Method, kit and device for detecting extracellular vesicles

Abstract

The purpose of the present invention is to simply detect extracellular vesicles with high sensitivity. Specifically, provided is a method for detecting an extracellular vesicle having a target enzyme, the method comprising: a dispensing step of dispensing an extracellular vesicle and a reagent containing a reporter molecule to be altered by the target enzyme to emit a signal into a plurality of separate separation chambers; a signal generation step of reacting the target enzyme and the reporter molecule with each other to generate a signal; a signal detection step of detecting the signal; and an identification step including determining a signal strength of each of the separate compartments based on a detection result obtained in the signal detection step, and identifying each of the separate compartments having a signal strength exceeding a predetermined threshold.

Description

Technical Field

[0001] The present invention relates to methods, kits, devices and procedures for detecting extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are a general term for non-nuclear vesicles secreted from cells and having a lipid bilayer structure. They are broadly divided into three types based on differences in their intracellular production mechanisms: exosomes, microvesicles, and apoptotic bodies. EVs have been noted to function as intercellular communication vehicles, delivering physiologically active functional molecules (such as proteins and nucleic acids) from cells to other cells. EVs are also found in bodily fluids (such as blood, urine, and cerebrospinal fluid), and therefore research into their medical applications in liquid biopsies is ongoing.

[0003] Vinita Gupta et al. of Genentech have disclosed an assay for detecting and / or quantifying membrane proteins (e.g., circulating CD20 (cCD20)) that incorporates an extracellular vesicle-based calibrant containing a membrane-associated tumor antigen, as well as the use of such an assay in detecting and treating hyperproliferative disorders (Patent Document 1). This document discloses an ELISA assay using a combination of a capture antibody and a detection antibody. The capture antibody binds to extracellular vesicles containing a membrane protein, thereby generating a capture antibody-extracellular vesicle complex and is immobilized on a solid phase. The detection antibody binds to the capture antibody-extracellular vesicle complex to form a detectable binding complex. Citation List Patent Literature

[0004] Patent Document 1: Japanese Patent Publication No. 2022-524327 Summary of the Invention Technical issues

[0005] Patent Document 1 discloses an ELISA assay for extracellular vesicles to detect the presence of membrane proteins serving as detection targets. However, when the detection target is an enzyme, an assay that utilizes the activity of the enzyme to detect the presence of active enzymes, and an assay that detects extracellular vesicles in which the active enzymes are present, are not disclosed.

[0006] The object of the present invention is to simply detect extracellular vesicles having enzyme activity with high sensitivity by detecting the activity of enzyme molecules included in the extracellular vesicles. Solution to the problem

[0007] Provided is a method for detecting extracellular vesicles with a target enzyme, the method comprising: a distribution step of distributing extracellular vesicles and reporter molecules to be changed by the target enzyme to emit signals into a plurality of independently separated compartments; a signal generating step of allowing the target enzyme and the reporter molecules to react with each other to generate a signal; a signal detecting step of detecting the signal; and an identification step, comprising determining the signal intensity of each of the independently separated compartments based on the detection result obtained in the signal detecting step, and identifying each of the independently separated compartments having a signal intensity exceeding a predetermined threshold.

[0008] A kit for detecting extracellular vesicles with a target enzyme in independent and isolated compartments is provided, the kit comprising: a reagent containing a reporter molecule, the reporter molecule to be changed by the target enzyme to emit a signal; and a container for providing a plurality of independent and isolated compartments.

[0009] Provided is an apparatus for detecting extracellular vesicles having a target enzyme, the apparatus comprising: a dispensing unit configured to dispense extracellular vesicles and a reagent containing a reporter molecule to be altered by the target enzyme and thereby emit a signal into a plurality of independently separated compartments; a signal generating unit configured to cause the target enzyme and the reporter molecule to react with each other to generate a signal; a signal detecting unit configured to detect the signal; and an identifying unit configured to determine the signal intensity of each of the independently separated compartments based on a detection result obtained using the signal detecting unit, and to identify each of the independently separated compartments having a signal intensity exceeding a predetermined threshold. [Beneficial effects of the present invention]

[0010] According to the present invention, a detection method, a kit, an apparatus, and a program for easily detecting extracellular vesicles having enzyme activity by detecting the activity of an enzyme contained in the extracellular vesicles can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a flowchart for explaining the process of the method for detecting extracellular vesicles according to an embodiment of the present invention. Figure 2 This is a flowchart illustrating the flow of a method for detecting extracellular vesicles using droplets as independent separation compartments. Figure 3 This is a flowchart for explaining the flow of a method for detecting extracellular vesicles using a well as an independent separation compartment. Figure 4 This is a flowchart for explaining the flow of a method for detecting extracellular vesicles in the case of performing background correction by using a mask image. Figure 5Ais a cross-sectional view of the hole array. Figure 5B is a cross-sectional view of a well array, the wells of which are filled with particle complexes formed by capture antibody-immobilized particles and extracellular vesicles. Figure 6 This is a functional block diagram for explaining an example of an extracellular vesicle detection device according to an embodiment of the present invention. Figure 7 This is a functional block diagram for explaining an example of an extracellular vesicle detection device according to an embodiment of the present invention. Figure 8 is a block diagram showing a hardware configuration example of an extracellular vesicle detection device according to an embodiment of the present invention. Figure 9A The results of Example 1 are shown, in which recombinant human MMP14 protein was measured with the SensoLyte 520 MMP-14 assay kit by using a 96-well plate. Figure 9B The results of Example 1 are shown, in which recombinant human MMP14 protein was measured with the SensoLyte 520 MMP-14 assay kit by using a 96-well plate. Figure 10 The results of Example 2 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with the SensoLyte 520 MMP-14 assay kit by using a 96-well plate. Figure 11 The results of Example 3 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with MMP-14 Substrate I by using a 96-well plate (time change). Figure 12 The results of Example 4 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1, Custom #2, or SensoLyte 520 MMP14 by using a 96-well plate (time course). Figure 13A The results of Example 5 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (time change). Figure 13B The results of Example 5 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (time change). Figure 14A The results of Example 6 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with Custom-made Product #1 (reaction solution composition, time changes were studied). Figure 14BThe results of Example 6 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with Custom-made Product #1 (reaction solution composition, time changes were studied). Figure 14C The results of Example 6 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with Custom-made Product #1 (reaction solution composition, time changes were studied). Figure 15A The results of Example 7 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with custom-made product #1 (reaction solution composition, time changes were studied). Figure 15B The results of Example 7 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with custom-made product #2 (reaction solution composition, time changes were studied). Figure 16A The results of Example 8 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (extracellular vesicle concentration varied, time varied). Figure 16B The results of Example 8 are shown, in which MDA-MB-231-derived extracellular vesicles (varying extracellular vesicle concentrations) were measured with Custom #1 or Custom #2 by using a 96-well plate. Figure 17A The results of Example 9 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 using a 96-well plate (varying reporter molecule concentrations, time variations). Figure 17B The results of Example 9 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #2 using a 96-well plate (varying reporter molecule concentrations, time variations). Figure 17C The results of Example 9 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (Linewerver-Burk plot). Figure 18A The results of Example 10 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (time change). Figure 18B The results of Example 10 are shown, in which A549-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (time change). Figure 18CThe results of Example 10 are shown, in which COLO201-derived extracellular vesicles were measured with Custom #1 or Custom #2 by using a 96-well plate (time change). Figure 19 The results of Example 11 are shown, in which AChE was measured with the Amplite Fluorescent Acetylcholinesterase Assay Kit by using a 96-well plate (varying AChE concentration, time variation). Figure 20A The results of Example 12 are shown, in which MDA-MB-231-derived extracellular vesicles were measured (time course) using an Amplite Fluorescent Acetylcholinesterase Assay Kit using a 96-well plate. Figure 20B The results of Example 12 are shown, in which A549-derived extracellular vesicles were measured (time course) using an Amplite Fluorescent Acetylcholinesterase Assay Kit using a 96-well plate. Figure 21 The results of Example 13 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using an Amplite Fluorescent Acetylcholinesterase Assay Kit using a 96-well plate (varying reporter molecule concentration, time variation). Figure 22 The results of Example 14 are shown, in which AChE was measured with a SensoLyte 520 acetylcholinesterase activity assay kit by using a 96-well plate (varying AChE concentration, time variation). Figure 23A Shown are the results of Example 15, in which MDA-MB-231-derived extracellular vesicles were measured with a SensoLyte 520 acetylcholinesterase activity assay kit by using a 96-well plate (time course). Figure 23B Shown are the results of Example 15, in which A549-derived extracellular vesicles were measured with a SensoLyte 520 acetylcholinesterase activity assay kit by using a 96-well plate (time change). Figure 24 The results of Example 16 are shown, in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with a SensoLyte 520 acetylcholinesterase activity assay kit (varying reporter molecule concentration, time variation). Figure 25A The results of Example 17 are shown, in which MMP14 was measured with Custom #1 by using a 96-well plate (varying buffer, varying time). Figure 25B The results of Example 17 are shown, in which MMP14 was measured with Custom #2 by using a 96-well plate (varying buffer, varying time). Figure 25C The results of Example 17 are shown, in which AChE was measured with the Amplite Fluorescent Acetylcholinesterase Assay Kit by using a 96-well plate (varying buffer, varying time). Figure 25D The results of Example 17 are shown, in which AChE was measured with a SensoLyte 520 Acetylcholinesterase Activity Assay Kit by using a 96-well plate (varying buffer, varying time). Figure 26A The results of Example 18 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom-made #2 by using a 96-well plate (time course). Figure 26B Shown is a fluorescence microscope image of the pores in Example 18 (sealed with AsahiKlin AE-3000 and Fomblin Y25 serving as a hydrophobic solvent). Figure 26C Shown are fluorescence microscopy images of the wells in Example 18 (sealed with Simoa SR-X sealing oil acting as a hydrophobic solvent). Figure 26D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 18 (sealed with AsahiKlin AE-3000 and Fomblin Y25 serving as a hydrophobic solvent). Figure 26E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 18 (sealed with Simoa SR-X sealing oil acting as a hydrophobic solvent). Figure 27 The results of Example 19 (fluorescence intensity from the reporter molecule, time course) in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with custom-made product #2 are shown. Figure 28A Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 3 hours of reaction). Figure 28B Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 3 hours of reaction). Figure 28C Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 3 hours of reaction). Figure 28D Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 3 hours of reaction). Figure 28E Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 3 hours of reaction). Figure 28FShown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 3 hours of reaction). Figure 28G A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 3 hours of reaction). Figure 28H A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 3 hours of reaction). Figure 28I A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 3 hours of reaction). Figure 28J A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 3 hours of reaction). Figure 28K A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 3 hours of reaction). Figure 28L A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 3 hours of reaction). Figure 29A Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 7 hours of reaction). Figure 29B Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 7 hours of reaction). Figure 29C Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 7 hours of reaction). Figure 29D Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 7 hours of reaction). Figure 29E Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 7 hours of reaction). Figure 29F Shown are fluorescence microscopy images of the wells in Example 19 (custom product #2, after 7 hours of reaction). Figure 29G A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 7 hours of reaction). Figure 29H A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 7 hours of reaction). Figure 29I A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 7 hours of reaction). Figure 29JA fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 7 hours of reaction). Figure 29K A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 7 hours of reaction). Figure 29L A fluorescence microscope image of the wells in Example 19 is shown (standard fluorescent substance, after 7 hours of reaction). Figure 30A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 3 hours of reaction). Figure 30B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 3 hours of reaction). Figure 30C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 3 hours of reaction). Figure 30D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 3 hours of reaction). Figure 30E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 3 hours of reaction). Figure 31A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 7 hours of reaction). Figure 31B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 7 hours of reaction). Figure 31C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 7 hours of reaction). Figure 31D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 7 hours of reaction). Figure 31E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 19 (after 7 hours of reaction). Figure 32 A graph showing the expected value λ calculated from the results of Example 19 versus the extracellular vesicle concentration is shown. Figure 33 The results of Example 20 (fluorescence intensity from the reporter molecule, time course) in which MDA-MB-231-derived extracellular vesicles were measured using a 96-well plate with custom-made product #1 are shown. Figure 34A Shown are fluorescence microscopy images of the wells in Example 20 (custom product #1, after 5 hours of reaction). Figure 34B Shown are fluorescence microscopy images of the wells in Example 20 (custom product #1, after 5 hours of reaction). Figure 34C Shown are fluorescence microscopy images of the wells in Example 20 (custom product #1, after 5 hours of reaction). Figure 34D Shown are fluorescence microscopy images of the wells in Example 20 (custom product #1, after 5 hours of reaction). Figure 34E Shown are fluorescence microscopy images of the wells in Example 20 (custom product #1, after 5 hours of reaction). Figure 34F Shown are fluorescence microscopy images of the wells in Example 20 (custom product #1, after 5 hours of reaction). Figure 34G A fluorescence microscope image of the wells in Example 20 is shown (standard fluorescent substance, after 5 hours of reaction). Figure 34H A fluorescence microscope image of the wells in Example 20 is shown (standard fluorescent substance, after 5 hours of reaction). Figure 34I A fluorescence microscope image of the wells in Example 20 is shown (standard fluorescent substance, after 5 hours of reaction). Figure 34J A fluorescence microscope image of the wells in Example 20 is shown (standard fluorescent substance, after 5 hours of reaction). Figure 34K A fluorescence microscope image of the wells in Example 20 is shown (standard fluorescent substance, after 5 hours of reaction). Figure 34L A fluorescence microscope image of the wells in Example 20 is shown (standard fluorescent substance, after 5 hours of reaction). Figure 35A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 20 (after 5 hours of reaction). Figure 35B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 20 (after 5 hours of reaction). Figure 35C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 20 (after 5 hours of reaction). Figure 35D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 20 (after 5 hours of reaction). Figure 35E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 20 (after 5 hours of reaction). Figure 35FShown are histograms obtained from fluorescence microscopy images of the wells in Example 20 (after 5 hours of reaction). Figure 36 A graph showing the expected value λ calculated from the results of Example 20 versus the extracellular vesicle concentration is shown. Figure 37 The results of Example 21 are shown, in which A-549-derived extracellular vesicles (fluorescence intensity from a reporter molecule, time change) were measured with Custom-made Product #1 by using a 96-well plate. Figure 38A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 21 (after 5 hours of reaction). Figure 38B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 21 (after 5 hours of reaction). Figure 38C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 21 (after 5 hours of reaction). Figure 38D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 21 (after 5 hours of reaction). Figure 38E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 21 (after 5 hours of reaction). Figure 38F Shown are histograms obtained from fluorescence microscopy images of the wells in Example 21 (after 5 hours of reaction). Figure 39 A graph showing the expected value λ calculated from the results of Example 21 versus the extracellular vesicle concentration is shown. Figure 40A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 or Custom #2 using Simoa Discs (after 1 hour of reaction, Custom #1). Figure 40B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #1). Figure 40C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #1). Figure 40D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #1). Figure 40E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #1). Figure 40F Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #1). Figure 40G Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #1). Figure 40H Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #1). Figure 41A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #2). Figure 41B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #2). Figure 41C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #2). Figure 41D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 1 hour of reaction, Custom #2). Figure 41E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #2). Figure 41F Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #2). Figure 41G Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #2). Figure 41H Shown are histograms obtained from fluorescence microscopy images of the wells in Example 22 (after 2 hours of reaction, Custom #2). Figure 42A A plot showing the expected value λ calculated from the results of Example 22 versus the extracellular vesicle concentration (Custom #1). Figure 42B A plot showing the expected value λ calculated from the results of Example 22 versus the extracellular vesicle concentration (Custom #2). Figure 43 The results of Example 23 are shown, in which MDA-MB-231-derived extracellular vesicles (fluorescence intensity from a reporter molecule, time change) were measured with the SensoLyte 520 MMP-14 assay kit by using a 96-well plate. Figure 44A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 23 (after 1 hour of reaction). Figure 44B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 23 (after 1 hour of reaction). Figure 44C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 23 (after 1 hour of reaction). Figure 44D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 23 (after 1 hour of reaction). Figure 44E Shown are histograms obtained from fluorescence microscopy images of the wells in Example 23 (after 1 hour of reaction). Figure 45 A graph showing the expected value λ calculated from the results of Example 23 versus the extracellular vesicle concentration is shown. Figure 46 The results of Example 24 are shown, in which MDA-MB-231-derived extracellular vesicles (fluorescence intensity from a reporter molecule, time change) were measured using a 96-well plate with an Amplite Fluorescent Acetylcholinesterase Assay Kit. Figure 47A A fluorescence microscope image of the wells in Example 24 is shown (after 3 hours of reaction, reporter molecule). Figure 47B A fluorescence microscope image of the wells in Example 24 is shown (after 3 hours of reaction, reporter molecule). Figure 47C A fluorescence microscope image of the wells in Example 24 is shown (after 3 hours of reaction, reporter molecule). Figure 47D A fluorescence microscope image of the wells in Example 24 is shown (after 3 hours of reaction, using a standard fluorescent substance). Figure 47E A fluorescence microscope image of the wells in Example 24 is shown (after 3 hours of reaction, using a standard fluorescent substance). Figure 47F A fluorescence microscope image of the wells in Example 24 is shown (after 3 hours of reaction, using a standard fluorescent substance). Figure 48A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 24 (after 3 hours of reaction, reporter molecule). Figure 48B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 24 (after 3 hours of reaction, reporter molecule). Figure 48CShown are histograms obtained from fluorescence microscopy images of the wells in Example 24 (after 3 hours of reaction, reporter molecule). Figure 49 A graph showing the expected value λ calculated from the results of Example 24 versus the extracellular vesicle concentration is shown. Figure 50 The results of Example 25 are shown, in which A549-derived extracellular vesicles (fluorescence intensity from a reporter molecule, time change) were measured using a 96-well plate with the Amplite Fluorescent Acetylcholinesterase Assay Kit. Figure 51A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 25 (after 1.5 hours of reaction). Figure 51B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 25 (after 1.5 hours of reaction). Figure 51C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 25 (after 1.5 hours of reaction). Figure 51D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 25 (after 1.5 hours of reaction). Figure 52 A graph showing the expected value λ calculated from the results of Example 25 versus the extracellular vesicle concentration is shown. Figure 53 The results of Example 26 are shown, in which A549-derived extracellular vesicles (fluorescence intensity from a reporter molecule, time change) were measured with the SensoLyte 520 acetylcholinesterase activity assay kit by using a 96-well plate. Figure 54A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 26 (after 5 hours of reaction). Figure 54B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 26 (after 5 hours of reaction). Figure 54C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 26 (after 5 hours of reaction). Figure 54D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 26 (after 5 hours of reaction). Figure 55 A graph showing the expected value λ calculated from the results of Example 26 versus the extracellular vesicle concentration is shown. Figure 56The results of Example 27 are shown, in which A549-derived extracellular vesicles (fluorescence intensity from reporter molecules, time changes) were measured using a 96-well plate with custom-made product #2. Figure 57A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 27 (after 5 hours of reaction). Figure 57B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 27 (after 5 hours of reaction). Figure 57C Shown are histograms obtained from fluorescence microscopy images of the wells in Example 27 (after 5 hours of reaction). Figure 57D Shown are histograms obtained from fluorescence microscopy images of the wells in Example 27 (after 5 hours of reaction). Figure 58 A graph showing the expected value λ calculated from the results of Example 27 versus the extracellular vesicle concentration is shown. Figure 59A Shown are fluorescence microscopy images of the wells in Example 28, in which MDA-MB-231-derived extracellular vesicles were measured using antibody-immobilized particles and Simoa Discs with Custom #2 (reporter molecule after 5 hours of reaction). Figure 59B A fluorescence microscope image of the wells in Example 28 is shown (after 5 hours of reaction, reporter molecule). Figure 59C Shown is a fluorescence microscope image of the pores in Example 28 (particles after 5 hours of reaction). Figure 59D Shown is a fluorescence microscope image of the pores in Example 28 (particles after 5 hours of reaction). Figure 60A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 28 (after 5 hours of reaction). Figure 60B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 28 (after 5 hours of reaction). Figure 61A Shown are fluorescence microscopy images of the wells in Example 29, in which MDA-MB-231-derived extracellular vesicles were measured with the Amplite Fluorescent Acetylcholinesterase Assay Kit using antibody-immobilized particles and Simoa Discs (reporter molecule after 3 hours of reaction). Figure 61B A fluorescence microscope image of the wells in Example 29 is shown (after 3 hours of reaction, reporter molecule). Figure 61CShown is a fluorescence microscope image of the pores in Example 29 (particles after 3 hours of reaction). Figure 61D Shown is a fluorescence microscope image of the pores in Example 29 (particles after 3 hours of reaction). Figure 62A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 29 (after 3 hours of reaction). Figure 62B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 29 (after 3 hours of reaction). Figure 63A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 30, in which MDA-MB-231-derived extracellular vesicles were measured with the SensoLyte 520 Acetylcholinesterase Activity Assay Kit (after 5 hours of reaction) using antibody-immobilized particles and Simoa Discs. Figure 63B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 30 (after 5 hours of reaction). Figure 64A Shown are histograms obtained from fluorescence microscopy images of the wells in Example 31, in which MDA-MB-231-derived extracellular vesicles were measured using antibody-immobilized particles and Simoa Discs with Custom #2 (after 5 hours of reaction). Figure 64B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 31 (after 5 hours of reaction). Figure 65A Fluorescence microscopy images of Example 32 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with Custom #1 by using droplets (droplets generated with an SPG pumping connector (pore size: 10 μm), reaction for 4 hours, bright field). Figure 65B Shown is a fluorescence microscopy image of Example 32 (droplets generated using an SPG pumping linker (pore size: 10 μm), reaction time 4 hours, custom product #1). Figure 65C Shown is a fluorescence microscope image of Example 32 (droplets generated using an SPG pumping linker (pore size: 10 μm), reaction for 4 hours, standard fluorescent substance). Figure 66A Shown is a fluorescence microscope image of Example 32 (droplets generated using an SPG pumping linker (pore size: 5 μm), reaction for 4 hours, bright field). Figure 66BShown is a fluorescence microscopy image of Example 32 (droplets generated using an SPG pumping linker (pore size: 5 μm), reaction time 4 hours, custom product #1). Figure 66C Shown is a fluorescence microscope image of Example 32 (droplets generated using an SPG pumping linker (pore size: 5 μm), reaction for 4 hours, standard fluorescent substance). Figure 67 Shown is a histogram of droplet diameter distribution obtained from fluorescence microscopy images of droplets in Example 32 (droplets generated using an SPG pumping linker (pore size: 10 μm), reaction for 4 hours, standard fluorescent substance). Figure 68A The results of Example 33 are shown, in which MDA-MB-231-derived extracellular vesicles (fluorescence intensity from reporter molecules, time changes) were measured using a 96-well plate with custom-made product #2. Figure 68B The results of Example 33 are shown, in which MDA-MB-231-derived extracellular vesicles were measured with resorufin β-D-galactopyranoside by using a 96-well plate (fluorescence intensity from a reporter molecule, time change). Figure 69A Shown are fluorescence microscopy images of the wells in Example 33, in which MDA-MB-231-derived extracellular vesicles were measured using antibody-immobilized particles and Simoa Discs with Custom #2 (reporter molecule after 4 hours of reaction). Figure 69B Shown are fluorescence microscopy images of the wells in Example 33, in which MDA-MB-231-derived extracellular vesicles were measured with resorufin β-D-galactopyranoside using antibody-immobilized particles and Simoa Discs (reporter molecule after 4 hours of reaction). Figure 69C Shown is a fluorescence microscope image of the pores in Example 33 (particles after 4 hours of reaction). Figure 70A Shown are histograms obtained from fluorescence microscopy images of wells in Example 33, where measurements were made using Custom #2 (after 4 hours of reaction). Figure 70B Shown are histograms obtained from fluorescence microscopy images of the wells in Example 33, in which the measurement was performed using resorufin β-D-galactopyranoside (after 4 hours of reaction). Figure 71A A graph showing the expected value λ calculated from the results of Example 33 versus the extracellular vesicle concentration is shown. Figure 71B A graph showing the expected value λ calculated from the results of Example 33 versus the extracellular vesicle concentration is shown. Figure 72 A graph showing the expected value λ calculated from the results of Example 49 versus the extracellular vesicle concentration, in which MDA-MB-231-derived extracellular vesicles were measured using Tim4-immobilized magnetic particles (PS Capture Exosome Flow Cytometry Kit) and Simoa Discs using Custom #1. Figure 73 A graph showing the expected value λ calculated from the results of Example 49 versus the extracellular vesicle concentration, in which MDA-MB-231-derived extracellular vesicles were measured using Tim4-immobilized magnetic particles (PS Capture Exosome Flow Cytometry Kit) and Simoa Discs using Custom #2. Figure 74 A graph showing the expected value λ calculated by BG correction from the results of Example 49 versus the extracellular vesicle concentration, in which MDA-MB-231-derived extracellular vesicles were measured using Tim4-immobilized magnetic particles (PS Capture Exosome Flow Cytometry Kit) and SimoaDiscs using Custom #1. Figure 75 A graph showing the expected value λ calculated by BG correction from the results of Example 49 versus the extracellular vesicle concentration, in which MDA-MB-231-derived extracellular vesicles were measured using Tim4-immobilized magnetic particles (PS Capture Exosome Flow Cytometry Kit) and SimoaDiscs using Custom #2. Figure 76 A graph showing the expected value λ calculated from the results of Example 50 versus the extracellular vesicle concentration, in which MDA-MB-231-derived extracellular vesicles were measured using Tim4-immobilized magnetic particles (MS300S) and Simoa Discs using Custom #2. Figure 77 A graph showing the expected value λ calculated by BG correction from the results of Example 50 versus the extracellular vesicle concentration, wherein MDA-MB-231-derived extracellular vesicles were measured using Tim4-immobilized magnetic particles (MS300S) and Simoa Discs using custom #2. DETAILED DESCRIPTION

