provided as a sample. The possibility of using exosome nanovesicles as biomarkers depends on how highly selected markers can be enriched in exosome isolation. If not concentrated, the target exosomes represent only a small fraction of the total proteomic/transcriptome in body fluid. As such, it is necessary to classify inclusions such as biomarkers according to exosomes, so the enrichment of diagnostic markers in resources such as body fluids containing exosomes helps to find biomarkers that are expressed relatively low enough to be difficult to detect in general. Accordingly, a technology for separating and enriching the target exosome is required. Conventional exosome separation technology includes a method of separation using ultracentrifugation, sedimentation separation, size exclusion chromatography, or microfluidics, but these prior techniques are still all exo present in body fluid. There is a limitation in selectively detecting a trace amount of a target exosome derived from a specific cell, such as cancer, because it provides a small amount in the form of a single population.

Accordingly, as shown on the right side of FIG. 1 , the present invention provides protein markers highly correlated with cancer on the surface of the exosome, for example, Caveolin 1 (Cav1) for melanoma cancer, HER-2/neu for gastric cancer, and ovarian cancer. In the case of cancer, paying attention to the presence of L1CAM, by adopting an immunoaffinity separation method for a specific marker, a specific disease-related exosome subgroup and a subpopulation by sequentially extending beyond it are to be separated. That is, the target disease association is relatively low, but the exosome subpopulation for the exosome marker (eg, CD63 for melanoma cancer) of a comprehensive supergroup that includes disease-related exosomes is first isolated, 1 Exosomes containing biomarkers (eg, specific miRNAs) involved in the spread of target diseases through exosome subpopulation isolation for exosome-specific markers (eg, Cav1 in case of melanoma cancer) in the tea isolation solution Mosses can be prepared as liquid biopsy samples in concentrated form.

Exosome profiling can provide personalized information for an individual patient and is important for accurate prognosis or differentiated diagnosis. In addition, changes in exosome-derived biomarkers may indicate a response to more than a single condition, as well as exosomes in body fluids, for example, plasma exosomes are secreted by various cellular resources from both blood cells and surrounding tissues.

During a liquid biopsy, various proteins are heterogeneously distributed on the surface of exosomes in body fluids for each exosome. In particular, specific surface proteins associated with cancer disease exist complexly in exosomes of normal individuals although their expression ratios are different. Cell type-specific molecules such as Tetraspanins (CD9, CD63, CD81, CD82, CD151, etc.), histocompatibility complex (MHC) class-I and class-II, adhesion molecules, HSP60, HSP70, etc. It has been reported that it can increase or decrease in relation to the characteristics of For example, CD63, a tetraspanin-type exosome surface protein, is a protein contained in the lysosomal membrane of exosomes derived from B cells and is reported to be increased in samples of patients with several cancer diseases, including melanoma cancer. In addition, since the expression level of CD63 appears to change depending on the type of parental cell compared to CD81 and CD9 of the same family, studies to use it as an optimal exosome marker for disease diagnosis have also been reported. Furthermore, for accurate quantitative analysis of exosome surface proteins, invariant housekeeping markers independent of the characteristics of hair cells can be used for standardization purposes when quantifying. For this standardization purpose, it is predicted that CD9 is more suitable than CD63 in the tetraspanin family.

Under this background, immunoaffinity separation into exosome subpopulations based on exosome surface proteins can provide the following advantages. First, unlike the separation methods based on density, size, precipitation, etc., since the antigen-antibody adhesion reaction is used, the inclusion of impurities other than the target exosome in the solution after separation is minimized. As described above, as the amount of impurities in the analyte sample during diagnosis decreases, the sample maternal effect such as a non-specific reaction decreases, so that the diagnostic sensitivity and specificity can be improved. Second, if immunoaffinity separation is performed on exosome markers of a comprehensive supergroup that includes exosomes associated with a specific disease but has relatively low disease association, the concentration of specific disease-related exosomes in the isolated subpopulation may be increased. can Such an approach is useful when the concentration of disease-related exosomes in the exosome population is initially low enough to be difficult to detect in body fluids. In this case, the sensitivity can be improved when diagnosing a specific disease by using the isolated subpopulation as a diagnostic sample.

Furthermore, if the isolation of the exosome subgroup is further possible through the specific exosome surface protein associated with a specific disease such as cancer, a high concentration of a specific disease marker contained in the specific exosome, for example, mRNA and miRNA, etc. This included exosome subpopulation can be provided as a diagnostic sample. In particular, it is known that exosomes released from cancer cells contain various types of RNAs that play a role in local environmental composition such as angiogenesis in the pre-cancer metastasis stage and execution roles in the metastasis stage. is being reported

In fact, an increased number of exosome-derived proteins have been identified as potential biomarkers for various diseases such as hepatitis, liver, kidney, and degenerative diseases as well as cancer diseases. Exosome proteins are reported to be related to specific diseases, and thus can be used as diagnostic or isolation targets in future product development. For example, there is CD81 as an exosomal protein marker associated with chronic hepatitis C in plasma, and CD63, caveolin-1, TYRP2, VLA-4, HSP70, and HSP90 as markers associated with melanoma cancer, prostate cancer and There is survivin as an associated marker, and L1CAM, CD24, ADAM10, EMMPRIN, and claudin as markers associated with ovarian cancer. In addition, there are Fetuin-A and ATF 3 as exosomal protein markers associated with acute kidney injury in urine, CD26, CD81, S1c3A1, and CD10 as markers associated with liver injury, EGF, resistin, and EGF as markers associated with bladder cancer. retinoic acid-induced protein 3 and PSA and PCA3 as markers associated with prostate cancer. Furthermore, there are amyloid peptides as exosome peptide markers associated with Alzheimer's in brain tissue and Tau phosphorylated Thr181 as markers associated with Alzheimer's in cerebrospinal fluid.

Tetraspanins, a type of skeletal membrane protein, are abundantly present in exosomes. For example, plasma CD63+ exosomes are significantly increased in melanoma cancer patients compared to healthy controls. Another exosome marker of the Tetraspanin family, CD81, plays a crucial role in hepatitis C virus adhesion and/or entry into cells. In addition, the concentration of serum exosome-containing CD81 is elevated in chronic hepatitis C patients and is also associated with exacerbation of inflammation and fibrosis. This suggests that exosome-containing CD81 could be a potential marker for hepatitis C diagnosis and therapeutic response. Moreover, many exosome-containing protein biomarkers are reported to be potentially useful for the diagnosis of central nervous system diseases. EGFRvIII (glioblastoma-specific epidermal growth factor receptor vIII) was found in serum exosomes from glioblastoma patients, which may be useful for the diagnostic detection of glioblastoma. In addition, it has been reported that exosome-containing amyloid peptides accumulate in brain plaques of Alzheimer's disease patients. Tau protein phosphorylated at Thr-181 is a well-established biomarker of Alzheimer's disease, and it is present in elevated concentrations in exosomes isolated from cerebrospinal fluid samples from mild Alzheimer's patients. From these findings, the potential value of exosomes for the early diagnosis of Alzheimer's is being highlighted. Currently, there is no diagnostic test method that can confirm early Alzheimer's disease.

In addition, the potential usefulness of proteins contained in urine exosomes that can be obtained by non-invasive means in diagnosis is being explored, particularly for urinary tract diseases. For example, urinary exosome-containing fetuin-A is increased in patients with acute kidney injury in the intensive care unit compared with normal subjects. ATF3 is also found only in exosomes isolated from patients with chronic kidney disease or acute kidney injury compared to normal individuals. Furthermore, urinary exosome proteins are being studied for use as potential biomarkers for bladder and prostate cancer. According to the research results, two types of prostate cancer biomarkers, PCA-3 and PSA, are present in exosomes isolated from the urine of prostate cancer patients. Overall, exosome-containing proteins identified in other bodily fluids are expected to be used as target platforms in the future development of diagnostic analysis systems.

