KR20210072672A - Diagnosis method by aqueous two-phase composition - Google Patents

Abstract

Disclosed is a diagnostic method capable of accurately diagnosing various diseases including cancer with high reliability by recovering a sample used for diagnosis from a biological sample with high purity and high yield.

Description

Disease diagnosis method using aqueous phase separation composition {Diagnosis method by aqueous two-phase composition}

The present invention relates to a disease diagnosis method using an aqueous phase separation composition.

Although cancer prevention and treatment methods have been developed remarkably in recent years, many people still recognize cancer as the most feared and incurable disease that modern medicine has not solved. The exact cause of cancer is still unknown. However, according to what is known so far, it is known that mutations occur in genes of normal cells or genes that suppress cancer.

Cancer can have a high cure rate when it is detected as early as possible and treated appropriately. Methods for diagnosing cancer that have been used up to now are physical methods, such as double imaging, compression imaging, or mucosal imaging. In addition, a biopsy (biopsy) can be performed to directly diagnose a suspected cancer. However, these methods have a sanitary problem and have disadvantages in that the patient has to suffer pain while the test is in progress, so an alternative diagnostic method is needed.

Meanwhile, exosomes, one of the representative biomarkers, are a kind of nanometer-sized spherical vesicles naturally secreted from cells in the body. It is understood that the exosomes are specifically secreted for the purpose of intercellular signal transduction, and contain functionally active substances such as proteins, lipids, and RNA contained in specific cells as they are. Therefore, it is possible to easily diagnose diseases by isolating exosomes from blood or urine and analyzing them to analyze characteristics related to various diseases.

The amount of exosomes affects the sensitivity of the diagnosis, and the purity affects the specificity of the diagnosis. Accordingly, a technology capable of isolating a large amount of exosomes as much as possible and simultaneously separating exosomes of the same high purity is required for diagnosis or development of therapeutic agents. However, since there are too many substances (proteins) other than exosomes in body fluid and the amount of exosomes is relatively small, there is a limit to applying the existing nanoparticle separation method. Although various isolation methods have been developed so far, there are few cases of successful diagnosis using exosomes.

When diagnosing a disease using exosomes, it is often analyzed using an antigen-antibody reaction. For example, in the case of diagnosis by analyzing the membrane protein of exosomes, antigen-antibody reaction is used.

At this time, in order to measure the amount of the target membrane protein, a separation technology that removes the antibody not attached to the membrane protein of the exosome is required. As the separation technology, an ultracentrifugation process is mainly used (US9,921,223, and US7,897,356). This method causes a lot of particle loss during the process, which limits the analysis. In particular, when the antigen-antibody reaction is applied multiple times (secondary antibody), most of the exosomes to which the antibody is attached are lost because the separation process must be repeated several times. The low content of exosomes due to this loss does not reflect information about the entire population. In addition, substances (proteins) other than exosomes that have not been separated still exist. As a result, when diagnosing a disease using the existing exosome separation method, the purity and content are low, thereby reducing the accuracy of measurement and analysis.

In addition, US 9952215 B2 discloses a separation technique through electrophoresis and a method of washing with saline (CA2453198), but this also still could not solve the problems in terms of purity and content.

U.S. Patent No. 9,921,223, 2018.03.20, Analysis of genomic DNA, RNA, and proteins in exosomes for diagnosis and theranosis U.S. Patent No. 7,897,356, 2011.03.01, Methods and systems of using exosomes for determining phenotypes U.S. Patent No. 9,952,215, 2018.04.24, Exosome analysis method, exosome analysis apparatus, antibody-exosome complex, and exosome electrophoresis chip Canadian Patent Publication No. 2453198, July 7, 2005, Quantification and generation of immune suppressive exosomes

Accordingly, the present inventors have conducted various studies to selectively remove only antibodies with little or no damage or loss of exosomes, and as a result of designing a two-phase phase separation system with a new composition of two aqueous solutions, high selection when using this system It was confirmed that it is possible to remove only the antibody in a short period of time, and it is possible to diagnose with high reliability when applied to the diagnosis of cancer, especially prostate cancer.

Accordingly, it is an object of the present invention to provide a method for diagnosing a disease in which antibody removal is performed using an aqueous phase phase separation composition.

In order to solve the above problem, the present invention

(S1) separating the extracellular vesicles from the biological sample;

(S2) binding the extracellular ER and a primary antibody specifically binding thereto through an antigen-antibody reaction;

(S3) binding a secondary antibody having colored or fluorescent properties through an antigen-antibody reaction for identification;

(S4) detecting information on the obtained complex; and

(S5) comparing the detection result with a normal control; provides a disease diagnosis method comprising a.

At this time, it is characterized in that after the steps (S2) and (S3), the step of removing impurities including unreacted antibodies using an aqueous phase-separation composition is performed.

The unreacted antibody may be a primary antibody and/or a secondary antibody.

In particular, the impurities are treated with an aqueous phase-separation composition such that the tension (γ) at the interface at which the first aqueous phase and the second aqueous phase are phase-separated satisfies the following Equation 1:

[Equation 1]

2 X 10 ^-6 J/m ² ≤ γ ≤ 500 X 10 ^-6 J/m ²

The first aqueous phase flows in a bulk form in the second aqueous phase, which is a continuous phase, by gravity or buoyancy, and by diffusion, impurities including unreacted antibody are removed from the first aqueous phase to the second aqueous phase.

The aqueous phase-separation composition according to the present invention can effectively remove impurities including unreacted antibodies after antigen-antibody reaction, thereby recovering antigen and antibody-bound extracellular vesicles with high purity and high yield.

This method can block the problem of damage or loss of extracellular vesicles when removing unreacted antibodies through the conventional centrifugation process, and has the advantage of reducing the pre-diagnosis preparation time from 7 hours to 3 hours.

The antigen and the antibody-bound extracellular vesicles are recovered with high purity, and thus can be preferably applied to diagnosis, monitoring and prediction of various diseases, particularly cancer.

1 is a flowchart showing the sequence of a disease diagnosis method according to the present invention.
2 is a schematic diagram for explaining the separation mechanism of the aqueous phase-separation composition presented in the present invention.
3 is a schematic diagram showing energy barriers in a first aqueous solution phase and a second aqueous solution phase.
4 is a simulation result showing movement according to particle size using three types of bead particles having different particle sizes.
FIG. 5 is a schematic diagram showing movement according to particle size using three types of bead particles having different particle sizes of FIG. 4 .
6 is a schematic diagram showing the energy barrier according to the interfacial tension and particle size of the first aqueous solution phase and the second aqueous solution phase.
7 is a graph showing the change in the critical particle size according to the interfacial tension between the first aqueous solution phase and the second aqueous solution phase.
8 is a graph showing a change in the escape velocity at the interface according to the particle size.
9 is a graph showing a change in a critical particle size according to a change in temperature.
10 is a photograph showing the movement of fluorescent beads after forming the first aqueous phase on the second aqueous solution in Example.
11 is a total reflection micrograph of the exosome-CD9-Alexa 488 complex obtained in Example 1 (a) and Comparative Example 1 (b).
12 is a graph showing changes in the content of Alexa-488 compared to the content of exosomes.
13 is a graph showing the change in the content of Alexa-488 compared to the CD9 concentration.
14 is a graph showing the expression level of PSMA compared to the expression level of CD9 in patients with benign prostatic hyperplasia (BPH) and prostate cancer (Pca).
15 is an ROC curve when diagnosis using the existing diagnostic method PSA and when diagnosis using APIF and PSA diagnosis are applied together.

The present invention relates to a method for diagnosing cancer with high reliability, in which impurities including unreacted antibodies are effectively removed within a short time after an antigen-antibody reaction in the stage of preparing a sample for diagnosis.

How to diagnose a disease

1 is a flowchart showing the sequence of a disease diagnosis method according to the present invention.

Referring to Figure 1, the disease diagnosis is

(S1) separating the extracellular vesicles from the biological sample;

(S2) binding the extracellular ER and a primary antibody specifically binding thereto through an antigen-antibody reaction;

(S3) binding a secondary antibody having colored or fluorescent properties through an antigen-antibody reaction for identification;

(S4) detecting information on the obtained complex; and

(S5) comparing the detection result with a normal control; provides a disease diagnosis method comprising a.

Hereinafter, each step will be described in detail.

(S1) extracellular vesicle isolation step

First, for disease diagnosis, a biological sample is collected and the extracellular vesicles are isolated therefrom.