[0012] Exemplary embodiments of the present invention will now be described with reference to the accompanying drawings, but the present invention is not limited to the following embodiments. The same elements or corresponding elements are denoted by the same reference numerals in the drawings, and descriptions thereof may be omitted or simplified. In the following sections, not only bonds (such as covalent bonds and coordinate bonds) but also binding (such as affinity interactions and surface adsorption) and binding modes including combinations thereof are described as "binding".

[0013] According to one embodiment of the present invention, a method for detecting extracellular vesicles with a target enzyme is provided, the method comprising: a dispensing step of dispensing extracellular vesicles and a reagent comprising a reporter molecule to be changed by the target enzyme and thereby emit a signal into a plurality of independently separated compartments; a signal generating step of allowing the target enzyme and the reporter molecule to react with each other to generate a signal; a signal detecting step of detecting the signal; and an identification step comprising determining the signal intensity of each independently separated compartment based on the detection result obtained in the signal detection step, and identifying each independently separated compartment having a signal intensity exceeding a predetermined threshold.

[0014] Figure 1 is a flowchart for explaining the flow of a method for detecting extracellular vesicles according to an embodiment of the present invention. The method for detecting extracellular vesicles according to this embodiment includes: a distribution step S101 of distributing extracellular vesicles and a reagent containing a reporter molecule to be changed by a target enzyme to emit a signal into a plurality of independent separation compartments; a signal generation step S102 of allowing the target enzyme and the reporter molecule to react with each other to generate a signal; a signal detection step S103 of detecting the signal; and an identification step S104, including determining the signal intensity of each independent separation compartment based on the detection result obtained in the signal detection step, and identifying each independent separation compartment having a signal intensity exceeding a predetermined threshold.

[0015] The signal detection step may comprise an image acquisition step of acquiring an image comprising the individually isolated compartments. The identifying step may include identifying independently separated compartments having a signal intensity exceeding a predetermined threshold by processing the image acquired in the image acquiring step. The identifying step may include identifying each independently isolated compartment having a signal intensity exceeding a predetermined threshold based on at least either a ratio or a difference between a signal intensity in a reference compartment and a signal intensity in each independently isolated compartment. The reference compartment may be a compartment that does not contain extracellular vesicles and contains a reagent sample containing a reporter molecule.

[0016] Additionally, additional steps may be added before or simultaneously with the dispensing step. An example of the additional step is a step of mixing the extracellular vesicles and the capture protein to be bound to the extracellular vesicles to form a complex. Another example of an additional step may be a recovery step, which includes mixing the extracellular vesicles and a capture protein to be bound to the extracellular vesicles to form a complex, and recovering the resulting complex. In addition, a step of mixing the complex with a second capture protein to be bound to the extracellular vesicles may be added.

[0017] According to another embodiment of the present invention, a kit for detecting extracellular vesicles with a target enzyme in independent separation compartments is provided, the kit comprising: a reagent containing a reporter molecule to be changed by the target enzyme to emit a signal; and a container for providing a plurality of independent separation compartments.

[0018] The above embodiments are described in further detail below. (Reagent) The reagent includes an aqueous liquid containing a reporter molecule that can be converted to emit a signal by the activity of the target enzyme. An example of an aqueous solution is a buffer. The reporter molecule and the buffer are described below.

[0019] When there are multiple enzymes to be detected, the reagent may contain two or more reporter molecules. In this case, the two or more reporter molecules may each generate a different signal, and the signal intensities of the signals emitted by the two or more reporter molecules may be detected separately. For example, when the signal to be emitted is fluorescence, signals based on multiple enzyme activities may be obtained by varying the wavelengths of the fluorescence emitted by the multiple reporter molecules.

[0020] When using such reagents, for example, when diagnosing a disease by detecting various enzyme activities as described above, the origin of extracellular vesicles can be more accurately determined, making it easier to identify the primary site. Furthermore, improvements in the sensitivity and specificity of the test can be expected.

[0021] The reagent may further comprise a capture protein to be bound to the extracellular vesicle. Alternatively, in a further step, a further reagent comprising a capture protein to be bound to the extracellular vesicle may be used. In this case, the extracellular vesicle and the capture protein bind to each other to form a complex. Therefore, in addition to enzyme activity, proteins included in the extracellular vesicles can also be detected. In this case, a capture protein, such as an antibody, having affinity for proteins included in the extracellular vesicles (capture target) can be used. By using the capture protein to simultaneously detect the enzymatic activity of the target enzyme and the capture target, an improvement in detection accuracy can be expected. In this case, the capture target can be the target enzyme, or the capture target can be a molecule different from the target enzyme. When the capture target is the target enzyme, for example, the ratio of the active enzyme to its inactive enzyme can be detected. The capture protein will be described later. In addition, a second capture protein to be bound to the extracellular vesicle can further bind to the complex formed by the capture protein and the extracellular vesicle.

[0022] In addition, a capture protein labeled with particles (capture protein immobilized particles) can be used as the capture protein. For example, an aqueous liquid in which an antibody labeled with useful particles (antibody immobilized particles) is dispersed can be used. In this case, from the perspective of capture efficiency, the capture protein is preferably an antibody in which the capture target is a universal protein, sugar chain, etc. expressed in many extracellular vesicles, or a mixture of multiple such antibodies. The use of particles enables recovery by magnetic force or centrifugal force, thereby promoting B / F separation (separation of bound form and free form). The particles will be described later. The second capture protein to be bound to the extracellular vesicle can be further bound to the particle complex recovered by the particles.