In addition, protein markers highly correlated with cancer on the exosome surface, for example, Caveolin 1 (Cav1) for melanoma cancer, HER-2/neu for gastric cancer, and L1CAM for ovarian cancer, are present. has been reported However, since the content of this specific exosome is very low, especially in the sample for early diagnosis, the introduction of the sequential and repeated separation process as described above is inevitable in order to selectively isolate it.

Figure 2 is a configuration diagram schematically showing an exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention.

As shown in Figure 2, the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention is a fixed member (10) of a solid state, exosomes secreted from a plurality of cells (1) The first capture material 20, the fixing member 10, and the first capture material that specifically binds to the first separation marker (m1) of the disease-related exosome (1a) and captures the disease-related exosome (1a) The first reversible linker 30 that separably binds the material 20, and the first capture material 20 are coupled to the unbound fixing member 10, and is a group of disease-related exosomes 1a. It includes a second capture material 40 that specifically binds to the second separation marker (m2) of the target exosome (1b) in the exosome subgroup (S) to capture the target exosome (1b).

Specifically, the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention includes a fixing member 10, a first capture material 20, a first reversible linker 30, and a second capture material 40 ) is included. In addition, as a means for coupling the second capture material 40 to the fixing member 10 , a second reversible linker 50 may be further included.

The fixing member 10 is a member in a solid state, and the first reversible linker 30 , the second capture material 40 and the second reversible linker 50 may be selectively coupled and fixed to the surface thereof. The first reversible linker 30 may be bonded and fixed on one fixing member 10 and then separated, and then the second capture material 40 or the second reversible linker 50 may be bonded and fixed. In addition, the fixing member 10 to which the first reversible linker 30 is fixed and the fixing member 10 to which the second capture material 40 or the second reversible linker 50 is fixed may be different from each other. The fixing member 10 may include any one or more selected from the group consisting of a hydrogel, a magnetic bead, a latex bead, a glass bead, a nanometal structure, a porous membrane, and a non-porous membrane. However, the fixing member 10 is not necessarily limited to the above, and if it is a material that can be combined with each of the first reversible linker 30 , the second capture material 40 or the second reversible linker 50 , there is a special limitation. none.

The first capture material 20 and the second capture material 40 are materials for selectively capturing a predetermined exosome (1). The first capture material 20 and the second capture material 40 capture the exosome 1 by specifically binding to the separation marker m of the exosome 1 . Here, the separation marker (m) is a component that is relatively increased in the body fluid compared to a normal person when a specific disease occurs, and is a protein, nucleic acid, lipid component, sugar component, peptide, and its present on the surface of the exosome (1). Other biochemical components may be included. These separation markers (m) may be specifically bound to the first capture material 20 and the second capture material 40 through an immunochemical interaction such as an antigen-antibody reaction or the like. The first capture material 20 and the second capture material 40 may be a predetermined antibody that reacts with the surface protein of the exosome 1 and the like antigen-antibody.

Among the separation markers (m), the first separation marker (m1) that specifically binds to the first capture material (20) is present on the surface of a predetermined disease-related exosome (1a). Therefore, from the bulk exosome population (B), which is a population of exosomes (1) isolated from a plurality of cells, the disease-related exosomes (1a) having the first separation marker (m1) are transferred to the first capture material (20). It can be separated by being captured. Here, the isolated group of disease-related exosomes (1a) becomes the exosome subgroup (S). The target exosome (1b) having the second separation marker (m2) among the disease-related exosomes (1a) in the exosome subpopulation (S) generated in this way specifically reacts with the second separation marker (m2) to capture it Thus, the exosome subpopulation (SA) is separated within the exosome subgroup (S). The first separation marker (m1) on the exosome (1) exhibits relatively low accuracy when used for diagnosing a specific disease compared to the second separation marker (m2), but is present in a relatively high concentration in the body fluid, exo It has more comprehensive characteristics as it is located higher in quantitative system classification according to group markers (1).

The present invention provides a technique for increasing the sensitivity of a diagnostic index by selectively separating an exosome subpopulation (S) containing a biomarker specific for a specific cancer disease or the like from a diagnostic sample. Here, the exosome subpopulation (S) to be isolated is a part of the bulk subpopulation (B) including all exosomes (1) present in the body fluid, and also includes the analysis target biomarker. In the present invention, after the primary separation, concentration, and recovery of the upper exosome surface protein marker (first separation marker) positive exosome subpopulation (S) containing the disease-specific marker predicted to have a low concentration in the body fluid, Sequentially, it is possible to recover the exosome subpopulation (SA) by secondary separation and concentration for the disease-specific marker (second separation marker). Here, the number of repetitions of the separation, concentration, and recovery processes that can be sequentially performed for other exosome surface protein markers can be extended to two or more times depending on the purpose. Biomarkers for diagnosis of specific diseases contained in exosomes isolated and concentrated in the form of subpopulations are components that increase relatively in body fluids compared to normal people when a specific disease occurs, and are involved in the biosynthesis of exosomes in cells, or exosomes from cells. Proteins, mRNAs, tRNAs, miRNAs, long non-coding RNAs, DNAs, lipid components, sugar components, peptides, and other biochemical components involved in the release step or in exosome reception by recipient cells. have.

When performing a liquid biopsy using exosomes in body fluids, the conversion from simply concentrated exosome samples in the form of bulk populations to the use of disease-relevant exosome samples selectively isolated for the disease to be tested can improve diagnostic accuracy. . In the case of melanoma cancer, which is a skin cancer that occurs in melanocytes, Caveolin 1 (Cav1), a surface protein of exosomes, is significantly increased in the blood of melanoma cancer patients compared to normal people, so Cav1-positive (Cav1+) exosome-based diagnosis The sensitivity was 68%, which is relatively higher than the 43% sensitivity of CD63-based diagnostic diagnostics, which is another melanoma cancer marker. Selective separation and concentration of target exosomes such as Cav1+ exosomes are required for clinical use through improved performance of liquid biopsy. However, since the ratio of the total exosomes in the body fluid is very low, conventional known general processes difficult to realize

Therefore, the present invention is a method of selectively separating exosomes in body fluids, as an example, obtaining a subgroup or subpopulation containing Cav1+ exosomes, and using this as a test sample to increase the accuracy and sensitivity of melanoma cancer diagnosis. want to use In consideration of the yield and reproducibility of exosome isolation, Cav1+ exosomes were not first targeted as an isolation target, but the upper exosome population associated with cancer was used as the primary isolation target. For example, compared to other surface proteins, CD63 protein, a house-keeping protein, which is abundant in common exosomes, is increased and expressed on the surface of exosomes during the occurrence of several cancers including melanoma cancer, so this surface protein is not recommended. 1 Using the isolation marker (m1), the CD63+ exosome subpopulation (S) was isolated, and using Cav1 as the second isolation marker (m2), the CD63+/Cav1+ exosome subpopulation from the CD63+ exosome subpopulation (S) was used. (SA) can be isolated.

Meanwhile, the first capture material 20 may be fixed to the fixing member 10 via the first reversible linker 30 and then separated. The second capture material 40 may be directly coupled and fixed to the fixing member 10 without separation, or may be fixed and then separated from the fixing member 10 by introducing the second reversible linker 50 .

The first reversible linker 30, which is any one of the reversible linkers, separably binds the first capture material 20 to the fixing member 10, and the other second reversible linker 50 is a second capture material ( 40) is detachably coupled to the fixing member 10 . Here, the first capturing material 20 is not coupled to the fixing member 10 to which the second capturing material 40 is fixed. That is, the second capture material 40 is fixed on the fixing member 10 to which the first capture material 20 is not coupled through the second reversible linker 50 . The first reversible linker 30 and the second reversible linker 50 may be made of the same material and structure, or may be implemented differently, and the specific material and structure will be described later.