The biological sample may be, but is not limited to, tissues, fractions, fluids, and cells isolated from mammals, including humans, for cancer diagnosis. More specifically, the biological sample is a material that can be a sample for diagnosing cancer, cell culture solution that can be dissolved in water, blood, plasma, serum, intraperitoneal fluid, semen, amniotic fluid, breast milk, saliva, One selected from the group consisting of bronchoalveolar fluid, tumor effusion, tears, runny nose and urine is possible. Such biological samples include proteins.

An extracellular vesicle is isolated from the biological sample for disease diagnosis.

The extracellular vesicle (EV) is an endoplasmic reticulum surrounded by a phospholipid bilayer having a size of 50 to 500 nm, and transports various substances such as proteins, nucleic acids, lipids, and metabolites at once. The extracellular vesicle includes exosomes, ectosomes, viruses, proteins, LDLs, HDLs, external vesicles microvesicles, microparticles, apoptosis, membrane particles, membrane vesicles, exosome-like vesicles, and ectosome-like vesicles. have.

Among them, exosomes refer to nano-sized extracellular vesicles secreted from cells and released into the extracellular space. The exosomes are separated from the inside and outside by a lipid bilayer composed of a cell membrane component, and membrane proteins, genetic material, and cytoplasmic components, allowing us to indirectly determine the properties and states of cells. In addition, exosomes bind to other cells and tissues to deliver membrane components, mRNAs, miRNAs, proteins (growth hormones, cytokines, etc.), and mediate cell-cell communication by delivering these messengers to recipient cells. acts as an extracellular transporter.

Isolation of the extracellular vesicles from the biological sample is not particularly limited in the present invention, and known as ultra-centrifugation isolation, size exclusion, immunoaffinity isolation, and microfluidics. Technology (microfluidics chip) and a method using a polymer (polymeric method), etc. are possible. Preferably, the separation process using the aqueous phase separation composition described in the present invention may be used.

(S2) primary antibody binding step

Next, a step of binding the extracellular ER and the primary antibody specifically binding thereto through an antigen-antibody reaction is performed.

A primary antibody specifically binds to the antigen of the extracellular endoplasmic reticulum, and includes all polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and parts thereof as long as they have antigen-binding properties. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody.

In one example, the primary antibody may be an exosome-specific marker, A33, ABL2, ADAM10, AFP, ALA, ALIX, ALPL, ApoJ / CLU, ASCA, ASPH (A-10), ASPH (D01P), AURKB, B7H3, B7H3, B7H4, BCNP, BDNF, CA125 (MUC16), CA-19-9, C-Bir, CD10, CD151, CD24, CD41, CD44, CD46, CD59 (MEM-43), CD63, CD63, CD66eCEA, CD81, CD81, CD9, CD9, CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3 (ErbB3), HSP70, HSPB1, hVEGFR2, iC3b, IL-1B, IL6R, IL6Unc, IL7Ralpha/CD127, IL8, INSIG-2, Integrin, KLK2, LAMN, Marmoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Muc1, MUC17, MUC2, Ncam, NDUFB7, NGAL, NK-2R(C-21), NT5E (CD73), p53 , PBP, PCSA, PCSA, PDGFRB, PIM1, PRL, PSA, PSA, PSMA, PSMA, RAGE, RANK, RegIV, RUNX2, S100-A4, Seprase/FAP, SERPINB3, SIM2(C-15), SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, TFF3, TGM2, TIMP-1, TMEM211, Trail-R2, Trail-R4, TrKB(poly), Trop2, Tsg101, TWEAK, UNC93A, VEGFA, wnt-5a(C-16 ), and the like.

The antigen-antibody reaction for binding the primary antibody is not particularly limited in the present invention, and a known method may be used.

In one example, the primary antibody may be treated by dilution in a ratio of 1:10 to 1:5000, and reacted at a temperature of 4 to 37° C. for 30 minutes to 12 hours. More preferably, the treatment is diluted in a ratio of 1:100 to 1:500, and the reaction may be performed at a temperature of 4 to 27° C. for 60 to 12 hours. If the dilution ratio, temperature, and time conditions are out of the above ranges, the primary antibody response may be reduced and the cell staining process may not be performed normally.

In this case, a cancer-specific antigen (or tumor marker) may be further included to diagnose a disease, particularly cancer.

The cancer-specific antigen may vary depending on the type of cancer, and any specific antigen known in the art may be used. For example, the prostate cancer-specific antigen is PSA, PSMA, PCSA, PSCA, B7H3, EpCam, TMPRSS2, mAB 5D4, XPSM-A9, XPSM-A10, galectin-3, E-selectin, galectin-1, or binding agents including, but not limited to, one or more binding agents for E4 (IgG2a kappa), or any combination thereof.

The complex obtained after this antigen-antibody reaction is used in the next step after removing the unreacted primary antibody. At this time, the unreacted primary antibody is removed using an aqueous phase separation composition described below.

(S3) secondary antibody binding step

Next, for identification, a step of binding a secondary antibody having colored or fluorescent properties through an antigen-antibody reaction is performed.

A secondary antibody is an antibody that detects a target protein captured by the primary antibody. Due to the use of the secondary antibody, it can be quantitatively measured through the magnitude of the signal of the detection label. .

Suitable detection labels include magnetic labels, fluorescent moieties, enzymes, chemiluminescent probes, metal particulates, non-metallic colloidal particulates, polymer dye particulates, pigment molecules, pigment particulates, electrochemically active species, semiconductor nanocrystals or quantum dots or gold particles, fluorophores, quantum dots, or other nanoparticles comprising radioactive labels. Protein labels include green fluorescent protein (GFP) and variants thereof (eg, cyan fluorescent protein and yellow fluorescent protein) as described below; and a luminescent protein such as luciferase. Labeled radioactive isotopes (radionuclides), such as 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu, 211 At, or 213 Bi. Fluorescent labels include, but are not limited to, rare earth chelates (eg, europium chelates), rhodamine, fluorescein, eg, FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; TAMRA; single room; Lissamine; cyanine; phycoerythin; Texas Red; Cy3, Cy5, Dapoxyl, NBD, Cascade Yellow, Dansyl, PyMPO, Pyrene, 7-diethylaminocoumarin-3-carboxylic acid and other coumarin derivatives, Marina Blue®, Pacific Blue®, Cascade Blue® , 2-anthracenesulfonyl, PyMPO, 3,4,9,10-perylene-tetracarboxylic acid, 2,7-difluorofluorescein (Oregon Green® 488-X), 5-carboxyfluorescein Shin, Texas Red-X, Alexa Fluor 430, 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), BODIPY FL, Bimane, and Alexa Fluor 350, 405, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, and 750, and derivatives thereof. For example, "The Handbook--A Guide to Fluorescent Probe and Labeling Technologies," Tenth Edition, available on the internet at probe (dot) invitrogen (dot) com/handbook. The fluorescent label may be one or more of FAM, dRHO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ, Gold540 and LIZ.

The antigen-antibody reaction for binding the secondary antibody is not particularly limited in the present invention, and a known method may be used.

For example, the secondary antibody may be treated by diluting in a ratio of 1:100 to 1:10000, and reacted at a temperature of 4 to 37°C for 30 minutes to 12 hours. More preferably, the treatment is diluted in a ratio of 1:1000 to 1:5000, and the reaction may be performed at a temperature of 4 to 28° C. for 60 minutes to 12 hours. If the dilution ratio, temperature, and time conditions are out of the above ranges, the secondary antibody response may be reduced and the cell staining process may not be performed normally.

The complex obtained after this antigen-antibody reaction is used in the next step after removing unreacted secondary antibody. At this time, the unreacted secondary antibody is removed using an aqueous phase-separation composition described below.

(S4) information detection step

Next, the complex to which the primary antibody and the secondary antibody are bound is applied to an analysis device to detect information obtained therefrom.

The assay device measures the mRNA level of the extracellular vesicles of the complex or the expression level of the protein.

Analytical methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, and DNA chip.

In addition, as an analysis method for measuring the expression level of a protein, Western blot (Western blot), ELISA (Enzyme Linked Immunosorbent Assay), radioimmunoassay (otA: badioimmunoassay), radioimmuno-diffusion method, Oukteroni immune diffusion method, Methods such as rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip may be used, and the present invention is not particularly limited.

(S5) Disease diagnosis stage through comparison with normal control group

Next, the disease is diagnosed by comparing the obtained detection result with a normal control group.

In the present invention, the disease may be cancer, and cancer may be diagnosed, monitored, or predicted through the above steps.

As used herein, the term "diagnosis" refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, or a specific disease or disorder determining the prognosis (e.g., identification of a pre-metastatic or metastatic cancer state, staging the cancer, or determining the responsiveness of the cancer to treatment) of a subject with monitoring the condition of the subject to provide information on efficacy).

