Crispr-cas12a-based exosomal mirna one-pot analysis method and device using hourglass structure

Abstract

The present invention relates to a CRISPR-Cas12a-based exosomal miRNA one-pot analysis method and device using an hourglass structure. The present invention provides an miRNA analysis method and device which can continuously perform the isolation and amplification of exosomes and the analysis of CRISPR Cas signals in a single reaction vessel by using multifunctional nanoparticles and an hourglass structure. The analysis method according to the present invention is characterized by: using an exosome isolation method using magnetic nanoparticles and an immune response without external equipment such as a centrifuge, thereby enabling selective isolation of only human cell-derived exosomes in a simple manner and at low cost; amplifying miRNA with CRISPR RNA (crRNA) instead of an aptamer, thus simplifying the amplification process; and analyzing a signal generated when a chromogenic enzyme immobilized via DNA is cleaved by Cas protein, thereby enabling analysis with the naked eye without separate fluorescence analysis equipment.

Description

CRISPR-CAS12A-based exosomal MIRNA one-pot analysis method and device using an hourglass structure

[0001] The present invention was made with the support of the Ministry of Science and ICT under the grant number 2710061804 and grant number RS-2022-NR069336. The research management organization of the above-mentioned project is the National Research Foundation of Korea, the research project name is “Individual Basic Research”, the research project title is “Development of an Isothermal Nucleic Acid Amplification Biochip Platform for Simultaneous Detection of Multiple Biomarkers of Tumor-Derived Exosomes”, the lead organization is Sogang University, and the research period is from March 1, 2022 to February 28, 2026.

[0002] This invention was made with the support of the Ministry of Trade, Industry and Energy under the project identification number 2050000493 and project number RS-2021-KP002505. The research management specialized organization of the project is the Korea Institute of Energy Technology Evaluation and Planning, the research project name is “Energy Human Resources Development Project”, the research project name is “CCUS Professional Human Resources Development for Green Growth”, the main organization is the KCCUS Promotion Group, and the research period is from April 1, 2021 to December 31, 2025.

[0003] The present invention relates to a CRISPR-Cas12a-based exosome miRNA one-pot analysis method and device using an hourglass structure, and more specifically, to a miRNA analysis method and device that can sequentially perform exosome separation and amplification and CRISPR Cas signal analysis in a single reaction vessel using multifunctional nanoparticles and an hourglass structure.

[0004] MicroRNA (miRNA) is a short, non-coding nucleic acid molecule consisting of approximately 22 nucleotides found in plants, animals, and viruses. It is known to regulate gene expression after transcription, including through RNA silencing and RNA interference. miRNA is encoded by DNA and can form complementary base pairs with messenger RNA (mRNA) transcribed from DNA, thereby degrading mRNA or inhibiting its translation.

[0005] Exosomes reflect the characteristics of the parent cells from which they are secreted. Composed of a phospholipid bilayer, exosomes can exist stably in the body. Unlike liposomes, which have the same form, exosomes are labeled with cell-derived proteins on their surface, making them useful for studying cell membrane proteins. Furthermore, they stably protect proteins and nucleic acids from proteases, RNases, and DNases, and they particularly stably contain miRNAs, which play a crucial role in tumor development, making them widely used in cancer research.

[0006] In particular, exosomal miRNAs are generally protected from RNAse degradation and are therefore more readily available than serum miRNAs. Exosomes are typically isolated through a series of ultracentrifugations (100,000 g), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most widely used method for amplifying exosomal miRNAs.

[0007] CRISPR-Cas12a is a protein derived from the defense system of prokaryotes. When CRISPR RNA (crRNA) and Cas protein meet complementary ssDNA, it cuts it and secondarily cuts other ssDNAs in the vicinity. Because of this trans-cleavage property, it has been used as a system that can detect specific target DNA. Cas12 has also been widely used in exosome research, and most studies consist of a system that isolates exosomes, synthesizes the extracted miRNA into DNA to activate Cas12a, and then detects electrochemical and fluorescent changes using the cleavage property of the activated Cas12a.

[0008] Analysis of exosomal miRNAs involves complex processes such as exosome isolation, miRNA extraction, miRNA extension and amplification, and detection of amplified nucleic acids, and the overall analysis requires specialized personnel and specialized equipment.

[0009] Research using the CRISPR-Cas system typically involves amplifying miRNA into DNA and reacting it with CRISPR RNA and Cas proteins to obtain a signal. However, CRISPR RNA is expensive and difficult to handle over long periods of time. Furthermore, most CRISPR cleavage experiments utilize fluorescent signals from quantum dots, which requires fluorescence analysis equipment, a disadvantage.

[0010] Therefore, it is necessary to lower the difficulty of miRNA research by developing a highly sensitive detection technology for exosomal miRNA that can be easily used.

[0011] Against this backdrop, the present inventors have invented a method and device for miRNA analysis that can sequentially perform exosome separation and amplification and CRISPR Cas signal analysis in a single reaction vessel using multifunctional nanoparticles and an hourglass structure.

[0012] The analysis method according to the present invention can selectively isolate only human cell-derived exosomes in a simple and low-cost manner by using an exosome separation method using nano magnetic particles and an immune reaction without external equipment such as a centrifuge.

[0013] The amplification process is simplified by amplifying miRNA with CRISPR RNA (crRNA) rather than aptamer.

[0014] By analyzing the signal produced when a chromogenic enzyme fixed to DNA is cleaved by a Cas protein, a method is provided that can be analyzed with the naked eye without a separate fluorescence analysis device.

[0015] The purpose of the present invention is to provide a one-pot exosome miRNA analysis method based on CRISPR-Cas12a using an hourglass structure.

[0016] Another object of the present invention is to provide a device for one-pot analysis of exosome miRNA based on CRISPR-Cas12a using an hourglass structure.

[0017] The present invention relates to a method and device for analyzing miRNA, which can sequentially perform exosome separation and amplification and CRISPR Cas signal analysis in a single reaction vessel using multifunctional nanoparticles and an hourglass structure.

[0018] Hereinafter, the present invention will be described in more detail.

[0019] One aspect of the present invention is a method for analyzing exosome miRNA, comprising the following steps:

[0020] (1) An exosome separation step in which exosomes in a cell culture medium are immunoreacted with a magnetic particle-based probe and then the exosomes are separated using magnetism;

[0021] (2) miRNA extraction step of crushing the separated exosomes and extracting miRNA;

[0022] (3) Amplification step of synthesizing target miRNA into crRNA by adding a synthetic solution;

[0023] (4) an activation step of contacting the crRNA and Cas12a protein to activate the CRISPR-Cas12a protein; and

[0024] (5) miRNA chromogenic detection step including a cleavage reaction on a chromogenic detection platform.