With the existing immunoseparation technology for exosome surface proteins, it is difficult to preserve the original form of exosomes due to the use of severe conditions such as acidic pH when recovering exosomes after separation, as well as low recovery rates. Therefore, it is difficult to use existing technologies such as chromatography, magnetic separation, FACS, and microfluidic separation for sequential immunoisolation two or more times. In fact, in addition to a simple separation method based on surface protein, fractionation based on particle size and density is still only in a rudimentary stage, and details on the separation of exosome subpopulations through a continuous immunoaffinity separation process have not been reported yet. .

Exosome immunoaffinity separation under mild conditions is possible using a reversible linker based on ligand-binder adhesion reaction. The reversible linker initially serves to immobilize a specific exosome (1) capture material (antibody) on the fixing member 10, but after capturing the exosome (1) in the sample to the fixing member 10, reaction conditions It is possible to recover the dissociated and captured exosomes (1) through the change. For example, sugar-sugar receptor binding reaction through hydrophilic and hydrophobic bonding, interion or ion-ion receptor binding reaction, matrix-enzyme reaction, antigen-antibody reaction, nucleic acid conjugation reaction, hormone-cell receptor reaction, and ligand- In addition to the nanostructure reaction, in particular, polyhistidine-tag, biotin-avidin reaction, and attachment reaction using a chelator may be used as a reversible linker. Such attachment reactions can be dissociated by adjusting the binder-ligand affinity of the linker, such as a change in the structure of the binder after binding or a competition reaction for the binder. Therefore, the use of a reversible linker enables the isolation and recovery of the target exosome 1b under mild conditions after immunological capture of the exosome 1 in the sample without using harsh conditions such as acidic pH.

In addition, it is an immunoaffinity separation process using the above-described attachment reaction-based reversible linker, which enables separation and recovery using physical, biological, and chemical properties such as density, size, solubility, membrane permeability, molecular structure, and magnetism. process can be used. In particular, using a specially designed immunoaffinity separation process such as chromatography or magnetic separation, it is possible to separate and recover a trace amount of exosomes in body fluids by controlling the affinity between the binder-ligand in the linker. For example, by using the 'switched attachment reaction' of a recognition material that recognizes the structural change of the binder in the linker induced during ligand attachment, it is possible to isolate and recover exosomes through the immunoseparation process only depending on the presence or absence of ligand ( 3 to 4). In addition, by introducing a competitive attachment reaction method using a ligand-like structure that competes with a ligand for a binder in a ligand-binder reaction during immunoseparation, it is possible to separate and recover exosomes depending on the presence or absence of a competitive attachment reaction (FIGS. 6).

Using such an immunoseparation process makes it possible to sequentially and repeatedly separate exosome samples and maintain reproducibility. This is because it does not use harsh conditions such as acidic pH or the inclusion of a surfactant to dissociate the specific exosomes 1a and 1b trapped in the fixing member 10, thereby reducing the damage and yield of the exosomes 1 Because the fundamental problem is solved. Therefore, as existing technologies overcome the problems of difficulty in preserving the original form and low recovery rate when recovering exosome subpopulations, it is possible to isolate a target exosome subpopulation associated with a specific disease through sequential multiple separation.

As described above, when it is possible to separate exosome subpopulations through specific exosome surface proteins associated with specific diseases such as cancer diseases, high concentrations of specific disease biomarkers contained in specific exosomes, such as mRNA and miRNA, etc. can be provided as a liquid biopsy sample. In recent studies, in particular, exosome-containing miRNAs have emerged as an ideal biomarker for clinical diagnosis as it is known that miRNAs containing exosomes are protected from degradation by RNase and can be stably detected in circulating plasma or serum. Examples of miRNAs (miRs) found in exosomes in plasma include miR-21, -141, -200a, -200c, -203, -205, and -214 associated with ovarian cancer, and miR- associated with lung cancer. 17, -3p, -21, -20b, -223, -301, and let-7f, miR-141 and miR-375 associated with prostate cancer, miR-21 associated with breast cancer, and cardiovascular disease miR-1 and miR-133a are associated, and miR-122 and miR-155 are associated with alcoholic liver disease. Also, examples of miRNAs found in exosomes in urine include miR-29c and CD2AP mRNA associated with renal fibrosis.

Most of the research on exosome-derived miRNAs has been focused on cancer diagnosis. According to the study results, specific miRNAs are found at similar concentrations in ovarian cancer biopsy specimens or in serum exosomes isolated from the same ovarian cancer patient. However, such exosome-containing miRNA has not been found in the normal group, suggesting that exosome-derived miRNA can be used as a potential diagnostic marker when collecting biopsy data. Early detection and diagnosis of prostate cancer can be performed using prostate-specific antigen (PSA). However, PSA-based tests show low diagnostic specificity and high false-positive rates, which may lead to overtreatment of prostate cancer. Therefore, there is a need for the development of new markers that show higher diagnostic accuracy when diagnosing prostate cancer. According to several studies, increased concentrations of miR-141 and miR-375 as miRNA (miR) markers are associated with tumor progression in prostate cancer. Since exosome-derived miRNA has RNase resistance in serum or plasma specimens, exosome-derived miR-141 and miR-375 may be valuable markers in the diagnosis of prostate cancer.

The miRNA contained in exosomes also has potential as a biomarker in the diagnosis of sophageal squamous cell cancer (ESCC). According to the study results, the concentration of exosome-derived miR-21 is elevated in serum from ESCC patients compared to that of patients with benign tumors without systemic inflammation, and the concentration is clearly associated with tumor progression and aggression. Moreover, exosome-derived miRNAs also have potential as diagnostic biomarkers for cardiovascular disease and renal fibrosis.

In addition to miRNA, exosome-containing mRNA has potential utility as a biomarker in clinical diagnosis. Indeed, urine exosome samples from prostate cancer patients contain mRNA of PCA3 and mRNA of TMPRSS2, a product of v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusion chromosomal rearrangement. Similarly, diagnostic and prognostic markers of renal ischemia/reperfusion injury or antidiuretic hormone action (eg, aquaporin-1 and -2) were also found in urinary exosomes.

From the above, it is clear that there are specific exosome markers associated with specific types of cancer, and these data represent a huge commercial opportunity for the development of diagnostic test methods in the oncology-related market.

Hereinafter, the above-described first reversible linker 30 and the second reversible linker 50 will be described in detail. The reversible linker may be implemented according to the following first to fourth embodiments, and the first reversible linker (30, 30a ~ 30d), and the second reversible linker (50, 50a ~ 50d) may consist of any one of them. have. 3 to 6 are schematic diagrams illustrating the first reversible linker and the second reversible linker shown in FIG. 2 according to various embodiments.

3, the reversible linkers 30a and 50a according to the first embodiment of the present invention may include a ligand 31a, a binder 33a, and a recognition material 35a.

The ligand 31a is a material detachably coupled to the binder 33a, and may include any one or more selected from the group consisting of sugar molecules, ions, substrates, antigens, peptides, vitamins, growth factors, and hormones. .

The binder 33a is a material that causes a conformation change by an adhesion reaction with the ligand 31a. The binder (33a) may include any one or more selected from the group consisting of a sugar-binding protein, an ion-binding protein, an enzyme, an antibody, an aptamer, a cell receptor, and a nanostructure. Accordingly, the binder 33a and the ligand 31a are a sugar-binding protein-sugar molecule pair, an ion-binding protein-ion pair, an enzyme-substrate pair, an antigen-antibody pair, an aptamer-ligand pair, a cell receptor-ligand A binder-ligand pair such as a pair, a nanostructure-ligand pair, etc. may be formed, and the most representative examples of the binder 33a and the ligand 31a include calcium binding protein (CBP) and calcium ions. . However, the binder 33a and the ligand 31a are not necessarily limited to the above materials, and may be any material as long as they are detachably coupled to each other to cause a structural change. In the reversible linker according to the first embodiment, the binder 33a is polymerized with the first capture material 20 and/or the second capture material 40 to form the binder-trapping material polymer (C).