[0023] (Target enzyme) Target enzymes include all enzymes included on the surface of extracellular vesicles or inside extracellular vesicles. Examples of target enzymes may include: hydrolases represented by various proteases, including the MMP family, such as matrix metalloproteinases (MMPs), disintegrin and metalloproteinases (ADAMs), and ADAMs with thrombospondin motifs (ADAMTSs), matriptase, β-secretase, endothelin-converting enzyme (ECE), calpain, dipeptidyl peptidase-4 (DPPIV), angiotensin-converting enzyme 1 (ACE1) and angiotensin-converting enzyme 2 (ACE2); various esterases, including acetylcholinesterase (AChE), autotaxin, , lipase, phospholipase and phosphatase; With various glycosidases, such as beta-galactosidase; Oxidoreductase, typically various oxidases, including monoamine oxidase (MAO), various peroxidases, including myeloperoxidase (MPO), catalase and superoxide dismutase; The transferase represented by various acetylases / deacetylases, including histone acetyltransferase (HAT) and histone deacetylase (HDAC), kinases, protein kinases and glycosyltransferases; With the isomerase represented by various cis-trans isomerases, including peptidyl prolyl isomerase (PPIase, Pin1), racemase and mutase. Any enzyme can be selected as target enzyme according to purpose, and target enzyme is not limited to above-mentioned example. Hereinafter, some enzymes that can be used in embodiments of the present invention are described in detail.

[0024] MMP is a family of enzymes that play a role in the remodeling of extracellular matrix proteins. It is an enzyme necessary for various normal physiological processes (such as embryogenesis, morphogenesis, reproduction, tissue absorption and tissue reconstruction), and is also associated with pathological processes (such as cancer, inflammation, arthritis, cardiovascular disease and fibrosis). MMP includes the following types: MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP27 and MMP28. In particular, the expression of MMP14 (MT1-MMP) is known to be highly expressed in highly invasive cancer cells. In cancer cells, the dysregulation of gene expression with epithelial-mesenchymal transition leads to the abnormal expression of MMP14. As a result, it is known that the protease-dependent and -independent effects of MMP14 promote the proliferation, invasion, and metastasis of cancer cells. Specific inhibition of MMP14 activity significantly suppresses cancer cell proliferation, invasion, metastasis, and angiogenesis, and thus humanized antibodies are increasingly expected to be used for treatment.

[0025] The ADAM family has 25 kinds of genes identified by human genome analysis, of which 4 are pseudogenes. Therefore, 21 kinds of ADAM proteins are produced in the known human race, of which 13 are protease type ADAM molecules, and the remaining 8 are non-protease type ADAM molecules. ADAM9, ADAM10 and ADAM17 are proteases belonging to the ADAM family and are found to be α-secretases. ADAM9, ADAM10 and ADAM17 serve as shedding enzymes and cut the cell surface proteins relevant to neuropathology, inflammatory response and tumor progression. In addition, ADAM9, ADAM10 and ADAM17 are physiologically associated as α-secretases for amyloid precursor protein, and play a key role in the molecular etiology of Alzheimer's disease. In addition, it is reported that ADAM28 is highly expressed in a cancer tissue-specific manner in human breast cancer and non-small cell lung cancer tissues.

[0026] Aggrecanase is a protease belonging to the ADAMTS family and degrades aggrecan, a major component of cartilage. The degradation of the core protein of cartilage causes the difference in normal dynamic properties (such as compressibility and elasticity), and is known to be one of the causes of various arthritis symptoms. ADAMTS4 is a key aggrecanase in human osteoarthritis cartilage.

[0027] Matriptase is a membrane protein and a trypsin-like protease belonging to the type II transmembrane serine protease family. Its substrates include extracellular matrix proteins, cell adhesion molecules, ion channels, growth factor-like proteins, or other proteases. Matriptase's actions lead to protein processing, activation, or degradation. Matriptase is known to be associated with the onset of ovarian, prostate, and cervical cancers.

[0028] β-Secretase is a membrane protein, a transmembrane aspartic protease present in acidic intracellular vesicles, and plays a key role in the production of β-amyloid peptide by cleaving β-amyloid precursor protein. Accumulation of neurotoxic β-amyloid peptide is observed in senile plaques in the brains of patients with Alzheimer's disease, an age-related cognitive disorder.

[0029] ECE degrades amyloid β peptide. It has been reported that Aβ degradation is reduced in the brains of ECE homozygous knockout mice, and Aβ-40 and Aβ-42 levels are significantly increased.

[0030] Calpains are a family of intracellular cysteine ​​proteases that regulate the biological activity of many proteins through calcium-dependent selective cleavage. Previous studies have shown that calpains are involved in a variety of calcium-regulated intracellular mechanisms, such as platelet activation, apoptosis, cell cycle, cell proliferation, differentiation, and signal transduction. Calpain dysfunction is also known to be involved in various diseases, such as atherosclerosis, Alzheimer's disease, diabetes, and cancer.

[0031] DPPIV is a protease called a prolyl protease that cleaves proteins and is a serine-dipeptidyl protease that cleaves alanine and proline at the N-terminus of target polypeptides (such as chemokines or peptide hormones). The relationship between DPPIV and the extracellular matrix that affects T cell activation and between DPPIV and adenosine deaminase has been noted, and it has been suggested that DPPIV plays a key role in diabetes, cancer, and autoimmune diseases.

[0032] ACE1 is a zinc metalloproteinase that degrades angiotensin-1 (AT-1) to produce angiotensin-II (AT-II). It is a membrane protein present on cell membranes. AT-II is responsible for blood pressure regulation, where AT-II binds to AT receptors in biological tissues to express vasoconstrictor activity. Overexpression of ACE activity is known to cause extreme vasoconstriction due to the production of large amounts of AT-II, leading to abnormally high blood pressure and, as a result, inducing vascular diseases such as myocardial infarction, cerebral infarction, and cerebral hemorrhage.

[0033] ACE2 is an enzyme of the renin-angiotensin system (RAS), and is known to be a zinc metalloproteinase that plays a central role in the control of angiotensin peptides. ACE2 is directly related to cardiac function and is mainly expressed in heart and kidney vascular endothelial cells. In addition, it has been proposed that ACE2 can protect the kidneys in the early stages of diabetes. ACE2 is known to play a key role in the regulation of hypertension. In addition, ACE2 is known to be the target receptor of the coronavirus that causes severe acute respiratory syndrome (SARS).

[0034] AChE is a serine hydrolase belonging to the carboxylesterase family that hydrolyzes acetylcholine in the synaptic cleft into choline and acetic acid. Decreased acetylcholine levels are recognized in Alzheimer's disease and myasthenia gravis, and AChE inhibitors have been developed to alleviate symptoms. AChE is inhibited by organophosphorus compounds and other drugs.

[0035] Autotaxin hydrolyzes the phosphodiester bonds of various nucleotides and nucleotide derivatives. The expression of known autotaxins increases melanoma, breast cancer, renal cell carcinoma, non-small cell lung cancer, neuroblastoma, hepatocellular carcinoma, glioblastoma multiforme and thyroid cancer. In addition, it has been reported that autotaxin participates in the invasion and attack of breast cancer cells and is one of the top 40 genes highly expressed in highly metastatic breast cancer. In addition, it has also been reported that autotaxin induces the motility of some cultured cancer cell lines by increasing the generation of LPA.

[0036] MAO belongs to the flavin-containing amine oxidoreductase family and catalyzes the oxidation of monoamines. MAO plays a major role in the inactivation of neurotransmitters. MAO dysfunction is known to be associated with depression, drug dependence, migraines, schizophrenia, attention deficit disorder, Parkinson's disease, Alzheimer's disease, and other conditions.

[0037] MPO is a heme-based peroxidase that exhibits antimicrobial activity against various organisms and is present in neutrophils, monocytes, and macrophages in soft tissue. MPO is often expressed in stimulated neutrophils and catalyzes the production of hypohalous acids, such as hypochlorous acid, from hydrogen peroxide and chloride ions or other halides. MPO is associated with the symptoms of many diseases including arthritis, cancer, kidney disease, and cystic fibrosis, and MPO deficiency, a known genetic disease, causes immunodeficiency. The relationship between an increase in MPO levels and coronary artery disease has been demonstrated, and it has been reported that MPO serves as a predictor of myocardial infarction in specific patients.

[0038] HAT regulates the acetylation of histones and non-histones, and the ε-amino group of lysine on the histone tail is not only related to transcriptional activity, but also to DNA replication, DNA repair and protein-protein interactions. HAT plays a major role in the control of cell fate, and it is known that the dysfunction of HAT is associated with tumorigenesis. P300 / CBP-associated factor (pCAF) specifically acetylates the lysine residues at the N-terminus of histone H3 and histone H4. pCAF is also known as the transcriptional coactivator of the tumor suppressor p53.

[0039] HDACs belong to a large family of epigenetic enzymes that play a key role in gene silencing, which controls gene expression, and are important for maintaining chromatin structure and gene transcription. HDACs play a key role in cellular processes such as cell proliferation, cell cycle regulation, apoptosis, and cell differentiation. Therefore, changes in HDAC expression or mutations are associated with many human diseases, such as cancer, inflammation, neuropsychiatric diseases, bone formation, and cardiovascular growth.

[0040] Pin1 catalyzes the cis-trans isomerization of the peptide bond in phosphoserine / threonine-proline. Pin1 is known to play a key role in regulating the cell cycle, and its expression is increased in various cancers. Furthermore, Pin1 has been reported to be associated with the onset of Alzheimer's disease. It has been proposed that Pin1 binds to the phosphorylated Thr-212 / 231 residues of tau protein to promote dephosphorylation, thereby inhibiting the formation of neurofibrillary tangles.

[0041] (Reporter) The reporter molecule is not particularly limited, as long as the reporter molecule is changed by the target enzyme to emit a signal. The emission of the signal includes a change from a state in which the signal does not exist to a state in which the signal exists, a change from a state in which the signal exists to a state in which the signal does not exist, or a change in the type or intensity of the signal. For example, when the signal is luminous, a reporter can be provided by any one of a change in luminous intensity or a change in luminous wavelength. In addition, the change in the signal can be a change in the signal of a multistep reaction based on a reaction product utilizing an enzyme reaction. In this case, a variety of reporter molecules can be used simultaneously. In this case, it is desirable to select a combination in which the reaction mechanisms do not interfere with each other.

[0042] The reporter molecule whose structural change caused by enzyme activity increases luminous intensity can be a molecule such as a molecule labeled with a fluorescent substance and a quencher. Such a molecule can be, for example, a molecule (FRET substrate) that has a fluorescent substance and a quencher on its two ends or amino acid residues that can be modified in the reporter molecule, and a peptide containing a substrate that serves as a target enzyme therebetween. In the FRET substrate, before enzyme cleavage, the fluorescent substance is close enough to the quencher so that the fluorescent substance is quenched, and when the peptide is cut by enzyme activity, the quenching caused by the quencher is released, thereby emitting fluorescence. When using the FRET substrate as a reporter molecule, the presence or absence of the target enzyme can be observed as a large change in fluorescence brightness. Preferably, the peptide included in the FRET substrate has more than 3 residues from the viewpoint of reactivity, and has less than 30 residues to obtain a quenching effect. In addition, from the viewpoint of realizing large brightness changes, it is also preferred that the chemical structure change caused by enzyme activity converts non-fluorescent substances into reporter molecules of fluorescent substances.

[0043] For example, the reporter molecule of each of the following commercially available kits can be used as such a reporter molecule, but the reporter molecule is not limited to the following examples. SensoLyte (trademark) 390 / 490 / 520 / 570 series (AnaSpec, Inc.) MMP activity measurement kit SensoLyte (AnaSpec, Inc.) SensoLyte 520 ADAM10 Activity Assay Kit (AnaSpec, Inc.) SensoLyte 520 Aggrecanase-1 Activity Assay Kit (AnaSpec, Inc.) SensoLyte Rh110 matriptase activity assay kit (AnaSpec, Inc.) SensoLyte 520 β-secretase measurement assay (AnaSpec, Inc.) SensoLyte 520 ECE activity assay kit (AnaSpec, Inc.) SensoLyte 520 calpain activity measurement assay (AnaSpec, Inc.) SensoLyte Rh110 DPP4 activity measurement assay kit (AnaSpec, Inc.) SensoLyte 390 ACE2 Activity Assay Kit (AnaSpec, Inc.) SensoLyte 520 acetylcholinesterase activity assay kit (AnaSpec, Inc.) SensoLyte HAT(p300) / HAT(pCAF) assay (AnaSpec, Inc.) SensoLyte 520 HDAC Activity Assay Kit (AnaSpec, Inc.) SensoLyte Green Pin1 Activity Assay Kit (AnaSpec, Inc.) Amplite series (AAT Bioquest, Inc. (trademark)) Universal MMP activity measurement kit Amplite (AAT Bioquest, Inc.) Amplite (trademark) Universal Fluorescent MMP Activity Assay Kit (Green Fluorescence) (AAT Bioquest, Inc.) Amplite Fluorescent Acetylcholinesterase Assay Kit (Green Fluorescence) (AAT Bioquest, Inc.) Amplite monoamine oxidase assay kit (AAT Bioquest, Inc.) other DPPIV / CD26 Measurement Assay Kit (Enzo Life Sciences Inc.) Angiotensin 1 converting enzyme (ACE1) activity measurement kit (Life Laboratory Company) Autotaxin activity measurement kit (Echelon Biosciences Inc.) OxiSelect Myeloperoxidase Chloride Activity Measurement Assay (Cell Biolabs, Inc.) FLUOR DE LYS (trademark) HDAC activity assay kit (Enzo Life Sciences Inc.)

[0044] In addition, synthetic reporter molecules can be custom-made based on the substrate sequence of the target enzyme to have a combination of fluorescent substances and quenchers at both ends of the substrate sequence or within the substrate sequence. In addition, examples of reporter molecules for MMP14 detection can include custom-synthesized FRET peptides used in the following examples. Custom #1 and Custom #2 used in the following examples are each reporter molecules for MMP14 detection, including substrates having FAM and TQ2 (which are a FRET pair) conjugated to both ends of the peptide sequence. Custom #1: [5-FAM]GRIGFLRTAK(TQ2)[OH] Custom #2: [5-FAM]GGPLGLAGGK(TQ2)[OH] Customized #3 and Customized #4 are each reporter molecules for MMP14 detection, having different fluorescence wavelengths obtained by replacing FAM and TQ2 (which are the FRET pairs of Customized #1 and Customized #2) with TAMRA and TQ3. These reporter molecules can be shared with another reporter molecule that emits fluorescence in the same wavelength range as FAM and can be used for multiplex detection. Custom #3: [5-TAMRA]GRIGFLRTAK(TQ3)[OH] Custom Compound #4: [5-TAMRA]GGPLGLAGGK(TQ3)[OH]

[0045] In addition, the reporter molecule can be, for example, a peptide that serves as a substrate for the target enzyme and is labeled with a fluorescent substance or a chromogenic dye. Examples of such dyes are p-nitroaniline derivatives. Substrates containing p-nitroaniline are nearly colorless, but p-nitroaniline cleaved by the enzyme is yellow. Furthermore, examples of fluorescent dyes are coumarin derivatives that act as fluorescence quenching agents. Substrates containing methylcoumarinamide do not emit fluorescence, but aminomethylcoumarin cleaved by the enzyme reaction emits strong fluorescence. An example of a similar compound is aminotrifluoromethylcoumarin.

