KR20240029314A - Label-free Method for Detecting Exosomal Surface Biomarker Based on Microarray of Polydiacetylene Liposome - Google Patents

Abstract

According to an embodiment of the present disclosure, an exosome sample obtained by separating and concentrating exosomes present with various impurities in the sample is applied to a microarray platform of polydiacetylene liposomes formed on a substrate to identify exosome surface biomarkers. A label-free method that is simple and can be detected with high accuracy is described.

Description

Label-free method for detecting Exosomal Surface Biomarker Based on Microarray of Polydiacetylene Liposome}

The present disclosure relates to a method for label-free detection of exosome (extracellular vesicle) surface biomarkers based on polydiacetylene liposome array. More specifically, the present disclosure provides a simple way to identify exosome surface biomarkers by applying an exosome sample obtained by separating and concentrating exosomes present with various impurities in the sample to a microarray platform of polydiacetylene liposomes formed on a substrate. It is about a label-free method that can detect with high accuracy.

Cells are known to secrete extracellular vesicles surrounded by lipid double membranes of various sizes. These extracellular vesicles are classified into exosomes, microvesicles, and large oncosomes depending on their size. Among them, exosomes are small vesicles with a membrane structure secreted by various types of cells. The diameter of exosomes is reported to be approximately 30 to 100 nm, and studies using electron microscopy have shown that rather than being released directly from the plasma membrane, they originate from specific intracellular compartments called multivesicular bodies (MVBs) and are released and released outside the cell. A secretion point was observed. In other words, when fusion of the multivesicular body and the plasma membrane occurs, these vesicles are released into the environment outside the cell, and are referred to as exosomes.

Although it has not yet been clearly identified by what molecular mechanism the above-mentioned exosomes are formed, various types of immune cells including not only red blood cells, but also B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, etc. It has been reported that tumor cells, etc., also produce and secrete exosomes while alive. Exosomes are known to be isolated and released from a number of different cell types under both normal and pathological conditions.

Recently, exosomes, which are newly emerging in the field of disease diagnosis, are released outside the cells and contained in various types of body fluids such as blood, saliva, urine, etc., and contain various proteins and genetic materials derived from cells, making them useful for disease diagnosis. It is gaining popularity as a medium. In particular, biomarkers present on the surface of exosomes can be analyzed without additional decomposition or separation of exosomes and have high potential because no complicated processes are required. In this regard, a method for diagnosing diseases by analyzing exosomes present in body fluids and identifying specific cell-derived biomarkers has been reported.

As an example, exosomes are known to contain approximately 4,500 types of proteins, lipids, and genetic information (e.g., microRNA, mRNA, tRNA, rRNA, DNA, etc.) that are indicators of drug response. Method for diagnosing diseases by detecting the presence or absence and amount of various microRNAs present in the body (e.g., Domestic Patent Publication No. 2010-0127768, etc.), samples resulting from cancer (blood, saliva, tears, etc.) By detecting exosomes and measuring the amount of change in microRNA, a method has been developed to predict and diagnose the relationship with a specific disease when it increases or decreases compared to the control group (e.g., WO2009/015357 A, etc.).

Alternatively, sensors that selectively detect or detect exosomes in body fluids are being studied. Most of these exosome detection methods use chips that allow liquid containing exosomes to flow into a passage, and exosomes that bind to receptors present on the surface of the passage are detected using electrical resistance, current, or surface plasmon resonance. It can be detected (for example, Korean Patent Publication No. 87594, etc.). However, in the process of producing a separate chip system, complex procedures and processes must be performed, and expensive equipment is required to obtain a detectable signal. For diagnosis, labeling substances are used, etc., making compatibility and It is disadvantageous in terms of versatility. In particular, the production of a chip system for exosome detection involves separate expensive manufacturing equipment and complicated procedures, so its use for commercial purposes is limited and mass production is difficult. In addition, the surface plasmon resonance-based device used to obtain exosome capture signals is an expensive device and has low versatility.

As such, due to issues related to simplification and cost reduction of complex diagnostic systems, a simple detection principle is essential for commercialization. However, in general, fluorescence and colorimetric principles involving target-specific light sources, filters, prisms and visualization devices are used, and these methods require complex methods and equipment, creating a need to improve diagnostic convenience. there is.

In response to this, the present inventors have developed a technology that can detect exosomes label-free in an aqueous medium using polydiacetylene liposomes immobilized with receptors specific to biomarkers present on the surface of exosomes (domestic patent) Publication number 2021-0067916). However, when using an aqueous medium, a solution containing a high concentration of the detection target is required to generate an optical change that can be observed with the naked eye, and there is room for improvement in that a relatively large amount of sample is required.

Meanwhile, in the case of samples such as blood, the concentration of biomarkers for specific diseases such as cancer and Alzheimer's present in the samples is quite low, making it difficult to distinguish patients from normal people using conventional detection techniques. Moreover, since the sample contains interfering substances such as many proteins in addition to the biomarker, a decrease in sensitivity due to non-specific reaction during detection is a problem.

Therefore, there is a need for a method that can easily and accurately detect exosomes, specifically surface biomarkers of exosomes, even when exosomes are contained with various interfering substances in a sample at a concentration so low that detection is difficult.

In one embodiment according to the present disclosure, it is intended to provide a method (or platform) that can easily and accurately detect or diagnose exosome surface biomarkers for various types of samples.

One embodiment according to the present disclosure seeks to provide a method for detecting surface biomarkers of exosomes in a sample without separate labeling.

According to the first aspect of the present disclosure,

a) preparing a receptor bead to which at least one receptor having specific binding ability to at least one surface biomarker of the target exosome is attached;

b) contacting the exosome-containing sample with the receptor bead to form an exosome-receptor complex on the bead, and then concentrating the exosomes by separating or dissociating the exosomes from them;

c) providing a diacetylene-phospholipid liposome solution separately from steps a) and b); and

d) forming a microarray of diacetylene-phospholipid liposomes on an amine-modified substrate using the diacetylene-phospholipid liposome solution; and

e) attaching at least one receptor having specific binding ability to at least one surface biomarker of the target exosome to the microarray of the diacetylene-phospholipid liposome;

f) forming a microarray of modified polydiacetylene liposomes by polymerizing the diacetylene-phospholipid liposomes to which the at least one receptor is attached; and

g) Contacting the solution containing the exosomes concentrated in step b) with a microarray of polydiacetylene liposomes, thereby optically induced by a ligand-receptor reaction between the concentrated exosomes and the modified polydiacetylene liposomes in the microarray. measuring characteristic changes;

A method for detecting exosome surface biomarkers in a sample comprising a is provided.

According to an exemplary embodiment, step a) further includes surface modifying the bead to attach a functional group capable of binding to at least one receptor, and attaching the at least one receptor to the surface-modified bead. Receptor beads for target exosomes can be manufactured.

According to an exemplary embodiment, the bead may be at least one selected from the group consisting of resin beads, metal beads, and glass beads.

According to an exemplary embodiment, the beads have a particle size ranging from 1 to 100 μm, and

The structure may be spherical, rod-shaped, cubic-shaped, hexagonal, triangular, hollow and/or flower-shaped.

According to an exemplary embodiment, the bead is a magnetic bead, and the magnetic bead is hematite (α-Fe ₂ O ₃ ), maghemite (γ-Fe ₂ O ₃ ), or magnetite (magnetite, Fe ₃ O ₄ ). It may be an iron oxide material selected from the group consisting of at least one.

According to an exemplary embodiment, the substrate may be at least one selected from the group consisting of glass, silicon wafer, and transparent plastic.

According to an exemplary embodiment, the amine-modified substrate may have the surface of the substrate modified with amino-terminated silane.

According to an exemplary embodiment, the amino-terminated silane is 3-aminopropyltriethoxysilane, 3-aminopropyl trimethoxysilane, N-(2-aminoethyl) -3-Aminopropyl trimethoxysilane (N-(2-aminoethyl)-3-aminopropyl trimethoxysilane), N-(2-aminoethyl)-3-aminopropyl triethoxysilane (N-(2-aminoethyl)- 3-aminopropyl triethoxysilane), 3-2-(2-aminoethylamino)ethylaminopropyl trimethoxysilane), 3-2-(2-aminoethylamino)enyl It may be at least one selected from the group consisting of aminopropyl triethoxysilane (3-2-(2-aminoethylamino)ethylaminopropyl triethoxysilane) and 3-aminopropyl methyldimethoxysilane.

According to an exemplary embodiment, the microarray may be formed in a checkerboard pattern.

According to an exemplary embodiment, the size (diameter) of the individual polydiacetylene liposomes constituting the microarray is in the range of 100 to 600 ㎛, and the spacing between arrays can be adjusted in the range of 0.1 to 0.6 times the array diameter. there is.

According to an exemplary embodiment, a plurality of biomarkers are present on the surface of the exosome, and the receptor may be a multi-receptor specific for the plurality of biomarkers.

According to an exemplary embodiment, the diacetylene monomer is at least one selected from the group consisting of pentacosadiyonic acid (PCDA), trocosadiyonic acid (TCDA), heneicosadiynoic acid (HCDA), and heptadecadiynoic acid (HDDA), and

The phospholipids include dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidyl glycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroyl ethylphosphocholine ( DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleoyloxy)-3 -(Trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidyl acid (DMPA), dipalmitoylphosphatidyl acid (DPPA), distearoylphosphatidyl acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), and distearoylphosphatidylinositol (DSPI) It may be at least one selected from the group consisting of thearoylphosphatidylserine (DSPS).