As used herein, the term “prognosis” refers to the prospect of future symptoms or course determined by diagnosing a disease. In cancer patients, the prognosis usually refers to whether or not metastasis or survival period within a certain period of time after cancer onset or surgical operation. Prediction of prognosis is a very important clinical task because it provides clues about the future direction of colorectal cancer treatment, including whether or not chemotherapy is used in colorectal cancer patients.

As used herein, the term “metastasis” refers to a state in which a tumor is transplanted from its primary site to another body site along various routes, and settled and proliferated there. Since cancer metastasis is not only determined by the unique characteristics of the cancer, but also an event that is the most important clue in determining the prognosis of cancer, it is treated as the most important clinical information related to the survival of cancer patients.

Applicable cancers include breast cancer, colorectal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, eye tumor, peritoneal cancer, uterine cancer, ovarian cancer, rectal cancer , perianal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , prostate cancer, testicular cancer, oral cancer, gallbladder cancer, cholangiocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and at least one selected from the group consisting of pituitary adenoma, preferably prostate cancer.

In this step, by using normal cells that do not have cancer as a control, and comparing the expression level of the protein with the control, it is possible to diagnose, monitor, or predict cancer.

In addition, after treatment, the patient's cells can be used as a control to monitor the patient's condition.

In particular, when the complex isolated according to the present invention is applied to cancer diagnosis as a biomarker, there is an advantage that analysis is possible only with a trace amount of a sample obtained in a non-destructive way from a patient. In addition, specificity and sensitivity for cancer can be combined at the same time. In addition, there is an advantage that the time from preparation of the sample to diagnosis can be significantly reduced and the cost can also be lowered.

This effect is affected by the extent to which unreacted antibodies are removed after the antigen-antibody reaction, and the extent to which the antibody-bound extracellular vesicles are lost and damaged. After the conventional antigen-antibody reaction, the unreacted antibody is subjected to ultracentrifugation at 160,000 x g. This ultracentrifugation process takes a long preparation time of at least 7 hours or more as the antigen-antibody reaction over two steps of the primary antibody and the secondary antibody is performed, and the extracellular vesicles are damaged or lost. A problem occurred.

Therefore, in the present invention, the unreacted antibody can be separated within a short time and removed using an aqueous phase phase separation composition to inhibit damage or loss of the extracellular vesicles.

In the present invention, impurities including unreacted primary antibody and/or secondary antibody after the antigen-antibody reaction of steps (b) and (c) are removed using an aqueous phase phase separation composition.

Removal of unreacted antibody using aqueous phase separation composition

The aqueous solution phase system referred to in the present invention means that aqueous solutions having different densities are phase-separated in a liquid-liquid state.

Therefore, the aqueous phase phase separation composition proposed in the present invention is two types of aqueous solutions are present in a phase-separated form, and impurities and targets are present at the interface (ie, interface) of the phase-separated state, and at this time, through the escape of the impurities It means that separation between them is possible.

The unreacted primary antibody and secondary antibody represented by impurities have a size of about 5 to 40 nm. In contrast, the complex obtained through (S2) is a primary antibody bound to the extracellular ER. Considering that the minimum size of the extracellular ER is 50 nm, the complex has a size larger than this. In addition, the complex obtained through (S3) is a secondary antibody is additionally bound, and has a size in a larger range than the above size.

Accordingly, in consideration of the difference in particle size between the unreacted primary antibody, the secondary antibody, and the complex, if a filter capable of passing only a specific range of sizes is used, impurities including the complex and the unreacted antibody can be effectively separated.

Separation mechanism using aqueous phase phase separation composition

A mechanism for removing impurities through separation of impurities/complexes including unreacted antibodies using an aqueous phase-separation composition is described as follows.

In this case, the impurities include unreacted antibody, a small amount (negligible) of antigen and extracellular endoplasmic reticulum, and the unreacted antibody is predominantly present (dominant). In addition, the complex refers to an extracellular vesicle to which a primary antibody is bound or an extracellular vesicle to which a primary and a secondary antibody are bound. Since the impurities and complexes are particles having a nano-level size, they are described below as the expression of nanoparticles.

2 is a schematic diagram for explaining the separation mechanism of the aqueous phase-separation composition presented in the present invention.

First, two types of nanoparticles to be separated are prepared. The two types of nanoparticles are for explanation, and all nanoparticles having different particle sizes are mixed for separation.

Next, two aqueous solutions are prepared to form an aqueous phase phase system. At this time, the object that needs to be separated (mixed nanoparticles, that is, impurities) is included in any one of the first aqueous phase (P1) and the second aqueous phase (P2). In FIG. 2, for convenience of explanation, the mixture was mixed in the first aqueous solution phase (P1).

In the formation of an aqueous phase phase, two aqueous solutions exist, and these aqueous solutions are phase-separated into a first aqueous solution phase (P1) and a second aqueous solution phase (P2) by contact as shown in FIG. 2(a).

The first aqueous phase P1 exists in a state of flowing in the second aqueous phase P2 due to gravitational force or buoyancy. When the first aqueous phase (P1) has a higher density than the second aqueous phase (P2), it moves in the direction of gravity, but does not sink to the bottom due to buoyancy and is floating in the second aqueous phase (P2).

In particular, the first aqueous solution phase (P1) is present in the form of a bulk (bulk), not in the state of being in the form of fine particles or droplets or the upper and lower layers are separated like the upper/lower layer. In conventional particle separation using an aqueous phase phase system, a process such as vortexing is performed to split the first aqueous solution phase into fine particles and separation of nanoparticles occurs, but in the present invention, separation of nanoparticles in the bulk form is performed. . In addition, in the conventional aqueous phase phase system, the energy barrier of the aqueous solution interface is changed by the external force applied during the vortexing process, and the particles can freely pass through the interface by this external force, so the filtering of the particles by the phase interface The separation effect due to the affinity between the particle surface and each aqueous phase is more pronounced than the effect.

Referring to FIG. 2(b), the mixed nanoparticles present in the first aqueous solution phase P1 in the bulk state actively undergo Brownian motion, which moves irregularly in the aqueous phase, and by this motion, the first It is in contact with the interface between the aqueous solution phase (P1) and the second aqueous solution phase (P2).

At this time, the first aqueous phase (P1) and the second aqueous phase (P2) come into contact and are trapped at the interface, and the second aqueous phase (P2) or the second aqueous phase (P2) or the second aqueous phase (P2) by the diffusion coefficient of the trapped nanoparticles 1 It moves to the aqueous phase (P1) or remains at the interface. As a result, as shown in FIG. 2(c) , some of the mixed nanoparticles of the first aqueous solution phase P1 move to the second aqueous solution phase P2 through the pores. As a result, after a certain period of time, some of the mixed nanoparticles remain in the second aqueous phase (P2), the rest remain in the first aqueous phase (P1) or the interface, and the second aqueous phase (P2) includes an interface Thus, the separation of nanoparticles can be performed through the recovery of nanoparticles present therein.

In particular, in the process of moving the first aqueous solution phase P1 to the lower side by gravity after the first aqueous solution phase P1 is injected into the second aqueous solution phase P2, the first aqueous solution phase P1 continuously interacts with the new second aqueous solution phase P2. Since they meet to form an interface, the movement of nanoparticles at the interface between the two phases P1 and P2 may be accelerated while continuously occurring. In the phase-separated form, which is one of the phase-separated forms, the continuous movement of nanoparticles cannot occur because it is impossible to continuously form a new interface. In addition, in the form of phase-separated fine droplets, a new interface may be formed by stirring, etc., but the energy barrier at the interface is changed by the external force applied during the stirring process, and the particles can freely pass through the interface by this external force. However, it is not suitable because many particles are lost through the interface during the stirring process. In this method, the separation effect due to the affinity between the particle surface and each aqueous phase (P1, P2) is prominent rather than the filtering effect at the interface, and the separation occurs by a mechanism different from the present invention.

Separation between impurities/complexes through the aqueous phase separation composition of the present invention is affected by the tension at the interface of the aqueous solution forming the first aqueous phase (P1) and the second aqueous phase (P2).

Interfacial tension refers to the tension generated at this interface when two or more different objects form a layer without mixing and contact. The first aqueous phase (P1) and the second aqueous phase (P2) have different densities including different solutes, and tension is formed at the interface due to the size difference and physical properties of the different solutes. By this tension, an energy barrier is formed on the surface that particles must cross, and this energy barrier acts like a pore. If the magnitude of the tension is large, the height of the energy barrier increases and the pores tend to be small, The size of the pores at the interface, that is, pores having a threshold diameter (or threshold diameter) are formed. A part of the mixed nanoparticles moves to the first aqueous solution phase P1 through the pores thus formed.