The recognition material (35a) is a material that specifically binds to the binder (33a) whose structure is changed by binding to the ligand (31a), an antibody, a protein receptor, a cell receptor, an aptamer, an enzyme, a nanoparticle, a nanostructure, and It may include any one or more selected from the group consisting of heavy metal chelators. However, since the material is only an example of the recognition material 35a, the scope of the present invention should not be limited thereto. As an example, when describing the binding reaction between the ligand 31a, the binder 33a, and the recognition material 35a, calcium ions (ligands) bind to the calcium-binding protein (binder) and the structural change of the calcium-binding protein is An antigen-antibody reaction occurs between the calcium-binding protein and the antibody (recognition material), which is caused and changed in structure, and can be bound to each other. In the reversible linker according to the first embodiment, the recognition material 35a is fixed to the surface of the fixing member 10 .

On the other hand, when the exosome 1 is captured by the first capture material 20 and/or the second capture material 40 coupled to the fixing member 10 via the reversible linkers 30 and 50, reaction conditions By dissociating the reversible linker through the change, the captured exosomes (1a, 1b) can be recovered. In the case of the reversible linkers 30a and 50a according to the first embodiment, the binder 33a having a changed structure may be dissociated when it is restored to its original structure. For example, when an antibody (recognition material) is bound to a calcium-binding protein (binder) whose structure is changed due to attachment of calcium ions (ligand) and the antibody captures the exosome (1), recovery without calcium ions By adding the solution, calcium ions are desorbed from the calcium-binding protein, thereby restoring the structure of the calcium-binding protein and separating the calcium-binding protein and the antibody.

Referring to FIG. 4 , the reversible linkers 30b and 50b according to the second embodiment of the present invention may include a ligand 31b, a binder 33b, and a recognition material 35b.

The ligand 31b, the binder 33b, and the recognition material 35b of the reversible linkers 30b and 50b according to the second embodiment are the ligands 31a of the reversible linkers 30a and 50a according to the first embodiment, respectively. , the binder 33a, and the bar corresponding to the recognition material 35a, the differences will be mainly described below.

In the reversible linkers 30a and 50a according to the first embodiment, the binder 33a is polymerized with the capture materials 20 and 40 to form a polymer (C), and the recognition material 35a is the fixing member 10 . In contrast to being fixed to the surface, in the case of the reversible linkers 30b and 50b according to the second embodiment, the binder 33b is fixed to the surface of the fixing member 10, and the recognition material 35b is the capture material 20, 40 ) to form a recognition material-capturing material polymer (C). Other than that, it is the same as the reversible linkers 30a and 50a according to the first embodiment, and a detailed description thereof will be omitted.

As an example of the reversible linkers 30 and 50 according to the first and second embodiments that enable the recovery of exosome subpopulations or subpopulations in a sample under mild conditions, binders 33a and 33b such as calcium binding proteins are Recognition materials (35a, 35b) such as antibodies that specifically recognize structural changes caused by binding to ligands (31a, 31b) such as calcium ions that react specifically may be used. Binder-recognition material reactions such as these antigen-antibody reactions are rapidly combined or dissociated depending on the presence or absence of ligands (31a, 31b) such as calcium ions, and are similar to the principle of turning on and off a light by manipulating a switch. described as 'switch-like reversible binding'.

As shown in FIG. 5 , the reversible linkers 30c and 50c according to the third embodiment of the present invention may include a ligand 31c and a binder 33c.

The ligand 31c of the reversible linkers 30c and 50c according to the third embodiment is polymerized with the first capture material 20 and/or the second capture material 40 to form a capture material-ligand polymer (C). .

The binder 33c of the reversible linkers 30c and 50c according to the third embodiment is fixed to the surface of the fixing member 10, and the capture material specifically bound to the exosomes 1a and 1b-ligand polymer (C) ), thereby trapping the exosomes (1a, 1b) in the fixing member (10). The binder 33c binds to the ligand 31c, but when the competing ligand 32 competing with the ligand 31c is added, the competing ligand 32 is attached. Due to this, as the ligand 31c is detached, the exosomes 1a and 1b may be separated from the fixing member 10 and recovered. Here, the ligand 31c is His-tag (polyhistidine-tag), vitamins such as biotin, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones selected from the group consisting of It may include any one or more. In addition, the binder 33c includes at least one selected from the group consisting of divalent metal ions, avidins, sugar-binding proteins, ion-binding proteins, enzymes, antibodies, aptamers, protein receptors, cell receptors, and nanostructures. can do. The competitive ligand 32 may include any one or more selected from the group consisting of vitamins such as imidazole and biotin, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones. can

For example, Hisstack may be used as the ligand 31c, divalent metal ions may be used as the binder 33c, and imidazole may be used as the competitive ligand 32 . Hisstack is an amino acid motif composed of at least 6 histidine (His) residues in a protein. It is usually located at the N-terminus or C-terminus of a protein and is mainly used for purification after expression of a recombinant protein. Here, the His-stack-labeled protein is captured using an affinity with nickel or cobalt, which is a divalent ion bound to a chelator on sepharose/agarose resin, and then recovered by adding a recovery solution containing imidazole. can be

Referring to FIG. 6 , the reversible linkers 30d and 50d according to the fourth embodiment of the present invention may include a ligand 31d and a binder 33d.

The ligand 31d, and the binder 33d of the reversible linkers 30d and 50d according to the fourth embodiment are the ligands 31c, and the binder 33c of the reversible linkers 30c and 50c according to the third embodiment, respectively. , and the differences will be mainly described below.

In the reversible linkers 30c and 50c according to the third embodiment, the ligand 31c is polymerized with the capture materials 20 and 40, and the binder 33c is fixed to the surface of the fixing member 10, whereas the fourth embodiment In the case of the reversible linkers 30d and 50d according to the example, the ligand 31d is fixed to the surface of the fixing member 10, the binder 33d is polymerized with the capture materials 20 and 40, and the capture material-binder polymer ( C) is formed. Other than that, the ligand 31d is bound to the binder 33d in the same manner, but is separated from the binder 33d by a competitive reaction with the competing ligand 32 .

In the present invention, for the separation of exosomes according to surface protein markers, an immunoseparation method using an antibody specific to a target marker can be applied. According to this method, the target exosome can be separated by reacting the exosome sample with an antibody immobilized on a solid surface, such as magnetic beads or gel beads, and dissociating it. Since the antigen-antibody reaction has very high selectivity, it is more suitable for isolating exosomes related to surface proteins compared to conventional separation methods such as ultracentrifugation, accelerated sedimentation, and size separation. However, in order to recover the exosome bound by the antigen-antibody reaction from the solid surface, severe conditions such as acidic pH must be used, in which case it may damage the exosome, and there is a problem in that it is difficult to recover the exosome in high yield. . However, using the above-described attachment reaction-based reversible linker technology, exosome subpopulations and subpopulations including target markers can be separated and concentrated with high efficiency under non-severe conditions.

On the other hand, the exosome subpopulation liquid biopsy sample preparation apparatus according to an embodiment of the present invention may further include a reactor (not shown). The reactor may accommodate the fixing member 10 therein. At this time, when the exosome (1) sample is added to the reactor, the disease-related exosome (1a) is transferred to the fixing member 10 via the first reversible linker 30 and the first capture material 20 therein. The captured exosome subpopulation (S) is separated, and the first reversible linker 30 is dissociated to recover the exosome subpopulation (S). In addition, the recovered exosome subpopulation (S) sample is added to the reactor, and the target exosome 1b is captured by the fixing member 10 using the second reversible linker 50 and the second capture material 40. An exosome subpopulation (SA) is isolated. The isolated exosome subpopulation (SA) is recovered as the second reversible linker 50 is dissociated. Here, by concentrating the fixing member 10 using any one or more of magnetic force, gravity, and centrifugal force, the exosome subpopulation (S) and/or the exosome subpopulation (SA) can be concentrated. For example, when a magnetic bead is used as the fixing member 10, the upper layer in a state where the fixing member 10 coupled to the disease-related exosome 1a and/or the target exosome 1b is captured using a magnet. By removing the liquid and removing the magnetic beads, the exosome subpopulation (S) and/or the exosome subpopulation (SA) can be concentrated.