According to an exemplary embodiment, the molar ratio of diacetylene monomer:phospholipid in step c) may range from 2 to 10:1.

According to an exemplary embodiment, the receptor may be a whole antibody or at least one selected from the group consisting of a partial antibody containing an antigen binding region and an aptamer.

According to an exemplary embodiment, the biomarker may be a tetraspanin protein.

According to an exemplary embodiment, the tetraspanin protein may be at least one selected from the group consisting of CD-9, CD-63, CD-81, and CD-82.

According to an exemplary embodiment, the change in optical properties may be measured with the naked eye or a measuring device.

According to an exemplary embodiment, the device for measuring the change in optical properties may be a UV-Vis spectrometer or a fluorescence spectrometer.

The method for detecting exosome surface biomarkers based on polydiacetylene liposomes according to an embodiment of the present disclosure can detect samples containing target exosomes present in low concentrations along with other interfering substances easily and with high accuracy. And, in particular, label-free detection can be implemented. Furthermore, by being based on a microarray of polydiacetylene liposomes, the amount of sample required for detection can be reduced, the microarray system production and signal analysis process are relatively easy, and analysis is performed using a fluorescence observation device without complex or expensive analysis equipment. The advantage of this being possible can be realized.

1 is a diagram schematically showing a series of processes for selecting and concentrating exosomes using beads according to an exemplary embodiment of the present disclosure;
Figure 2A is a schematic diagram of manufacturing a polydiacetylene (PDA)-based array detection platform for exosome detection according to an exemplary embodiment, (a) diacetylene/phospholipid liposomes on an amine-modified substrate are EDC/ It shows the process of activation and immobilization by the NHS reaction, and (b) the process of the receptor being conjugated (attached) to the PDA surface to provide specificity to the array detection platform to selectively detect exosomes;
FIG. 2B schematically illustrates the process of forming a diacetylene/phospholipid liposome array on a substrate using a lithographic technique according to an exemplary embodiment;
Figure 3 is a graph showing the results of nanoparticle tracking assay (NTA) of exosome samples separated using separation particles;
Figure 4 is a photograph showing the PDA liposome array substrate produced in Example;
Figure 5 is an SEM image of a PDA liposome array arrayed on an amine-modified substrate, showing (a) a bare PDA liposome array, and (b) an exosome attached to a receptor-introduced PDA liposome;
Figure 6 shows the characterization results of PCDA/DMPC liposomes, including (a) TEM image of PCDA/DMPC liposomes, (b) particle distribution of PDA liposomes, (c) surface zeta potential, and (d) blue state and red state. is the UV-vis spectrum of PDA;
Figure 7 shows the characterization results of the amine-modified glass substrate, (a) the XPS spectrum of the amine-modified glass substrate, (b) the fluorescence micrograph of the red form of PDA attached to the glass substrate, and (c) the glass substrate. Fluorescence intensity of PDA liposomes immobilized on the surface according to (under the same experimental conditions, two groups change to a reddish form by introduction at 75 ° C for 10 minutes after attachment);
Figure 8 is a graph showing the SEM image and size distribution of exosomes as a result of exosome characterization;
Figure 9 shows the results of an optimization experiment of receptor concentration for efficient exosome detection (to determine the appropriate linkage concentration of the receptor, both antibodies and aptamers target CD-63 of exosomes), (a) Fluorescence intensity depending on the concentration of anti-CD-63 receptor (the concentration of antibodies and aptamers to be incubated ranges from 0.1-1000 ng/mL, and fluorescence was measured after adding 1 × 10 ¹⁰ vesicles/mL of exosomes to each sample. measured), and (b) a fluorescence picture of PDA liposomes after addition of exosomes (fluorescence intensity was measured based on the largest fluorescence value using an excitation wavelength of 560 nm);
Figure 10 shows the results of an optimization experiment of the incubation concentration of the receptor bound to PDA liposomes, showing the fluorescence of the array incubated with (a) antibody and (b) aptamer exposed to a sample with an exosome concentration of 1 × ¹⁰ 10 vesicles/mL. In the picture (antibody concentrations are 1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL and 0.1 ng/mL, respectively; scale bar is 250 μm);
Figure 11 is a graph showing the change in UV-Vis spectrum of the PDA liposome array according to exosome concentration, (a) each concentration is 1 × 10 ¹⁰ vesicles/mL, 1 × 10 ⁹ vesicles/mL, and 1 × 10 ⁸ Absorption spectrum of PDA liposomes when vesicles/mL of exosomes are added to blue PDA containing CD-63 and CD-81 antibodies, and (b) absorption spectrum of red form of PDA according to exosome incubation concentration ( 500-550 nm);
Figure 12 shows (a) the fluorescence intensity of the PDA array as a function of exosome concentration, and (b) the calibration curve between arrayed PDA liposomes and exosome concentration (fluorescence intensity was calculated at the maximum value, and the excitation wavelength was 560 nm (n =4) is);
Figure 13 is a calibration curve between fluorescence intensity and exosome concentration;
Figure 14 is a graph showing (a) the ratio of exosomes captured by receptor type, and (b) the fluorescence intensity of fluorescent exosomes by receptor type, as the results of a quantification experiment of exosomes captured on the surface of PDA liposomes;
Figure 15 is a graph showing the results of testing the specificity of the PDA liposome array for exosomes using plasma proteins (anti-CD-63 and anti-CD-81 simultaneously contain antibodies and aptamers for exosome targeting, respectively) is);
Figure 16 is a diagram showing the results of fluorescence signal analysis of the PDA liposome array according to the exosome selection method;
Figure 17 is a diagram showing the results of fluorescence signal analysis of the PDA liposome array according to exosome concentration;
Figure 18 is a diagram comparing the fluorescence signal measurement results of the PDA liposome array between the disease group (AD) and the normal group (NC);
Figure 19 is a diagram showing the results of reproducibility analysis of the PDA liposome array system;
Figure 20 shows the Area Under the Curve (AUC)-Receiver Operation Characteristic (ROC) curves for each of the disease group and the normal group; and
Figure 21 is a fluorescence microscope image according to the polydiacetylene array manufacturing method, showing a contact array (top) and a lithography-based array (bottom), respectively.

The present invention can be achieved by the following description with reference to the attached drawings. It should be understood that the following description describes preferred embodiments of the present invention, and that the present invention is not necessarily limited thereto.

In addition, the attached drawings may be somewhat exaggerated compared to the actual thickness (or height) of the layer or the ratio to other layers to aid understanding, and the meaning will be properly understood based on the specific purpose of the related description described later. You can.

Terms used in this specification may be defined as follows.

“Binding or binding” may mean bound or connected to a surface in a covalent or non-covalent manner.

“Immobilization” can mean the attachment of any substance or bioactive agent to a substrate, covalently or non-covalently, in a direct or indirect manner.

In a broad sense, “array” may mean a set of a plurality of elements, and in a narrow sense it may mean a set of elements arranged or arranged in a predetermined pattern (for example, a regular pattern).

“Sample” is not limited to a specific type or form, as long as it can contain the target pathogen to be detected. Illustratively, the sample may be a biological sample, such as biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, and amniotic fluid. Biological tissue is a collection of cells, usually intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures, as well as specific types of tissue, such as connective tissue, epithelial tissue, muscle tissue and nerves. This may include organizations, etc. Examples of biological tissues may also include organs, tumors, lymph nodes, arteries, and individual cell(s).

“Antigen” is any substance that can cause a specific immune response, and may specifically mean any molecule or group of molecules that can be recognized by at least one antibody. An antigen contains at least one epitope (a specific biochemical unit that can be recognized by an antibody). In addition, “antibody” refers not only to a complete polyclonal (or polyclonal) or monoclonal (monoclonal) antibody, but also to fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv), ), diabodies, multispecific antibodies formed from antibody fragments, mutations thereof, fusion proteins comprising antibody portions, any other modified arrangement of an immunoglobulin molecule containing an antibody recognition site of the desired specificity. . Antibodies may include antibodies that fall into any category, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies that do not belong to a particular class.

“Receptor” can refer to a molecular group or receptor capable of recognizing a specific substance, and by introducing it, a unique optical characteristic change can be induced due to the conjugated structure of polydiacetylene.

The expression “specifically binds” refers to the specificity of a binding reagent, for example, an antibody, and may mean preferentially binding to a defined sample or target substance. Recognition by a binding reagent or antibody of a specific specimen or target substance in the presence of other potential targets may be a characteristic of such binding. In some embodiments, a binding reagent that specifically binds to a sample may avoid binding to other interfering moieties or portions in the sample to be tested.

The expressions “on” and “above” may be understood as being used to refer to the concept of relative position. Accordingly, not only may other components or layers be present directly in the mentioned layer, but also other layers (intermediate layers) or components may be interposed or present between them. Similarly, the expressions “below,” “in the lower part,” “below,” and “between” may also be understood as relative concepts of location. Additionally, the expression “sequentially” can also be understood as a relative position concept.

Selection and enrichment of exosomes

A series of processes for selecting and concentrating exosomes using beads according to an exemplary embodiment are schematically shown in FIG. 1.

Referring to the drawing, a series of processing processes are performed to select and concentrate exosomes to be detected in the sample (i.e., target exosomes) or biomarkers present on the surface (i.e., exosome surface biomarkers). . At this time, there may be one target exosome, or there may be two or more target exosomes.