[0025] In the present invention, the immune response may be a binding of the antigen protein on the surface of the exosome of the cell culture medium and the magnetic particle-based probe, but is not limited thereto.

[0026] In the present invention, the magnetic particle-based probe may be, but is not limited to, a gold-magnetic nanoparticle (Au-MNPs) comprising nanoparticles composed of iron oxide nanoparticles and reduced gold ions; an anti-CD63 antibody; and a promoter.

[0027] In the present invention, the magnetic particle-based probe may react with the CD63 antigen of the nitrocellulose membrane in the LFIA system to produce a color dot signal, but is not limited thereto.

[0028] In the present invention, the average size of the gold-magnetic nanoparticles may be 30 to 36 nm, for example, 34 nm, but is not limited thereto.

[0029] In the present invention, the promoter may be a promoter for synthesis of the crRNA, but is not limited thereto.

[0030] In the present invention, the exosome separation step may be performed under conditions of 20 to 26°C, 22 to 26°C, 24 to 26°C, or 24°C, but is not limited thereto.

[0031] In the present invention, the synthetic solution may include, but is not limited to, T7 RNA polymerase, DNA ligase, acceptor oligonucleotide, and donor oligonucleotide.

[0032] In the present invention, the amplification step is a single-stranded nucleic acid molecule elongation step including a base sequence complementary to the target miRNA and a base sequence complementary to the crRNA; and

[0033] The step of T7 polymerase transcribing crDNA comprising a base sequence complementary to the single-stranded nucleic acid molecule may include, but is not limited to, this.

[0034] In the present invention, the amplification step may include a template generation step for crDNA generation and a crRNA generation step, but is not limited thereto.

[0035] In the present invention, the cleavage reaction may be a cleavage of the ssDNA linker by the CRISPR-Cas12a protein, but is not limited thereto.

[0036] In the present invention, the color detection platform may include, but is not limited to, a gold plate (Au plate), an ssDNA-biotin linker, and a multi-enzyme nanoparticle (MENP).

[0037] In the present invention, the color detection platform may be installed on the upper part of the reaction vessel, but is not limited thereto.

[0038] In the present invention, the miRNA color detection step may be initiated by flipping the reaction vessel upside down, but is not limited thereto.

[0039] In the present invention, the miRNA color detection step may additionally include a step of administering a substrate capable of reacting with the multi-enzyme nanoparticles, but is not limited thereto.

[0040] In the present invention, the miRNA color detection step may include a catalytic reaction using TMB as a substrate and the multi-enzyme nanoparticle as a catalyst, but is not limited thereto.

[0041] In the present invention, the multi-enzyme nanoparticle may be an enzyme ball capable of carrying out multiple enzyme reactions with multiple substrates by carrying multiple enzymes therein, but is not limited thereto.

[0042] In the present invention, the average size of the multi-enzyme nanoparticles may be 100 to 140 nm, for example, 120 nm, but is not limited thereto.

[0043] In the present invention, the enzyme is a chromogenic enzyme, and may be, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), lactoperoxidase, β-galactosidase, or acetylcholinesterase, but is not limited thereto.

[0044] In the present invention, the miRNA extraction step, amplification step, activation step or miRNA color detection step may be performed under isothermal conditions of 36 to 42°C, but is not limited thereto.

[0045] In the present invention, the exosome miRNA analysis method may be a one-pot analysis method performed in one reaction vessel, but is not limited thereto.

[0046] Another aspect of the present invention is a gold-magnetic nanoparticle (Au-MNPs) comprising nanoparticles composed of iron oxide nanoparticles and reduced gold ions, an anti-CD63 antibody, and a promoter; and

[0047] The present invention relates to an exosome miRNA analysis device comprising a colorimetric detection platform including a gold plate (Au plate), an ssDNA-biotin linker, and multi-enzyme nanoparticles (MENPs) in a single reaction vessel.

[0048] The description of the above exosome miRNA analysis device is omitted in consideration of the complexity of the present specification to the extent that it overlaps with the above exosome miRNA analysis method.

[0049] Additionally, the device of the present invention may further include a user guide describing optimal reaction conditions. The guide is a printed document explaining how to use the kit, such as how to prepare a buffer solution, the proposed reaction conditions, etc. The guide may include instructions in the form of a pamphlet or leaflet, a label attached to the kit, or on the surface of the package containing the kit. Furthermore, the guide may include, but is not limited to, information disclosed or provided through electronic media such as the Internet.

[0050] In one embodiment of the present invention, the target miRNA to be detected comprises a base sequence represented by SEQ ID NO: 1.

[0051] In one embodiment of the present invention, the acceptor oligonucleotide comprises a base sequence represented by SEQ ID NO: 2.

[0052] In one embodiment of the present invention, the donor oligonucleotide comprises a base sequence represented by SEQ ID NO: 3.

[0053] In one embodiment of the present invention, the crRNA comprises a base sequence represented by SEQ ID NO: 5.

[0054] In one embodiment of the present invention, the CRISPR / Cas activating DNA comprises a base sequence represented by SEQ ID NO: 6.

[0055] In one embodiment of the present invention, the 6-FAM tag ssDNA comprises a base sequence represented by SEQ ID NO: 7.

[0056] In one embodiment of the present invention, the linker ssDNA comprises a base sequence represented by SEQ ID NO: 8.

[0057] In one embodiment of the present invention, the promoter comprises a base sequence represented by SEQ ID NO: 9.

[0058] Throughout this specification, materials, methods, and examples are illustrative only and do not limit the present invention. To facilitate implementation of various embodiments of the present invention, explanations of specific terms are provided:

[0059] [Terminology]

[0060] The term "miRNA" in this specification refers to a short, non-expressed nucleic acid molecule consisting of about 22 nucleotides found in plants, animals, and viruses, which functions to regulate gene expression after transcription, such as RNA silencing and RNA interference, or may refer to a nucleic acid molecule that can be used as a biomarker for various diseases.

[0061] The term "nucleic acid" in this specification may refer to a molecule that is essential to life, including both DNA and RNA, and that plays a role in storing and transmitting genetic information in the cells of a living organism, and may be used interchangeably with "nucleotide."