The critical diameter is usually at the nm level, and the interfacial tension between the first aqueous solution phase P1 and the second aqueous solution phase P2 must have a certain range to maintain the nm level. That is, the small interfacial tension means that the first aqueous solution phase P1 and the second aqueous solution phase P2 can be miscible with each other, so particle separation cannot occur. However, the critical diameter is so small that the separation of the complex cannot be performed. The relationship between the interfacial tension and the critical diameter is a new concept that has not yet been proposed in the field of research on aqueous phase systems.

The numerical value of the interfacial tension set by the first aqueous phase (P1) and the second aqueous phase (P2) can be designed by the composition of the first aqueous phase (P1) and the second aqueous phase (P2), and such a composition The design of is dependent on whether impurities and/or complexes in the biological sample to be separated can diffuse beyond the energy barrier at the interface formed by the contact of the first aqueous phase (P1) and the second aqueous phase (P2).

3 is a schematic diagram showing the energy barrier of the first aqueous phase (P1) and the second aqueous phase (P2).

Referring to FIG. 3 , the first aqueous solution phase P1 and the second aqueous solution phase P2 are aqueous solutions containing different solutes, and energy barriers having different heights exist. The mixed nanoparticles present in the first aqueous solution phase P1 escape to the second aqueous solution phase P2 by crossing the energy barrier between the interface and the second aqueous solution phase P1 for particle separation. In this case, when the device of FIG. 2 is used, some nanoparticles escaping to the second aqueous solution phase P2 do not go back to the first aqueous solution phase P1. The passage of the energy barrier at the interface, that is, the escape energy in the first aqueous phase P1, is affected by the tension at the interface at the interface between the first aqueous phase P1 and the second aqueous phase P2. .

The higher the surface tension of the interface, the higher the energy barrier that the particles at the interface must cross to escape to another phase, thereby reducing the critical size of the particles that can escape the interface. This means that the interfacial tension of the interface can be used as a variable depending on how the phases of the first and second aqueous solutions to be used are selected.

In addition, the movement and escape of nanoparticles from the first aqueous solution phase P1 to the second aqueous solution phase P2 at the interface is explained through Fick's laws of diffusion. The fixed diffusion law is two laws representing the diffusion process in thermodynamics. There are a first law and a second law, and in the present invention, it can be described as Fick's second law related to continuous diffusion.

Particle movement was predicted using Fick's second law and the escaping rate of particles trapped at the interface. Equation (1) is satisfied for the rate at which particles trapped at the interface escape.

------------------ 식 (1)

------------------ Equation (1)

(In Equation (1), Γ: ratio of particles escaping from the interface, J: flux of particles, n is the concentration of particles at the interface, α: proportionality constant, ΔΕ: the energy difference between the particles at the interface and the particles escaping from the interface, κ: Boltzmann’s constant, T: temperature.)

The energy barrier (ΔΕ) of particles at the interface between the first aqueous solution phase P1 and the second aqueous solution phase P2 can be expressed by the following formula (2).

---식 (2)

---Equation (2)

(In the above formula (2), γ _Phase/Phase2 : Tension acting at the interface of the first aqueous phase and the second aqueous phase, R: Radius of the _{particle, γ particle/Phase1} : Acting on the surface of particles in the first aqueous phase tension, γ _{particle/Phase2} : tension acting on the surface of particles in the second aqueous phase, κ: Boltzmann constant, T: absolute temperature, K: separation coefficient)

In the above equation, ΔΕ means an energy barrier that particles at the interface must cross to escape from the interface, and the larger the value, the lower the escape rate of the particles.

In addition, the flow of particles near the interface between the first aqueous solution phase P1 and the second aqueous solution phase P2 satisfies the following equation.

----------------- 식 (3)

----------------- Equation (3)

--------------식 (4)

--------------Equation (4)

(In the above formulas (3) and (4), J _{Phase1-interface} : flow of particles between the first aqueous phase and the interface, J _{Interface-Phase2} : flow of particles between the second aqueous phase and the interface, C _Phase1 : interface the concentration of particles near the first aqueous phase, C _Phase2: the concentration of particles in the invention near the interface 2 aqueous phase, C _interface: the concentration of particles in the boundary surface, κ1, κ2: proportional constant Γ _Phase1: first receiving at the interface The ratio of particles escaping to the liquid phase, Γ _Phase2 : It is the ratio of particles escaping from the interface to the second aqueous phase)

Looking at the above formula, the flow of particles between the second aqueous phase P2 and the interface is the flow of particles moving from the second aqueous phase P2 to the interface and the flow of particles escaping from the interface to the second aqueous phase P2. is the sum of Similarly, the flow of particles between the first aqueous phase P1 and the interface is the sum of the flow of particles moving from the first aqueous phase P1 to the interface and the flow of particles escaping from the interface to the first aqueous phase P1.

Simulation was performed by applying Equation (1, 2, 3, 4) to Fick's second law.

). 이에, 제2수용액상(P2)으로 탈출한 비드 입자의 양을 측정하여 지배 방정식과 경계 조건의 계수를 결정했다. 결정된 계수를 바탕으로 입자의 크기, 여과 시간, 분리 계수(partition coefficient), 온도, 경계면의 표면 장력에 따른 입자의 분리를 시뮬레이션 했다. For the particles, three types of mixed bead particles of 10 nm, 50 nm and 100 nm were applied and the amount of bead particles escaping to the second aqueous phase (P2) was measured to determine the governing equation and coefficient of boundary conditions. Dextran aqueous solution (1% concentration) was used as the first aqueous phase (P1), and polyethylene glycol (3% concentration) was used as the second aqueous phase (P2). In addition, since the first aqueous solution phase P1 continuously contacts the new second aqueous solution phase P2 in the above equation, it is assumed that the concentration of particles in the second aqueous solution phase P2 near the interface is close to zero. (

). Accordingly, by measuring the amount of bead particles escaping to the second aqueous phase (P2), the governing equation and the coefficient of the boundary condition were determined. Based on the determined coefficient, particle separation according to particle size, filtration time, partition coefficient, temperature, and surface tension of the interface was simulated.

4 is a simulation result showing movement according to particle size using three types of bead particles having different particle sizes.

4(a) and 4(b), the critical size of the bead particles passing through the interface gradually increased over time, and based on this, it was found that the small bead particles escape to the second aqueous solution phase (P2). Able to know.

Specifically, referring to FIG. 4( a ), the increase in the critical size of the bead particles that can pass through the interface over time abruptly occurs within 200 seconds and then converges to a certain size. In addition, referring to FIG. 4(b), it can be seen that all nanoparticles having a size of 10 nm passed after 60 minutes, and 50 nm and 100 nm remained at the interface. This means that the aqueous phase separation composition serves as a filter through which only particles having a specific size or less pass therethrough.

In particular, in the case of 10 nm particles, it can be seen that they completely escape after 60 minutes, and it can be seen that nanoparticles can be separated with high purity, that is, a yield close to 100% or close to 100%, without loss of nanoparticles from the mixed nanoparticles. From these results, when a biological sample containing an actual impurity and complex is applied to the separation process, problems such as loss of the complex occurring in the separation process such as a conventional separation membrane are fundamentally blocked, and the aqueous phase phase separation composition according to the present invention is used to It can be seen that the advantage of being able to isolate a complex from a biological sample with high purity can be secured.

This can be seen in more detail through the schematic diagram of FIG. 5 .

FIG. 5 is a schematic diagram showing movement according to particle size using three types of bead particles having different particle sizes of FIG. 4 .

As shown in FIG. 5 , the particles move from the first aqueous solution phase P1 to the interface formed by contacting the second aqueous solution phase P2. Part of the particles in contact with the interface are trapped at the interface, and at this time, escape from the first aqueous solution phase P1 to the second aqueous solution phase P2 according to the size of the particles.

FIG. 5 shows three types of interfaces, and in the case of Interface A, only the smallest sized particles pass, in Interface B up to medium sized particles, and in the case of Interface C, all of them can pass. Through this, nanoparticles can be selectively separated from mixed nanoparticles according to particle size.

More specifically, when the smallest particle among the three particles is to be separated, it is designed like Interface A, and the small particle is passed through the second aqueous phase (P2), and then it can be recovered and separated. In addition, when the largest particle size is to be separated, it is designed like Interface B, and the large size particles remaining in the first aqueous solution phase (P1) can be recovered and separated. In addition, in the case of medium-sized particles, after designing like Interface B, the second aqueous phase (P2) is recovered, and it is designed to have an Interface α (not shown) again, and then in the second aqueous phase (P2). Only medium-sized particles can be selectively recovered through a process of once again separating small-sized particles and medium-sized particles.