For this purpose, first, prepare a receptor bead on which a receptor having specific binding ability to the target exosome (or exosome surface biomarker) is attached or immobilized on the bead.

According to an exemplary embodiment, the above-described beads may be made of a material selected from the group consisting of resin, metal, and glass. In this regard, when using resin, various natural resins (eg, agarose) or synthetic resins (eg, polystyrene) can be used, and can also be applied in the form of resin moldings, gels, etc. Additionally, the beads may have a regular or irregular shape and may also have a symmetric or asymmetric shape. More typically, the beads may be spherical, rod-shaped, cubic, hexagonal, triangular, hollow and/or flower-shaped, and in certain embodiments may be spherical beads as shown. At this time, the particle size of the bead can be adjusted considering the material constituting the bead or the manufacturing environment, and may range from nanoscale to micron scale. In an exemplary embodiment, the particle size of the beads may be set, for example, in the range of about 1 to 100 ㎛, specifically 5 to 80 ㎛, and more specifically 10 to 50 ㎛. The particle size of the beads can vary depending on the material and shape of the particles. In addition, considering the ease of separation and stability after formation of the receptor beads, it may be desirable for the beads to have a density above a certain level, for example, about 2 to 10 g/cm3, specifically about 4 to 8 g/cm3. ㎤, more specifically, it may be in the range of about 5 to 7 g/㎤, but the present invention is not limited thereto.

In certain embodiments, the beads can be metal beads, specifically magnetic beads. These magnetic beads may have ferromagnetic or superparamagnetic properties. For example, magnetic particles containing iron, cobalt, nickel, iron oxide, iron hydroxide and/or other iron alloys, rare earth magnetic particles, etc. may be used.

As an example, the magnetic bead may be made of iron oxide, specifically hematite (α-Fe ₂ O ₃ ), maghemite (γ-Fe ₂ O ₃ ), or magnetite (magnetite, Fe ₃ O ₄ ). It may optionally further contain at least one of manganese (Mn), cobalt (Co), nickel (Ni), zinc (Zn), and gadolinium (Gd). In this way, when using magnetic beads, the bead to which the receptor is bound (receptor bead) can be more easily separated from other components (for example, receptors that are not attached or fixed) using a means such as a magnet.

Meanwhile, in the illustrated embodiment, the receptor may be a substance capable of ligand-receptor interaction, such as an antibody or aptamer. At this time, the target may include, for example, an exosome surface biomarker protein (CD-9, CD-63, CD-81, CD-82, etc.), and may also include a biomarker protein for various diseases (CD-82, etc.) -44, beta-amyloid, tau), etc. As an example, the receptor may be an antibody corresponding to a target protein, for example, when the target protein is CD-63. The receptor may be an anti-CD-63 antibody.

In order to attach or bind these receptors to beads, they can be activated, for example, by applying a chemical method using a chemical functional group. According to an exemplary embodiment, in the case of a chemical functional group that can be introduced into a bead, it can be attached or bound to the surface of the bead and binds or couples to a receptor (or a corresponding chemical functional group bound to a receptor) (e.g. , covalent bond, etc.) can be selected from a variety of types. As an example, chemical methods may use EDC (N-hydroxysuccinimide)/NHS (N-hydroxysuccinimide) reaction, avidin (and/or streptavidin)/biotin combination, sulfide-maleimide, etc. Specifically, avidin ( and/or streptavidin)/biotin combination may be used. In this regard, it can be used by activating the bead surface with avidin (or streptavidin) and conjugating biotin to the receptor. In this regard, biotin is a type of vitamin, specifically a B-complex vitamin consisting of a ureido (tetrahydroimidizalone) ring fused to a tetrahydrothiophene ring (hexahydro-2-oxo-lH-thieno[3,4 -d]imidazoline-4-valeric acid), has a molecular weight of about 244 g/mol, and has a valeric acid substituent attached to one of the carbon atoms of the tetrahydrothiophene ring. Biotin can specifically bind to avidin or streptavidin due to its strong affinity. For example, four biotin molecules can bind to one streptavidin molecule. Since attachment by the above-mentioned chemical method is widely known in the art, detailed description will be omitted.

According to an exemplary embodiment, when there are multiple target exosomes, there may also be multiple receptors attached to or bound to the beads.

In an exemplary embodiment, the step of attaching the receptor to the beads may be performed under stirring conditions, for example, at 4 to 30° C., specifically at room temperature. The concentration of the beads upon attachment (reaction) with the receptor can be adjusted, for example, in the range of about 0.1 to 1 mg/mL, specifically about 0.2 to 0.5 mg/mL. In certain embodiments, attaching the receptor to the beads may be performed at room temperature over about 30 to 60 minutes. Beads may be stored at approximately 4°C except when the bead receptor reaction described above is performed.

In addition, when the reaction (or processing) step for forming receptor beads is completed, post-processing known in the art, such as removal of remaining receptors that did not participate in the reaction, washing, etc., can be performed.

Meanwhile, as described above, when beads to which a receptor having specific binding ability to target exosomes is attached (i.e., receptor beads) are prepared, a step of contacting the exosome-containing sample can be performed. At this time, the receptor beads may be contacted in a dry state, or may be contacted in the form of a receptor bead solution (dispersion).

As shown, receptor beads can be introduced into an exosome-containing sample (for example, it may be a body fluid, and more specifically, it may be blood or serum separated therefrom). In this regard, the concentration of exosomes in the above-described exosome-containing sample (initial sample or sample before pretreatment) is, for example, about 10 ⁸ to 10 ¹² vesicles/mL, specifically about 10 ⁹ to 5×10 ¹¹ vesicles. /mL, more specifically, it may range from about 10 ¹⁰ to 10 ¹¹ vesicles/mL, and in some cases, the level may be below the detection limit through additional dilution of body fluids.

In this regard, the amount of receptor beads introduced into the exosome-containing sample can be determined by considering the volume of the exosome-containing sample (specifically, body fluid) to be mixed, for example, mixed per 10 μL of the sample. The amount (concentration) of the receptor beads may be, for example, set in the range of about 0.2 to 0.5 mg/mL, specifically about 0.25 to 0.45 mg/mL, and more specifically about 0.3 to 0.4 mg/mL. Additionally, the volume of the receptor beads may be, for example, about 100 to 300 μL, specifically about 150 to 200 μL, but this can be understood as an example.

According to an exemplary embodiment, the reaction conditions of the receptor beads and the exosome-containing sample are not particularly limited, but after combining the exosome-containing sample and the receptor beads, a relatively low temperature (e.g., about 1 to 10 The reaction may be carried out at ℃, specifically about 3 to 5℃, more specifically about 4℃) for about 5 to 12 hours (specifically, about 6 to 10 hours, more specifically about 7 to 9 hours). Alternatively, it may be carried out at a relatively high temperature, for example about 20 to 35° C., specifically at room temperature, with stirring for about 0.5 to 3 hours, specifically about 0.8 to 2 hours.

According to the above-mentioned procedure, the exosomes in the sample can bind to the receptor attached to the bead, and then the exosome bound to the bead is dissociated or separated, that is, the bond between the exosome and the receptor bead is cut to form the exosome. A step of preparing a concentrated exosome solution can be performed by dissociating the exosomes in solution.

According to an exemplary embodiment, means known in the art (for example, means for dissociation of an antigen-antibody complex) can be applied as long as they can cleave the bond between the exosome and the receptor of the receptor bead. In this regard, representative examples include methods using glycine-hydrochloric acid buffer, citrate buffer, acetate buffer, and phosphate buffer. As an example, the concentration of the buffer solution can be determined considering the concentration and separation state of exosomes, for example, about 10 to 200 mM, specifically about 50 to 150 mM, more specifically about 100 to 200 mM. It may be in the range of, and the pH of the buffer solution may be adjusted, for example, in the range of about 2.5 to 4, specifically in the range of about 3 to 3.5. According to a specific embodiment, a glycine-hydrochloric acid buffer solution may be used, which may be advantageous in providing a pH and osmotic environment suitable for dissociation of exosomes. The concentration and/or pH of the above-mentioned buffer solution can be changed depending on the type, reaction time, reaction temperature, etc. of each exosome and receptor.

In addition, in an exemplary embodiment, the content (concentration) of the exosome-receptor complex beads in the buffer solution is, for example, about 2 to 10 g/cm3, specifically about 4 to 8 g/cm3, more specifically about 5 to 5 g/cm3. It can be adjusted in the range of 7 g/cm3, but this can be understood as an example.

In the above-described embodiment, the dissociation reaction may be performed, for example, at about 3 to 6°C (specifically, about 4 to 5°C) for about 500 to 1000 minutes (specifically, about 600 to 900 minutes). Alternatively, it may be performed at higher temperature conditions, for example, about 20 to 30° C. (specifically, about 23 to 25° C.) over about 20 to 100 minutes (specifically, about 30 to 60 minutes).

In this way, the exosome solution generated (pretreated) through dissociation or separation may be in a state in which various interfering components or impurities contained in the initial exosome-containing sample are substantially removed, and also in the pretreated liquid sample. The concentration of exosomes is, for example, at least about 50%, specifically at least about 100%, more specifically about 200 to 500%, compared to the initial exosome-containing sample (exosome-containing solution before pretreatment). May be in a state of increased concentration. As an example, exosomes can be concentrated by adjusting the amount (volume) of the buffer solution (e.g., glycine-hydrochloric acid buffer solution) added during dissociation of exosomes to a relatively low level, and subsequent polydiacetylene It can be applied for detection in a concentrated state on a liposome array platform.