[0062] The term “target miRNA” in this specification may refer to various miRNA molecules present in a sample isolated from a subject.

[0063] The term "promoter" in this specification refers to an upstream region of a gene involved in the initiation of transcription, and may refer to a site on a DNA strand where RNA polymerase binds or a site where transcription begins by synthesizing RNA complementary to a DNA strand.

[0064] The sample may be, but is not limited to, a cultured cell, a nucleic acid, or a biological pharmaceutical product containing the same, a tissue obtained from the brain, eye, heart, intestine, kidney, liver, lung, muscle, spleen, or testis of an animal or human, or a body fluid such as blood, plasma, serum, urine, saliva, sweat, semen, or mucus.

[0065] The term "DNA ligase" in this specification may refer to an enzyme that connects two DNA strands by catalyzing a reaction that forms a phosphodiester bond, and may play a role in repairing DNA damage in a living organism, and in particular, may connect the 5'-terminal portion of one strand of DNA to the 3'-terminal portion of the other strand of DNA.

[0066] The term "RNA polymerase" in this specification may mean an enzyme that uses DNA as a template to synthesize RNA having a complementary base sequence, catalyzes a reaction that synthesizes nucleotides in the direction of the 5'-terminal site to the 3'-terminal site, and is particularly essential for the transcription process of synthesizing RNA from DNA.

[0067] The term "crRNA (CRISPR RNA)" in this specification may refer to a nucleic acid molecule that constitutes a guideRNA used in the CRISPR / Cas system, along with tracrRNA, as an RNA transcript of the bacterial CRISPR locus. Typically, pre-crRNA is transcribed from the CRISPR locus and then processed into mature crRNA by Cas proteins. The mature crRNA can form a complex with various Cas proteins and induce a reaction in which the Cas protein cleaves a nucleic acid molecule having a base sequence recognized by the crRNA.

[0068] The term "CRISPR / Cas12a" in this specification may refer to one of the Cas proteins, an RNA-guided nuclease that constitutes the CRISPR system in some bacteria or archaea. CRISPR / Cas12a can form a complex with crRNA to target and cleave a nucleic acid molecule with a specific base sequence.

[0069] The present invention relates to a CRISPR-Cas12a-based exosome miRNA one-pot analysis method and device using an hourglass structure.

[0070] The present invention provides a miRNA analysis method that can sequentially perform exosome separation and amplification and CRISPR Cas signal analysis in a single reaction vessel using multifunctional nanoparticles and an hourglass structure.

[0071] The analysis method according to the present invention can selectively isolate only human cell-derived exosomes in a simple and low-cost manner by using an exosome separation method using nano magnetic particles and an immune reaction without external equipment such as a centrifuge, and simplifies the amplification process by amplifying miRNA with CRISPR RNA (crRNA) rather than an aptamer, and analyzes a signal that appears when a chromogenic enzyme fixed through DNA is cleaved by a Cas protein, so that it can be analyzed with the naked eye without a separate fluorescence analysis device.

[0072] Figure 1a is a schematic diagram showing the exosome isolation and miRNA amplification process.

[0073] Figure 1b is a schematic diagram showing the CRISPR-Cas12a activation and color signal detection process.

[0074] Figure 1c shows the base sequence of the nucleic acid used in the example.

[0075] Figures 1d and 1e illustrate the structures of nucleic acids used in the examples.

[0076] Figure 2a is a schematic diagram showing the manufacturing process of Au-MNP.

[0077] Figures 2b to 2e show the results of characterization of Au-MNP.

[0078] Figure 3a is a schematic diagram showing the manufacturing process of Pr / CD63Ab / Au-MNP.

[0079] Figures 3b and 3c show the results of characterization of Pr / CD63Ab / Au-MNP.

[0080] Figure 4 shows the results of analyzing the separated exosomes using TEM images.

[0081] Figures 5a and 5b show the template for crRNA production and the results of electrophoresis analysis to confirm whether crRNA was produced.

[0082] Figure 6 shows the results of confirming whether the produced crRNA can cause cleavage of ssDNA.

[0083] Figures 7a and 7b are schematic diagrams showing the manufacturing process of MENP and Au plate immobilized with color-developing particles (Au plate-ssDNA-MENPs).

[0084] Figure 7c shows the results of TEM analysis, DLS measurement, and color signal for the size and shape of MENP.

[0085] Figure 7d shows the analysis results for each stage of the manufacturing process of the Au plate.

[0086] Figure 8 shows the structure of a device including a color detection platform, the analysis process, and the color detection results.

[0087] Figure 9a shows the results of selecting miRNAs that exhibit the strongest signals using various miRNA sequences.

[0088] Figure 9b shows the results of color signal confirmation using various concentrations of miRNA-21.

[0089] Figures 10a to 10c show the results of standardizing the miRNA analysis results and comparing them with the results from RT-qPCR.

[0090] Exosome miRNA analysis method including the following steps:

[0091] (1) An exosome separation step in which exosomes in a cell culture medium are immunoreacted with a magnetic particle-based probe and then the exosomes are separated using magnetism;

[0092] (2) miRNA extraction step of crushing the separated exosomes and extracting miRNA;

[0093] (3) Amplification step of synthesizing target miRNA into crRNA by adding a synthetic solution;

[0094] (4) an activation step of contacting the crRNA and Cas12a protein to activate the CRISPR-Cas12a protein; and

[0095] (5) miRNA chromogenic detection step including a cleavage reaction on a chromogenic detection platform.

[0096] The present invention provides a CRISPR-Cas12a-based exosome miRNA one-pot analysis method and device using an hourglass structure.

[0097] Hereinafter, the present invention will be described in detail through experimental examples.

[0098] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise stated.

[0099] Additionally, when a numerical range is disclosed herein, such range is continuous and includes all values ​​from the minimum value to the maximum value inclusive, unless otherwise specified.

[0100] Additionally, the term “or” in this specification is intended to mean an inclusive “or” rather than an exclusive “or.”

[0101] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless specifically stated otherwise.

[0102] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0103] [Experimental Overview]

[0104] The present invention is designed to perform all exosome miRNA analysis from separation to detection in a single reaction vessel without complex devices.