Whether the nanoparticles are trapped or passed through Interface A, Interface B, and Interface C is, as mentioned above, an energy barrier according to each composition constituting the first aqueous phase (P1) and the second aqueous phase (P2); It depends on the tension (ie, interfacial tension) at the interface they come into contact with and form. When the energy barrier of the first aqueous phase (P1) and the second aqueous phase (P2) is low, the interfacial tension formed by contacting them decreases, and as the interfacial tension decreases, the size of particles passing through the interface (that is, critical particle size) may increase.

6 is a schematic diagram showing the energy barrier between the first aqueous solution phase (P1) and the second aqueous solution phase (P2) according to interfacial tension and particle size.

In FIG. 6 , the higher the interfacial tension between the first aqueous phase P1 and the second aqueous phase P2 is, the higher the energy barrier is. At this time, there is a difference in the energy barrier that each of 10 nm, 50 nm, and 100 nm nanoparticles must cross, and has the lowest energy barrier at 10 nm. That is, the smaller the particle size, the lower the energy barrier that must be crossed when escaping from the interface to another phase. When the particle exceeds a certain size, the energy barrier is too high, and the particle cannot cross this interface in a trapped state at the interface. .

When the first aqueous phase (P1) and the second aqueous phase (P2) come into contact to form an interface, the nanoparticles with a size of 10 nm having a low energy barrier escape to the second aqueous phase (P2) first. Accordingly, large-sized nanoparticles with a size of 100 nm are trapped at the interface, and in order to pass at the interface, the interfacial tension of the first aqueous phase (P1) and the second aqueous phase (P2) must be lowered in proportion to the energy barrier. It can be seen that the lowered, escape to the second aqueous solution phase (P2) may occur.

This result means that the critical particle size through which the trapped nanoparticles can pass through the interface can be dominantly determined according to the interfacial tension. In other words, the low interfacial tension means that the size of the nanoparticles passing through it can be increased.

7 is a graph showing the change _{in the critical particle size (D T} ) according to the interfacial tension of the first aqueous solution phase (P1) and the second aqueous solution phase (P2).

Referring to FIG. 7 , it can be seen that the critical particle size at which the particles trapped at the interface escape varies according to the interfacial tension.

Specifically, it can be seen that the higher the interfacial tension, the smaller the critical particle size of particles capable of escaping the interface tends to be finally reduced. In addition, it can be seen that the critical particle size of the particles is changed according to time until a specific time is reached, but the critical particle size finally reached is the same. In addition, it can be seen that the critical particle size of the particles increases with time. By analyzing this, it is possible to know the time it takes to remove the desired size. For example, it can be seen that in the case of an aqueous phase separation composition designed to have an interfacial tension of 5X10 ^-6 J/m ² , nanoparticles having a critical particle size of 40 nm can be separated within 30 minutes.

Through these results, in the present invention, in order to separate the complex from the biological sample containing the impurities and the complex, the first aqueous solution phase (P1) and the second aqueous solution phase (P2) have interfacial tension satisfying Equation 1 below (γ) must have a range.

[Equation 1]

2 X 10 ^-7 J/m ² ≤ γ ≤ 50 X 10 ^-5 J/m ²

The size of pores that can be implemented through the aqueous phase separation composition according to the present invention is 1 to 500 nm, preferably 3 to 450 nm, more preferably 5 to 400 nm, more preferably 5 to 350 nm, even more preferably 10 to 250 nm , most preferably in the range of 30 to 180 nm.

In order to have the above pores, the aqueous phase separation composition has an interfacial tension formed at the interface of 2 X 10 ^-7 J/m ² to 50 X 10 ^-5 J/m ² , preferably 2 X 10 ^-7 J/m ² to 40 X 10 ^-6 J/m ² , more preferably 3 X 10 ^-6 J/m ² to 350 X 10 ^-6 J/m ² , more preferably 4 X 10 ^-6 J/m ² to 270 X 10 ^-6 J/m ² , even more preferably 5 X 10 ^-6 J/m ² to 150 X 10 ^-6 J/m ² , most preferably 10 X 10 ^-6 J/m ² to 60 X 10 ^It has a range of -6 J/m ^{2 .}

At this time, if the interfacial tension is not within the range shown in Equation 1, but the numerical value is decreased and out of the limit to some extent, phase separation does not occur and mixing of the first aqueous phase (P1) and the second aqueous phase (P2) occurs. If the aqueous phase phase separation composition is not configured, and the interfacial tension is higher than a certain value, impurities or complexes are trapped at the interface and escape does not occur. This is data that can prove that a stable aqueous phase phase-separation composition can be constructed only when the interfacial tension numerical range of Equation 1 presented in the present invention is capable of being separated through the escape of impurities or complexes.

In addition, looking at the separation time, it can be seen that the smaller the size of the impurity or the complex, the faster the rate of escaping from the interface.

According to the change of the critical particle size according to the interfacial tension shown in FIG. 7, when the compositions of the first aqueous phase (P1) and the second aqueous phase (P2) are designed to have a specific interfacial tension, the complex can be easily separated with high purity. It can be predicted that there is

Preferably, the aqueous phase separation composition according to the present invention controls the interfacial tension through the design of the composition, and the critical particle size is 1 to 500 nm, 3 to 450 nm as the critical particle size can be controlled by adjusting the interfacial tension , more preferably from 5 to 400 nm, more preferably from 5 to 350 nm, still more preferably from 10 to 250 nm, and most preferably from 30 to 180 nm. In this case, the aqueous phase-separation composition can be separated up to a size difference of about 10 nm between the impurity and the complex in separation ability. At this time, the resolution of 10 nm means that nanoparticles can be separated even from those having a difference of 10 nm, such as 10 nm and 20 nm, and separation in the case of showing a difference of 90 nm, such as 10 nm and 100 nm, can be self-evidently performed do.

On the other hand, FIG. 7 is a schematic diagram divided into a case in which the first aqueous phase P1 is a stationary phase and a case in which the first aqueous phase P1 is a moving phase. When the time is infinity, the first aqueous phase (P1) showed the same results in both the stationary phase and the fluidized phase, but within a predetermined time, when the first aqueous phase (P1) has a fluidized phase, it is advantageous for the separation of nanoparticles. Able to know. Considering that nanoparticle separation is typically performed within a predetermined time rather than an infinite time, when the first aqueous solution phase P1 has a fluidized phase, it can be seen that it is preferable to apply it to the actual separation process.

Design of aqueous phase separation composition

The tension at the interface between the first aqueous solution phase P1 and the second aqueous solution phase P2 may be achieved by designing the composition of the first aqueous solution phase and the second aqueous solution phase.

In order to have interfacial tension between the first aqueous phase P1 and the second aqueous phase P2, phase separation between them must be preceded. The phase separation may be performed in various other methods, but in the present invention, it may be achieved through a two phase diagram between the first aqueous phase P1 and the second aqueous phase P2. In addition, in the separation of impurities and complexes, the separation rate and separation ability may be improved depending on whether these surfaces have affinity for any one of the first aqueous phase (P1) and the second aqueous phase (P2). In consideration of these details, a specific composition constituting the first aqueous phase (P1) and the second aqueous phase (P2) may be selected through a two phase diagram to constitute an aqueous phase phase separation composition.

The first aqueous phase (P1) and the second aqueous phase (P2) are basically aqueous solutions in which a solute is dissolved in water.

Depending on the type of the solute, the first aqueous phase (P1) and the second aqueous phase (P2) may be an aqueous polymer solution or an aqueous salt solution in which a polymer and/or a salt are present.

The polymer as the solute may be a hydrophilic polymer.

Hydrophilic polymers that can be used include polyarginine, polylysine, polyethylene glycol, polypropylene glycol, polyethyleneimine, chitosan, protamine, polyvinyl acetate, hyaluronic acid, chondroitin sulfate, heparin, alginate, hydroxyoxypropyl methylcellulose, It may be one type of hydrophilic polymer selected from the group consisting of gelatin, starch, poly(vinyl methyl ether ether), polyvinylpyrrolidone, and combinations thereof.

In addition, the polymer as a solute may be a polymer polysaccharide. The polymer polysaccharide is cyclodextrin, glucose, dextran, mannose, sucrose, trehalose, maltose, ficoll, inositol, mannitol, sorbitol, sucrose-mannitol, glucose-mannitol, trehalose-polyethylene glycol, It may be one type of hydrophilic polymer selected from the group consisting of sucrose-polyethylene glycol, sucrose-dextran, and combinations thereof.