Hereinafter, a liquid biopsy sample preparation method using the exosome subpopulation liquid biopsy sample preparation apparatus according to the present invention will be described. As described above for the exosome subpopulation liquid biopsy sample preparation apparatus, the overlapping matters will be omitted or simply described.

7 to 11 are flowcharts of a method for preparing a liquid biopsy sample of exosome subpopulation according to a first embodiment of the present invention.

Referring to Figure 7, the exosome subpopulation liquid biopsy sample preparation method according to the first embodiment of the present invention is (a) a first separation marker of a predetermined disease-related exosome among exosomes secreted from a plurality of cells and A step (S100) of binding a first capture material that specifically binds to a fixation member in a solid state using a first reversible linker (S100), (b) a sample solution containing a population of exosomes, and a fixation member Separating the exosome subpopulation, which is a group of disease-related exosomes, by reacting the first capture material bound to the disease-related exosomes to capture the disease-related exosomes (S200), (c) the captured disease-related exosomes are not fixed to dissociate the first reversible linker to recover the exosome subpopulation (S300), (d) the second capture that specifically binds to the second separation marker of the target exosome in the exosome subpopulation The step of binding the material to the fixing member (S400), and (e) reacting the exosome subpopulation solution containing the recovered exosome subpopulation with the second capture material bound to the fixing member to form the target exosome By capturing, it includes a step (S500) of separating the exosome subpopulation, which is a group of target exosomes. Here, the second capture material may be coupled to the fixing member by a second reversible linker.

The exosome subpopulation liquid biopsy sample preparation method according to the present invention uses a bulk exosome group or body fluid, which is a group of exosomes secreted from a plurality of cells, as a sample solution, and primarily separates and recovers the exosome subgroup After secondly isolating the exosome subpopulation from the exosome subpopulation solution, it is provided as a liquid biopsy sample.

First, in order to separate and recover the exosome subpopulation, the first capture material and the first reversible linker are reacted with the fixing member (S100). In this case, the first capture material is coupled to the fixing member via the first reversible linker.

Next, by adding the sample solution, it reacts with the first capture material (S200). Here, the first capture material specifically binds to the first separation marker of the disease-related exosomes contained in the sample solution. Thereby, the exosome subpopulation targeting the disease-related exosomes is captured by the fixing member and separated from the sample solution.

After the exosome subpopulation is isolated, the first reversible linker is dissociated (S300). At this time, since the disease-related exosomes are separated from the fixing member, a subpopulation of exosomes can be recovered.

When the disease-related exosomes are separated from the fixing member, the fixing member is recycled, or the second capture material is combined on the fixing member using a new fixing member (S400). In this case, the second capture material may be directly coupled to the fixing member, or may be coupled to the fixing member using a second reversible linker.

Next, the exosome subpopulation solution containing the recovered exosome subpopulation is reacted with the second capture material (S500). At this time, since the second capture material specifically binds to the second separation marker of the target exosome in the exosome subpopulation, the target exosome is fixed to the fixing member. Thereby, the exosome subpopulation, which is a set of target exosomes, is separated. It can be provided as a liquid biopsy sample in the form of a subpopulation of exosomes fixed to a fixing member without being recovered in this way. That is, if the biomarker for diagnosis of a specific disease is separated into a sub-group form and then the biomarker is directly analyzed without recovering the exosome or the biomarker eluted by dissolving the exosome is performed, sequential multiple In the final separation step of the immunoaffinity separation process, only the biomarker can be used as a sample without recovering the subpopulation exosomes.

Hereinafter, the present invention will be described in more detail with reference to specific examples of the first reversible linker and the second reversible linker.

Figure 8 shows a method for preparing a liquid biopsy sample of the exosome subpopulation using the first reversible linker (30a) and the second reversible linker (50a) according to the first embodiment described above. Referring to FIG. 8 , first, the recognition material 35a is fixed to the surface of the fixing member 10 , and the ligand solution containing the ligand 31a, the binder 33a, and the first capture material 20 are reacted. At this time, the binder 33a and the first capture material 20 form the binder-first capture material polymer (C), and the recognition material 35a is combined with the polymer (C). Here, the ligand 31a contained in the ligand solution is bound to the binder 33a, causing a structural change of the binder 33a, and the recognition material 35a specifically recognizes and binds the changed structure of the binder 33a. do. Next, the sample solution containing the bulk exosome (1) population is reacted. At this time, while the disease-related exosome 1a having the first separation marker m1 specifically bound to the first capture material 20 is captured by the fixing member 10, the exosome subpopulation S is separated. do. When the exosome subgroup (S) is separated, a recovery solution is added. The recovery solution is a ligand-free solution that does not contain the ligand 31a. When the recovery solution is added, the ligand 31a bound to the binder 33a) is removed, and as a result, the changed structure of the binder 33a is restored to its original state, and the coupling between the binder 33a and the recognition material 35a is released. Therefore, the polymer (C) to which the disease-related exosome (1a), which is the object of the exosome subgroup (S) is bound, is separated from the recognition material (35a), and the exosome subgroup (S) can be recovered.

Next, the disease-related exosomes 1a are separated and the fixing member 10 to which the recognition material 35a is fixed is recycled to react the second reversible linker 50a and the second capture material 40 . At this time, the second reversible linker 50a shares the recognition material 35a of the first reversible linker 30a, and compared with the first reversible linker 30a, the first capture material 20 is compared to the second capture material ( 40) and coupled to the fixing member 10 . When the recovered exosome subgroup (S) solution is added here, the second capture material 40 is specific to the second separation marker (m2) of the target exosome 1b in the exosome subgroup (S) solution. , and the exosome subpopulation (SA) is isolated. At this time, since the exosome subgroup (S) contains the binder 33a of the first reversible linker 30a, the recognition material 35a coupled to the fixing member 10 and the binder of the first reversible linker 30a Since (33a) can react with each other, after reacting the second reversible linker (50a) and the second capture material (40), the remaining attachment site on the recognition material (35a) coupled to the fixing member (10) is replaced with a binder (33a) ) can be blocked with an appropriate reactive component such as

As an example, when describing a method for isolating a CD63+/Cav1+ exosome subpopulation (SA) according to the present invention having a first reversible link and a second reversible link according to the first embodiment, the antibody (reversible recognition material) is solid (Fixed member) It is immobilized on the bead surface, and calcium binding protein (CBP, binder) is polymerized through biotin-streptavidin linkage with a specific antibody (first capture material) against the CD63 exosome surface protein (CBP-capture). antibody polymer). When a CBP-capturing antibody polymer dissolved in a solution containing calcium ions (eg >10 mM Ca ^{2 +} ) is added to the immobilized reversible recognition material, the polymer is immobilized on the solid surface by a 'switch-on' recognition reaction. When the prepared bulk exosome samples are sequentially added to the same solution, the exosomes containing the CD63 marker react with the capture antibody on the solid surface and are captured. After washing with the same solution, when calcium ion-bound calcium ions are desorbed and become 'switch-off', the exosomes that have been captured can be recovered into the solution in a state bound to the capture antibody. can As a result, the CD63+ exosome subpopulation is separated and recovered, and it is possible to concentrate the exosomes according to the volume ratio of the sample solution and the recovery solution used. Additionally, the solid surface can be regenerated and recycled for the separation of other samples.