Preparation of diacetylene-phospholipid liposomes and microarray formation of liposomes on amine-modified substrates

The procedure for manufacturing a polydiacetylene (PDA)-based microarray (array) detection platform for exosome detection according to an exemplary embodiment is shown in FIG. 2A. The process (a) in which diacetylene/phospholipid liposomes are activated and immobilized on an amine-modified substrate by EDC/NHS reaction is shown.

Referring to the drawing, diacetylene-phospholipid liposomes are prepared separately from the selection and concentration steps of exosomes described above.

According to an exemplary embodiment, the diacetylene monomer may be a compound containing a diacetylene group, for example, consisting of pentacosadiyonic acid (PCDA), trocosadiyonic acid (TCDA), heneicosadiynoic acid (HCDA), and heptadecadiynoic acid (HDDA). It may be at least one selected from the group. Specifically, the diacetylene monomer may be PCDA and/or TCDA, and more specifically, may be PCDA.

Additionally, phospholipids include, for example, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), and distearoylphosphatidylglycerol. Glycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidyl glycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroyl Ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleic acid) Oiloxy)-3-(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidyl acid (DMPA), dipalmitoylphosphatidyl acid (DPPA), distearoylphosphatidyl Acid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine ( It may be at least one selected from the group consisting of DPPS) and distearoylphosphatidylserine (DSPS). Specifically, it may be at least one selected from the group consisting of DMPC and DMPA, and more specifically, it may be DMPC.

In this regard, inserting phospholipids may affect the sensitivity of polydiacetylene (PDA), which will be described later, so it may be advantageous to take this into account and combine them in an appropriate ratio. Illustratively, the molar ratio of diacetylene monomer:phospholipid may range, for example, from about 2 to 10:1, specifically from about 3 to 8:1, and more specifically from about 4 to 6:1.

According to an exemplary embodiment, the diacetylene/phospholipid liposome complex can be prepared by a thin film hydration method, a syringe method, etc., and specifically, the thin film hydration method can be applied. As an example, when applying the thin film hydration method, solutions of each diacetylene monomer and phospholipid can be prepared first, and at this time, at least one selected from chloroform, tetrahydrofuran, ethanol, acetone, etc. can be used as a solvent. In addition, the concentration of each solution may be adjusted, for example, in the range of about 5 to 30 mg/mL, specifically about 10 to 25 mg/mL, and more specifically about 15 to 20 mg/mL. It can be set around 20 mg/mL. Afterwards, the two solutions are combined in consideration of the molar ratio of the above-mentioned diacetylene monomer:phospholipid, then dried with an inert gas (e.g., nitrogen, argon, etc.) to form a lipid thin film, and water is subsequently added. In this case, the amount of water added can be adjusted so that the concentration of lipids and phospholipids is in the range of, for example, about 0.2 to 2mM, specifically about 0.5 to 1mM. However, the above-mentioned conditions may be understood as illustrative purposes.

In addition, after addition of water, it can be uniformly dispersed or dissolved using a dispersing means known in the art (e.g., sonicator, homogenizer, etc.). At this time, the temperature is, for example, about 60 to 95 degrees Celsius. It can be adjusted in the range of ℃, specifically about 70 to 90 ℃, more specifically about 75 to 85 ℃. In a subsequent step, the dispersion (or solution) is crystallized by cooling to, for example, about 0 to 10° C., specifically about 2 to 8° C., more specifically about 3 to 5° C., thereby forming diacetylene/phospholipid liposomes (lipid vesicles). It can be induced to be reassembled.

According to one embodiment, an amine-modified substrate is provided to form an array of diacetylene/phospholipid liposomes. At this time, the material of the substrate may be, for example, at least one selected from glass, silicon wafer, transparent plastic, etc., and more specifically, it may be a substrate that provides a two-dimensional plane made of glass.

According to an exemplary embodiment, the amine-modified substrate may be one in which the surface of the substrate is modified with amino-terminated silane. In this regard, amino-terminated silanes include 3-aminopropyltriethoxysilane, 3-aminopropyl trimethoxysilane, N-(2-aminoethyl)-3-amino Propyl trimethoxysilane (N-(2-aminoethyl)-3-aminopropyl trimethoxysilane), N-(2-aminoethyl)-3-aminopropyl triethoxysilane (N-(2-aminoethyl)-3-aminopropyl triethoxysilane ), 3-2-(2-aminoethylamino)ethylaminopropyl trimethoxysilane (3-2-(2-aminoethylamino)ethylaminopropyl trimethoxysilane), 3-2-(2-aminoethylamino)enenylaminopropyl triethylene It may be at least one selected from 3-2-(2-aminoethylamino)ethylaminopropyl triethoxysilane, 3-aminopropyl methyldimethoxysilane, etc., and more specifically, 3-aminopropyl triethoxysilane. Toxysilane can be used, which can be advantageous in that it provides a cleaning action and an amine group so that polydiacetylene liposomes can be effectively attached to the surface.

In order to modify the substrate surface with amino-terminated silane, a solution of an aminosilane-based compound can be prepared, and the solvent that can be used in this case is an aliphatic alcohol (e.g., an aliphatic alcohol with 1 to 3 carbon atoms such as methanol, ethanol, etc.) , aromatic hydrocarbons (eg, toluene, benzene), etc. Additionally, the concentration of the aminosilane compound solution may be, for example, in the range of about 0.5 to 4% by weight, specifically about 1 to 3% by weight, and more specifically about 2 to 2.5% by weight.

Thereafter, the aminosilane compound solution can be modified by contacting the substrate surface. The contact temperature is, for example, in the range of about 20 to 30 ° C. (specifically, about 23 to 27 ° C.), and the contact time is, for example, For example, it can be adjusted in the range of about 0.5 to 3 hours (specifically, about 1 to 2 hours).

According to an exemplary embodiment, prior to contacting the diacetylene/phospholipid liposome solution onto an amine-modified substrate, a method known in the art to facilitate binding to a specific receptor as shown, e.g., the EDC/NHS couple, is used. The surface of diacetylene/phospholipid liposomes can be activated using chemical functional groups that can implement ring reactions, sulfone-maleimide binding reactions, avidin-biotin binding reactions, etc. These chemical functional groups can be attached or bound to the surface of lipid endoplasmic reticulum and can be selected from a variety of types that can bind or couple (for example, covalently bond, etc.) with a receptor. More specifically, the EDC/NHS coupling reaction can be applied, in which case EDC and NHS can be added to the diacetylene/phospholipid liposome solution, where the sum of the concentrations of EDC and NHS is, for example, about 10. It may be adjusted in the range of from 200 mM, specifically about 20 to 100 mM, and more specifically about 30 to 50 mM.

According to an exemplary embodiment, the diacetylene/phospholipid liposome solution prepared as described above is used to array on an amine-modified substrate, for example, through tips or pins containing the diacetylene/phospholipid liposome solution. A method of forming an array by contacting a substrate, or a non-contact method of forming an array by dropping a droplet-shaped diacetylene/phospholipid liposome solution on the substrate can be adopted. According to an exemplary embodiment, the liposome array (microarray) formed as described above may be in a checkerboard pattern.

According to an alternative embodiment, the array can be made using a lithography technique that is advantageous for commercial scale manufacturing, as schematically shown in FIG. 2B.

Referring to the drawing, first, photoresist is applied on the substrate and then irradiated with ultraviolet rays using a mask to engrave a pattern at the point where the polydiacetylene liposome is attached, while the remaining portion is removed through development. At this time, the substrate surface can be optionally hydrophobically modified, for example, FDTS (Trichloro(1H,1H,2H,2H-perfluorooctyl)silane) vapor in a reduced pressure dryer can be used. Accordingly, photoresist is selectively left only in the areas corresponding to the array, and other areas have a hydrophobic surface. Thereafter, as described above, after performing the amine modification treatment, cleaning may be performed by immersing in a cleaning solution (for example, an aliphatic alcohol having 1 to 3 carbon atoms, specifically ethanol) and selectively applying ultrasonic irradiation.

Next, the activated diacetylene/phospholipid liposome solution as described above is brought into contact with the amine-modified substrate to react, and then subjected to a washing process to form a diacetylene/phospholipid liposome array on the substrate.

In the case of the above-mentioned lithography method, the water droplet-based array method (contact or non-contact) can solve the problems that are limited in producing polydiacetylene microarrays with a size (diameter) of less than 100 ㎛, providing higher sensitivity. It can provide achievable advantages.

In this embodiment, when the diacetylene/phospholipid liposome solution is arrayed on an amine-modified substrate to form a microarray of the diacetylene/phospholipid liposome solution on the substrate and the polymerization reaction is subsequently performed, it is based on an aqueous medium. Compared to the polydiacetylene liposome detection platform (or system), the volume (or amount) of sample required for measurement can be reduced and the ratio of detection material to liposome can be increased, providing the advantage of increasing detection sensitivity. can do.

According to an exemplary embodiment, the diacetylene/phospholipid liposome solution may array diacetylene/phospholipid liposomes on an amine-modified substrate using, for example, an aminosilane-based compound. As an example, as shown in FIG. 4, which will be described later, a plurality of reactors or wells may be arranged at predetermined intervals on a substrate, and a plurality of liposome arrays may be formed within individual reactors or wells.

Formation of polydiacetylene liposome array

According to one embodiment, when an array of diacetylene/phospholipid liposomes is formed on a substrate, a target biomarker, that is, a specific molecule (specifically, present on the surface of exosomes, which are mediators for disease diagnosis or drug delivery), is placed on the liposomes. A receptor with specific binding characteristics or selectivity for a biomarker) can be introduced.