[0105] In the first step, exosomes were isolated through immunoreactivity of magnetic nanoparticles (Pr / CD63Ab / Au-MNP). After extracting miRNAs from the isolated exosomes, they were directly amplified using a one-pot miRNA amplification system using T7 polymerase, SplintR ligase, and a promoter. If target miRNAs were present, crRNAs for CRISPR-Cas12a activation were synthesized, followed by activation of the Cas12a protein. Multi-enzyme encapsulated nanoparticles (MENPs) for colorimetric signaling were attached to the Au plate via single-stranded DNA in the cap of the reaction vial.

[0106] The activated CRISPR-Cas12a protein, upon amplification of the target miRNA, cleaved the reactive ssDNA in a bottom-up manner, dispersing the enzyme nanoparticles in the solution. After the cleavage reaction, the vial was inverted to stop the cleavage reaction, and colorless H2O2 / TMB was added to the solution. Finally, the target exosomal miRNA was identified by analyzing the color change using a microplate reader or colorimetric card.

[0107] The novel analytical device, which designs the reaction steps for the entire exosome miRNA in an hourglass-shaped reaction vessel, amplifies miRNA into crRNA through one-step isothermal amplification, and utilizes the Cas12a slicing system, can simplify the entire process of exosome miRNA analysis using multifunctional nanoparticles such as Pr / CD63Ab / Au-MNP and MENP. The overall analytical process is illustrated in Figures 1A and 1B.

[0108]

[0109] [Base sequence and structure of nucleic acids used]

[0110] The base sequences and structures of nucleic acids used in the examples of the present invention are shown in Figures 1c to 1e and Table 1 below.

[0111] Sequence number name base sequence (5' → 3') 1Target miRNA (miRNA-21)UAG CUU AUC AGA CUG AUG UUG A2Acceptor (A1 for miRNA-21)TGA ACA AAA GGA AGA AAA TCA TCT ACA AGA GTA GAA ATT ATC AAC ATC AGT C3Donor (B1 for miRNA-21)Phospho-TGA TAA GCT ACC CTA TAG TGA GTC GTA TTA4A1 + B1TGA ACA AAA GGA AGA AAA TCA TCT ACA AGA GTA GAA ATT ATC AAC ATC AGT CTG ATA AGC TAC CCT ATA GTG AGT CGT ATT A5Core crRNAUAA UUU CUA CUC UUG UAG AUG AUU UUC UUC CUU UUG UUC A6Activation ssDNATTA TGA ACA AAA GGA AGA AAA TCA76-FAM ssDNA6-FAM - AAA AAA AAA ATA GCT TAT CAG AAA AAA AAA8Linker ssDNAThiol- AAA AAA AAA ATA GCT TAT CAG AAA AAA AAA-Biotin9PromoterThiol- (A) 15 TAA TAC GAC TCA CTA TAG GG

[0112] [Experimental Example]

[0113] Example 1. Exosome isolation using magnetic particles from cell culture medium

[0114] (1) Fabrication of multifunctional magnetic particles (Pr / CD63Ab / Au-MNP)

[0115] Anti-CD63 antibody and thiolate single-stranded promoter sequence were coated on gold-magnetic nanoparticles (Au-MNPs) to synthesize Pr / CD63Ab / Au-MNP (promoter / CD63Ab / Au-magnetic nanoparticle).

[0116] 100 μL of 30 nm amine-functionalized magnetic nanoparticles (MNPs) were mixed with 100 μL of 20 mM sodium citrate solution. The mixture was mixed with 2 mL of distilled water, boiled, and 20 μL of 100 mM HAuCl4 solution was added to the solution. When the gold nanoparticles are reduced on the surface of the magnetic particles, the color changes to red. After the color of the solution changed from brown to deep red, the solution was cooled to 24°C. The fabricated Au-MNPs were collected using a permanent magnet and redispersed in distilled water. This process was repeated twice more. The pure Au-MNP solution was stored in a dark room at 24°C until use. The Au-MNP fabrication process is shown in Figure 2a.

[0117] Next, 5 μL of 200 μg / mL anti-CD63 antibody was mixed with 1 mL of Au-MNPs solution. After incubating the mixture (CD63Ab / Au-MNPs) at 25°C for 15 min, 15 μL of SH-promoter (10 μM) was mixed with the CD63Ab / Au-MNPs solution with vigorous stirring at 25°C for 30 min. To prevent non-specific protein absorption, 10 μL of 80 μM TEG was mixed with the Pr / CD63Ab / Au-MNPs mixture solution with vigorous stirring at 25°C for 60 min.

[0118] After the TEG reaction, the unbound promoter ssDNA sequence and antibody were separated by centrifugation at 6,000 rpm (10 min) at 25°C to remove the Pr / CD63Ab / Au solution. The supernatant was discarded, and the pellet was dispersed in 1 mL of DEPC water. The washing step was repeated once more, and the final Pr / CD63Ab / Au-MNPs solution was stored in the dark at 4°C.

[0119] (2) Analysis of magnetic particle characteristics

[0120] Au-MNP characterization

[0121] The synthesis of Au-MNPs was performed by attaching Au on the surface of citrate-capped amine-functionalized MNPs. 3+ was performed by reduction. Since negatively charged AuNPs can be easily synthesized on the surface of MNPs, -NH3+ on the surface of MNPs can electrostatically interact to form solid Au-MNPs. The synthesized Au-MNPs had a deep red color, in contrast to the dark brown color of MNPs. When a permanent magnet was placed on the completed Au-MNPs, it was confirmed that all particles were attracted, confirming that the gold nanoparticles were well attached to the MNPs. For more accurate characterization of Au-MNPs, the following analysis was performed.

[0122] The UV-vis spectra of Au-MNP and MNP were compared, and the results are shown in Fig. 2b. As a result, maximum absorption was observed at 535 nm in Au-MNP due to the introduction of the Au component.

[0123] Additionally, X-ray crystallography (XRD) analysis of Au-MNPs was performed at a scanning speed of 10° / min and a 2θ range from 5° to 90°, and the results are shown in Fig. 2c. As a result, peaks at (111), (200), (220), and (222) appeared in Au-MNPs. The XRD spectrum confirmed that Au-MNPs were successfully synthesized.

[0124] TEM analysis was performed to investigate the morphology of Au-MNPs, and the results are shown in Fig. 2d. MNPs had an average size of 30 nm, and Au-MNPs had an average size of 34 nm. Au-MNPs exhibited a core-satellite shape, with a single MNP core surrounded by multiple small Au particles (3–4 nm). UV, XRD, and TEM analyses confirmed that gold nanoparticles were well immobilized on iron oxide nanoparticles.