The salt used in the aqueous salt solution is (NH ₄ ) ₂ SO ₄ , Na ₂ SO ₄ , MgSO ₄ , K ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, KCl, NaBr, NaI, LiCl, n-Bu ₄ NBr, n _{-Pr 4} NBr, Et ₄ NBr, Mg(OH) ₂ , Ca(OH) ₂ , Na ₂ CO ₃ , ZnCO ₃ , Ca ₃ (PO ₄ ) ₂ , ZnCl ₂ , (C ₂ H ₃ ) ₂ Zn, ZnCO ₃ , CdCl ₂ , HgCl ₂ , CoCl ₂ , (CaNO ₃ ) ₂ , BaCl ₂ , MgCl ₂ , PbCl ₂ , AlCl ₃ , FeCl ₂ , FeCl ₃ , NiCl ₂ , AgCl, AuCl ₃ , CuCl ₂ , sodium dodecyl sulfate ( sodium dodecyl sulfate, sodium tetradecyl sulfate, dodecyltrimethylammonium bromide, dodecyltrmethylammonium chloride, tetradecyltrimethylammonium bromide, and combinations thereof It may be one type of hydrophilic polymer selected from the group.

In addition, a polymer salt may be used as the solute, and for example, a combination of the polymer and salt as described above may be used.

The selection of the first aqueous solution phase (P1) and the second aqueous solution phase (P2) among the above-mentioned polymers and salts depends on the characteristics (eg, surface characteristics) of the nanoparticles to be separated, and the characteristics and concentrations that enable phase separation. may vary depending on

Among them, each combination that can be used as the first aqueous phase (P1) and the second aqueous phase (P2) may have a hydrophilicity-hydrophobic property in the case of a polymer. Since the polymer is basically dissolved in an aqueous solution, it exhibits hydrophilicity, but when the two compositions are combined, it may have relatively hydrophilicity or relatively hydrophobicity. For example, in the case of dextran and polyethylene glycol, dextran has a relatively hydrophilicity, molecular structure is more dense, polyethylene glycol has relatively hydrophobicity, and molecular structure is less dense. There are characteristics. Accordingly, dextran/polyethylene glycol may be used as the first aqueous phase/second aqueous phase (P1/P2), respectively.

In addition, in the case of a polymer, molecular weight and concentration may be important selection reasons for each combination that can be used as the first aqueous phase (P1) and the second aqueous phase (P2).

As the molecular weight of the polymer increases and the concentration increases, the first aqueous solution and the second aqueous solution phase are stably formed. If the molecular weight of the polymer is too small, the first aqueous solution and the second aqueous solution are easily mixed.

The molecular weight of the polymer should exist in a dissolved (or swollen) state in the minimum aqueous solution, which has a difference in solubility in water depending on the type of polymer, so it is not easy to limit the range. However, in the case of the hydrophilic polymer as described above, the weight average molecular weight is 200 to 2,000,000, preferably 500 to 1,000,000, more preferably 1000 to 500,000. For example, in the case of polyethylene glycol in combination with dextran, those having a weight average molecular weight in the range of 200 to 60000, preferably 500 to 40000 are used. In addition, the dextran has a weight average molecular weight in the range of from 15 to 1000,000, preferably from 1000 to 500,000.

In this case, the concentration of the polymer or the aqueous polymer salt solution may be 0.001 to 20% by weight, preferably 0.01 to 15% by weight, although there is a difference in solubility in water. If the concentration is too low, the aqueous polymer solution of the first aqueous solution phase (P1) and the second aqueous solution phase (P2) exhibits fluidity similar to that of water, and the two are miscible with each other, making it difficult to form an aqueous phase ideal system. On the other hand, if it is too high, it takes time to dissolve the polymer, and the tension at the interface of the aqueous phase phase is too high, so that the critical particle size is small, which makes it difficult to separate nanoparticles.

When a salt is used instead of a polymer, a high concentration of salt is required to form an aqueous solution phase. As described above, as the molecular weight of the polymer increases, the first aqueous phase (P1) and the second aqueous phase (P2) are stably formed. In the case of salt, since the molecular weight is smaller than that of the polymer, the aqueous phase phase can be formed only at high concentration. Preferably, the high concentration salt is used in an amount of 1 to 70% by weight, more preferably 5 to 50%.

When a low concentration of salt is added to a system that can already form the first aqueous phase and the second aqueous phase, the salt is dissociated into an ionic state in the aqueous solution and serves to change the movement speed of the nanoparticles. In this case, the salt preferably has an average molecular weight of 10 to 1000 parts by weight.

Specifically, the combination of the first aqueous phase / second aqueous phase (P1/P2) in the aqueous phase separation composition according to the present invention is a combination of a polymer-polymer, and a polymer-high concentration salt, as shown in Table 1 below. Can be used. .

1st aqueous phase (P1, dispersed phase) 2nd aqueous phase (P2, continuous phase) Polymer-Polymer Combination 1% Dextran 3% polyethylene glycol (MW= 35000) 2% Dextran 5% polyvinylpyrrolidone (MW=5000) 2% Dextran 2% polyvinyl alcohol (MW=130000) 5% Dextran 5% Ficoll (MW=400) 3% polyvinylmethyl ether (MW=5000) 5% polyethylene glycol (MW=35000) Polymer-High Concentration Salt Combination 10% (NH ₄ ) ₂ SO ₄ 20% polyethylene glycol (MW=35000) 10% Na ₂ SO ₄ 20% polyethylene glycol (MW=35000) 10% MgSO ₄ 20% polyethylene glycol (MW=35000) 10% K ₂ HPO ₄ 20% polyethylene glycol (MW=35000) 10% KH ₂ PO ₄ 20% polyethylene glycol (MW=35000) 10% Na ₂ CO ₃ 20% polyethylene glycol (MW=35000)

The combination shown in Table 1 is an example, and any combination may be used as long as the various combinations using the composition as described above satisfy the interfacial tension presented in Equation (1).

Since the complex has a larger particle size than the impurities, impurities having a relatively small particle size are not trapped at the interface and move to the second aqueous solution phase, and only the biological target remains in the first aqueous solution phase. Then, the biological target may be recovered by separating the first aqueous phase from the aqueous phase using equipment such as a pipette or a dropper.

The extracellular vesicles have a size of 50 to 500 nm, and impurities including unreacted antibodies have a size of 30 nm or less. Therefore, when designing a composition having interfacial tension so that the critical particle diameter at the interface has a size exceeding 30 nm using the aqueous phase-separation composition presented in the present invention, the antibody-bound extracellular vesicles are separated from the first aqueous phase and Trapped at the interface between the second aqueous phases and remaining in the first aqueous phase, the remaining impurities (unreacted primary antibody, unreacted secondary antibody, etc.) migrate to the second aqueous phase, resulting in a pure antibody-bound extracellular vesicle (i.e., , complex) can be selectively isolated.

In particular, the aqueous phase-separation composition of the present invention can be separated even from particles having a difference of 10 nm in particle size, so that the complex can be separated with high purity.

In addition, in order to increase the resolution and the separation rate during the separation process, a temperature may be applied or an ultrasonic wave may be applied.

Temperature is a parameter related to the separation rate, and as the temperature is increased, the separation rate of the complex and impurities increases.

8 is a graph showing a change in the escape velocity at the interface according to the particle size. In FIG. 8, Γ _P1 : means the ratio of particles escaping from the interface to the first aqueous solution phase, and Γ _P2 : means the ratio of particles escaping from the interface to the second aqueous phase.

Referring to FIG. 8 , as the temperature increases, the Brownian motion of the particles is accelerated, so that the escape velocity of the particles trapped at the interface increases. For example, when the particle size of the impurity in the biological sample is smaller than the size of the complex, it means that the escape rate of the impurity trapped at the interface may be increased. From these results, it can be seen that temperature can be used as a variable of process conditions that can be usefully used when it is necessary to separate in a very accurate size within a short time.

9 is a graph showing a change in a critical particle size according to a change in temperature.

Referring to FIG. 9 , it can be seen that the critical particle size tends to increase linearly as the temperature increases. However, since the size difference between the critical particle sizes is within 10 nm, it can be seen that even if the temperature is increased during separation of impurities and complexes, only the separation rate is increased, and the critical particle size is not increased more than necessary, so that impurities and complexes can be separated with high purity. .

In addition, when the first aqueous phase P1 is a stationary droplet and a moving droplet, it can be seen that the minute critical particle size, which differs only in the separation rate, is finally the same.

In addition, through the application of ultrasonic waves, a physical force from the outside lowers the energy barrier of the interface to change the critical particle size or to help the diffusion of particles, thereby increasing the separation rate of impurities and complexes.