These receptors can be selected from the range of receptors used previously in the selection and enrichment process of exosome samples. Specifically, the receptor may typically be at least one selected from the entire antibody or a partial antibody (F _ab , F _(ab')2 , scFv, etc.) containing an antigen binding region, an aptamer, etc. . At this time, the aptamer can function as a nucleic acid receptor that can accommodate proteins such as CD-9, CD-63, CD-81, and CD-82, which are biomarkers of exosomes. More typically, it may be an antibody and/or aptamer.

According to an exemplary embodiment, the receptor may be a single receptor that corresponds to a specific biomarker in exosomes or includes a specific biomarker receptor. According to an alternative embodiment, the receptor may be a multiple receptor containing two or more diverse biomarker receptors that correspond to or are specific for a plurality of biomarkers in exosomes. For this purpose, any one of the multiple receptors can be first introduced or attached to the diacetylene/phospholipid liposome, and then the remaining receptors can be introduced or attached sequentially. In this way, when introducing multiple receptors, polydiacetylene liposomes modified with multiple receptors, prepared in the subsequent procedure, not only show specificity for exosomes, but also induce a more efficient ligand-receptor reaction to amplify the measurement signal. This can be advantageous compared to the case where a single receptor is applied.

In the case of diacetylene/phospholipid liposomes in which no receptors are introduced, they can exhibit a relatively large negative charge (e.g., about -30 to -20 mV) due to the functional group (e.g., carboxyl group) attached thereto. , when modified with an acceptor, it may exhibit a relatively small negative charge (for example, about -10 to 0 mV).

Additionally, the receptor can be prepared in solution form and introduced or conjugated to liposomes by applying it to the surface of a liposome microarray and then incubating it. As an example, the solvent for preparing the receptor solution may be at least one selected from, for example, phosphate buffer solution, albumin solution, distilled water, etc., and the receptor concentration thereof is, for example, about 0.1 to 100 ng/mL. , specifically about 0.5 to 50 ng/mL, and more specifically about 1 to 10 ng/mL. However, the concentration of the solution can be changed depending on the type of receptor. For example, when the receptor is an antibody, it may be, for example, about 0.1 to 50 ng/mL (specifically, about 0.5 to 5 ng/mL). When the receptor is an aptamer, it may be in the range of, for example, about 1 to 100 ng/mL, specifically about 5 to 50 ng/mL, but this can be understood as an example.

Additionally, the incubation temperature may range, for example, from about 4 to 27 °C (specifically from about 22 to 25 °C), and the incubation time may range from, for example, about 0.5 to 8 hours (specifically from about 1 to 2 hours). ) can be adjusted within the range. According to an exemplary embodiment, the fixed amount of receptors (i.e., single receptor or multiple receptors) is about 0.01 to 0.5%, specifically about 0.05 to 0.3%, more specifically about 0.1% of the total molar concentration of the diacetylene/phospholipid liposome. It may be set in the range of 0.2%, but this can be understood as an example.

Meanwhile, it is worth noting that in this specific example, the diacetylene/phospholipid liposome complex is arrayed on a substrate, a receptor is introduced, and then a polymerization reaction is performed to form a microarray of polydiacetylene liposomes. Compared to the method of preparing polydiacetylene liposomes by performing a polymerization reaction in advance and then arranging them, liposomes and receptors are competitively attached to the substrate, so compared to the case where liposomes and receptors are mixed to form an array, liposomes are This can be advantageous because it can be densely attached to the substrate.

According to an exemplary embodiment, the polymerization reaction of diacetylene/phospholipid liposomes arrayed on an amine-modified substrate may be a photopolymerization reaction by ultraviolet irradiation or a polymerization reaction by gamma-ray irradiation, and more specifically, the polymerization reaction by ultraviolet irradiation. It may be a polymerization reaction using irradiation.

According to a specific embodiment, the polymerization reaction may be a photopolymerization reaction by irradiating ultraviolet rays, and the irradiated ultraviolet ray wavelength is, for example, about 100 to 400 nm, specifically about 150 to 350 nm, more specifically about 200 to 200 nm. It may have a wavelength in the 300 nm range. In addition, the ultraviolet ray intensity can be adjusted within a range of, for example, about 100 to 1000 μW/cm2, specifically about 200 to 600 μW/cm2, and more specifically about 300 to 500 μW/cm2. In addition, the ultraviolet irradiation time may be selected within a range of, for example, about 0.5 to 10 minutes, specifically about 1 to 5 minutes, and more specifically about 2 to 3 minutes. Additionally, the ultraviolet irradiation energy can be adjusted within a range of, for example, about 0.02 to 0.2 J/cm2, specifically about 0.05 to 0.1 J/cm2. The polymerization conditions by ultraviolet irradiation described above may be understood as exemplary.

In this way, as the polymerization reaction is performed, a polydiacetylene liposome array (microarray) that can specifically bind to the target exosome or the biomarker contained therein is formed. At this time, as described above, polydiacetylene liposomes can be formed in a grid pattern similar to a checkerboard. In the case of this alignment pattern, when observing the fluorescence of a polydiacetylene liposome array, measurement is made by including as many arrays as possible in one image. It can reduce the time needed.

On the other hand, polydiacetylene has a stable structure with an internal space isolated by a lipid layer membrane (specifically, a double layer). In addition, it does not develop color before irradiating ultraviolet rays, but after polymerization, it develops a blue color with a maximum absorption wavelength of 640 nm. Afterwards, the absorption spectrum may change due to external stimuli such as temperature, pH, solvent, and molecular recognition, resulting in a color transition to red with a maximum absorption wavelength of 540 nm. In this way, the optical properties of polydiacetylene liposomes arrayed on a substrate can change without a separate label.

According to an exemplary embodiment, the area of the individual polydiacetylene liposomes constituting the microarray is, for example, about 5000 to 500000 μm ² (specifically about 7000 to 100000 μm ² , more specifically about 10000 to 50000 μm ² ). It may be in the range of . In addition, the size (diameter; can be measured by fluorescence microscopy) of the macroarrayed polydiacetylene liposomes may be somewhat reduced compared to the diacetylene/phospholipid liposome array, for example, about 100 to 600 ㎛, Specifically, it may range from about 200 to 500 μm, more specifically about 250 to 400 μm (when droplet-based contact or when using a non-contact array method).

In this regard, improved sensitivity can be expected because the smaller the size of the polydiacetylene liposome array, the higher the reaction ratio of polydiacetylene liposomes in a sample of the same concentration and volume. In this regard, when the above-described lithography method is adopted, a size (diameter) of, for example, about 100 μm or less, specifically about 50 μm or less, more specifically about 10 μm or less, especially about 2 to 5 μm, is obtained. It can be applied to fabricate a polydiacetylene microarray.

In addition, the spacing between arrayed polydiacetylene liposomes may be adjusted, for example, in the range of about 0.1 to 0.6 times, specifically about 0.2 to 0.5 times, and specifically about 0.3 to 0.4 times the array diameter. As an example, the spacing between arrayed liposomes may be adjusted, for example, in the range of about 20 to 300 ㎛, specifically about 30 to 200 ㎛, and more specifically about 50 to 100 ㎛.

Meanwhile, in the case of a polydiacetylene microarray formed as in the embodiment shown in FIG. 4, the diameter of one reaction furnace is, for example, about 2 to 7 mm, specifically about 3 to 6 mm, and more specifically about 4 mm. It may range from to 5 mm. Additionally, about 2 to 10 polydiacetylene arrays, specifically about 4 to 6 polydiacetylene arrays, may be formed in one reactor.

In this way, when polydiacetylene liposomes are arranged on a substrate in the form of an array (microarray), the exosome-containing sample (solution) at a concentration lower than the measurable range on an aqueous medium and a small amount of sample are used as described above. Detection is also possible. In particular, the size of the polydiacetylene liposome array can be reduced, thereby achieving high sensitivity characteristics. As an example, in the case of one reactor or well, when forming an array with a size of about 100 to 600 ㎛ inside a circle of about 3 to 5 mm, the reactor can store about 5 to 10 ㎕ of sample. is required. On the other hand, in the case of an aqueous medium-based polydiacetylene liposome platform, approximately 20 μl of sample is required.

In addition, in the case of polydiacetylene liposomes using an aqueous medium, an exosome concentration of about 3 × 10 ⁸ vesicles/mL is required for observation using a fluorescence signal, whereas the microarray-based platform according to this embodiment requires about Detection is possible even at exosome concentrations as low as 1×10 ⁷ vesicles/mL.

Detection of exosomes

Figure 2 shows the process (b) of selectively detecting exosomes by conjugating (attaching) the receptor to the surface of polydiacetylene (PDA) to provide specificity to the array detection platform.

As shown, the purified and concentrated exosome solution was used as a sample and brought into contact with the polydiacetylene liposome array on the substrate, so that the exosomes specifically interact with the receptors (single receptors or multiple receptors) attached to the liposomes. It combines, and as a result, the optical properties of the polydiacetylene liposome change. This change in optical properties can be measured with the naked eye or by a measuring means or device commonly used in the art to qualitatively and/or quantitatively detect exosomes in the sample. According to an exemplary embodiment, changes in optical properties may be measured using a device, and such measuring device may be, for example, a UV-Vis spectrometer or a fluorescence spectrometer.