[0046] (Reaction buffer) The reagent preferably contains a reaction buffer. In addition, a reaction buffer can be used in further steps. An example of a reaction buffer is a Tris-based buffer, which has been shown to be useful for enzymatic reactions using MMPs. A specific example is an incubation buffer (Abcam plc.) for MMP gelatin zymography having the following composition. 50 mM Tris-HCl (pH: 7.5) 5mM CaCl2 1μM ZnCl2 1% Triton X-100

[0047] Regarding AChE, an example of a buffer is a buffer having the following composition as described by Shirata et al. of Yamagata University in the following Scientific Research Grant-in-Aid: Research Category No. 24928013, "Study on Clinical Application of Acetylcholinesterase Measurement Using Selective Substrates". 0.1 M phosphate buffer (pH: 7.4) 0.1% Tween 20

[0048] When detecting multiple target enzymes, the composition of the reaction buffer needs to be optimized to simultaneously detect the enzymatic activity of each enzyme. Generally, the reaction temperature is preferably about 20° C. to about 37° C., but the optimal temperature only needs to be selected according to the enzyme. When detecting multiple target enzymes, the reaction temperature needs to be optimized so that the enzyme activity of each enzyme can be detected simultaneously.

[0049] For example, when MMP14 and AChE are detected simultaneously, a reaction buffer having the following composition can be used. 50 mM Tris-HCl (pH: 7.5) 10mM CaCl2 0.01% Brij35

[0050] (Capture protein and its detection reagent) In addition to the reporter molecule that can be converted by the activity of the target enzyme to emit a signal, the reagent may also contain a compound for detecting proteins, etc., contained in the extracellular vesicles. Alternatively, in a further step, another reagent containing a compound for detecting proteins, etc., contained in the extracellular vesicles may be used.

[0051] Examples of compounds such as detection proteins are capture proteins that bind to extracellular vesicles. Antibodies can be used as examples. Furthermore, by using two or more capture proteins, a complex having a so-called sandwich structure can be formed in which extracellular vesicles are captured by the two capture proteins.

[0052] Examples of molecules that are common proteins in extracellular vesicles and that become capture targets of capture proteins include actin, tubulin, GAPDH, flotillin (a lipid raft-associated protein), apoptosis linker gene 2 (ALG-2)-interacting protein X (ALIX) and syntenin, which are ESCRT-related proteins, tumor susceptibility gene 101 protein (TSG101), heat shock proteins (HSP70 and HSP90), soluble-N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) involved in enhanced extracellular vesicle secretion, and RABs and annexins involved in membrane transport and fusion. Examples also include tetraspanins, such as CD9, CD63, and CD81, which are membrane proteins known to be located on the surface of extracellular vesicles and are a family of four-transmembrane proteins; and cell adhesion molecules, such as integrins, selectins, and EpCAM, which are transmembrane proteins that mediate cell-cell adhesion. Examples also include disease-specific proteins included in extracellular vesicles.

[0053] Examples of capture proteins include anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, anti-rafts antibodies, anti-ALIX antibodies, anti-syndecan binding protein antibodies, anti-TSG101 antibodies, anti-HSP70 antibodies, anti-HSP90 antibodies, anti-SNARE antibodies, anti-Rab antibodies, anti-Annexin antibodies, anti-MMP antibodies, anti-ADAM antibodies, anti-AChE antibodies, anti-integrin antibodies, anti-selectin antibodies, and anti-EpCAM antibodies. In addition, for a large number of disease-specific proteins, antibodies that recognize each protein as a capture target can be used. When grasping the total number of extracellular vesicles, any of the following can be used: anti-CD9 antibodies, anti-CD63 antibodies, anti-CD81 antibodies, anti-rafts antibodies, anti-ALIX antibodies, anti-syndecan binding protein antibodies, anti-TSG101 antibodies, anti-HSP70 antibodies, anti-HSP90 antibodies, anti-SNARE antibodies, anti-Rab antibodies, anti-Annexin antibodies, anti-integrin antibodies, anti-selectin antibodies, and anti-EpCAM antibodies, or multiple thereof. In addition, when a disease-specific protein or the like is a capture target, the target enzyme may be the capture target, or the capture target may be different from the target enzyme. When the capture target is the target enzyme, for example, the ratio of its active enzyme to inactive enzyme or the like may be detected.

[0054] T cell immunoglobulin and mucin domain-containing protein 4 (Tim-4), which has an affinity for the phospholipid (phosphatidylserine) of extracellular vesicles, can also be used as a capture protein. In this case, extracellular vesicles can be broadly detected, so Tim-4 can be used to determine the total number of extracellular vesicles.

[0055] In addition, lectins can also be used as capture proteins to detect carbohydrate chains contained in extracellular vesicles. The captured protein can be labeled, for example, with a fluorescent substance, a dye, an enzyme, or a particle.

[0056] The reagent may also contain a detection reagent for detecting the label molecule. As an alternative, in a further step, a detection reagent for detecting the label molecule can be used. As a common method, it is known to use an enzyme such as alkaline phosphatase (AP), horseradish peroxidase (HRP) or beta-galactosidase (β-Gal) to label the captured protein, and a substrate for the enzyme containing the labeled antibody as a detection reagent. This enzyme or substrate can be used as a detection reagent. In addition, a reagent comprising a second antibody can also be used, the second antibody having an enzyme bound thereto, such as AP, HRP or beta-Gal, or a substrate for the enzyme.

[0057] (Staining Reagent) In addition to the reporter molecules described above that can be converted by the activity of the target enzyme to emit a signal, a staining reagent that can stain the membrane itself or proteins included in the extracellular vesicles to detect the proteins or membranes can be further added. In this case, a staining reagent having an affinity for proteins or a staining reagent having an affinity for membranes can be used. For example, the following staining reagents can be used. Exosome fluorescent staining reagent (ExoSparkler series) (manufactured by Dojindo Laboratories) Exo-Glow EV Labeling Kit Series (manufactured by System Biosciences, LLC) Lipid membrane staining fluorescent dye CellBrite Steady & CellBrite Fix series (manufactured by Biotium) ExoBrite EV Membrane Staining Kit Series (manufactured by Biotium) MemGlow series (manufactured by Cytoskeleton, Inc.) PKH series (manufactured by Sigma-Aldrich Co. LLC) Wheat Germ Agglutinin, Alexa Fluor series (manufactured by Thermo Fisher Scientific Inc.) Lipophilic carbocyanine dye series (e.g., Dilinoleyl DiI / DiO) (manufactured by PromoCell GmbH) Near-infrared fluorescent probe Sciforiem (trademark) FI7510 for exosome staining (manufactured by Toyo Ink Co., Ltd.) ACOERELA Lipid Membrane Fluorescent Labeling Kit (manufactured by ACOERELA) When using staining reagents, the number of extracellular vesicles with target enzyme activity can be measured, while also providing an understanding of the total number of extracellular vesicles. For example, staining reagents can be used to determine the fraction or ratio of extracellular vesicles with target enzyme activity.

[0058] (Particles) Capture protein labeled with particles (capture protein immobilized particles) can be used as the capture protein. The kind of particles may be primary particles or secondary particles in which primary particles are aggregated, as long as the capture protein can be bound to the particles.

[0059] The binding of the capture protein to the particle is not limited, and examples of binding include covalent bonding and physical adsorption. Covalent bonding is preferred. The capture protein or linker described later and the particle are easily bound to each other, so it is preferred that the binding is formed by a reaction utilizing the carboxyl groups originally present in the particle. In other words, the binding site between the capture protein and the particle preferably has a structure derived from the carboxyl groups of the particle.

[0060] The capture protein and the particle may be bound to each other via an amide bond. In this case, a structure is obtained in which C═O from the carboxyl group in the particle and NH from the amino group in the protein form an amide bond.

[0061] The capture protein and the particle may be bound to each other via a linker. In this context, "linker" refers to a structure that forms the bond between the capture protein and the particle. As the binding part between the capture protein and the capture protein side linker, there are listed: the ε-amino group of the lysine residue in the capture protein; the α-amino group at the N-terminus of the capture protein; various tag peptide sequences or tag proteins artificially inserted into the N-terminus of the capture protein; and the like. Examples of tag peptides include His tag, HA tag, and DDDDK tag (FLAG (trademark)). In addition, an example of a tag protein is Halo-tag (trademark).

[0062] As the bonding portion between the particle and the linker, various bonding portions are given depending on the kind of the linker. As an example, when the linker binds to the ε-amino group of a lysine residue of a capture protein or to the α-amino group of the N-terminus of a capture protein on the capture protein side, a carboxyl group, an aldehyde group, etc. are given as a structure for binding to the particle side linker. In addition, for example, when the linker causes an amino group in the capture protein and a carboxyl group in the particle to form an amide bond on the capture protein side, a condensation reaction of N-hydroxysuccinimide (NHS) / water-soluble carbodiimide (WSC) can be used for binding to the linker on the particle side.

[0063] Examples of linkers include: any of various tag peptide sequences or tag proteins and a structural moiety having affinity therefor; a complex of avidin and biotin; and PEG having any of various functional groups at its end. Furthermore, the capture protein can be bound to the particle by, for example, physical adsorption between the capture protein and the particle surface. The position in the capture protein to be bound to the particle is preferably a position that does not inhibit the function of the capture protein.

[0064] As the material of the particles, for example, polymer resin (eg, styrene resin or acrylic resin) particles, silicon oxide particles, resin particles, agar-based resin particles, metal particles, and latex particles are given. Examples of commercially available particles include magnetic particles measured with Magnosphere (trademark) MS300, Magnosphere MS160, PureProteome (trademark) Nickel Magnetic Beads, Simoa (trademark), and the Homebrew assay kit (Quanterix). As the material for the magnetic particles, particles containing paramagnetic materials, ferromagnetic materials, superparamagnetic materials, and the like, such as iron, nickel, and magnetite, are preferably used, but other materials may also be used. When using magnetic particles, recovery can be performed by applying a magnetic field.

[0065] The particle size of the particles is preferably 10 nm or more, more preferably 1 μm or more and 10 μm or less. Specific examples of particle size measurement methods include observation with an optical microscope or electron microscope, laser diffraction, dynamic light scattering, and centrifugal sedimentation, but other methods of measuring particle size may also be used.

[0066] When capture protein-immobilized particles are used, extracellular vesicles can be recovered by utilizing the properties of the particles. That is, in this embodiment, as a further step, a recovery step comprising mixing extracellular vesicles and a capture protein to be bound to the extracellular vesicles to form a complex and recovering the resulting complex can be further added before or simultaneously with the distribution step, and the capture protein can be labeled with the particles. When magnetic particles are used as the particles, B / F separation of the capture protein can be performed, and the recovery step can be performed more simply. The recovery step may also include a step of washing the complex. In addition, the recovered complex can be bound to a second capture protein to be bound to the extracellular vesicle, thereby forming a complex having a so-called sandwich structure, in which the extracellular vesicle is captured by two capture proteins.

[0067] In the recovery step, after the extracellular vesicles are recovered from the sample, the extracellular vesicles can be substantially concentrated by dispersing them in a medium having a volume smaller than that of the original sample. For example, when a recovery step is added, extracellular vesicles to be discarded can be recovered by allowing the capture protein labeled with particles to act on the extracellular vesicles without filling the independent separation chamber. After recovery, the extracellular vesicles can be detected by filling the independent separation chamber with particles (particle complexes) that have captured the extracellular vesicles. In addition, for example, trace amounts of extracellular vesicles dissolved in samples such as blood or aqueous solutions can be captured and recovered by the capture protein labeled with particles.

[0068] Furthermore, for example, when the sample containing extracellular vesicles is a large volume solution, the extracellular vesicles can be captured by using a capture protein labeled with particles before filling the tiny independent separation chamber, thereby filling the independent separation chamber with the particles (particle complex) that have captured the extracellular vesicles. As a result, the extracellular vesicles can be distributed to the independent separation chamber with reduced extracellular vesicle loss, and the extracellular vesicles can be detected with high sensitivity.

[0069] Furthermore, when magnetic particles are used as particles, extracellular vesicles can be easily recovered by utilizing magnetic force. Capture protein as described above.Even when using particle as mark, capture target also can be target enzyme, or capture target can be different from target protein.When the material beyond target protein is capture target, can detect capture target and target enzyme's colocalization (colocalization).When capture target is target enzyme, can expect the improvement of target enzyme's detection precision.

[0070] Tim-4, which has an affinity for the phospholipids (phosphatidylserine) of extracellular vesicles, can be used as a capture protein. In this case, extracellular vesicles can be extensively recovered, which is useful in the recovery of extracellular vesicles. Furthermore, lectins can be used to capture sugar chains contained in extracellular vesicles.

[0071] (Sealing agent) The reagent may further include a blocking agent. In a further step, a blocking agent can be used. For example, when the particle and the captured protein are bound to each other, the portion of the linker binding portion of the particle that has not yet bound the captured protein can be blocked by a blocking agent to inhibit nonspecific binding or undesirable binding. For example, when the amino group of the captured protein and the carboxyl group of the particle are condensed by utilizing NHS / WSC, the particle may have unreacted carboxyl groups after the reaction. Therefore, the carboxyl group that is not bonded to the captured protein can react with ethanolamine, PEG with an amino group, etc. that act as a blocking agent. Examples of blocking agents include Blockmaster (trademark) CE series (manufactured by Medical & Biological Laboratories Co., Ltd.). In addition, known substances can be used as blocking agents without restriction, and for example, albumin, casein, skim milk, Blockmaster PA series (manufactured by Medical & Biological Laboratories Co., Ltd.), Blockmaster DB series (manufactured by Medical & Biological Laboratories Co., Ltd.), etc. can be used. Furthermore, a blocking agent can be incorporated into the detection reagent containing the reporter molecule. This can inhibit nonspecific binding of the reporter molecule to the modified portion of the particle surface. Known substances can be used as blocking agents without limitation, and examples thereof include albumin, casein, and skim milk.

[0072] (Extracellular vesicle sample and recovery method) Examples of samples to be applied by the detection method of the present invention include blood (serum and plasma), urine, cerebrospinal fluid, ascites, amniotic fluid, milk, saliva and tears for liquid biopsy, and various samples can be applied. In addition, culture supernatant is also a target in the field of basic research. "Liquid biopsy" is widely defined as a method of diagnosis and inspection by biologically derived markers in body fluids, and is an inspection method with reduced invasiveness by using the above-mentioned body fluids compared to previous tissue biopsies. Liquid biopsy can obtain various information, such as genetic information, protein information and metabolic information, without sampling the tumor site, and perform diagnosis and prediction of treatment effects based on the information.

[0073] When extracellular vesicles are used for liquid biopsy, a step of recovering exosomes from body fluids is essentially required. Examples of major recovery methods include ultracentrifugation, density gradient centrifugation, polymer precipitation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity chromatography, immunoaffinity capture (magnetic beads), tangential flow filtration, and microfluidic separation.

[0074] The most commonly used exosome recovery method to date is ultracentrifugation, which is considered the gold standard for recovery methods. However, ultracentrifugation is time-consuming and involves complex operations, making it unsuitable for high-throughput processing. In addition, ultracentrifugation produces significant losses, making it unsuitable for recovery from small amounts of body fluids. Examples of methods that can recover exosomes in high yields through simple operations include: polymer precipitation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, immunoaffinity chromatography, immunoaffinity capture (magnetic beads), tangential flow filtration, and microfluidic separation.

[0075] In some embodiments, the present invention relates to the method for the precipitation of polymer particles.More specific examples of polymer precipitation methods include Total Exosome Isolation series (manufactured by ThermoFisher Scientific Inc.) and ExoQuick series (manufactured by System Biosciences, LLC).The example of ultrafiltration method includes Amicon Ultra series (manufactured by Merck KGaA).The example of size exclusion chromatography includes PURE-EV series (manufactured by HansaBioMed Life Sciences Ltd), EV Second series (manufactured by GL SciencesInc), SmartSEC series (manufactured by System Biosciences, LLC) and qEV series (manufactured by Izon ScienceLimited).The example of ion exchange chromatography includes CIM monolithic series (manufactured by Resonac Corporation) and EV Quick Filter series (manufactured by Human Metabolome Technologies, Inc.).The example of immunoaffinity chromatography includes Fab-TACS Exosome Isolation Kit series (manufactured by IBALifelsciences GmbH). The example of immunoaffinity capture method (magnetic beads) includes Exo-Flow Exosome IP Kit series (manufactured by System Biosciences, LLC) and MagCapture Exosome Isolation Kit series (manufactured by FUJIFILM Corporation). The example of tangential flow filtration includes TFF-EV series (manufactured by HansaBioMed Life Sciences Ltd), TFF-Easy series (manufactured by HansaBioMed Life Sciences Ltd) and tangential flow filtration Minimite EVO TFF system (manufactured by Cytiva). The development of microfluidic separation method is constantly advancing in research institutions.