Impurities and complexes with different particle sizes have differences in mechanical properties such as differences in their sizes. When the material or composition is different from the above size, mechanical properties such as material, density, and compressibility are significantly different. Here, when ultrasonic waves are applied to the mixed nanoparticles, the nanoparticles have different acoustic radiation forces depending on the particle size, and the nanoparticles move along the sound pressure node line of the ultrasonic waves. That is, when impurities and complexes are mixed, they can be separated and their movement speed can be increased. In particular, it is possible to increase the separation rate between impurities and complexes trapped at the interface between the first aqueous phase P1 and the second aqueous phase P2, thereby preventing a decrease in the separation rate due to the trapped particles.

This acoustic radiation force can be adjusted by controlling the frequency. Preferably, in the present invention, it is preferable to carry out for 1 minute to 240 minutes at an intensity of 200 W to 400 W of 0.01 to 100 kHz. At this time, if the intensity of the ultrasonic wave applied is too high, it affects the first aqueous solution phase P1 that should be in a bulk form, and the first aqueous solution phase P1 may form fine droplets, so it is properly performed within the above range. do. Also, the ultrasonic application may be performed through an ultrasonic generator.

[Example]

Hereinafter, the configuration and operation of the present invention will be described in more detail through preferred embodiments of the present invention. However, this is presented as a preferred example of the present invention and cannot be construed as limiting the present invention in any sense.

Experimental Example 1: Simulation test

In addition to theoretical considerations related to the interfacial tension, energy barrier, and critical particle size, a direct experiment was performed to determine whether the separation of nanoparticles in the present invention can be applied in an actual process through simulation results.

First, three types of fluorescent beads of 30 nm, 50 nm and 100 nm were prepared as mixed nanoparticles, an aqueous dextran solution was used as the first aqueous phase (P1), and polyethylene glycol (P2) was used as the second aqueous phase (P2). PEG) was used. Separation coefficient (K, partition coefficient) = 100, temperature = 20 °C, time = 300 s , boundary tension = 0.013 mJ/m ^{2 It} was performed under conditions.

At this time, by controlling the concentrations of the first aqueous solution phase (P1) and the second aqueous solution phase (P2), an aqueous solution phase filter having interfacial tension was designed as follows.

Configuration Interfacial tension ( X10 ^-6 J/m ² ) Composition of the first aqueous phase (P1) Composition of the second aqueous phase (P2) A 5.36 3% dextran 4% PEG B 3.57 2.5% Dextran 3.5% PEG C 1.65 2% Dextran 3% PEG D 30 3.5% Dextran 4.5% PEG E 55 4% Dextran 5% PEG

A total of 9 tubular test tubes were prepared, and three types of fluorescent beads of 30 nm, 50 nm, and 100 nm were respectively injected into the filters of components A, B, and C, and the movement of the fluorescent beads was confirmed with a fluorescence microscope. At this time, the filter having the configuration of D and E had a critical particle diameter of 30 nm or less, and there was no escape of nanoparticles.

For the measurement, first, 75 μL of polyethylene glycol aqueous solution forming the second aqueous phase (P2) was injected into a tubular test tube (inner diameter 2 mm). 5 ng of fluorescent beads were added to 3 μl of the dextran aqueous solution in the first aqueous solution, and the mixture was uniformly mixed. 3 μl of the obtained dispersion was slowly injected into the upper part of the tubular test tube containing the second aqueous phase (P2), and it was confirmed that the first aqueous phase (P1) was transferred to the lower side of the test tube.

The movement of the fluorescent beads was measured through a fluorescence spectrometer immediately after mixing the first aqueous phase (P1) and the second aqueous phase (P2) (T=0 min), after 30 min and after 60 min.

As a result, it can be seen that, when the interfacial tension is 5.36 X10 ^-6 J/m ² , only 30 nm nanoparticles among the nanoparticles of 30 nm, 50 nm and 100 nm migrated to the second aqueous solution phase P2. Similarly, in the case of composition B, the interfacial tension is 3.57 X10 ^-6 J/m ² , the nanoparticles of 30 nm and 50 nm move to the second aqueous phase (P2), and the nanoparticles of 100 nm are trapped at the interface and the first It was confirmed that it remained in the aqueous phase (P1). In addition, in the case of composition C, the interfacial tension is 1.65 X10 ^-6 J/m ² It can be seen that all of 30 nm, 50 nm and 100 nm move to the second aqueous phase (P2). Through this, as shown in Equation 1, it can be seen that the ^{interfacial tension must be at least 2 X10 -6} J/m ^{2 or more.}

Through these results, it can be seen that the critical particle size that can pass through the interface when the nanoparticles are separated can be controlled through the control of the interfacial tension.

Example 1: Separation using an aqueous phase-separation composition

(1) exosome-CD9-Alexa 488 complex preparation

CD9 (primary antibody, 150 kDa) was incubated overnight at 4° C. in exosomes (50 nm to 500 nm) separated from plasma by centrifugation. Thereafter, unreacted CD9 was removed from the obtained solution using an aqueous phase-separation composition (critical particle diameter of 42 nm).

The method using the aqueous phase-separation composition was performed as follows.

5 ml of polyethylene glycol aqueous solution was added to the horizontal * vertical test tube. Then, 500 μl of the sample was mixed with 0.5 ml of dextran aqueous solution and dissolved for 1 hour, and the resulting solution was added to the test tube to induce dextran/polyethylene glycol phase separation, and left for 30 minutes at 37° C. for unreacted CD9 It was allowed to migrate into this polyethylene glycol. Then, the dextran aqueous phase containing the exosome-CD9 complex was recovered and used in the next step.

Next, Alexa 488 (secondary antibody) was added to the exosome-CD9 complex and incubated at 4° C. overnight. Then, unreacted Alexa 488 was removed from the obtained solution using an aqueous phase-separation composition.

Example 2: Separation using an aqueous phase-separation composition

It was carried out in the same manner as in Example 1, except that the removal of impurities was carried out for 60 minutes instead of 30 minutes.

Comparative Example 1: Separation using ultracentrifugation process

Except that the ultracentrifugation process was performed at 160,000 for 2 hours to remove impurities, the same procedure as in Example 1 was performed.

Experimental Example 2: Exosome-CD9 and exosome-CD9-Alexa 488 complex analysis

(1) Exosome-CD9 complex recovery efficiency and CD9 removal rate measurement

Using the recovered exosome-CD9 complex, the recovery efficiency of the exosome-CD9 complex and the removal rate of the CD antibody were measured.

In order to compare the recovery efficiency of the exosome-CD9 complex, the isolated amount was confirmed compared to the amount of total exosomes or CD9 in the initial stage. To this end, the percentage (%) of the amount of isolated exosomes or CD9 with respect to the total amount was defined as recovery efficiency (E), and the recovery efficiency of the aqueous phase system was calculated according to Equation 2 below. At this time, the amount of exosomes was measured by the amount of RNA, and the amount of CD9 was measured using a Bradford assay.

[Equation 2]

Example 1 Example 2 Comparative Example 1 Exosome-CD9 complex recovery efficiency 97.39% 92.57% 18.8% Removal of unreacted CD9 93.14% 95% 86.53%

Referring to Table 3, it can be seen that the recovery efficiency of the exosome-CD9 complex using the aqueous phase-separation composition according to the present invention can be recovered in a high yield compared to the ultracentrifugation process of Comparative Example 1. In addition, unreacted It can be seen that the removal rate of CD9 is also high, so that the exosome-CD9 complex can be obtained with high purity.

(2) Microscopic analysis

The exosome-CD9-Alexa 488 complex prepared in Example 1 and Comparative Example 1 was measured using a total internal reflection fluorescence microscope ( TIRF).

11 is a total reflection micrograph of the exosome-CD9-Alexa 488 complex obtained in Example 1 (a) and Comparative Example 1 (b). Specifically, the lipid layer of the exosome of 300 ug was stained with DII (red), and then the membrane protein of the exosome was bound with CD9 and Alexa 488 (green). In FIG. 11 , the yellow part due to overlapping red and green means the exosome in which the exosome membrane protein and lipid are coexisted, and yellow particles were confirmed in both ultracentrifugation and APIF.

11 , it can be seen that the method using the aqueous phase-separation composition of Example 1 contains a higher amount of exosome-antibody complex than the ultracentrifugation process of Comparative Example 1.

(3) Change in the content of Alexa-488 compared to the content of exosomes

After treating different amounts of exosomes of 1ug, 2.5ug, 6ug, and 12.5ug with the aqueous phase-separation composition under the same conditions, the change in the content of Alexa-488 compared to the content of exosomes was measured.