[0125] For more precise particle analysis, energy dispersive spectroscopy (EDS) mapping was performed, and the results are shown in Fig. 2e. As a result, it was confirmed that the manufactured Au-MNPs contained Au and Fe components.

[0126] Pr / CD63Ab / Au-MNP Characterization

[0127] A probe that can be used for exosome isolation and miRNA amplification was produced by attaching an anti-CD63 antibody and a thiol promoter sequence to the surface through self-assembly using thiol-Au interactions on the manufactured gold nano-magnetic particles.

[0128] Lateral flow immunoassay (LFIA) and UV-vis spectroscopy were used to confirm whether antibodies were immobilized on Pr / CD63Ab / Au-MNPs. Pr / CD63Ab / Au-MNPs and Pr / Au-MNPs without added antibodies were prepared, and CD63 protein was immobilized at the center of a nitrocellulose membrane in the LFIA system. When the two nanoprobes flowed, the probe with the antibody reacted with the CD63 antigen on the nitrocellulose membrane and showed a colored dot signal. In contrast, the probe without the antibody did not react with the antigen and showed no signal. These results confirmed that the anti-CD63 antibody was immobilized on the Au-MNPs.

[0129] Comparison of the UV-vis spectra of Au-MNPs revealed that the spectrum of Pr / CD63Ab / Au-MNPs exhibited a 280 nm peak, indicating protein immobilization onto Pr / CD63Ab / AuNPs. Except for the antibody immobilization step, the Pr / CD63Ab / Au-MNP process was protein-free, confirming that this peak indicated antibody immobilization onto Pr / CD63Ab / Au-MNPs. The results of UV-vis analysis and LFIA analysis are shown in Figure 3b.

[0130] To confirm that the thiol-modified promoter was successfully immobilized on the Au-MNPs conjugated to the antibody (CD63Ab / Au-MNP), fluorescence analysis was performed using a Cyanine3-tagged anti-promoter ssDNA sequence, and the results are shown in Fig. 3c.

[0131] Two nanoprobes, promoter-modified CD63Ab / Au-MNP (Pr / CD63Ab / Au-MNP) and CD63Ab / Au-MNP, were prepared and mixed with a Cy3-modified ssDNA sequence capable of complementary binding to the promoter sequence. After washing to remove unbound Cy3 probes, the fluorescence intensities of the two nanoprobes were measured. Particles with the promoter sequence exhibited fluorescence due to complementary binding to the Cy3-tagged DNA. These results confirmed that the immobilization of the thiol promoter sequence was successfully achieved on Pr / CD63Ab / Au-MNPs.

[0132]

[0133] (3) Exosome separation using magnetic particles

[0134] To isolate exosomes from cell culture media, 1 mL of cell culture media was mixed with 200 μL of Pr / CD63Ab / Au-MNPs in a 4-mL vial. After an immunoreaction between exosomes and Pr / CD63Ab / Au-MNPs was performed at 24°C for 1 h, the complexes were separated using a permanent magnet. The supernatant was removed, and the separated complexes were dispersed in 1 mL of deionized water (DW).

[0135] The complex was separated once more with a permanent magnet, the supernatant was removed, 50 μL of 1% Triton X-100 was added, and the mixture was incubated at 37°C for 30 minutes to lyse the exosomes.

[0136] The isolated exosomes were analyzed using TEM images, and the results are shown in Fig. 4. The TEM images confirmed that Pr / CD63Ab / Au-MNPs bound to the exosomes, successfully isolating them. As a result, the present invention was confirmed to have the advantages of being simple, requiring a short time, and not requiring equipment required for exosome isolation.

[0137]

[0138] Example 2. miRNA extraction and one-pot isothermal nucleic acid amplification

[0139] (1) Experimental method

[0140] Acceptor and donor nucleotides (2 μL each, 10 μM, final concentration = 1 μM), miRNA-21 (2 μL at various concentrations), 10X SplintR ligase buffer (2 μL each), and SplintR ligase (1 μL each) were added to 13 μL of DEPC water. The mixture was heated at 37°C for 30 minutes to initiate the binding reaction of the acceptor and donor nucleotides via the target miRNA. The reaction was terminated by heating at 95°C for 5 minutes.

[0141] Next, the agarose powder was dissolved in 0.5 Х TAE buffer and the solution was heated. The heated agarose solution was 55 Х 60 Х 0.5 mm 3It was poured into a mold and cooled to room temperature to form a transparent gel. Then, with the loading well of the gel closed toward the cathode, the gel was carefully transferred to an electrophoresis chamber immersed in 0.5X TAE buffer, and 10 μL of sample and 5 μL of marker were carefully loaded into the well. The agarose gel was electrophoresed for 30 minutes at 100 V in the absence of light, and single-stranded DNA was stained with 1X SYBR Gold solution for 30 minutes. The RNA in vitro transcription reaction solution containing 10 μL of 5X T7 RNA polymerase reaction buffer, 5 μL rNTPs (25 mM each rNTP), 5 μL T7 polymerase, 1 μL RNase inhibitor, 5 μL template synthesized by ligating ssDNA via miRNA, and 29 μL DEPC water was incubated at 37°C for 90 minutes. The synthesized crRNA was examined by gel electrophoresis.

[0142] (2) Template for crRNA production, confirmation of crRNA production

[0143] The principle of miRNA-21-specific isothermal elongation is as follows. Based on the characteristics of SplintR ligase, a target RNA of a specific sequence is used as a splint, and a ssDNA sequence ligase (acceptor and donor nucleotides) is activated. The acceptor and donor nucleotides are designed to bind partially complementarily to miRNA-21, so that in the presence of miRNA-21, these two DNAs anneal to miRNA-21 and are joined by SplintR ligase.

[0144] The production of a template for crRNA production was analyzed by gel electrophoresis, and the results are shown in Figure 5a. The results showed that template synthesis occurred only when the miRNA-21 acceptor, donor, and SplintR ligase were all present. In the absence of any one of these, elongation did not occur, and no template was synthesized.

[0145] The synthesized template contains a single-stranded promoter sequence and a complementary crRNA sequence. This sequence can bind to the promoter of Pr / CD63Ab / Au-MNP, and T7 polymerase recognizes it to synthesize the crRNA sequence. Ultimately, Pr / CD63Ab / Au-MNP is a multifunctional nanoparticle that is first used to isolate exosomes through an immune response in the exosome isolation step, and secondly used to amplify crRNA together with the crRNA amplification template in the miRNA amplification step.