Although the present disclosure is not bound by a particular theory, this change in optical properties can be explained as follows: the biomarker present on the surface of the exosome and the receptor immobilized on polydiacetylene specifically bind or bind ( In other words, physical stimulation can be applied to the surface of the polydiacetylene polymer through ligand-receptor interaction. This physical stimulus is transmitted to the main chain of the conjugated polymer, and as the conjugated chain is twisted, it causes a change in the absorption spectrum and fluorescence. At this time, larger clusters can be formed by inducing a chain reaction through ligand-receptor interaction.

According to an exemplary embodiment, the size of the cluster formed by binding of the exosome and the modified polydiacetylene liposome is, for example, about 10 to 200 nm, specifically about 20 to 100 nm, more specifically about 30 to 50 nm. It may be in the nm range. However, the present disclosure is not limited to this numerical range, and may vary depending on the nature and type of the exosome.

According to an exemplary embodiment, the biomarker on the exosome may typically be a tetraspanin protein, which tetraspanin protein is a group consisting of CD-9, CD-63, CD-81, and CD-82. It may be at least one selected from. These biomarkers correspond to ligands and can specifically bind to receptors immobilized on modified polydiacetylene liposomes through ligand-receptor interaction (or reaction).

As previously explained, the receptor can be a single receptor or multiple receptors, and when fixed in the form of multiple receptors, more ligands are created between the polydiacetylene liposome and the exosome by inducing binding to multiple biomarkers present in the exosome. -It can provide a signal amplification effect by inducing a receptor response.

According to this specific example, the specific reaction between the biomarker of exosomes and the receptor of polydiacetylene liposomes is performed on a microarray platform of liposomes formed on a substrate, and by applying this liposome array-based platform, various detection substances can be detected. It can provide the advantage of being able to observe signals simultaneously and the detection process and signal analysis are relatively easy, especially compared to the method of detecting polydiacetylene liposomes in combination with exosomes in an aqueous medium, Not only is the amount of sample required for measurement small, but higher sensitivity can be achieved by increasing the reaction ratio of exosomes to liposomes.

According to an exemplary embodiment, the contact temperature between the sample (i.e., the exosome-containing aqueous solution previously subjected to the selection and concentration process) and the polydiacetylene liposome array is, for example, about 20 to 50 ° C., specifically about 25 to 45 ° C. ℃, more specifically, can be adjusted within the range of about 30 to 40 ℃, and the contact time can be adjusted, for example, in the range of about 10 to 120 minutes, specifically about 20 to 100 minutes, more specifically about 30 to 90 minutes. You can. However, the temperature and time conditions described above are provided as examples, and the present disclosure is not limited thereto.

Meanwhile, the detection platform according to this embodiment is useful as an exosome sensor because it can provide a simple and intuitive signal compared to a chip-based system, which is a conventional exosome detection technology. In particular, the presence or absence of exosomes (qualitative analysis) can be confirmed by measuring the color change and/or fluorescence expressed simply by contacting the exosome-containing sample with a polydiacetylene array, and further, the concentration of exosomes in the sample. (quantitative analysis) can be predicted, and is simple yet provides good reliability.

According to an exemplary embodiment, as a result of performing pretreatment (selection and enrichment) and exosome detection according to this embodiment, the detection limit of exosomes in the initial sample may be, for example, about 1×10 ⁷ vesicles/mL or less. there is.

Hereinafter, preferred examples are presented to aid understanding of the present invention, but the following examples are provided to facilitate understanding of the present invention and do not limit the present invention thereto.

Example

The materials used in this example are as follows:

- 10,12-pentacosadiyonic acid (PCDA) was purchased from GFS Chemicals (Powell, OH).

- 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), N-hydroxysuccinimide (NHS) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Purchased it from.

- (3-aminopropyl)triethoxysilane (APTES) and Tween ^® 20 were purchased from Sigma-Aldrich (St. Louis, MO).

- Anti-CD-9 monoclonal antibody, anti-CD-63 monoclonal antibody and anti-CD-81 monoclonal antibody were purchased from ThermoFisher Scientific (MA, USA).

- Anti-CD63 aptamer and anti-CD81 aptamer were purchased from Aptamer Sciences, Inc. (Gyeonggi-do, Korea).

-PBS buffer (pH 7.4) was purchased from Welgene, Inc. (Gyeongsan-si, Korea).

Example 1

Selection and enrichment of exosomes

- Preparation of receptor beads

Magnetic beads M-270 (invitrogen) surface-modified with streptavidin were stirred uniformly at a concentration of 10 mg/mL, 800 uL of which was transferred to a 1.5 mL tube, and the beads were allowed to settle on a magnetic stand for 10 minutes. Then, the supernatant was removed and dispersed in 1000 μl of PBS (pH 7.4). Thereafter, the bead washing process below is repeated two additional times.

The beads that had undergone the final washing process were dispersed in 160 uL PBS, and biotin-conjugated anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody against exosomal tetraspanin (CD9-biotin, CD63-biotin, A total of 3.3 ㎕ of CD81-biotin (1 mg/mL) solution was injected at the same concentration and volume, and then sufficiently stirred at room temperature for 30 minutes to allow avidin-biotin binding to occur.

A magnetic stand was used to precipitate receptor-bound beads (receptor beads) in a mixed dispersion (solution) of antibodies and beads, and the supernatant was removed to remove remaining unbound antibodies. The precipitated beads were washed with 1000 uL of 0.1% BSA-PBS solution and the same procedure was repeated one more time.

- Binding of exosomes in the sample to the receptor beads

The dispersion (solution) of the washed receptor beads was precipitated with a magnetic stand to remove the supernatant prior to mixing with the serum, and then mixed with the serum that had passed through a syringe filter (0.2 ㎛) in a 1.5 mL tube. The concentration of exosomes in serum diluted with buffer solution was 1×10 ⁹ vesicles/mL.

Next, the mixture of serum and aqueous bead dispersion was maintained at 4°C overnight to allow exosomes and receptor beads to bind.

- Dissociation (separation) of exosomes

The bead mixture that had undergone exosome-receptor binding was centrifuged at 100 rcf for 1 minute, then the beads were settled with a magnetic stand, and unbound proteins in the serum and supernatant were removed. Afterwards, it was dispersed in 500 ㎕ of 0.1% BSA-PBS solution, and further washed twice with the same amount of buffer solution.

After sinking the exosome-immobilized beads once more, 50 ㎕ of 50 mM glycine-HCl (pH 3.0) was added, stirred for 10 seconds, and reacted at 4°C for 10 minutes to promote the binding between exosomes and beads. By cutting, exosomes were dissociated into solution.

In the solution in which the exosomes were dissociated, the beads were precipitated on a magnetic stand, the supernatant was separated into a separate container, and 1M Tris-HCl (pH 8.0) solution was added in an amount of 1.5 μl. Next, the solution was stirred to stabilize the pH, and the final pH was confirmed to be in the range of 7.0 to 7.6. The concentration of exosomes in the dissociated solution was 5×10 ⁹ vesicles/mL.

Fabrication of polydiacetylene liposome array substrate

- Fabrication of amine-modified substrates

The slide glass was washed with bath sonication in chloroform, acetone, and ethanol for 5 minutes each, and then immersed in Piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3 : 1) for 1 hour. to remove residues, including organic substances, present on the surface.

The solution and surface residues were removed by washing with distilled water several times, and surface moisture was removed through drying. When the drying process was completed, silanization occurred on the glass surface while gently stirring in a 2% by weight 3-aminopropyltriethoxysilane (APTES)-ethanol solution for 2 hours. The silanized glass was washed with ethanol to remove unreacted APTES, and annealed in a vacuum oven at 110°C for 1 hour to complete the silanization reaction (preparation of amine-modified substrate). In order to improve the efficiency of the liposome array system and distinguish each reaction point (well), the surface of the slide glass was masked or taped to include several wells with a diameter of 5 mm.

- Formation of polydiacetylene liposome array

EDC/NHS was added to the diacetylene/phospholipid liposome solution to a concentration of 100 mM, and the mixture was arrayed on the amine-modified glass surface with a size of 200 to 500 ㎛ using a manual microarray spotter (Labnext Inc.). . Next, the reaction was performed overnight at 4°C in a humid environment to prevent evaporation, allowing the diacetylene/phospholipid liposome to attach to the surface of the glass substrate.

The glass on which the liposomes were arrayed in this way was washed twice with distilled water and 0.1% by weight Tween-20 PBS to remove residues, and the receptor (antibody) solution for the target biomarker (1 ng/mL for antibodies, and 15 μl (10 ng/mL for aptamer) was applied to the array surface and incubated at 4°C for approximately 24 hours.

Next, unreacted receptors were removed and dried by washing three times with distilled water and a buffer solution, and then polymerization was performed by irradiating UV light (400 μW/cm2) with a wavelength of 254 nm for 5 minutes.

Detection of exosomes

10 ㎕ of the previously prepared concentrated exosome solution was added dropwise to the polydiacetylene array and incubated at 30°C for about 1 hour, then the solution was removed and the absorbance and fluorescence intensity of the polydiacetylene liposome array were measured and analyzed respectively.

At this time, the color change of the polydiacetylene liposome array was calculated using UV-vis spectroscopy as shown in Equation 1 below within the range of 450 to 700 nm.

[Equation 1]

In the above formula, A refers to the relative absorbance for blue (640 nm) and red (540 nm) elements, and PB refers to before and after reaction, respectively.