12 is a graph showing changes in the content of Alexa-488 compared to the content of exosomes.

Referring to FIG. 12 , as a result of measuring whether the Alexa 488 fluorescence signal was linear with respect to the amount of exosomes, it was confirmed that the fluorescence signal was linear with R=0.9927. On the other hand, when the ultracentrifugation process was performed as in Comparative Example 1, the loss of exosomes was large (96% loss of exosomes), so fluorescence was not measured with a fluorescence meter, making analysis impossible. These results mean that it is advantageous to treat a small amount of exosome samples that cannot be analyzed by conventional methods with an aqueous phase-separation composition.

(4) Changes in Alexa-488 content compared to CD9 concentration

After fixing the amount of exosomes to 25 ug and binding different concentrations of CD9 antibody, Alexa 488 was bound to measure the change in the content of Alexa 488 according to the concentration of CD9.

13 is a graph showing the change in the content of Alexa-488 compared to the CD9 concentration.

Referring to FIG. 13 , it was confirmed that the fluorescence amount converges to a certain value at a CD9 concentration of 4 ng/ml or higher when Alexa 488 was stained. This phenomenon means that all exosomes are stained at a specific CD9 concentration or higher, and staining using the aqueous phase-separation composition according to the present invention is normal.

Looking at the results of (1) to (4) above, when using the aqueous phase-separation composition according to the present invention, the unreacted antibody CD9 is effectively removed, and the loss and damage of the exosomes are small, so that a high-purity exosome-antibody complex is produced. It can be seen that it can be prepared in a high yield. This high-yield and high-purity preparation method has the advantage that the entire population of exosomes in the initial sample can be analyzed.

Experimental Example 3: Evaluation of separation performance using an aqueous phase-separation composition

The exosome-CD9 complex was isolated using the method according to the present invention and a method of performing agitation/centrifugation, and the recovery efficiency was confirmed.

(1) recovery efficiency

In this case, as Comparative Example 2, it was performed using a method based on Korean Patent Application Laid-Open No. 10-2016-0116802. It was performed in the same manner as in Example 1, but in the separation process, an ultracentrifugation process was performed for 10 minutes at room temperature with 1,000 × g-force.

Example 1 Comparative Example 2 Exosome-CD9 complex recovery efficiency 97.39% 68.3% Removal of unreacted CD8 93.14% 77.5% Purity (exosome recovery efficiency/CD9 recovery efficiency) 14.2 3.04

Experimental Example 4: Prostate cancer diagnosis

In the present invention, a complex was prepared using an aqueous phase-separation composition, and the obtained complex was used for actual prostate cancer diagnosis to confirm the diagnostic ability.

Diagnosis using existing exosomes is difficult to extract from plasma with high purity and a large amount of exosomes, so the amount of isolated exosomes is less than the measurement limit of the measuring instrument, so the analysis itself is often impossible. In addition, when the antibody is stained for analysis after separating the exosome, exosome loss occurs in the process of removing the unstained antibody, making it difficult to analyze and impossible to analyze the entire exosome population.

Accordingly, in the present invention, an aqueous phase phase separation composition was used for all processes of separation of exosomes from plasma collected from patients and removal of impurities during the preparation of complexes. For the diagnosis of prostate cancer, prostate specific membrane antigen (PSMA), a membrane protein marker associated with prostate cancer, was used.

1. Pre-cleaning 200 uL of plasma from cancer patients (n=25) and patients with benign prostatic hyperplasia (n=25)

2. Plasma -> Exosome Separation

3. Preparation of exosome-PSMA-CD9 complex and removal of impurities

4. Preparation of exosome-PSMA-CD9-Alexa 488 complex and removal of impurities

5. Determination of the amount of PSMA with Alexa 488 fluorescence

14 is a graph showing the expression level of PSMA compared to the expression level of CD9 in patients with benign prostatic hyperplasia (BPH) and prostate cancer (Pca). Referring to FIG. 14 , it was confirmed that patients with prostate cancer had higher values than patients with benign prostatic hyperplasia. This is consistent with the result that PSMA expression in plasma exosomes of cancer patients is higher than in patients with benign prostatic hyperplasia.

At this time, for comparison, the process was performed through the ultracentrifugation process as in Comparative Example 1. The content of exosomes finally obtained is less than 0.2% of the initial amount (a total of 4 centrifugation steps are required, about 80% of exosomes are lost at a time), and the signals of PSMA and CD9 are weak, so it is measured with a fluorometer. couldn't

On the other hand, when diagnosing prostate cancer, the concentration of PSA in plasma is generally measured. When the diagnosis was made on the 50 patient groups previously tested using prostate specific antigen (PSA) in plasma, the diagnosis success rate was 70%.

15 is a receiver operating characteristics curve (ROC curve) when diagnosis with PSA, a conventional diagnostic method, and when diagnosis using an aqueous phase separation composition (AIF) according to the present invention and PSA diagnosis are applied together. The ROC curve is a curve that can confirm sensitivity and specificity. PSA + AIF is the method according to the present invention, Serum-PSA means the existing diagnostic method, and the Reference Line is the baseline.

Referring to FIG. 15 , it can be seen that the sensitivity and specificity of the diagnosis are improved when the diagnosis using the aqueous phase separation composition and the PSA diagnosis are applied together.

As a result, when exosome diagnosis was performed with PSA using the aqueous phase separation composition as in the present invention, the diagnosis success rate was 90%, which was significantly improved compared to the existing diagnosis success rate of about 70%. Moreover, when the aqueous phase-separation composition was applied to a diagnostic method using exosomes, it was possible to diagnose a small amount of sample that could not be diagnosed by the conventional method. In addition, it was confirmed that the diagnostic performance was improved when the diagnosis was made by combining the PSA diagnostic method and the aqueous phase separation composition.

Although the present invention as described above has been described with reference to the embodiments shown in the drawings, this is merely exemplary, and those of ordinary skill in the art will understand that various modifications and variations of the embodiments are possible therefrom. , such modifications should be considered to be within the technical protection scope of the present invention. Therefore, the true technical protection scope of the present invention should be defined by the technical spirit of the appended claims.

Claims (11)

(S1) separating the extracellular vesicles from the biological sample;
(S2) binding the extracellular ER and a primary antibody specifically binding thereto through an antigen-antibody reaction;
(S3) binding a secondary antibody having colored or fluorescent properties through an antigen-antibody reaction for identification;
(S4) detecting information on the obtained complex; and
(S5) comparing the detection result with a normal control; when diagnosing a disease, including
After the steps (S2) and (S3), the step of removing impurities including unreacted antibodies using an aqueous phase phase separation composition is performed, a disease diagnosis method.
According to claim 1,
The method for diagnosing a disease, wherein the unreacted antibody is at least one of a primary antibody and a secondary antibody.
According to claim 1,
The biological sample is one selected from the group consisting of cell culture fluid, blood, plasma, serum, intraperitoneal fluid, semen, amniotic fluid, breast milk, saliva, bronchoalveolar fluid, tumor effluent, tears, runny nose and urine, a disease diagnosis method.
According to claim 1,
A method for diagnosing a disease, further comprising an antigen.
According to claim 1,
In the aqueous phase-separation composition, the tension (γ) at the interface at which the first aqueous phase and the second aqueous phase are phase-separated satisfies the following Equation 1, a disease diagnosis method:
[Equation 1]
2 X 10 ^-6 J/m ² ≤ γ ≤ 50 X 10 ^-6 J/m ²
6. The method of claim 5,
The method for diagnosing a disease, wherein the first aqueous phase flows in bulk in the second aqueous phase, which is a continuous phase.
6. The method of claim 5,
The method for diagnosing a disease, wherein, in the aqueous phase-separation composition, the unreacted antibody is removed by moving from the first aqueous solution to the second aqueous phase by diffusion.
6. The method of claim 5,
The method for diagnosing a disease, wherein the first aqueous phase and the second aqueous phase are any one selected from a polymer-polymer, or a polymer-high-concentration aqueous solution system.
According to claim 1,
In addition, the method for diagnosing a disease, wherein the extracellular vesicles are isolated from a biological sample using an aqueous phase-separation composition.
According to claim 1,
Wherein the disease diagnosis is cancer diagnosis, monitoring or predicting, a method for diagnosing a disease.
11. The method of claim 10,
The cancer is breast cancer, colon cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, eye tumor, peritoneal cancer, uterine cancer, ovarian cancer, rectal cancer, Perianal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, testicular cancer, oral cancer, gallbladder cancer, cholangiocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and At least one selected from the group consisting of pituitary adenoma, a method for diagnosing a disease.

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