For sequential exosome subpopulation separation, a reversible recognition material is immobilized on a regenerated or new solid surface to polymerize a specific antibody against Cav1 exosome surface protein, which is highly related to a specific disease, with CBP, and then switch The CBP-capturing antibody polymer is immobilized on a solid surface through an on' recognition reaction. When the CD63+ exosome subpopulation recovered after the primary separation is added as a secondary separation sample while maintaining the same state, the CD63 and Cav1 marker-containing (CD63+/Cav1+) exosomes react with the capture antibody on the solid surface and are captured. After washing with the same solution, if a calcium-removed exosome recovery solution is added, the 'switched off' state is obtained, so that the captured exosomes can be recovered into the solution. As a result, the CD63+/Cav1+ exosome subpopulation is separated and recovered through secondary separation.

On the other hand, if recovery is not required after separation of the subpopulation of exosomes, the subpopulation can be separated by directly immobilizing the capture antibody on a solid surface without introducing a second reversible linker. In this case, mainly for the captured exosomes Immunoassay is performed, or the nucleic acid contained in the exosome is extracted and molecularly tested by lysing it immediately after being captured.

Figure 9 shows a method for preparing a liquid biopsy sample of the exosome subpopulation using the first reversible linker (30c) and the second reversible linker (50c) according to the third embodiment described above. Referring to FIG. 9 , the ligand 31c is polymerized with the first capture material 20 that specifically reacts with the first separation marker m1 of the disease-related exosome 1a, and is bound to the fixing member 10 . After immobilizing the ligand-capturing material polymer using the affinity with the binder 33c, the exosome (1) sample is added to capture and separate the exosome subpopulation (S), and recovery including the competing ligand (32) The solution is used to recover the exosome subpopulation (S).

Next, the second capture material 40 and the ligand 31c that specifically react to the second separation marker m2 of the target exosome 1b are polymerized, and the binder 33c coupled to the fixing member 10 . ) and its polymer (C), the exosome subpopulation (S) solution can be added to capture and isolate the exosome subpopulation (SA). At this time, since the exosome subpopulation (S) includes the ligand 31c of the first reversible linker 30c, the binder 33c bound to the anchoring member 10 and the ligand of the first reversible linker 30c ( 31c) can react with each other, and after reacting the second reversible linker 50c and the second capture material 40, the remaining attachment sites on the binder 33c bound to the fixing member 10 are replaced with the ligand 31c and the like. It can be blocked with an appropriate reactive component, such as.

As an example, when describing a method for isolating a CD63+/Cav1+ exosome subpopulation according to the present invention having a first reversible link and a second reversible link according to the third embodiment, a binder (nickel ion) is applied to a solid surface It is immobilized on the (bead surface), and the ligand (histack) is polymerized with a specific antibody against the CD63 exosome (1) surface protein as the first separation marker (m1) (histack-capturing antibody polymer). When a His-stack-trapping antibody polymer is added to the immobilized nickel ions, the polymer is immobilized on the solid surface by a binder-ligand reaction. When the prepared bulk exosome sample is added to the same solution, the CD63+ exosome reacts with the capture antibody on the solid surface and is captured. After washing with the solution, if an exosome recovery solution containing a high concentration of a competitive ligand (imidazole) is added, the hisstack that has been bound to nickel ions by the competition reaction is desorbed, so that the captured exosome can be recovered into the solution. As a result, the CD63+ exosome subpopulation is separated and recovered, and the solid surface is regenerated and can be recycled for the next sample separation.

The above-described separation process is similarly used for sequential separation of the CD63+/Cav1+ exosome subpopulation from the CD63+ exosome subpopulation recovered above. In this case, the ligand (histack) is polymerized with an antibody specific for the Cav1 exosome surface protein as a second separation marker. After the polymer is immobilized on the solid surface through a binder-ligand reaction, when a CD63+ exosome subpopulation sample is added, the CD63+/Cav1+ exosome reacts with the capture antibody on the solid surface and is captured. After washing, if an exosome recovery solution containing a high concentration of competitive ligand (imidazole) is added, the CD63+/Cav1+ exosomes bound to Con A can be recovered into the solution. As a result, the CD63+/Cav1+ exosome subpopulation is separated and recovered.

10 shows a method for preparing a liquid biopsy sample of exosome subpopulation using the first reversible linker 30a according to the first embodiment, and the second reversible linker 50c according to the third embodiment. Referring to FIG. 10, as described above in FIG. 8, the exosome subpopulation (S) is isolated and recovered using the first reversible linker 30a according to the first embodiment, and as described above in FIG. 9, the third The exosome subpopulation (SA) can be separated using the second reversible linker (50c) according to the embodiment. At this time, the exosome subpopulation (S) includes the binder 33a of the first reversible linker 30a, but when the exosome subpopulation SA is separated, it reacts with the binder 33a according to the first embodiment In the third embodiment, the other binder 33c is coupled to the fixing member 10, that is, the second reversible linker 50c that operates by a mechanism different from that of the first reversible linker 30a is used, so the above-described blocking process is unnecessary.

11 shows a method for preparing a liquid biopsy sample of exosome subpopulation using the first reversible linker 30c according to the third embodiment, and the second reversible linker 50a according to the first embodiment. Referring to FIG. 11, after isolating and recovering the exosome subpopulation (S) using the first reversible linker 30c according to the third embodiment as described above in FIG. The exosome subpopulation (SA) can be separated using the second reversible linker (50a) according to the first embodiment. At this time, the exosome subpopulation (S) contains the ligand 31c of the first reversible linker 30c, but when the exosome subpopulation SA is separated, the second reversible linker 30c is distinguished from the first reversible linker 30c. Since the reversible linker 50a is adopted, the above-described blocking process is unnecessary.

12 is a flowchart of a method for preparing a liquid biopsy sample of exosome subpopulation according to another embodiment of the present invention.

Referring to Figure 12, the exosome subpopulation liquid biopsy sample preparation method according to another embodiment of the present invention may further include the step of recovering the exosome subpopulation (S600). Here, the second capture material is bound to the anchoring member by a second reversible linker, and by dissociating the second reversible linker, the captured target exosomes are separated from the anchoring member, thereby recovering a subpopulation of exosomes. Analysis of biomarkers may be performed on the recovered exosomes, or analysis of biomarkers eluted by dissolving the exosomes may be performed.

13 is a flowchart of a method for preparing an exosome subpopulation liquid biopsy sample according to another embodiment of the present invention.

Referring to Figure 13, the exosome subpopulation liquid biopsy sample preparation method according to another embodiment of the present invention may further include the step of concentrating the exosome subpopulation (S250).

After adding the sample solution and before adding the recovery solution, the exosome subpopulation can be concentrated. Specifically, by using any one or more of magnetic force, gravity, and centrifugal force, the fixation member coupled to the exosome subpopulation target disease-related exosome is concentrated. For example, a magnetic bead is used as a fixing member, and the fixing member is captured in a predetermined space region through magnetic force in a mixed solution in which the ligand solution and the sample solution are mixed, and then a part of the mixed solution ( For example, by removing the supernatant), the exosome subpopulation can be enriched.

In the same way, by using any one or more of magnetic force, gravity, and centrifugal force, by concentrating the fixing member coupled to the target exosome, the exosome subpopulation may be concentrated (S550).

To support the usefulness of CD63+ subpopulation isolation in cancer diagnosis, a bulk exosome standard sample and an exosome subpopulation sample obtained after isolation were simultaneously measured for CD9 and Cav1 markers, respectively, using an Octet sensor (ForteBio). and the results are shown in FIGS. 14 to 17 .

14 to 17 are experimental results of simultaneous measurement of CD9 and Cav1 markers for each of the exosome standard sample and the sample obtained after immunoaffinity separation using an unlabeled sensor in order to verify the effect of separating the exosome subgroup according to the present invention. 14 is the CD9 expression concentration in a normal exosome sample, FIG. 15 is the Cav1 expression concentration in the normal exosome sample, FIG. 16 is the CD9 expression concentration in the cancer cell exosome sample, FIG. 17 is Cav1 in the cancer cell exosome sample Expression concentrations are respectively indicated.