The fluorescence intensity of the polydiacetylene liposome array was measured based on a fluorescence microscope (ZEISS) using a filter with an excitation wavelength of 540 to 580 nm and an emission wavelength of 600 to 660 nm, with an exposure time of 3000 ms. set. In the photos taken from the fluorescence microscope, the intensity of the array was measured based on the intensity of the pixels using the ImageJ program.

Results and Discussion

- Nanoparticle tracking assay (NTA) results of exosome samples separated using separated particles are shown in Figure 3.

According to the figure, the SEM image of exosomes extracted from plasma shows that the size of exosomes ranges from 10 nm to 150 nm.

- The external appearance of the manufactured PDA liposome array substrate is shown in Figure 4.

Referring to the drawing, the substrate is a slide glass (76 mm It can be seen that is formed in a checkerboard arrangement pattern.

- SEM images of the PDA liposome array arranged on an amine-modified substrate, (a) bare PDA liposome array, and (b) exosomes attached to the receptor-introduced PDA liposome are shown in Figure 5. In addition, (a) TEM image of PCDA/DMPC liposome, (b) particle distribution of PDA liposome, (c) surface zeta potential, and (d) UV-vis spectrum of PDA in blue state and red state are shown in Figure 6. It was.

Referring to the drawing, PCDA/DMPC liposomes are immobilized on an amine-modified glass substrate through EDC-NHS reaction, and the shape and size of PCDA/DMPC liposomes in solution are determined from the PCDA/DMPC liposomes immobilized on the substrate. It was similar to

In addition, according to Figure 6b, it indicates that the prepared polydiacetylene liposomes have a size distribution of approximately 30 to 400 nm. In addition, Figure 6c shows that the PDA liposome has a negative charge, and Figure 6d shows the results of comparing the UV-vis absorption spectra of the PDA liposome before (Blue) and after (Red) modification, that is, the shape and properties of polydiacetylene. shows.

- (a) It is shown in Figure 7.

According to the figure, as a result of XPS analysis, silanization was successfully achieved on the amine-modified substrate (FIG. 7a).

In addition, the PDA array immobilized on the amine-modified glass substrate is more densely and uniformly distributed compared to the PDA array immobilized on the unmodified glass substrate, so the polydiacetylene liposomes are attached more uniformly and at a higher density. It can be seen that (Figures 7b and 7c). Receptors for exosome biomarkers are introduced onto liposomes (endoplasmic reticulum) to provide selectivity and reactivity to exosomes, and due to the characteristics of PDA liposomes (a signal resulting from the interaction between the receptor and exosomes is generated) The sensitivity of the PDA sensor platform is controlled by the exosome capture efficiency of the receptor (concentration ratio of exosomes captured in a single PDA array dot (normalized surface area (N)) arrayed in a single well), expressed by Equation 2 below. You can see that it can be done.

[Equation 2]

In the above formula, C ₀ is the concentration of exosomes recovered from the control group from the PDA array dots into which the receptor was not introduced, and C ₁ is the concentration of the remaining exosomes recovered from the PDA array dots into which the receptor was introduced.

Meanwhile, as a result of exosome characterization, the SEM image and size distribution of exosomes are shown in Figure 8.

From the above figure, the binding between exosomes and receptors was confirmed. In particular, when multiple exosome ligands are applied instead of a single receptor, more efficient exosome capture can be achieved, and capture efficiency can be improved by increasing the probability of interaction with exosomes. In particular, it is noteworthy that the capture of exosomes can be confirmed through the label-free PDA array platform, and the improvement in sensitivity of the multi-target detection platform can be quantified from these results.

- Optimization of receptor density on PDA surface

For the interaction between immobilized liposomes and exosomes, protein-based antibodies and nucleic acid-based aptamers were used as receptors. Ligand-receptor interaction modifies the main chain of PDA incorporated into immobilized liposomes, causing changes in the optical properties of PDA. Sufficient receptor density is required on the PDA surface to effectively transmit stimulation to the conjugated yen-in bonds of the PDA backbone. For the purpose of optimizing the concentration of the exosome-targeting receptor, receptor solutions ranging in concentration from 0.1 ng/mL to 1000 ng/mL were evaluated. Exosome incubation was performed at the same exosome concentration (1 x 10 ¹⁰ vesicles/mL), and the receptor concentration showing the strongest signal was selected (see Figure 9). In the case of the above figure, the fluorescence intensity of the PDA liposome array is shown according to the concentration of the receptor introduced into the PDA liposome array for the receptors (antibodies and aptamers), 1 ng/mL for antibodies and 10 ng/mL for aptamers. It can be seen that mL shows the best fluorescence signal for exosomes. In other words, it indicates that the PDA liposome array most efficiently releases signals for exosomes at the corresponding concentration.

On the other hand, when the receptor density was too high, the polymerization reaction of the PDA liposome did not proceed smoothly, which could be attributed to the steric hindrance caused by the receptor molecule strongly bound on the liposome, which inhibited the self-assembly of the diacetylene structure (Figure 10).

Conversely, if the receptor density is too low, the probability of interaction with exosomes and binding efficiency decreases. When using the anti-CD63 receptor-based PDA platform, the fluorescence intensity of PDA was strongest at 1 ng/mL antibody solution and 10 ng/mL aptamer solution.

- Spectrometric transition of PDA liposomes through exosome interaction

If the ligand-receptor interaction occurs on the surface of the receptor-PDA complex, the interaction stimulus is transmitted to the PDA backbone and disrupts the conjugated yen-in bond. The modified conjugate bond shifted the energy absorption spectrum, causing a color change in the PDA supramolecule (see Figure 11). Blue PDA liposomes immobilized on an amine-modified glass substrate showed a maximum absorption peak around 610 nm (Figure 11a). When exosomes bound to receptors on the PDA surface, the intensity of the maximum absorption peak decreased and a new absorption peak appeared around 540 nm.

The absorption spectrum of blue PDA without exosomes in the 540 nm region, indicating the red-type modified PDA, did not significantly deviate from the baseline spectrum. However, when exosomes were introduced, the spectrum began to deviate from the baseline, and as the exosome concentration increased, the deviation area also increased. Under a rich exosome environment (1 x 10 ¹⁰ vesicles/mL), significant spectral changes were observed in the 540 nm region (see Figure 11b).

In addition to the color change, the fluorescence intensity derived from the PDA array according to the concentration of each type of exosome was also measured. The results are shown in Figure 12.

Referring to the figure, when the PDA liposome to which the receptor is attached changes to a red form due to the interaction induced by the addition of exosomes, it shows a fluorescence peak in the 640 nm region. As the exosome concentration increased, the fluorescence intensity of PDA induced by ligand-receptor interaction tended to increase. This phenomenon supports that the PDA array platform is a suitable method for exosome detection and quantification.

- Detection limits of PDA array platform

In order to confirm the change in detection limit of the PDA platform due to multiple targets, PDA signals were characterized for individual exosome concentrations, and the results are shown in Figure 13.

For LOD calculation, the sum of three times the background and surface deviations was set as the baseline. LOD corresponds to the exosome concentration at the point when the signal begins to cross the baseline. In the case ^of the antibody, in the single target array, the LOD was measured to be about ^3.5 On the other hand, the CD63 and CD81 ^multi -target platform required 4

In the case ^of the aptamer, in the single target array, the LOD was measured ^to be about 1.5 On the other hand, the CD63 and CD81 multi-target platform required ^3.5

- Analysis of the effect of multiple receptors on capture efficiency

As a result of the quantification experiment of exosomes captured on the PDA liposome surface, (a) the ratio of captured exosomes by receptor type, and (b) the fluorescence intensity of fluorescent exosomes by receptor type are shown in Figure 14.

Referring to the figure, in a relatively abundant exosome environment, the capture efficiency of exosomes was measured at a similar value regardless of the type of receptor or target ligand. However, in a rare exosome environment, the concentration of recovered exosomes showed differences even if the receptors were introduced at the same density. As such, the exosome binding efficiency was greater in the multi-target platform compared to the single-target platform. Exosome binding efficiencies for multiple target platforms were approximated to the sum of exosome efficiencies for individual single target platforms. Surprisingly, exosome binding efficiency in the mixed receptor system appeared to be increased for both antibodies and aptamers. This can be seen because the possibility of ligand-receptor interaction is increased due to the PDA array recognizing multiple targets on exosomes, supporting the advantages of the PDA array platform.

As an additional quantification method, exosomes labeled with a fluorescent dye were incubated following the same procedure, and the binding density of attached exosomes was assessed using fluorescence microscopy.

In the rich exosome solution, a relatively strong fluorescence signal appeared even if CD63 or CD81 existed as a single receptor, and the increase in fluorescence intensity due to the mixed receptor system was not significant (see Figure 14b). On the other hand, when a single receptor was present at a low concentration of exosomes, a relatively low fluorescence intensity was observed, indicating that the exosomes were not sufficiently attached to the surface of the PDA array. Meanwhile, when multiple target receptors were present, the exosome fluorescence signal increased compared to when a single receptor was present, and the increase was larger compared to a high concentration exosome environment. This phenomenon was observed in both aptamer- and antibody-based systems. That is, regardless of the type of receptor used, the multi-target platform increased exosome capture efficiency, and this effect was significant in low concentration environments. From the attachment experiment of fluorescent exosomes, it can be predicted that exosomes separated from plasma using centrifugation can also be applied to the PDA array system, and that the exosome collection efficiency can be increased by multiple receptors. In particular, it is believed to be advantageous for application at low biomarker concentrations.