[0076] (cell) The cells from which extracellular vesicles are collected in the present invention are not particularly limited. Mammalian cells are preferably used. Cells present in a living body, cultured cells, cells in tissue sections, cells extracted from tissues or biological samples, etc. become target cells. Normal cells, cancer cells, degenerated cells, mixed cultures of cancer cells and normal cells, mixed cultures of cancer cells, mixed cultures of normal cells, cells in mixed tissues or blood, etc. become target cells. In addition, slow-twitch muscle fibers and fast-twitch muscle fibers of the same differentiated cell type, or cells predicted to undergo the same type of cell phenotypic changes, such as epithelial-mesenchymal transition of cancer cells, differentiation induction from undifferentiated cells, cell degeneration or cell aging, etc., also become targets.

[0077] (Independent separation compartment) Extracellular vesicles and a reagent containing a reporter molecule to be changed by the target enzyme to emit a signal are distributed into multiple independent separation compartments. Therefore, the extracellular vesicles and the reagent are independently separated and isolated. When the extracellular vesicles are distributed into the independent separation compartments, there are independent separation compartments into which the extracellular vesicles are distributed and independent separation compartments into which the extracellular vesicles are not distributed. Therefore, the extracellular vesicles are concentrated in the independent separation compartments into which the extracellular vesicles are distributed, so that the weak activity of a small amount of enzyme in the extracellular vesicles can be detected, and the time for signal saturation can be shortened. In addition, when the volume of each compartment in the independent separation compartments is sufficiently reduced, the extracellular vesicles included in one compartment can be set to less than one extracellular vesicle, and when the number of compartments from which the signal is obtained is counted, the concentration of extracellular vesicles with target enzyme activity in the sample can be calculated.

[0078] The term "independently separated compartment" refers to an independently separated compartment. A chemical reaction is carried out in each compartment, and in principle, the substances in different compartments do not mix or affect each other. Each independently separated compartment preferably includes a solvent. Each independently separated compartment preferably includes a solvent of 0.1 fL or more and 1400 fL or less, more preferably includes a solvent of 0.8 fL or more and 600 fL or less. The solvent is preferably a water-soluble solvent. Droplets or holes can be used as independent separation compartments. Water-in-oil emulsions (W / O emulsions) are preferably used as each of the droplets. In addition, for example, Figure 5A and Figure 5B The holes included in the hole array of the structure shown can be used as the holes. In addition, specific examples of the holes include holes on Simoa Discs (Quanterix Corporation).

[0079] Figure 5A is a cross-sectional view of the hole array 200, and Figure 5BFIG2 is a cross-sectional view of a well array 200 having wells 204 filled with particle complexes 206, which are protein-capturing, immobilized particles that have captured extracellular vesicles. Well array 200 includes a lower substrate 201, an upper substrate 202, an injection port (not shown), and an outlet port (not shown). Hydrophobic partitions 203 are formed on lower substrate 201. Multiple wells 204 are separated from each other by partitions 203 on lower substrate 201.

[0080] The lower substrate 201 preferably has a hydrophilic surface, and glass, silicon or a polymer resin can be used as the material of the lower substrate 201, for example. In addition, the upper surface of the upper substrate 202 (the surface opposite to the lower substrate 201) is preferably hydrophobic. For example, a hydrophobic resin, a water-repellent resin or a fluorinated polymer resin can be used as the material of the partition wall 203. When the bottom surface of each hole 204 is hydrophilic and the top surface of each partition wall 203 is hydrophobic, the hole 204 can be effectively filled with a solution, and in the step of removing excess solution with a hydrophobic solvent, the hydrophobic solvent can be prevented from entering the hole 204. In addition, the lower substrate 201, the upper substrate 202 and the partition wall 203 can be formed of the same material, and an example of the material is a polymer resin, such as a cycloolefin polymer (COP).

[0081] The holes 204 are recessed portions for containing a solution and are separated from each other by the partition walls 203. The holes 204 use the lower substrate 201 as their bottom surface, and the shape of the area surrounded by the bottom surface and side surfaces of each hole 204 may be, for example, cylindrical or prismatic. Figure 5A and Figure 5B In the illustrated hole array 200 , the depth of each hole 204 is equal to the height of each partition wall 203 .

[0082] When the shape of each hole 204 is a cylinder with a circular bottom surface, it is preferable that the diameter of the bottom surface of each hole 204 is 0.5 μm or more and 12 μm or less, and the depth of each hole 204 is 0.5 μm or more and 12 μm or less. Furthermore, it is more preferable that the diameter of the bottom surface of each hole 204 is 1 μm or more and 9 μm or less, and the depth of each hole 204 is 1 μm or more and 9 μm or less. Upper substrate 202 faces the openings of wells 204 and the top surface of partition wall 203 across space 205. Space 205 serves as a channel for various liquids to flow through, allowing them to flow from the injection port to the discharge port. After wells 204 are filled with a solution, space 205 is filled with a hydrophobic solvent. When particle composite 206 is used, wells 204 are filled with particle composite 206, and space 205 is filled with a hydrophobic solvent. For example, fluorinated oil or aliphatic hydrocarbons can be used as the hydrophobic solvent.

[0083] The volume of each independent separation chamber is preferably 0.1 fL or more and 1400 fL or less, more preferably 0.8 fL or more and 600 fL or less. When the volume of each independent separation chamber is 0.1 fL or more, droplets or wells each having such a volume can be formed without difficulty. In addition, when the volume of each independent separation chamber is 1400 fL or less, the detection time can be made sufficiently short.

[0084] Specific examples of the method for detecting extracellular vesicles according to this embodiment are described below with respect to the case where the independently separated compartment is a droplet and the case where the independently separated compartment is a well.

[0085] Figure 2 is a flowchart for explaining the flow of the method for detecting extracellular vesicles according to this embodiment in the case where the independently separated compartments are droplets. (Step S201) Droplets are prepared, each containing a sample and a reporter molecule. The droplets are each preferably a water-in-oil emulsion. (Step S202) Droplets each containing a sample and a reporter molecule are placed in a tube and incubated in an incubator at 37° C. However, the reaction temperature can be set to any temperature and is not limited to 37° C. Upon incubation, the enzymatic reaction proceeds and a signal is generated from the reporter molecule. (Step S203) After a predetermined reaction time, the incubation is terminated and the observation chamber is filled with the droplets. The observation chamber is preferably a plate for sedimentation. (Step S204) An example in which the signal is fluorescence is described below. A fluorescence image of each droplet filling the observation chamber is acquired using a fluorescence microscope. A camera, such as a CCD camera, mounted on the fluorescence microscope acquires the fluorescence image of the droplet. Fluorescence is detected by acquiring the fluorescence image of the droplet.

[0086] (Step S205) Based on the detection results of the signal, the signal intensity of each droplet is determined, and each droplet having a signal intensity exceeding a predetermined threshold is identified. Each droplet having a signal intensity exceeding a predetermined threshold can be identified based on at least either a ratio or a difference between a signal intensity of a reference droplet and a signal intensity of each droplet containing a sample and a reporter molecule. The signal intensity of the reference droplet can be a signal intensity obtained for a droplet that does not contain extracellular vesicles and contains a reporter molecule. The signal intensity of each droplet is determined by performing predetermined image processing on the signal image of the droplet captured by the imaging device. For example, the signal intensity of each droplet can be determined by using Image J (manufactured by the National Institutes of Health) or the like as image processing software.

[0087] The case of using fluorescence as a signal is also described below. The predetermined image processing has a function of binarizing the fluorescent image based on brightness information. Negative droplets are droplets that do not contain extracellular vesicles and do not produce fluorescence derived from the fluorescent substance in the reporter molecule. Positive droplets are droplets that contain extracellular vesicles containing the target enzyme and produce fluorescence derived from the fluorescent substance in the reporter molecule. The fluorescence intensity of each droplet can be determined by binarizing the fluorescent image (grayscale image) using predetermined image processing.

[0088] (Step S206) After the enzymatic reaction is carried out by incubation in step S202, the concentration of extracellular vesicles is calculated based on the number of droplets that generate a signal. When the number of extracellular vesicles in the sample is large, a single droplet may contain two or more extracellular vesicles. Therefore, the number of extracellular vesicles may not match the number of droplets that generate a signal. For the above reasons, it is preferable to calculate the concentration of extracellular vesicles by calculation considering Poisson distribution. In Poisson distribution, when the average number of molecules per droplet is represented by λ, the ratio P(k) of droplets generating a signal can be expressed by the following formula (1). P(k)=(λk / k!)e -λ (k=0, 1, 2, ...) ... Formula (1)

[0089] From the number of droplets that generate a signal, P(k) can be determined and the expected value λ can be calculated. Therefore, by using Equation (1), the concentration of extracellular vesicles can be calculated from the number of droplets in which a signal is detected among all droplets.

[0090] Figure 3 is a flowchart for explaining the flow of the method for detecting extracellular vesicles according to this embodiment in the case where the independently separated compartment is a pore.

[0091] (Step S301) Figure 5A and Figure 5B The well array 200 schematically shown in FIG serves as the well array. In the well array 200 , an injection port (not shown) and an outlet port (not shown) are open, and a reaction solution containing a sample and a reporter molecule is supplied from the injection port into the space 205 .

[0092] (Step S302) The holes 204 are filled with a reaction solution. For example, as a reaction solution filling method, a method is provided that includes placing the hole array 200 under reduced pressure and degassing the space 205. Specifically, the hole array 200 is preferably placed in a vacuum desiccator at 0.1 atm for a predetermined period of time. By degassing, the air in the holes 204 is removed, so that the holes 204 can be effectively filled with the reaction solution. The degassing time is not particularly limited and can be freely set. The reaction solution filling method is not limited to a method based on degassing. For example, the holes can even be effectively filled with the reaction solution by performing a suction process from the discharge port.

[0093] (Step S303) The hydrophobic solvent to be sealed is supplied to space 205. That is to say, the reaction solution present in the space 205 above hole 204 is replaced by a hydrophobic solvent. As a hydrophobic solvent, for example, fluorinated oil, saturated aliphatic hydrocarbons, unsaturated aliphatic hydrocarbons, aromatic hydrocarbons and silicone oil can be used. The example of fluorinated oil can include Fluorinert (manufactured by 3M), AsahiKlin AE-3000 (manufactured by AGC Inc.), Fomblin (manufactured by Solvay SA) and Krytox (manufactured by DuPont). The example of saturated hydrocarbons includes Isopar (manufactured by Exxon Mobil Corporation) and mineral oil.

[0094] (Step S304) The well array 200 filled with the reaction solution is incubated in an incubator at 37° C. However, the reaction temperature may be set to any temperature without being limited to 37° C. Upon incubation, the enzymatic reaction proceeds and a signal is generated from the reporter molecule.

[0095] (Step S305) After the predetermined reaction time, the incubation is terminated, and a fluorescence image of each well 204 is acquired using a fluorescence microscope, for example, in the case where the signal is fluorescence.

[0096] (Step S306) The signal intensity of each well 204 is determined, and each well 204 having a signal intensity exceeding a predetermined threshold is identified. Each well 204 having a signal intensity exceeding the predetermined threshold can be identified based on at least either a ratio or a difference between the signal intensity of a reference well and the signal intensity of each well 204 containing a reporter molecule and a sample. The signal intensity of the reference well refers to the signal intensity obtained for each well 204 that does not contain extracellular vesicles and contains a reporter molecule. Predetermined image processing is used to determine the signal intensity of each well 204. As image processing software, the above-mentioned Image J can be used, and the signal intensity of each well 204 can be determined by an operation similar to the case where the independently separated compartment is a droplet.

[0097] (Step S307) The concentration of extracellular vesicles can be calculated by an operation similar to that in the case where the independently isolated compartments are droplets.

[0098] Figure 4 This is a flowchart for explaining the flow of the method for detecting extracellular vesicles according to this embodiment when background correction is performed by using a mask image. An example in which the signal is fluorescence is described.

[0099] (Step S401) A mask image indicating the positions of the independently separated compartments is generated from a fluorescence microscopy image of a standard fluorescent substance. (Step S402) Correct the background of the fluorescence microscopy images of the reporter molecules by using a mask image. (Step S403) The mean brightness value of each independently isolated compartment was obtained by using the corrected fluorescence image of the reporter molecule and the mask image, and a histogram was created. (Step S404) A predetermined threshold is applied to the histogram to determine negative or positive results, and the results are summarized.

[0100] (Other steps) The method for detecting extracellular vesicles according to this embodiment may include steps other than the above steps. An example of the additional step is a step of acquiring a fluorescence image serving as a reference (hereinafter referred to as a “reference fluorescence image”) in a case where the signal is fluorescence. For example, if the fluorescence image acquired in step S204 or step S305 includes fluorescence originating from a substance other than the fluorescent substance used to detect extracellular vesicles, the concentration of extracellular vesicles may not be correctly calculated. A conceivable example of fluorescence originating from a source other than the fluorescent substance used to detect extracellular vesicles is autofluorescence emitted from the pores.

[0101] The timing of acquiring the reference fluorescence image in the step of acquiring the reference fluorescence image only needs to be before the presence of the enzyme causes the above-mentioned reporter molecule to be cut and thus emit fluorescence. For example, the reference fluorescence image can be acquired before the hole or droplet is filled with a reagent or sample containing the reporter molecule, before filling with a sealing oil that seals an independent separation chamber (such as a hole or droplet), or before performing the incubation of step S202 or step S304 (before performing heating). In addition, when the timing of acquiring the reference fluorescence image is after the hole or droplet has been filled with the reporter molecule and before incubation is performed (before performing heating), the reference fluorescence image can be acquired after a predetermined time period from the time when the hole or droplet is started to be filled with the reporter molecule. When the timing for acquiring the reference fluorescence image is set to before incubation (before heating), the reference fluorescence image can be acquired before the heating device, having received the drive signal, begins heating. Furthermore, the timing for acquiring the reference fluorescence image can be determined by using a device for monitoring the state of the wells or droplets filled with the sample or reporter molecules.

[0102] When calculating fluorescence intensity based on the presence of the enzyme in each well or droplet in the fluorescence image obtained in step S204 or step S305, the reference fluorescence image can be used as a reference. Specifically, when calculating the degree to which fluorescence intensity increases from the fluorescence intensity of the previously acquired reference fluorescence image, fluorescence intensity due to components other than the fluorescent substance used to detect extracellular vesicles in each well or droplet can be excluded. Consequently, the fluorescence intensity derived from the fluorescent substance of the reporter molecule used to detect extracellular vesicles can be more accurately obtained. Furthermore, for example, for the fluorescence image obtained in step S204 or step S305, it is possible to more appropriately determine whether the pixel value (fluorescence intensity) of the target well or droplet is equal to or greater than a predetermined threshold, or less than a predetermined threshold. The predetermined threshold only needs to be determined before determining the number of wells or droplets. For example, the predetermined threshold can be a fixed value in the extracellular vesicle detection device, or can be a value set by user input. A determination of being equal to or greater than the predetermined threshold can be considered a positive determination, and a determination of being less than the predetermined threshold can be considered a negative determination.