[0146] To confirm crRNA production, gel electrophoresis analysis was performed, and the results are shown in Figure 5b. The results confirmed that CRISPR RNA was produced only when both the template and T7 polymerase were present.

[0147]

[0148] Example 3. Confirmation of CRISPR / Cas12a activation

[0149] Since extension and amplification (transcription) can be performed under the same isothermal conditions (37°C), the two reactions can proceed simultaneously in one pot. Therefore, miRNA-21 can be amplified into crRNA through one-pot miRNA amplification based on SplintR ligase and T7 polymerase, and the result can be used to activate CRISPR-Cas12a. In summary, an experiment was conducted to confirm whether the crRNA produced by one-step isothermal nucleic acid amplification of miRNA can induce ssDNA cleavage, and the results are shown in Fig. 6.

[0150] The amplified product from one-pot isothermal miRNA amplification was mixed with CRISPR-Cas12a and activation ssDNA that complementarily bind to crRNA, and this was reacted with 6-FAM-tagged ssDNA and analyzed by gel electrophoresis. As a result, the fluorescent dye-tagged ssDNA was cleaved only when all the necessary materials (target miRNA, SplintR ligase, activation DNA, and T7 polymerase) were present. This indicates that the CRISPR / Cas12a protein was activated to cleave the ssDNA, resulting in the disappearance of the fluorescent signal. Overall, the results confirmed that the cleavage of ssDNA could be controlled depending on the presence or absence of miRNA-21 using one-pot isothermal miRNA amplification and the CRISPR-Cas12a detection system.

[0151]

[0152] Example 4. Confirmation of miRNA detection using CRISPR / Cas and detection devices.

[0153] (1) Production of a platform for color detection

[0154] Fabrication of multienzyme nanoparticles

[0155] An enzyme ball capable of strong signal amplification and high-sensitivity detection compared to existing chromogenic enzymes was produced as follows.

[0156] MENP was produced using the ethanol dissolution method. First, a solution was prepared by mixing 10 μl of HRP-streptavidin solution (1.25 mg / ml), 100 μl of bovine serum albumin (BAS solution, 50 mg / ml), and 20 μl of HRP solution (25 mg / ml). Then, the solution was stirred at 850 rpm and 25°C, and 400 μl of 100% ethanol was added at a rate of 1 ml / min. At this time, ethanol lowers the solubility of proteins and helps them to aggregate with each other. When the proteins aggregate into nano-sized particles, the solution turns cloudy, and 10 μl of a 4% glutaldehyde solution was added and the reaction was allowed to proceed for 4 hours. Glutaldehyde acts as an adhesive that helps cross-link between proteins.

[0157] Then, the particles after the reaction were centrifuged at 9,000 rpm for 15 minutes, the supernatant was removed, and the MENPs pellet was dispersed in 0.01 M Tris buffer with 0.01% Tween 20. This step was repeated twice more. After the final centrifugation, the MENPs were dispersed in 1 mL of 0.01 M PBS solution and stored in a dark room at 4°C. The fabrication steps and structure of the multienzyme nanoparticles are shown in Figure 7a.

[0158] Fabrication of Au plates immobilized with chromogenic particles (Au plate- ssDNA- MENPs)

[0159] First, organic substances were removed and washed on a 5 mm X 15 mm Au plate using piranha solution (sulfuric acid:hydrogen peroxide = 7:3). The Au plate was then further washed using ethanol and DEPC water (DW).

[0160] The sliced ​​thiol-ssDNA-biotin linker (10 μM) was spread evenly on the gold substrate and reacted for 2 hours to allow the thiol groups present in the sliced ​​ssDNA sequence to attach to the gold substrate. After removing unreacted ssDNA linker through DW washing, the ssDNA-modified gold substrate was placed in the MENP solution and reacted for 2 hours. At this time, the streptavidin in the enzyme ball and the biotin in the sliced ​​ssDNA sequence react, allowing the multienzyme nanoparticles to be immobilized on the gold substrate via the sliced ​​ssDNA.

[0161] The MENPs-conjugated Au plate was attached to a 4 mL virus cap using an adhesive and stored in a darkroom at 4°C until use. The fabrication steps and structure of the chromogenic particle-immobilized Au plate are shown in Figure 7b.

[0162]

[0163] (2) Analysis of the platform for color detection

[0164] MENPs are produced through the aggregation and cross-linking of multiple HRP and BSA nanoparticles. Through aggregation, the nanoparticles can encapsulate large amounts of HRP. The size and shape of MENPs were analyzed by TEM. MENPs exhibited a spherical shape with an average diameter of approximately 120 nm.

[0165] Since TEM images provided information on the dried nanoparticle size, DLS measurements were performed on the hydrodynamic radius size of the dispersed MENPs.

[0166] The DLS results showed that the size of MENP was in the range of 70–240 nm, and the z-average value was 122.9 nm (polydispersity index, PI = 0.128). To determine how strong the signal could be generated by MENP, the number of single HRP and MENP required to generate a constant color signal for 10 min was calculated and compared. For a color intensity of 0.286, 16.3 χ 105 / mL and 8.7 χ 103 / mL of HRP and MENP were required, respectively, indicating that MENP showed a signal that was approximately 200 times stronger than that of HRP.

[0167] The results of TEM analysis for the size and morphology of MENPs, DLS measurement for the hydrodynamic size, and colorimetric signal are shown in Fig. 7c.

[0168] To analyze the layer formation process of Au plate-ssDNA-MENP, atomic force microscopy (AFM) (XE7, Park System) analysis was performed.

[0169] The parameters for AFM operation were set (scan size, 1 μm; scan rate, 0.999 Hz; aspect ratio, 1:1; integral gain, 0.2; proportional gain, 0.4; and amplitude set point, 0.252 V). The results of the three fabrication steps (i) Au plate, (ii) Au plate-ssDNA-biotin, and (iii) Au plate-ssDNA-MENP) were analyzed and the results are shown in Fig. 7d.

[0170] In Figure 7d, (i) shows a nonporous morphology with a uniform distribution of granular structures with a roughness of 1.2 nm in height. After conjugation of the ssDNA linker, (ii) shows that a well-ordered thiol-ssDNA self-assembled monolayer is formed on the Au surface, increasing the roughness (to 6.5 nm). Finally, (iii) combined with MENP shows a nanopatterned morphology with a ball-like structure with a roughness of 120 nm. The formation of Au plate-ssDNA-MENP can be confirmed through AFM analysis results.