First, in order to measure CD9-containing exosomes before and after separation in a normal sample, sensors with a specific capture antibody for CD9 were immersed in each exosome sample and measured in real time for 1,200 seconds (see FIG. 14 ). As a result of measuring the signal associated with CD9 for the original sample before separation, the average adhesion signal (converted to adhesion thickness) increased to 0.74 nm (red in FIG. 14 ). On the other hand, the signal for the sample after separation was only measured to be about 0.03 nm (blue in Fig. 14). The same experiment was repeated for melanoma cancer samples obtained before and after isolation (see FIG. 15 ). From this, the measured signal appeared as 0.04 nm (red) before separation and <0.01 nm (blue) after separation. Comparing these two results, the signal intensity for CD9-containing exosomes in normal and melanoma cancer standard samples before separation showed a large difference depending on the source of the sample, whereas after affinity separation for CD63, all of them were almost background signals. level was shown to decrease.

The same label-free sensing technology was applied to the analysis of the same samples described above using sensors immobilized with a Cav1-specific capture antibody. In the case of a normal sample, the Cav1-containing exosomes increased as small as 0.04 nm before separation to 0.06 nm after separation (see FIG. 16 ). In the case of melanoma cancer samples, the Cav1-containing exosomes increased more than 10-fold from <0.01 nm before isolation to 0.1 nm after isolation, which is thought to be due to the specific effect associated with affinity dissociation for CD63 (Fig. 17).

In summary, using the immunoaffinity separation method according to the present invention, the concentration of CD9-containing exosomes was significantly reduced regardless of the source of the exosome sample. In contrast, the isolation method used in the present invention increased the concentration of Cav1-containing exosomes, and the increase in concentration was particularly remarkable in melanoma cancer samples. A clear interpretation was possible in terms of the separation mechanism from the conflicting effects of these exosome markers.

The above results may actually vary depending on the degree of disease progression when diagnosing a cancer disease. According to published literature, the concentration of Cav1+ exosomes, which is reported to be increased during cancer development, is significantly increased at the stage of cancer metastasis rather than at the beginning of cancer development. Therefore, the measurement of Cav1+ exosomes in samples collected at the stage of cancer metastasis is easy without exosome separation and concentration. However, in the early stages of cancer, the concentration in body fluids such as blood is very low, so it is very difficult to directly measure it even with a sensitive analytical method such as immunoassay. Therefore, if the separation and enrichment process of the CD63+/Cav1+ exosome subpopulation presented in the present invention is followed, the biomarker contained in the exosome, for example, a specific miRNA (eg, miR222 in the case of melanoma cancer, etc.) This allows for early and accurate diagnosis of cancer.

Such early diagnosis is made possible by the synergistic effect of reducing the analysis observation window by isolating the target exosome and concentrating the target concentration. 18 is a schematic diagram showing the targeting synergistic effect according to the concentration of the target exosome and the reduction of the analysis window when sequential CD63+/Cav1+ exosome subpopulation is measured (blue curve in FIG. 16) using the CD63+ exosome subgroup as an analysis sample. .

Referring to FIG. 18, when the observation window size is compared, exosome species released from many different cells are mixed in the body fluid used as the analysis sample (bulk exosome population c in FIG. 18; see FIG. 1), the body fluid If the exosome subgroup (FIG. 18 a) or sub-subgroup (see FIG. 18 b ₁ )) containing the target exosome from is used as an analysis sample, unrelated exosome species are excluded from disease-related exosome species. Thus, the effect of reducing the observation window in a desired direction is obtained. In this case, it is possible to minimize interference caused by non-specific reactions of unrelated biomarkers during disease diagnosis. Furthermore, if the concentration process is used when separating into the subgroup or subgroup described above, an effect of increasing the concentration of the target exosome is obtained (refer to the comparison of target concentration in FIG. 18 ). Therefore, according to the present invention, when a subpopulation of exosomes is used as an analysis sample during diagnosis, the interference effect due to non-specific exosomes is minimized and the target exosome concentration is increased at the same time, thereby obtaining a positive synergistic effect on diagnostic accuracy.

In most existing exosome separation methods, as described above, since the entire bulk exosome population in the target body fluid is separated and concentrated, the target exosome subpopulation or subpopulation is mixed with other exosomes to provide only a concentration effect. can In this case, the concentration of all other exosomes also increases, so that there is still a sample matrix effect such as interference by non-specific reactions of other exosome components when analyzing the target exosome. As a method to overcome such existing problems, a change in the distribution (profiling) of a plurality of biomarkers contained in exosomes, that is, proteins and miRNAs, can be measured, but the specificity of the biomarkers used for the disease to be diagnosed Since this is fundamentally low, it is difficult to improve the diagnostic accuracy to the extent that it can be used for early diagnosis.

Exosome-derived proteins and nucleic acids that can be used as isolation and diagnostic markers and specific diseases in which exosome subgroup and sub-subgroup samples prepared using immunoaffinity separation technology that can be performed under mild conditions as described above can be applied to diagnosis are listed below. presented.

A number of exosome-derived breast cancer markers have recently been clinically identified, such as CD24 and EpCAM. In addition to breast cancer, exosomal proteins provide a useful resource for biomarkers for many other cancers. In several studies, one type of inhibitor of apoptosis (IAP) protein group was detected in plasma-derived exosomes from normal individuals and prostate cancer patients, but the relative content of exosome-containing survivin was significantly higher in the plasma of prostate cancer patients. appeared to be higher. Urinary exosomes, which are readily accessible by non-invasive means, are useful for developing biomarkers for prostate and bladder cancer. Protein profiling (label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis) of urine exosomes obtained from normal individuals and bladder cancer patients identified the protein contained in urine exosomes as potential biomarkers for bladder cancer. made to confirm For example, two known prostate cancer markers, PCA3 and TMPRSS2-ERG, are also present in urinary exosomes harvested from prostate cancer patients. Cell surface proteoglycans (protein-bound polysaccharides) were not found in benign pancreatic disease, but were found to be present in exosomes isolated by FACS from the serum of patients with early and late-stage pancreatic cancer. Exosomes isolated by ultracentrifugation from plasma from ovarian cancer patients contained TGF-1 and MAGE 3/6, but these two proteins were not present in exosomes obtained from patients with benign tumors.

In addition to proteins, miRNAs (miRs) and mRNAs were identified as major components of exosome loading in the initial analysis of nucleic acids isolated from exosomes. According to the results of further studies, exosomes were found to contain different types of RNAs such as transfer RNAs (tRNAs) and long non-coding RNAs. In addition to RNA species, single-stranded and double-stranded DNAs fragments were identified to be present in exosomes. Among exosome-derived nucleic acids, exosome miRNAs are receiving the most attention as cancer diagnostic biomarkers as their stability is secured from RNase-dependent degradation. Examples of exosome nucleic acid biomarkers found in preclinical and clinical studies include miR-21 and miR-1246 as exosomal nucleic acids in plasma applied to breast cancer diagnosis, and exosome nucleic acids in serum applied to ovarian cancer diagnosis and screening steps. miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214, and let-7a as an exosome nucleic acid in serum applied for colorectal cancer diagnosis, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a, and miR-141 and miR-375 as nucleosome nucleic acids in plasma and serum applied for prostate cancer diagnosis, There are miR-17-5p and miR-21 as nucleosome nucleic acids in serum and urine applied to the diagnosis of pancreatic cancer.