[0103] Furthermore, for example, a reference fluorescence image can be acquired to detect a state in which a well or droplet to be filled with a sample or reporter molecule is not filled with the sample or reporter molecule due to a malfunction in the well or droplet, or during an operation to fill the well or droplet. In this case, for example, a substance (hereinafter referred to as a "reference substance") emitting light at a wavelength different from the wavelength of the fluorescent substance used to detect extracellular vesicles can be filled with the well or droplet, and a fluorescence image thereof can be acquired to obtain a fluorescence distribution. In this case, the difference between the center wavelength of the emission wavelength (fluorescence wavelength) of the reference substance and the center wavelength of the emission wavelength (fluorescence wavelength) of the reporter fluorescent substance is preferably 30 nm or greater, more preferably 50 nm or greater, and even more preferably 100 nm or greater.

[0104] When fluorescence is not detected as a result of acquiring a reference fluorescence image, it may be appropriate to restart from the first step, display an error message, or determine whether to restart or continue measurement based on the number of wells or droplets in which fluorescence was not detected. When the number of wells or droplets in which fluorescence was not detected is equal to or less than a predetermined threshold, it may be appropriate not to count the fluorescence of the wells or droplets. It is only necessary to determine the predetermined threshold before determining the number of wells or droplets. For example, the predetermined threshold may be a fixed value in the extracellular vesicle detection device, or may be a value set by user input. In addition, for example, in the case where fluorescence is not detected as a result of acquiring a reference fluorescence image in the extracellular vesicle detection device used to implement the method for detecting extracellular vesicles of this embodiment, it may be appropriate to allow the user to make an appropriate selection regarding the operation.

[0105] The extracellular vesicle detection apparatus implementing the extracellular vesicle detection method of the present embodiment may be configured to be switchable between a mode including the step of acquiring the reference fluorescent image described above and a mode not including the step of acquiring the reference fluorescent image.

[0106] As described above, the step of acquiring a reference fluorescence image has been described in the extracellular vesicle detection method of this embodiment, but this step can also be applied to the extracellular vesicle detection device of the above-mentioned embodiment of the present invention. In this case, the reference fluorescence image can be acquired using the fluorescence detection unit or image acquisition unit in the extracellular vesicle detection device of this embodiment, or other means (such as a reference fluorescence image acquisition unit) can be used to acquire the reference fluorescence image.

[0107] In addition, according to another embodiment of the present invention, a device for detecting extracellular vesicles with a target enzyme is provided, which includes: a distribution unit configured to distribute extracellular vesicles and a reagent containing a reporter molecule to be changed by the target enzyme to emit a signal into a plurality of independent separation compartments; a signal generating unit configured to cause the target enzyme and the reporter molecule to react with each other to generate a signal; a signal detection unit configured to detect the signal; and an identification unit configured to determine the signal intensity of each of the independent separation compartments based on a detection result obtained using the signal detection unit, and identify each of the independent separation compartments having a signal intensity exceeding a predetermined threshold. refer to Figure 6 The detection device of this embodiment is further described in detail.

[0108] (Allocation Unit) When the independent separation compartments are droplets, for example, the distribution unit 101 includes an emulsification membrane or a microchannel. When the distribution unit 101 includes an emulsification membrane, the droplets can be prepared by, for example, using a direct membrane emulsification method or a pumping method, using a Shirasu porous glass (SPG) membrane of SPG Technology Co., Ltd. or the like. When using the distribution unit 101 including an emulsification membrane, for example, a combination of Isopar L (aliphatic hydrocarbon, manufactured by Exxon Mobil Corporation) and KF-6038 (surfactant, manufactured by Shin-Etsu Chemical Co., Ltd.) can be used to prepare droplets each having a diameter of 1 μm to 10 μm. In addition, when using an emulsification membrane having different pore sizes, the average droplet diameter can be changed.

[0109] When the dispensing unit 101 includes a microchannel, for example, a Dolomite Microfluidics microchannel can be used as the microchannel. When using the dispensing unit 101 including a microchannel, for example, nearly monodisperse droplets with a diameter of 1 μm to 10 μm can be produced using each of the following three combinations. Furthermore, when using microchannels with different nozzle sizes, the average droplet diameter can be varied. A combination of Isopar L (aliphatic hydrocarbon, manufactured by Exxon Mobil Corporation) and KF-6038 (surfactant, manufactured by Shin-Etsu Chemical Co., Ltd.) A combination of mineral oil (aliphatic hydrocarbon) and SPAN-80 (surfactant, manufactured by Tokyo Chemical Industry Co., Ltd.) Automated Droplet Generator oil (manufactured by Bio-Rad Laboratories, Inc.)

[0110] When the independent separation chambers are wells, the dispensing unit 101 includes an injection port, and injects the solution from the injection port of the well array into the wells via a nozzle, for example, by using the injection port. In the dispensing unit 101, the sample containing extracellular vesicles and the reagent containing the reporter molecule can be pre-mixed before being dispensed into the independent separation chamber and then dispensed as a reaction solution, or can be dispensed separately so as to be mixed with each other in the independent separation chamber. The sample containing extracellular vesicles and the reporter molecule are preferably pre-mixed before being dispensed into the independent separation chamber because a uniformly mixed reaction solution can be easily obtained.

[0111] (Signal Generation Unit) The signal generating unit 102 generates a signal by changing the reporter molecule with the enzyme by appropriately adjusting the environment such as temperature according to the sample, the type of target enzyme, the type of reporter molecule, etc. For example, an incubator can be used as the signal generating unit 102 .

[0112] (Signal detection unit) The signal detection unit 103 detects the signal generated by the signal generation unit 102. Any device can be used as the signal detection unit 103 as long as the device can detect a signal in an independent separation compartment, but examples thereof include a plate reader and a fluorescence microscope.

[0113] (Identification Unit) The identification unit 104 may include an extraction unit 105, a determination unit 106, a judgment unit 107, a calculation unit 108, a display unit 109, and a storage unit 110. In the extraction unit 105, the extraction unit 105 extracts information about the number and relative positional relationship of the plurality of independently separated compartments from the detection results obtained by the signal detection unit 103. The determination unit 106 determines the signal strength of each of the independently separated compartments identified by the extraction unit 105 based on the detection results obtained by the signal detection unit 103. The judgment unit 107 judges each independently separated compartment whose signal strength exceeds a predetermined threshold based on the signal strength determined by the determination unit 106. Thus, each independently separated compartment having a signal strength exceeding the predetermined threshold is identified.

[0114] The identification unit 104 is preferably configured to identify each independently isolated compartment having a signal intensity exceeding a predetermined threshold based on at least either a ratio or a difference between the signal intensity of the reference compartment and the signal intensity of each independently isolated compartment. Furthermore, the signal intensity of the reference compartment is preferably a signal intensity obtained using a sample free of extracellular vesicles and a reporter molecule. The signal intensity of the reference compartment can be determined in the same manner as for the independently isolated compartment, except that the reference compartment does not contain extracellular vesicles.

[0115] The ratio between the signal intensity of the reference compartment and the signal intensity of each independently separated compartment is calculated by, for example, using the signal intensity of the reference compartment as a denominator and using the signal intensity of each independently separated compartment as a numerator. The difference between the signal intensity of the reference compartment and the signal intensity of each independently separated compartment is calculated by, for example, subtracting the signal intensity of each independently separated compartment from the signal intensity of the reference compartment. When the value obtained based on the ratio and / or the difference between the signal intensity of the reference compartment and the signal intensity of the independently separated compartment is less than a predetermined threshold value, the determination unit 107 considers that the signal intensity of the independently separated compartment is equal to the signal intensity of the reference compartment, and determines that the independently separated compartment is negative. In addition, when the value obtained based on the ratio and / or the difference between the signal intensity of the reference compartment and the signal intensity of the independently separated compartment is equal to or greater than a predetermined threshold value, the determination unit 107 determines that the independently separated compartment is positive.

[0116] The calculation unit 108 calculates the concentration of extracellular vesicles in the sample based on the number of identified independent separation compartments having a signal intensity exceeding a predetermined threshold. When the concentration of extracellular vesicles in the sample is not calculated, the identification unit 104 does not need to include the calculation unit 108.

[0117] Display unit 109 displays information acquired or extracted by extraction unit 105, determination unit 106, judgment unit 107, and calculation unit 108. Storage unit 110 stores data acquired or extracted by extraction unit 105, determination unit 106, judgment unit 107, and calculation unit 108.

[0118] In the extracellular vesicle detection device according to this embodiment, the signal detection unit 103 may be an image acquisition unit. Figure 7 1 is a functional block diagram for explaining an extracellular vesicle detection device 20 including an image acquisition unit 111 as a signal detection unit 103 , and its configuration is similar to that of the above-described extracellular vesicle detection device 10 .

[0119] Image acquisition unit 111 acquires an image of a particle complex containing independent isolated compartments or extracellular vesicles and particles bound to captured proteins. The image acquired by image acquisition unit 111 includes the signal generated by signal generation unit 102 as image information. For example, if the signal is fluorescence, a fluorescence microscope can be used as image acquisition unit 111.

[0120] In the extracellular vesicle detection device 20, the extraction unit 105 included in the identification unit 104 preferably extracts information about the number and relative positional relationship of the plurality of independently separated compartments based on the image acquired by the image acquisition unit 111. That is, in the extracellular vesicle detection device 20, the identification unit 104 preferably identifies each independently separated compartment having a signal intensity exceeding a predetermined threshold by processing the image acquired by the image acquisition unit 111. For example, if the signal is fluorescence, extraction unit 105 uses brightness information to extract information about the independently separated compartments using a region extraction method. In particular, if the independently separated compartments are droplets, the regions corresponding to the droplets on the image each have a contour, so extraction unit 105 can extract the edges of these contours as closed curves. Furthermore, extraction unit 105 can extract the regions corresponding to the droplets on the image by binarizing the image based on brightness information.

[0121] Furthermore, the determination unit 106 determines the signal intensity of each independently separated compartment based on the brightness information that each independently separated compartment on the image has.

[0122] Figure 8 4 is a block diagram illustrating an example of the hardware configuration of the extracellular vesicle detection device 10 according to this embodiment. The extracellular vesicle detection device 10 includes a distribution device 401, a signal generation device 402, a signal detection device 403, and an information processing system 404. The information processing system 404 can be, for example, an independent separation compartment identification device.

[0123] The allocating device 401 , the signal generating device 402 and the signal detecting device 403 are devices for performing the functions of the allocating unit 101 , the signal generating unit 102 and the signal detecting unit 103 , respectively.

[0124] The information processing system 404 has the functionality of a computer. For example, the information processing system 404 can be integrated with a desktop personal computer (PC), a laptop PC, a tablet PC, a smartphone, or the like. The information processing system 404 has the functionality to identify each independent separation compartment having a signal strength exceeding a predetermined threshold. Furthermore, the information processing system 404 may also have the functionality to control the operations of the dispensing device 401, the signal generating device 402, and the signal detecting device 403 according to a predetermined program.

[0125] In order to realize the function as a computer that performs arithmetic operations and stores data, the information processing system 404 includes a central processing unit (CPU) 406, a random access memory (RAM) 407, a read-only memory (ROM) 408, and a hard disk drive (HDD) 409. In addition, the information processing system 404 also includes a communication interface (I / F) 410, a display device 411, and an input device 412. The CPU 406, RAM 407, ROM 408, HDD 409, the communication I / F 410, the display device 411, and the input device 412 are connected to each other via a bus 405. The display device 411 and the input device 412 can be connected to the bus 405 via a drive device (not shown) for driving those devices.

[0126] exist Figure 8 4, the components forming the information processing system 404 are shown as integrated devices, but some of the functions of these components may be implemented by external devices. For example, the display device 411 and the input device 412 may be external devices different from the components that implement the computer functions including the CPU 406.

[0127] The CPU 406 performs predetermined operations according to programs stored in, for example, the RAM 407 and the HDD 409, and also has a function of controlling each component of the information processing system 404. The RAM 407 is constructed from a volatile storage medium and provides a temporary memory area required for the operation of the CPU 406. The ROM 408 is constructed from a non-volatile storage medium and stores necessary information such as programs for the operation of the information processing system 404. The HDD 409 is a storage device constructed from a non-volatile storage medium and stores information on, for example, the number of independent separation compartments, their positions, and signal strengths.

[0128] Communication I / F 410 is a communication interface based on standards such as Wi-Fi (trademark) or 4G, and is a module for communicating with other devices. Display device 411 is, for example, a liquid crystal display or an organic light-emitting diode (OLED) display, and is used to display moving images, still images, characters, etc. Input device 412 is, for example, a key, a touch panel, a keyboard, or a pointing device, and is used by the user to operate information processing system 404. Display device 411 and input device 412 can be integrally formed as a touch panel.

[0129] Figure 8The hardware configuration shown is an example, and devices other than the devices shown can be added, or a portion of the devices shown can be omitted. In addition, some devices can be replaced with other devices having the same functions. In addition, some functions can be provided by other devices via a network, and the functions for implementing the embodiment can be shared and implemented by multiple devices. For example, HDD 409 can be replaced by a solid-state drive (SSD) using a semiconductor element (such as flash memory), or can be replaced by cloud storage.

[0130] The CPU 406 implements the functions of the extraction unit 105, the determination unit 106, the judgment unit 107, and the calculation unit 108 by loading a program stored in the ROM 408 or elsewhere onto the RAM 407 and executing the program. The CPU 406 also implements the function of the display unit 109 by controlling the display device 411. The CPU 406 also implements the function of the storage unit 110 by controlling the HDD 409.

[0131] As an example of the hardware configuration of the extracellular vesicle detection device 20 of this embodiment, an image acquisition device that performs the function of the image acquisition unit 111 in the hardware configuration example of the extracellular vesicle detection device 10 described above is shown as the fluorescence detection device 403. Except for the foregoing, the hardware configuration of the extracellular vesicle detection device 20 can be the same as the hardware configuration of the extracellular vesicle detection device 10.

[0132] According to another embodiment of the present invention, there is provided a program for causing a computer included in an extracellular vesicle detection device to execute the method for detecting extracellular vesicles, thereby causing the extracellular vesicle detection device to execute the method. [Example]

[0133] The present invention will be described in more detail below by way of examples. However, the present invention is by no means limited to the examples described below.

[0134] (Materials, etc.) The extracellular vesicles, reporter molecules, and standard fluorescent substances used in the examples are described.