[0171]

[0172] (3) Confirmation of target miRNA detection

[0173] To apply the fabricated one-pot isothermal miRNA amplification and CRISPR-Cas12a detection system to MENPs-ssDNA-Au plates for exosomal miRNA-21 detection, an analytical device mimicking an hourglass structure was designed. The hourglass-mimicking device can perform separation, amplification, CRISPR-Cas12a activation, and colorimetric detection in a single pot without complex processes or specialized skills.

[0174] Au plate-ssDNA-MENP was immobilized on the cap of the vial. First, crRNA was synthesized by one-pot isothermal amplification of miRNA-21 in the vial (90 min). After the reaction, the CRISPR-Cas12a activation solution, consisting of the CRISPR-Cas12a protein and activated ssDNA, was added to the vial. The vial was closed and sealed with a cap, and then the vial was inverted to cause ssDNA cleavage in the Au plate-ssDNA-MENP by the trans cleavage activity of CRISPR-Cas12a. After 30 min, the vial was inverted to stop ssDNA cleavage, and the cap was removed. For signal development, 1 mL of TMB solution was added to the vial and incubated for 10 min. As a result, a color reaction was confirmed to occur due to MENP cleaved from the Au plate-ssDNA-MENP in the cap.

[0175] The composition and content of the solution used in the one-pot experiment are shown in Table 2.

[0176] NameConcentrationVolumeAcceptor for miRNA-21 (A1)10 μM1 μLDonor for miRNA-21 (B1)10 μM1 μLSlintR ligase25,000 U / mL2 μL10×SlintR Ligase buffer10 μLT7 polymerase20 U / μL5 μL5×T7 polymerase buffer20 μLRNase inhibitor50 U2 μLrNTP25 mM of each rNTP (rG,rC,rA,rU)5 μLDEPC water2 μLTotal mixture50 μLExosome lysis (exosome miRNA / Pr / CD63Ab / Au)50 μLTotal One-pot miRNA amplification reaction volume100 μLCRISPR-Cas12a reactionCRISPR-Cas12a protein10 μM5 μLActivation ssDNA10 μM5 μLWater890 μLTotal CRISPR-Cas12a slicing solution1 mL

[0177] To verify the miRNA-21 detection capability of the hourglass-mimicking device, the entire process of the device was performed under the following conditions: A) using artificially synthesized crRNA (10 μM), B) one-pot isothermal amplification using 10 nM miRNA-21, C) one-pot isothermal amplification without miRNA-21, and D) using 10 nM miRNA-21 but in the absence of CRISPR-Cas12a protein. In cases A) and B), CRISPR-Cas12a was successfully activated by crRNA, resulting in the generation of a blue signal. On the other hand, in cases C) and D), CRISPR-Cas12a was not activated, resulting in no signal. In case C), crRNA was not amplified due to the absence of miRNA-21, and thus Cas12a was not activated, resulting in no color development by TMB. In case D), no color development occurred because the ssDNA of the Au plate-ssDNA-MENP was not cleaved in the absence of Cas12a protein. The structure of the analysis device, the analysis process, and the color development confirmation results are shown in Figure 8.

[0178] As expected, the above results confirmed that in the absence of miRNA or cas12a, protein activity did not occur, cleavage did not occur, and thus no signal was generated, but in the presence of crRNA and crRNA produced through miRNA, a color reaction occurred.

[0179] (4) Confirmation of target specificity and measurement sensitivity

[0180] The specificity of this analysis system for miRNA-21 detection was tested. Using various miRNA sequences, we aimed to select the miRNA with the strongest signal. The RNA sequences used in the experiment and the experimental results are shown in Table 3 and Figure 9a.

[0181] Target miRNA base sequence (5'-> 3')miRNA-21UAG CUU AUC AGA CUG AUG UUG AmiRNA-21(miss_1)UAG CUU AUC ACA CUG AUG UUG AmiRNA-21(miss_2)UAG CUU AUC ACAUUG AUG UUG AmiRNA-U6CGC AAG GAU GAC ACG CAA AUU CGU GAA GCG UUC CAU AUU UUUmiRNA-25bUCC CUG AGA CCC UAA CUU GUG AmiRNA-146aCCU CUG AAA UUC AGU UCU UCA G

[0182] When using various miRNAs (miRNA-21, 1-base mismatch miRNA-21, 2-base mismatch miRNA-21, miRNA-U6, miRNA-25b, miRNA-146a), it was confirmed that miRNA-21 showed the highest blue signal. However, it was also found that blue color was generated even with 1-2 base mismatch miRNA-21. For accurate analysis, the signal was analyzed using a microplate reader. The analysis results showed that it was difficult to detect one or two base differences completely clearly. This was expected to be due to the limitation of sequence-specific amplification. However, considering the signal difference between miRNA-21 and 1-base or 2-base mismatch miRNA-21, it was confirmed that the hourglass-mimicking analysis system of the present invention showed excellent specificity for the target miRNA. Next, the sensitivity of the system was verified by applying various concentrations of miRNA-21 to the analysis device. The colorimetric signal results were obtained according to various concentrations of miRNA-21 (1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 100 aM, and 0) and are shown in Figure 9b.

[0183] As shown in Fig. 9b, it was visually confirmed that the color signal increased as the concentration of the target miRNA increased. To analyze the color intensity, the TMB reaction was stopped with 1 M HCl, and the signal was analyzed using a microplate reader. A calibration curve of the color intensity at 450 nm according to the miRNA-21 concentration was created. The correlation coefficient (R2) of the graph was 0.9509, which corresponds to the equation y = 0.4936 x + 1.175, which is a linear relationship between the absorbance intensity of the color at 450 nm (au) and the log value of the miRNA concentration (pM). The limit of detection (LOD) value was estimated to be 132 aM based on 3 σ / s (σ is the standard deviation of the blank sample, s is the linear calibration slope), and the limit of quantification (LOQ) was estimated to be 396 aM based on 3 Х LOD. The hourglass-mimicking biosensor was confirmed to exhibit superior sensitivity compared to other Cas12a-based biosensors because it amplified miRNA-21 signals using a double amplification method that combines one-pot isothermal nuclear amplification and MENP, a highly sensitive nanoprobe.