- Evaluation of exosome specificity of PDA array

To confirm the selectivity or specificity of the PDA array platform in exosome detection, the response characteristics of PDA to various proteins present in plasma, such as BSA, immunoglobulin G (IgG), and fibrinogen, were evaluated. Using the same protocol for exosome detection, 1 x PBS buffer, BSA, IgG and fibrinogen solutions were applied to the PDA array. Each plasma protein was injected into the array at a concentration of 20 μg/mL without exosomes, and PDA fluorescence was measured after incubation. PDA fluorescence signals for various plasma proteins without exosomes did not show significant deviations around the LOD (see Figure 15). This suggests that plasma proteins do not interact with receptors on the PDA surface and that the array has sufficient selectivity for exosome detection.

- The results of fluorescence signal analysis of the PDA liposome array according to the exosome selection method are shown in Figure 16. The exosome selection method includes a method of selecting exosomes using beads into which the CD63 receptor is introduced, a method of selecting exosomes using exoquick®, an exosome isolation kit from SYSTEM BIOSCIENCES, and a method of selecting exosomes using Exoquick and CD63 receptor sequentially. Exosomes were classified using a method of selection. The exosome solution separated through each method was applied to the polydiacetylene liposome array into which the amyloid beta receptor was introduced, and the fluorescence intensity signal of the polydiacetylene array was analyzed.

According to the figure, among the exosome selection methods, the highest fluorescence intensity signal of the polydiacetylene array was observed in the exosome solution from the exosome selection method using ExoQuick and CD63 sequentially. This means that in the exosome selection method using Exoquick and CD63 sequentially, the polydiacetylene array incorporating the amyloid beta receptor interacts the most with exosomes containing amyloid beta. Moreover, the microbead-based exosome isolation technology according to this example has an advantage over other separation technologies in a polydiacetylene array-based measurement system, and can bring about a greater effect when used in combination with other separation technologies. Instruct. Furthermore, considering that a larger signal was measured with the exosome isolation method applied to the PDA liposome array compared to the existing commercial separation method (exoquick, EQ), a greater synergy effect can be derived when linked with these. suggests.

- The exosome solution separated by ExoQuick and bead-based separation technology was applied to the PDA liposome array at different exosome concentrations, and the results of fluorescence signal analysis are shown in Figure 17.

According to the figure, when diluted starting from the undiluted solution, the fluorescence intensity of the array gradually decreased, and the detection limit was confirmed to be a discernable signal even in the exosome solution diluted 10 times from the initial concentration.

- The results of measuring the fluorescence signal of the PDA liposome array between the disease group (AD) and the normal group (NC) are shown in Figure 18. The exosome solution was separated from the plasma of each experimental subject in the same manner as above, applied to a polydiacetylene array into which amyloid beta was introduced, and the resulting fluorescence intensity was expressed.

According to the figure, the fluorescence intensity in the disease group was approximately 20% higher than that in the normal group, and there were some overlapping areas between the disease group and the normal group, but in some samples, groups showing significantly higher fluorescence intensity were observed. It has been done. In other words, the fluorescence intensity in the normal group was approximately 15 to 33 units, but in the disease group, some indicators exceeding this were observed.

- To confirm the reproducibility of the PDA liposome array system, the same sample was independently performed in different environments and the fluorescence intensity in each experiment was listed. The results of the analysis are shown in Figure 19.

According to the figure, as a result of independently measuring the fluorescence intensity of the array in the normal group (top) and the disease group (bottom), it was observed that the array signals measured independently and repeatedly were similar in a significant number of samples. Through this, it can be seen that the PDA liposome array platform does not show large errors even when measured in different environments, and overall produces a signal similar to the measurement target.

- The AUC-ROC curves for each of the disease group and normal group are shown in Figure 20. According to the above figure, the accuracy of classifying the disease group and the normal group was predicted to be at the 0.76 level.

- As a fluorescence microscope image according to the polydiacetylene array manufacturing method, the results for the contact array (top) and lithography-based array (bottom) are shown in FIG. 21.

According to the figure, in the case of a lithography-based array, a PDA liposome microarray of a smaller size can be implemented compared to a contact array, and detection accuracy is also good.

Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (20)

a) preparing a receptor bead to which at least one receptor having specific binding ability to at least one surface biomarker of the target exosome is attached;
b) contacting the exosome-containing sample with the receptor bead to form an exosome-receptor complex on the bead, and then concentrating the exosomes by separating or dissociating the exosomes from them;
c) providing a diacetylene-phospholipid liposome solution separately from steps a) and b); and
d) forming a microarray of diacetylene-phospholipid liposomes on an amine-modified substrate using the diacetylene-phospholipid liposome solution; and
e) attaching at least one receptor having specific binding ability to at least one surface biomarker of the target exosome to the microarray of the diacetylene-phospholipid liposome;
f) forming a microarray of modified polydiacetylene liposomes by polymerizing the diacetylene-phospholipid liposomes to which the at least one receptor is attached; and
g) Contacting the solution containing the exosomes concentrated in step b) with a microarray of polydiacetylene liposomes, thereby optically induced by a ligand-receptor reaction between the concentrated exosomes and the modified polydiacetylene liposomes in the microarray. measuring characteristic changes;
A method for detecting exosome surface biomarkers in a sample comprising a. The method of claim 1, wherein step a) further includes surface modifying the bead to attach a functional group capable of binding to at least one receptor, and attaching the at least one receptor to the surface-modified bead to target the bead. A method for detecting exosomes, comprising producing exosome receptor beads. The method of detecting exosomes according to claim 1, wherein the beads are at least one selected from the group consisting of resin beads, metal beads, and glass beads. The method of claim 1, wherein the beads have a particle size ranging from 1 to 100 μm, and
A method for detecting exosomes, characterized in that they are spherical, rod-shaped, cubic-shaped, hexagonal, triangular, hollow-shaped and/or flower-shaped. The method of claim 3, wherein the metal bead is a magnetic bead, and the magnetic bead is hematite (α-Fe ₂ O ₃ ), maghemite (γ-Fe ₂ O ₃ ), or magnetite (magnetite, Fe ₃ O ₄ ). A method for detecting exosomes, characterized in that at least one iron oxide material is selected from the group consisting of. The method of detecting exosomes according to claim 1, wherein the substrate is at least one selected from the group consisting of glass, silicon wafer, and transparent plastic. The method for detecting exosomes according to claim 1, wherein the surface of the amine-modified substrate is modified with amino-terminated silane. The method of claim 7, wherein the amino-terminated silane is 3-aminopropyltriethoxysilane, 3-aminopropyl trimethoxysilane, N-(2-aminoethyl)- 3-Aminopropyl trimethoxysilane (N-(2-aminoethyl)-3-aminopropyl trimethoxysilane), N-(2-aminoethyl)-3-aminopropyl triethoxysilane (N-(2-aminoethyl)-3 -aminopropyl triethoxysilane), 3-2-(2-aminoethylamino)ethylaminopropyl trimethoxysilane (3-2-(2-aminoethylamino)ethylaminopropyl trimethoxysilane), 3-2-(2-aminoethylamino)enylamino Of the exosome, characterized in that it is at least one selected from the group consisting of propyl triethoxysilane (3-2-(2-aminoethylamino)ethylaminopropyl triethoxysilane) and 3-aminopropyl methyldimethoxysilane (3-aminopropyl methyldimethoxysilane) Detection method. The method of detecting exosomes according to claim 1, wherein the microarray is formed in a checkerboard pattern. The method of claim 1, wherein the area of the individual polydiacetylene liposomes constituting the microarray and the distance between them are adjusted in the range of 5000 to 500000 ㎛ ² and 20 to 300 ㎛, respectively. The method of claim 10, wherein the size of individual polydiacetylene liposomes constituting the microarray is set in the range of about 100 to 600 ㎛. The method of claim 1, wherein a plurality of biomarkers are present on the surface of the exosome during step e), and the receptor is a multi-receptor specific for the plurality of biomarkers. method. The method of claim 1, wherein the diacetylene monomer is at least one selected from the group consisting of pentacosadiyonic acid (PCDA), trocosadiyonic acid (TCDA), heneicosadiynoic acid (HCDA), and heptadecadiynoic acid (HDDA), and
The phospholipids include dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), Dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidyl glycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroyl ethylphosphocholine ( DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), 1,2-bis(oleoyloxy)-3 -(Trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidyl acid (DMPA), dipalmitoylphosphatidyl acid (DPPA), distearoylphosphatidyl acid (DSPA), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), and distearoylphosphatidylinositol (DSPI) A method for detecting exosomes, characterized in that it is at least one selected from the group consisting of thearoylphosphatidylserine (DSPS). The method of detecting exosomes according to claim 1, wherein the molar ratio of diacetylene monomer:phospholipid in step c) is in the range of 2 to 10:1. The method of claim 1, wherein the receptor is at least one selected from the group consisting of a full antibody, a partial antibody containing an antigen binding region, and an aptamer. The method of detecting exosomes according to claim 1, wherein the biomarker is a tetraspanin protein. The method of claim 16, wherein the tetraspanin protein is at least one selected from the group consisting of CD-9, CD-63, CD-81, and CD-82. The method of detecting exosomes according to claim 1, wherein the change in optical properties is measured with the naked eye or a measuring device. The method of claim 18, wherein the device for measuring changes in optical properties is a UV-Vis spectrometer or a fluorescence spectrometer. The method of detecting exosomes according to claim 1, wherein the method is performed without labeling.