[0135] Extracellular vesicles (sometimes abbreviated as "EVs") As extracellular vesicles, the following commercially available purified exosomes (manufactured by System Biosciences, LLC) were used. EXOP-105A-1: Frozen exosomes from MDA-MB-231 (concentration: >1×10 6) (Human Breast Cancer, Aggressive / Invasive / Metastatic Cell Line): EXOP-120A-1: Frozen exosomes from A549 (concentration: >1×10 6 ) (Human Non-small Cell Lung Cancer Cell Line): In addition, the following commercially available purified exosomes (manufactured by FUJIFILM Wako Pure Chemical Corporation) were also used as extracellular vesicles. 052-09301, exosomes, from COLO201 cells, purified (50 μL): Exosome concentration: 10 μg / mL (protein concentration estimated by CD63 signal value)

[0136] <Reporter molecules for MMP detection> To detect the enzyme activity of MMP, the following commercially available MMP activity measurement kit SensoLyte (manufactured by AnaSpec, Inc.) and MMP-14 Substrate I available from Merck KGaA were used. SensoLyte's reporter molecule, included in SensoLyte 520 MMP-14 Assay Kit (Fluorimetric) AS-72025, is a FRET peptide having 5-FAM as a fluorescent substance and QXL520 (trademark) as a quencher, and emits fluorescence at Ex / Em = 490 nm / 520 nm upon peptide chain cleavage by MMP14. Furthermore, SensoLyte 520 MMP Substrate Sampler Kit (Fluorimetric) AS-71170 was used to detect various MMPs. Each substrate is a FRET peptide containing 5-FAM as a fluorescent substance and QXL520 as a quencher, and emits fluorescence at Ex / Em = 490 nm / 520 nm upon peptide chain cleavage of various MMPs. Among 16 substrates, the following 6 were used. SB2 MMP-1 / 7 / 8 / 12 / 13 QXL520-Pro-Leu-Ala-Leu-Trp-Ala-Arg-Lys(5-FAM)-NH2 SB6 MMP-2 / 13 QXL520-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys(5-FAM)-NH2 SB7 MMP-7 / 12 / 13 QXL520-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-Lys(5-FAM)-NH2 SB9 MMP-1 / 2 / 7 / 8 / 12 / 13 QXL520-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Lys(5-FAM)-NH2 SB10 MMP-13 QXL520-Pro-Leu-Ala-Tyr-Trp-Ala-Arg-Lys(5-FAM)-NH2 SB14 MMP-1 / 2 / 3 / 7 / 8 / 9 / 10 / 12 / 13 / 14 / 15 / 16 / 24 QXL520-γ-Abu-Pro-Cha-Abu-Smc-His-Ala-Dab(5-FAM)-Ala-Lys-NH2 (Smc = S-methyl-L-cysteine) The MMP-14 Substrate I, Fluorogenic (Calbiochem: 444258) reporter molecule (MMP-14 Substrate I) is a FRET peptide containing MCA as a fluorescent substance and DPA as a quencher. It emits fluorescence at Ex / Em = 325 nm / 392 nm upon cleavage of the MMP14 peptide chain. Specifically, the reporter molecule has the following sequence. Peptide sequence: MCA-Pro-Leu-Ala-Cys(p-OMeBz)-Trp-Ala-Arg(Dpa)-NH2 Dpa stands for N 3 -(2,4-dinitrophenyl)-L-2,3-diaminopropionyl. Bz represents a benzyl group. DMSO was added to the freeze-dried product of each of the peptides to prepare a 1 mM solution. In addition, the following custom-synthesized FRET peptides were used, obtained through contract synthesis by Cosmo Bio Co., Ltd. These peptides are designated Custom #1, Custom #2, Custom #11, Custom #12, Custom #13, Custom #14, Custom #15, and Custom #16. Each of these peptides is a FRET peptide having 5-FAM as a fluorescent substance and TQ2 as a quencher, and emits fluorescence at Ex / Em = 490 nm / 520 nm upon peptide chain cleavage by MMP14. Custom Compound #1: [5-FAM]GRIGFLRTAK(TQ2)[OH] Custom #2: [5-FAM]GGPLGLAGGK(TQ2)[OH] Custom #11: TQ2-LAPLGLQRRK(5-FAM) Custom #12: TQ2-FMPLGLRGIK (5-FAM) Custom #13: TQ2-WYPAGLRMVK(5-FAM) Custom #14: TQ2-DLPAGLQARK (5-FAM) Custom #15: TQ2-YPPRPLLARK(5-FAM) Custom #16: TQ2-WPHGSLQAAK (5-FAM) In addition, the following custom-synthesized FRET peptides, obtained through contract synthesis by Cosmo Bio Co., Ltd., were used as different FRET dye pairs for Custom #2. These peptides are designated Custom #2T and Custom #2TF3, respectively. Custom #2T is a FRET peptide with TAMRA as a fluorescent substance and TQ3 as a quencher, and emits fluorescence with an Ex / Em = 553 nm / 575 nm upon peptide chain cleavage by MMP14. Furthermore, Custom #2TF3 is a FRET peptide with TF3 as a fluorescent substance and TQ3 as a quencher, and emits fluorescence with an Ex / Em = 555 nm / 584 nm upon peptide chain cleavage by MMP14. Custom #2T:TAMRA-GGPLGLAGGK(TQ3) Custom #2TF3:TF3-GGPLGLAGGK(TQ3) TF3 (Tide Fluor 3) TQ3 (Tide Quencher 3) DMSO was added to the freeze-dried product of each of the peptides to prepare a 1 mM solution.

[0137] <Reporter molecules for AChE> In order to detect the enzyme activity of AChE, the following commercially available AChE activity measurement kits SensoLyte (manufactured by AnaSpec Corporation) and Amplite (manufactured by AAT Bioquest Inc.) were used. SensoLyte's reporter molecule, included in the 520 Acetylcholinesterase Activity Assay Kit (AS-72242), is a FRET peptide having 5-FAM as a fluorescent substance and QXL520 as a quencher, and emits fluorescence of Ex / Em=490nm / 520nm upon peptide chain cleavage by AChE. Amplite's reporter molecule is included in Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence) 11401 and reacts with hydrolyzed thiocholine reflecting the AChE activity in the sample solution to allow the green fluorescent dye Tiolite Green to emit fluorescence at Ex / Em=510nm / 525nm.

[0138] <Reporter Molecules for ADAM> To detect ADAM enzyme activity, the following commercially available ADAM activity measurement substrates (manufactured by BioZyme Inc.) were used. Each of the substrates is a FRET peptide having 5-FAM as a fluorescent substance and Dabcyl as a quencher, and emits fluorescence of Ex / Em=490nm / 520nm upon cleavage of the ADAM peptide chain. ADAM Substrate II(Fluorogenic)-PEPDAB010 Dabcyl-SPLAQAVRSSK(5FAM)-NH2 (Dabcyl = 4-((4-(dimethylamino)phenyl)azo)benzoic acid) ADAM17 Selective Substrate(Fluorogenic)-PEPDAB014 Dabcyl-EHADLLAVVAK(5FAM)-NH2 DMSO was added to the freeze-dried product of each substrate to prepare a 1 mM solution.

[0139] <Reporter Molecules for Cathepsin D> In addition, to detect the enzymatic activity of cathepsin D, a commercially available Cathepsin D Assay Kit, Fluorimetric, SensoLyte 520AS-72170 (manufactured by AnaSpec, Inc.) was used. The reporter molecule is a FRET peptide having HiLyteFluor (trademark) 488 as a fluorescent substance and QXL520 as a quencher, and emits fluorescence at Ex / Em = 497 nm / 525 nm upon cleavage of the peptide chain of cathepsin D.

[0140] <Standard fluorescent substances> Alexa Fluor (trademark) 647 (manufactured by Molecular Probes (trademark)), which is a standard fluorescent substance for identifying droplets or wells described later, was dissolved in DMSO to prepare a standard fluorescent substance solution (1 mM) to be used.

[0141] Example 1 (Detection of MMP-14 activity using SensoLyte 520 MMP-14 Assay Kit (Fluorimetric)) Recombinant human MMP14 protein (Active) (manufactured by Abcam plc): ab285993 was used as an MMP14 standard, and 10 μg of the MMP14 freeze-dried product was dissolved in 100 μL of purified water. 5 μL of the solution was taken and added to the assay buffer (component D) included in the kit to obtain a 120 μL solution. The MMP14 concentration of this solution was 4.17 μg / mL. The concentration of this solution was set to x1, and a 3-fold serial dilution was performed to prepare standard samples with seven concentrations. In addition, a sample without MMP14 was similarly prepared and used as a sample for background (BG) measurement. Furthermore, to detect MMP14 activity, 4.95 mL of assay buffer (component D) was added to 50 μL of MMP-14 substrate (component A) included in the kit to provide 5 mL of MMP14 detection solution. 50 μL of the MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C. to evaluate the MMP14 activity.

[0142] (Evaluation of MMP14 activity using a 96-well plate) MMP14 activity was assessed using a 96-well plate (manufactured by Thermo Fisher Scientific Inc., model number: 137101). Fluorescence intensity was measured every 5 minutes for 1 hour at 37°C using a fluorescence microplate reader (Synergy MX, manufactured by BioTek Instruments, Inc.). An excitation wavelength of 485 ± 20 nm and a fluorescence wavelength of 528 ± 20 nm were used for measurement. The relative fluorescence unit difference ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 9A The concentration and fluorescence intensity of MMP14 after 1 hour are shown. Figure 9A As shown, MMP14 activity can be detected depending on the MMP14 concentration. Figure 9B Five points on the low-concentration side that are approximately linear are shown. It was found that the low-concentration region has satisfactory linearity.

[0143] Example 2 (Detection of MMP-14 Activity in Extracellular Vesicles Derived from MDA-MB-231 Using the SensoLyte 520 MMP-14 Assay Kit (Fluorimetric)) 5 μL (equivalent to 5 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the assay buffer (component D) included in the kit was added to the aliquot to provide a 120 μL solution. The concentration of this solution was 41.7 μg / mL. The concentration of this solution was set to x1, and a 2-fold serial dilution was performed to prepare standard samples with six concentrations. In addition, a sample without extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP14 activity, 4.95 mL of assay buffer (component D) was added to 50 μL of MMP-14 substrate (component A) included in the kit to provide 5 mL of MMP14 detection solution. 50 μL of the MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C. to evaluate the MMP14 activity.

[0144] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 10 The concentration and fluorescence intensity of MDA-MB-231-derived extracellular vesicles after 1 hour are shown. Figure 10 As shown, MMP14 activity can be detected depending on the extracellular vesicle concentration. Satisfactory linearity was observed in the evaluated concentration range. Compared to the slope of the approximate straight line in Example 1, for the recombinant MMP14 with a molecular weight of 24 kDa in Example 1, it can be estimated that approximately 0.86% of the weight of extracellular vesicle protein corresponds to MMP14. Furthermore, for intact MMP14 with a molecular weight of 70 kDa, it can be estimated that 2.5% of the weight corresponds to MMP14.

[0145] Example 3 (Detection of MMP-14 activity in extracellular vesicles derived from MDA-MB-231 using Calbiochem MMP-14 Substrate I, Fluorogenic) A 2 μL aliquot (equivalent to 2 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg protein in 50 μL of sterile 1x PBS containing 50 μg of exosome protein) was taken, and the assay buffer (Component D) included in the SensoLyte 520 MMP-14 Assay Kit was added to the aliquot to provide a 200 μL solution. The concentration of this solution was 10 μg / mL. A sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP14 activity, 1 mM MMP-14 Substrate I was diluted with the assay buffer (component D) included in the SensoLyte 520 MMP-14 Assay Kit to prepare 20 μM, 10 μM, and 5 μM MMP14 detection solutions. 50 μL of each MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C., and the MMP14 activity was evaluated. In this case, the concentration of the extracellular vesicles in each reaction solution was 5 μg / mL, and the concentration of the reporter molecule in each reaction solution was 10 μM, 5 μM, or 2.5 μM.

[0146] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1, except that the excitation wavelength was set to 325 ± 20 nm, the fluorescence wavelength was set to 392 ± 20 nm, and the reaction time was set to 2 hours. ΔRFU was calculated by subtracting the measured value of the BG measurement sample from the measured value of each sample. Figure 11 The figure shows the temporal changes in the fluorescence intensity of MMP-14 Substrate I from MDA-MB-231-derived extracellular vesicles. Figure 11 As shown, it was found that the fluorescence intensity increased with the reaction time, and the MMP14 activity could be detected.

[0147] Example 4 (Detection of MMP-14 Activity in MDA-MB-231-Derived Extracellular Vesicles Using Reporter Molecules: Customized #1 or Customized #2) A 2 μL aliquot (equivalent to 2 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was taken, and the assay buffer (Component D) included in the SensoLyte 520 MMP-14 Assay Kit was added to the aliquot to provide a 200 μL solution. The concentration of this solution was 10 μg / mL. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP14 activity, 1 mM Customized Product #1 or Customized Product #2 was diluted with the assay buffer (Component D) included in the SensoLyte 520 MMP-14 Assay Kit to prepare a 10 μM MMP14 detection solution. Furthermore, the SensoLyte 520 MMP-14 detection solution of Example 1 was used as a control. 50 μL of each MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C. to evaluate the MMP14 activity. In this case, the concentration of the extracellular vesicles in each reaction solution was 5 μg / mL, and the concentration of the reporter molecule in each reaction solution was 5 μM.

[0148] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 12 The time-dependent changes in fluorescence intensity from various reporter molecules via MDA-MB-231-derived extracellular vesicles are shown. Figure 12 As shown, the fluorescence intensity was found to increase with the reaction time, and MMP14 activity could be detected. In addition, the increase in fluorescence intensity from each of Custom #1 and Custom #2 was more significant than the increase in fluorescence intensity from SensoLyte 520MMP-14.

[0149] Example 5 (Detection of MMP-14 Activity in MDA-MB-231-Derived Extracellular Vesicles Using Reporter Molecules: Customized #1 or Customized #2) 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the assay buffer (component D) included in the SensoLyte 520MMP-14 Assay Kit was added to the aliquot to provide a 200 μL solution. The concentration of this solution was 20 μg / mL. The concentration of this solution was set to x1, and a 3-fold serial dilution was performed to prepare a standard sample with three concentrations. In addition, a sample without extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP14 activity, 1 mM of Customized Substance #1 or Customized Substance #2 was diluted with the assay buffer (Component D) included in the SensoLyte 520 MMP-14 Assay Kit to prepare a 10 μM MMP14 detection solution. 50 μL of the MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C. to evaluate the MMP14 activity. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 10 μg / mL, and the concentration of the reporter molecule in each reaction solution was 5 μM.

[0150] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 13A The temporal changes in fluorescence intensity of various reporter molecules are shown based on the differences in extracellular vesicle concentrations derived from MDA-MB-231. Figure 13A As shown, it was found that the fluorescence intensity varied with the extracellular vesicle concentration. In addition, it was found that at the same extracellular vesicle concentration, the fluorescence intensity from Customized Product #1 was higher than that from Customized Product #2. The time-dependent increase in fluorescence intensity within 1 hour of the reaction was approximately linear, and the slope (reaction rate) of the increase in each reporter molecule and each extracellular vesicle concentration was plotted at Figure 13B From the approximate straight line, the reaction rates per 1 μg / mL for Custom #1 and Custom #2 can be estimated to be 101.5 ΔRFU / min and 83.3 ΔRFU / min, respectively.

[0151] Example 6 (Study on the composition of the reaction solution for detecting MMP-14 activity using the reporter molecule: Customized compound #1) 5 μL (equivalent to 5 μg) of MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg protein in 50 μL PBS) were aliquoted, and the following buffer (1) was added to the aliquot to provide a 500 μL solution. Buffer (1) 50 mM Tris-Cl (pH: 7.6) 10mM CaCl2 The concentration of this solution was 10 μg / mL. In addition, a sample not containing extracellular vesicles was similarly prepared and used as a sample for BG measurement. In addition, to detect MMP14 activity, 1 mM custom compound #1 was diluted with buffer (1) to prepare a 10 μM MMP14 detection solution. 500 μL of MMP14 detection solution was added to 500 μL of sample and mixed, and the mixture was then aliquoted into 100 μL portions. NaCl and Brij35 were added to each portion as described below. The amounts (μL) of NaCl and Brij35 added to 100 μL of each reaction solution are described below. After mixing, the reaction was carried out at 37°C, and the MMP14 activity was evaluated. In this case, the concentration of the extracellular vesicles in each reaction solution was 5 μg / mL, and the concentration of the reporter molecule in each reaction solution was 5 μM.

[0152] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 14A The figure shows the temporal change of the fluorescence intensity of Custom #1 by the difference in Brij35 concentration in the case of containing NaCl at a final concentration of 150 mM. It was found that the fluorescence intensity decreased even when the Brij35 concentration was high or zero. In addition, Figure 14B The figure shows the temporal change in fluorescence intensity of Custom #1 by the difference in Brij35 concentration in the absence of NaCl. It was found that the fluorescence intensity decreased even when the Brij35 concentration was high or zero, similar to the case of containing NaCl at a final concentration of 150 mM. Figure 14C The graph shows the temporal change in fluorescence intensity of Custom #1 in the presence or absence of NaCl in the case of containing Brij35 at a final concentration of 0.0125%. It was found that the fluorescence intensity in the case of not containing NaCl became higher.