In summary, cancer cell-derived exosomes contribute to cancer progression by enhancing intercellular delivery of substances including proteins, lipids, and nucleic acids in the tumorigenic microenvironment. Since the exosome loading component reflects the changed state from the original cancer, exosomes are highly valuable as a minimally invasive biomarker for early detection, diagnosis, and prognosis. In fact, exosome-based diagnosis provides higher sensitivity and specificity compared to conventional tissue biopsy or other liquid biopsy biomarkers because the stability of exosomes is maintained in body fluids.

In addition, exosome markers can be easily obtained from most body fluids, and recent advances in exosome isolation technology have provided an opportunity to effectively make exosome-based diagnostic costs and labor. However, a major obstacle to be overcome in the field of exosome diagnostics is to develop an optimal method to isolate a pure exosome population. Microvesicles themselves may be a useful diagnostic tool to detect cancer, but techniques to separate the two major extracellular vesicles into pure exosomes and other microvesicles populations only allow comparative studies. Another challenge is to understand the mechanisms by which cancer-derived exosome loading components differ in regulating the heterogeneity of cancer exosomes and thus affecting the reproducibility of diagnostic results. Despite this importance, exosome-based cancer diagnosis is at the next level by future efforts that combine next-generation sequencing of exosome RNAs and DNAs, proteomic analysis of exosome surface proteins, and immunoaffinity-based capture technology. step will be reached.

Although the present invention has been described in detail through specific examples, this is for the purpose of describing the present invention in detail, and the present invention is not limited thereto, and by those of ordinary skill in the art within the technical spirit of the present invention. It is clear that the modification or improvement is possible.

All simple modifications and variations of the present invention belong to the scope of the present invention, and the specific scope of protection of the present invention will be made clear by the appended claims.

1: exosome 1a: disease-related exosome
1b: target exosome 10: fixation member
20: first capture material 30: first reversible linker
31a to 31d: ligand 32: competitive ligand
33a ~ 33d: binder 35a, 35b: recognition material
40: second capture material 50: second reversible linker
m: separation marker m1: first separation marker
m2: first isolation marker B: bulk exosome population
S: exosome subpopulation SA: exosome subpopulation
C: polymer

Claims (18)

a solid state fixing member;
a first capture material that specifically binds to a first separation marker of a predetermined disease-related exosome among exosomes secreted from a plurality of cells to capture the disease-related exosome;
a first reversible linker separably coupling the fixing member and the first capture material; and
The first capture material is coupled to the unbound fixing member, and specifically binds to a second separation marker of the target exosome in the exosome subgroup, which is a group of disease-related exosomes, to capture the target exosome. A second capture material comprising; exosome subpopulation liquid biopsy sample preparation device.
The method according to claim 1,
Exosome subpopulation liquid biopsy sample preparation apparatus further comprising a; a second reversible linker for separably coupling the fixing member to which the first capture material is not bound and the second capture material.
3. The method according to claim 2,
At least one of the first reversible linker and the second reversible linker,
ligand;
a binder that is polymerized with the capture material to form a polymer, the ligand is detachably attached, and the structure is changed by an attachment reaction with the ligand; and
Exosome subpopulation liquid biopsy sample preparation apparatus comprising a; a recognition material fixed to the surface of the fixing member and specifically binding to the binder whose structure is changed, and is separated from the binder when the ligand is desorbed from the binder .
3. The method according to claim 2,
At least one of the first reversible linker and the second reversible linker,
ligand;
a binder fixed to the surface of the fixing member, to which the ligand is detachably attached, and whose structure is changed by an attachment reaction with the ligand; and
Exosome subpopulation liquid biopsy sample comprising; a recognition material that polymerizes with the capture material to form a polymer, binds specifically to the binder with a changed structure, and is separated from the binder when the ligand is desorbed from the binder manufacturing equipment.
5. The method according to claim 3 or 4,
The ligand is a sugar molecule, an ion, a substrate, an antigen, a peptide, a vitamin, a growth factor, and an exosome subpopulation liquid biopsy sample preparation device comprising any one or more selected from the group consisting of.
5. The method according to claim 3 or 4,
The binder is a sugar-binding protein, an ion-binding protein, an enzyme, an antibody, an aptamer, a cell receptor, and an exosome subpopulation liquid biopsy sample preparation device comprising any one or more selected from the group consisting of nanostructures.
5. The method of claim 3 or 4,
The recognition material is an exosome subpopulation liquid biopsy sample comprising at least one selected from the group consisting of antibodies, protein receptors, cell receptors, aptamers, enzymes, nanoparticles, nanostructures, and heavy metal chelators manufacturing equipment.
3. The method according to claim 2,
At least one of the first reversible linker and the second reversible linker,
a ligand that polymerizes with the capture material to form a polymer; and
Exosome subpopulation liquid biopsy sample preparation apparatus comprising a; a binder fixed to the surface of the fixing member, coupled to the ligand, and separated from the ligand by a competition reaction with a competing ligand.
3. The method according to claim 2,
At least one of the first reversible linker and the second reversible linker,
a ligand fixed to the surface of the fixing member;
Exosome subpopulation liquid biopsy sample preparation apparatus comprising a; polymerized with the capture material to form a polymer, and bound to the ligand, the binder separated from the ligand by a competitive reaction with the competing ligand.
10. The method according to claim 8 or 9,
The binder includes at least one selected from the group consisting of divalent metal ions, avidins, sugar-binding proteins, ion-binding proteins, enzymes, antibodies, aptamers, protein receptors, cell receptors, and nanostructures,
The ligand includes at least one selected from the group consisting of His-tag, polyhistidine-tag, vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones, ,
The competitive ligand is an exosome subpopulation comprising at least one selected from the group consisting of imidazole, vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones. Liquid biopsy sample preparation device.
The method according to claim 1,
The fixing member is an exosome subpopulation liquid biopsy sample preparation device comprising at least one selected from the group consisting of a hydrogel, a magnetic bead, a latex bead, a glass bead, a nanometal structure, a porous membrane, and a non-porous membrane .
The method according to claim 1,
Exosome subpopulation liquid biopsy sample preparation device further comprising; a reactor for accommodating the fixing member therein.
The method according to claim 1,
The disease-related exosomes or the fixing member coupled to the target exosomes is concentrated by any one or more of magnetic force, gravity, and centrifugal force, exosome subpopulation liquid biopsy sample preparation device.
(a) A first capture material that specifically binds to a first separation marker of a predetermined disease-related exosome among exosomes secreted from a plurality of cells is immobilized in a solid state using a first reversible linker coupling to the member;
(b) reacting the sample solution containing the population of exosomes with the first capture material bound to the fixing member to capture the disease-related exosomes, thereby forming the disease-related exosome population separating the population;
(c) recovering the exosome subpopulation by dissociating the first reversible linker so that the captured disease-related exosomes are separated from the fixing member;
(d) binding a second capture material that specifically binds to a second separation marker of the target exosome in the exosome subpopulation to the fixing member; and
(e) reacting the exosome subpopulation solution containing the recovered exosome subpopulation and the second capture material bound to the fixing member to capture the target exosome, thereby forming the target exosome population Separating the exosome subpopulation; exosome subpopulation liquid biopsy sample preparation method comprising.
15. The method of claim 14,
In the step (d), the exosome subpopulation liquid biopsy sample preparation method in which the second capture material is coupled to the fixing member by a second reversible linker.
16. The method of claim 15,
The exosome subpopulation liquid biopsy sample preparation method further comprising; dissociating the second reversible linker to recover the exosome subpopulation so that the captured target exosome is separated from the fixing member.
15. The method of claim 14,
Between step (b) and step (c),
Using any one or more of magnetic force, gravity, and centrifugal force, concentrating the fixing member in which the disease-related exosomes are captured; exosome subpopulation liquid biopsy sample preparation method further comprising a.
15. The method of claim 14,
After step (e), using any one or more of magnetic force, gravity, and centrifugal force, concentrating the fixing member in which the target exosome is captured; exosome subpopulation liquid biopsy sample preparation method further comprising a.

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