[0184]

[0185] Example 5. Comparison of results with RT-qPCR

[0186] We performed exosome extraction and detection from cell culture media, and compared the results of quantitative analysis using the device of the present invention and the existing RT-qPCR method to confirm the validity of the system. MCF-10A normal cells and MCF-7 and SK-BR-3 cancer cells were selected to create two different cell groups. In addition, to standardize the miRNA analysis results in the hourglass-mimicking device of the present invention, the isolated exosomes were counted using FL-NTA (fluorescent-NTA), and the analysis process is shown in Figure 10a.

[0187] The results of the hourglass-mimicking device are compared with those of RT-qPCR, as shown in Figure 10b. The results show that the diagnostic results are similar.

[0188] To standardize the results of miRNA analysis, the results of counting using FL-NTA (fluorescence-NTA) are shown in Figure 10c.

[0189] First, the number of isolated exosomes was counted using FL-NTA. The concentrations of exosomes extracted from cell cultures of MCF-7, SK-BR-3, and MCF-10A were 3.96 х 10, respectively. 9 , 1.55 Х 10 9 , 2.99 Х 10 8 (particles / mL) and are shown in Table 4.

[0190] Cell line Exosome concentration (Particles / mL) MCF-7 3.96 × 10 9 SK-BR-31.55 × 10 9 MCF-10A2.99 × 10 8

[0191] Based on these results, the concentration of miRNA-21 was normalized in the form of moles / number of exosomes (mol / particle). Specifically, the miRNA-21 per exosome unit was analyzed to be 2.86 times more in MCF-7 exosomes than in MCF-10A exosomes, and 1.2 times more in SK-BR-3 exosomes than in MCF-10A exosomes. This confirmed that the difference in the normalized miRNA-21 values ​​between exosomes derived from cancer cells and normal cells was significant, and confirmed that exosome-derived cell type analysis and cancer diagnosis are possible using normalized miRNA-21 analysis.

[0192] Sintering

[0193] In the present invention, we developed promoter-antibody gold magnetic particles to isolate and amplify exosomes and their target miRNAs. The crRNA generated through this process was used to activate Cas12a, resulting in a visual chromatic signal. Furthermore, to facilitate this process, we implemented a system operating in an hourglass configuration. The hourglass-based device fabricated in this manner was able to detect target miRNA-21 with very high sensitivity and selectivity.

[0194]

[0195] The present inventors have invented a method and device for miRNA analysis that can sequentially perform exosome separation and amplification and CRISPR Cas signal analysis in a single reaction vessel using multifunctional nanoparticles and an hourglass structure.

[0196] The analysis method according to the present invention can selectively isolate only human cell-derived exosomes in a simple and low-cost manner by using an exosome separation method using nano magnetic particles and an immune reaction without external equipment such as a centrifuge.

[0197] The amplification process is simplified by amplifying miRNA with CRISPR RNA (crRNA) rather than aptamer.

[0198] By analyzing the signal produced when a chromogenic enzyme fixed to DNA is cleaved by a Cas protein, a method is provided that can be analyzed with the naked eye without a separate fluorescence analysis device.

[0199] The purpose of the present invention is to provide a one-pot exosome miRNA analysis method based on CRISPR-Cas12a using an hourglass structure.

[0200] Another object of the present invention is to provide a device for one-pot analysis of exosome miRNA based on CRISPR-Cas12a using an hourglass structure.

Claims

1. Exosome miRNA analysis method including the following steps: (1) An exosome separation step in which exosomes in a cell culture medium are immunoreacted with a magnetic particle-based probe and then the exosomes are separated using magnetism; (2) miRNA extraction step of crushing the separated exosomes and extracting miRNA; (3) Amplification step of synthesizing target miRNA into crRNA by adding a synthetic solution; (4) an activation step of contacting the crRNA and Cas12a protein to activate the CRISPR-Cas12a protein; and (5) miRNA chromogenic detection step including a cleavage reaction on a chromogenic detection platform.

2. In paragraph 1, A method for analyzing exosome miRNA, wherein the above immune response is a binding of the antigen protein on the surface of the exosome in the cell culture medium to the magnetic particle-based probe.

3. In paragraph 1, A method for analyzing exosome miRNA, wherein the magnetic particle-based probe is a gold-magnetic nanoparticle (Au-Magnetic Nanoparticle, Au-MNP) comprising a nanoparticle composed of iron oxide nanoparticles and reduced gold ions; an anti-CD63 antibody; and a promoter.

4. In paragraph 3, A method for analyzing exosome miRNA, wherein the above promoter is a promoter for synthesis of the above crRNA.

5. In paragraph 1, A method for analyzing exosome miRNA, wherein the above synthetic solution comprises T7 RNA polymerase, DNA ligase, acceptor nucleotides, and donor nucleotides.

6. In paragraph 1, The above amplification step is a single-stranded nucleic acid molecule elongation step including a base sequence complementary to the target miRNA and a base sequence complementary to the crRNA; and A method for analyzing exosome miRNA, comprising a step of transcribing crDNA containing a base sequence complementary to the single-stranded nucleic acid molecule by T7 polymerase.

7. In paragraph 1, The above cleavage reaction is a method for analyzing exosome miRNA, wherein the ssDNA linker is cleaved by the CRISPR-Cas12a protein.

8. In paragraph 1, An exosome miRNA analysis method, wherein the above color detection platform comprises a gold plate (Au plate), an ssDNA-biotin linker, and a multi-enzyme nanoparticle (MENP).

9. In paragraph 1, An exosome miRNA analysis method, wherein the above color detection platform is installed on the upper part of the reaction vessel.

10. In paragraph 1, An exosome miRNA analysis method, wherein the above miRNA color detection step is initiated by flipping the reaction vessel upside down.

11. In paragraph 8, An exosome miRNA analysis method, wherein the above miRNA color detection step includes a catalytic reaction using TMB as a substrate and the above multi-enzyme nanoparticle as a catalyst.

12. In paragraph 1, The above exosome miRNA analysis method is a one-pot analysis method performed in one reaction vessel.

13. It is performed by an exosome miRNA analysis method according to any one of clauses 1 to 12, Gold-magnetic nanoparticles (Au-MNPs) comprising nanoparticles composed of iron oxide nanoparticles and reduced gold ions, anti-CD63 antibodies, and promoters; and An exosome miRNA analysis device comprising a colorimetric detection platform including a gold plate (Au plate), a ssDNA-biotin linker, and a multi-enzyme nanoparticle (MENP) in a single reaction vessel.

14. In paragraph 13, An exosome miRNA analysis device, wherein the above promoter is a promoter for the synthesis of crRNA.

Images