[0153] Example 7 (Study on the composition of the reaction solution for detecting MMP-14 activity using the reporter molecule: Customized Compound #1 or Customized Compound #2) 5 μL (equivalent to 5 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg protein in 50 μL PBS) was aliquoted, and the above buffer (1) was added to the aliquot to provide a 500 μL solution. The concentration of this solution was 10 μg / mL. In addition, a sample not containing extracellular vesicles was similarly prepared and used as a sample for BG measurement. In addition, to detect MMP14 activity, 1 mM custom compound #1 or custom compound #2 was diluted with buffer (1) to prepare a 10 μM MMP14 detection solution. 500 μL of MMP14 detection solution was added to 500 μL of sample and mixed, and the mixture was then aliquoted into 100 μL portions. A 0.5% Brij35 solution was added to each portion as described below. The amount (μL) of 0.5% Brij35 solution added to 100 μL of each reaction solution was as described below. After mixing, the reaction was carried out at 37°C, and the MMP14 activity was evaluated. In this case, the concentration of the extracellular vesicles in each reaction solution was 5 μg / mL, and the concentration of the reporter molecule in each reaction solution was 5 μM.

[0154] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 15A The time variation of the fluorescence intensity of Customized Product #1 by the difference of Brij35 concentration is shown. It is found that the fluorescence intensity decreases when the Brij35 concentration is high. In addition, Figure 15B The temporal change in fluorescence intensity of Custom #2 is shown by the difference in Brij35 concentration. Unlike Custom #1, it was found that the difference in Brij35 concentration within this range only slightly affected the fluorescence intensity.

[0155] Example 8 (Detection of MMP-14 activity in extracellular vesicles derived from MDA-MB-231 using reporter molecules: Customized #1 or Customized #2) 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the following buffer (2) was added to the aliquot to provide a 200 μL solution. The concentration of this solution was 20 μg / mL. The concentration of the solution was set to x1, and a 3-fold serial dilution was performed to prepare standard samples having three concentrations. In addition, a sample without extracellular vesicles was similarly prepared and used as a sample for BG measurement. Buffer (2) 50 mM Tris-Cl (pH: 7.6) 10mM CaCl2 0.0125% Brij35 In addition, to detect MMP14 activity, 1 mM of Customized Compound #1 or Customized Compound #2 was diluted with buffer (2) to prepare a 10 μM MMP14 detection solution. 50 μL of the MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C. to evaluate the MMP14 activity. In this case, the concentration of the extracellular vesicles in the reaction solution was 10 μg / mL, and the concentration of the reporter molecules in the reaction solution was 5 μM.

[0156] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 16A The temporal changes in fluorescence intensity of various reporter molecules are shown based on the differences in extracellular vesicle concentrations derived from MDA-MB-231. Figure 16A As shown, it was found that the fluorescence intensity varied with the extracellular vesicle concentration. In addition, it was found that at the same extracellular vesicle concentration, the fluorescence intensity from Customized Product #1 was higher than that from Customized Product #2. The time-dependent increase in fluorescence intensity within 1 hour of the reaction was approximately linear, and the slope (reaction rate) of the curve with respect to the increase in concentration of each reporter molecule and each extracellular vesicle was approximately linear. Figure 16B . From the approximate straight line, the reaction rates per 1 μg / mL for Customized Compound #1 and Customized Compound #2 can be estimated to be 198 ΔRFU / min and 99.0 ΔRFU / min, respectively. Compared to 101.5 ΔRFU / min and 83.3 ΔRFU / min in the assay buffer (Component D) included in the SensoLyte 520 MMP-14 Assay Kit in Example 5, the reaction rates for both Customized Compound #1 and Customized Compound #2 in buffer (2) were found to be improved.

[0157] Example 9 (Detection of MMP-14 activity in extracellular vesicles derived from MDA-MB-231 using reporter molecules: Customized #1 or Customized #2) 4 μL (equivalent to 4 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the following buffer (3) was added to the aliquot to provide a 500 μL solution. The concentration of this solution was 8 μg / mL. Buffer (3) 50 mM Tris-Cl (pH: 7.6) 10mM CaCl2 0.005% Brij35 In addition, to detect MMP14 activity, 1 mM custom product #1 or custom product #2 was diluted with buffer (3) to prepare a 20 μM MMP14 detection solution. The concentration of the solution was set to x1, and 2-fold serial dilutions were performed to prepare MMP14 detection solutions having five concentrations. 50 μL of each MMP14 detection solution was added to 50 μL of the sample, and the reaction was carried out at 37° C., and the MMP14 activity was evaluated. In this case, the extracellular vesicle concentration in each reaction solution was 4 μg / mL, and the reporter molecule concentration in each reaction solution was 10 μM, 5 μM, 2.5 μM, 1.25 μM, or 0.625 μM.

[0158] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value at 0 minute from the measured value of each sample. Figure 17A The time variation of fluorescence intensity due to the difference in the concentration of Customized Substance #1 is shown. Figure 17A As shown, the fluorescence intensity was found to vary depending on the concentration of Customized Substance # 1. Furthermore, the fluorescence intensity was found to vary significantly below 2.5 μM. Figure 17B The time variation of fluorescence intensity due to the difference in the concentration of Customized Substance #2 is shown. Figure 17B As shown, the fluorescence intensity was found to vary depending on the concentration of Customized Substance #2. Furthermore, the fluorescence intensity was found to vary significantly below 5 μM. The increase in fluorescence intensity over time was approximately linear within 1 hour of the reaction, and Figure 17CThe Linewaver-Burk plot of the slope (reaction rate) with respect to the increase in each reporter molecule concentration is shown in . In addition, the values ​​of Vmax, Km, and Vmax / Km calculated from the Linewaver-Burk plot are shown below.

[0159] [Table 1] Table 1 reporter molecules Custom Item #1 Custom Item #2 Vmax(ΔRFU / min) 1278.77 582.411 Km (μM) 0.44306 0.51116 <![CDATA[Vmax / Km(ΔRFU / min · μM)]]> 2886.23 1139.38 It was found that under the above reaction conditions, Customized Compound #1 was about 2.5 times easier to cut than Customized Compound #2.

[0160] Example 10 (Detection of MMP-14 activity in extracellular vesicles derived from MDA-MB-231, A549, or COLO201 using reporter molecules: Customized #1 or Customized #2) For each of the MDA-MB-231-derived extracellular vesicles (50 μg of protein in 50 μL of PBS), A549-derived extracellular vesicles (50 μg of protein in 50 μL of PBS), and COLO201-derived extracellular vesicles (10 μg of protein / mL) described in the "Materials, etc." section, 2 μL (corresponding to 2 μg for MDA-MB-231- and A549-derived extracellular vesicles and 0.4 μg for COLO201-derived extracellular vesicles) was aliquoted, and buffer (3) was added to the aliquot to provide 200 μL of each solution. The concentration of the extracellular vesicles in the solution was as follows: 10 μg / mL for each of the MDA-MB-231- and A549-derived extracellular vesicles and 2 μg / mL for the COLO201-derived extracellular vesicles. In Example 8, the concentration of 20 μg / mL was set to x1. Therefore, for each of the extracellular vesicles derived from MDA-MB-231 and A549, the concentration of the solution was set to x1 / 2, and three-fold serial dilutions were performed to prepare standard samples having three concentrations. For the extracellular vesicles derived from COLO201, the concentration of the solution was set to x1 / 10, and three-fold serial dilutions were performed to prepare standard samples having three concentrations. In addition, to detect MMP14 activity, 1 mM of Customized Compound #1 or Customized Compound #2 was diluted with buffer (3) to prepare a 10 μM MMP14 detection solution. 50 μL of the MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 37° C. to evaluate the MMP14 activity. In this case, the maximum concentration of extracellular vesicles in the reaction solution was 5 μg / mL for MDA-MB-231- and A549-derived extracellular vesicles and 1 μg / mL for COLO201-derived extracellular vesicles, and the concentration of each reporter molecule in the reaction solution was 5 μM.

[0161] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 18A The temporal changes in fluorescence intensity of various reporter molecules are shown based on the differences in extracellular vesicle concentrations derived from MDA-MB-231. Figure 18A As shown, the fluorescence intensity was found to vary with the extracellular vesicle concentration. Furthermore, at the same extracellular vesicle concentration, the fluorescence intensity from Customized Product #1 was found to be higher than that from Customized Product #2. This result satisfactorily replicated the results of Example 8.

[0162] Figure 18B The temporal changes in fluorescence intensity of various reporter molecules are shown by the difference in A549-derived extracellular vesicle concentrations. Figure 18B As shown, it was found that the fluorescence intensity varied depending on the extracellular vesicle concentration. It was found that at the same extracellular vesicle concentration, the fluorescence intensity from Customized Product #2 was higher than that from Customized Product #1. In this case, in the case of Customized Product #2, a fluorescence intensity change almost equivalent to that in the extracellular vesicles derived from MDA-MB-231 was obtained.

[0163] Figure 18C The temporal changes in fluorescence intensity of various reporter molecules are shown as a function of the concentration of COLO201-derived extracellular vesicles. Figure 18C As shown, no change in fluorescence intensity was observed. No literature related to MMP14 was found in the PubMed search for COLO201 (only one literature related to MMP-7 was found by searching for COLO201×MMP), so the results are considered reasonable.

[0164] Example 11 (AChE activity was detected using the Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence)) First, prepare the following reagents. Thiolite Green stock solution (200x) 50 μL of DMSO (Component E) was added to Thiolite Green (Component A) to provide a 200-fold stock solution. Acetylcholine stock solution (500x) 0.6 mL of purified water was added to acetylcholine (Component C) to provide a 500x stock solution. Acetylcholinesterase standard stock solution (50U / mL) 100 μL of purified water containing 0.1% BSA was added to the acetylcholinesterase standard (Component D) to provide a 50 U / mL acetylcholinesterase standard solution. 980 μL of Assay Buffer (Component B) was added to 20 μL of the above-mentioned AChE standard solution to prepare a 1000 mU / mL AChE solution. Furthermore, a 10-fold dilution was performed with Assay Buffer (Component B) to provide a 100 mU / mL AChE solution. The concentration of the AChE solution was set to x1, and a 3-fold serial dilution was performed to prepare standard samples having seven concentrations. Furthermore, a sample without AChE was similarly prepared and used as a BG measurement sample. Furthermore, to detect AChE activity, 1 μL of acetylcholine stock solution (500-fold) and 2.5 μL of Thiolite Green stock solution (200-fold) were added to 500 μL of Assay Buffer (Component B) to provide 503.5 μL of AChE detection solution. 50 μL of the AChE test solution was added to 50 μL of each sample, and the reaction was carried out at 30° C. to evaluate the AChE activity. In this case, the maximum AChE concentration in the reaction solution was 50 mU / mL.

[0165] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 19 The time variation of AChE concentration and fluorescence intensity from the reporter molecule is shown. Figure 19 As shown, it was found that the fluorescence intensity varied with the AChE concentration. The concentrations of x1 / 9, x1 / 27, x1 / 81, and x1 / 243 at which the activity could be satisfactorily detected corresponded to 5.56 mU / mL, 1.85 mU / mL, 0.62 mU / mL, and 0.21 mU / mL, respectively.

[0166] Example 12 (AChE activity of extracellular vesicles derived from MDA-MB-231 or A549 was detected using the Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence)) For each of the extracellular vesicles derived from MDA-MB-231 (50 μg protein in 50 μL of PBS) and the extracellular vesicles derived from A549 (50 μg protein in 50 μL of PBS) described in the "Materials, etc." section, 4 μL (equivalent to 4 μg for each of the extracellular vesicles derived from MDA-MB-231 and A549) was taken, and the Assay Buffer (Component B) used in Example 11 was added to the aliquot to provide 200 μL of each solution. The concentration of each solution of the extracellular vesicles derived from MDA-MB-231 and A549 was 20 μg / mL. In Example 8, the concentration of 20 μg / mL was set to x1, so for each of the extracellular vesicles derived from MDA-MB-231 and A549, the concentration of the solution was set to x1, and a 2-fold serial dilution was performed to prepare a standard sample with seven concentrations. In addition, a sample not containing extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect AChE activity, 2 μL of acetylcholine stock solution (500-fold) and 5 μL of Thiolite Green stock solution (200-fold) were added to 1000 μL of Assay Buffer (Component B) to provide 1007 μL of AChE detection solution. 50 μL of AChE test solution was added to 50 μL of each sample, and the reaction was performed at 30°C to evaluate AChE activity. In this case, the maximum concentration of extracellular vesicles in the reaction solution was 10 μg / mL for each of MDA-MB-231- and A549-derived extracellular vesicles.

[0167] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 20A The time changes of the concentration of extracellular vesicles derived from MDA-MB-231 and the fluorescence intensity from the reporter molecule are shown. Figure 20A As shown, the fluorescence intensity was found to vary with the concentration of extracellular vesicles. Figure 20B The time changes of A549-derived extracellular vesicle concentration and the fluorescence intensity from the reporter molecule are shown. Figure 20BAs shown, the fluorescence intensity was found to vary with the concentration of extracellular vesicles. Comparison of extracellular vesicles derived from MDA-MB-231 and A549 revealed that extracellular vesicles derived from A549 had stronger AChE activity.

[0168] Example 13 (AChE activity of MDA-MB-231-derived extracellular vesicles was detected using the Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence)) A 1 μL aliquot (equivalent to 1 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg protein in 50 μL of PBS) was taken, and the Assay Buffer (Component B) used in Example 11 was added to the aliquot to provide a 250 μL solution. The concentration of this solution was 4 μg / mL. Since the concentration of 20 μg / mL in Example 8 was set to x1, the concentration of this solution was x1 / 5. In addition, to detect AChE activity, 1 μL of acetylcholine stock solution (500 times) and 2.5 μL of Thiolite Green stock solution (200 times) were added to 250 μL of Assay Buffer (Component B) to provide 253.5 μL of AChE detection solution. The concentration of this solution was twice the concentration specified in the test kit. The concentration of this solution was set to x2, and a 2-fold serial dilution was performed to provide an AChE detection solution with four concentrations. 50 μL of each AChE test solution was added to 50 μL of the sample, and the reaction was carried out at 30° C. to evaluate the AChE activity. In this case, the concentration of extracellular vesicles in the reaction solution was 2 μg / mL.

[0169] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 21 The time variation of fluorescence intensity due to the difference in reporter molecule concentration is shown. Figure 21 As shown, the fluorescence intensity was found to vary with the concentration of the reporter molecule. The change in fluorescence intensity can be controlled by changing the concentration of the reporter molecule.

[0170] Example 14 (AChE activity was detected using the SensoLyte 520 Acetylcholinesterase Activity Assay Kit (Fluorimetric)) In the same manner as in Example 11, 980 μL of Assay Buffer (Component B) was added to 20 μL of AChE standard solution to prepare a 1000 mU / mL AChE solution. Furthermore, a 10-fold dilution was performed with Assay Buffer (Component B) to provide a 100 mU / mL AChE solution. The concentration of the AChE solution was set to x1, and a 3-fold serial dilution was performed to prepare standard samples having seven concentrations. Furthermore, a sample without AChE was similarly prepared and used as a sample for BG measurement. The solution in the SensoLyte 520 Acetylcholinesterase Activity Assay Kit was used for the following AChE assay solution. To assay AChE activity, 5 μL of acetylcholine solution (Component C) and 5 μL of AChE substrate solution (Component A) were added to 490 μL of Assay Buffer (Component D) to provide 500 μL of AChE assay solution. 50 μL of the AChE test solution was added to 50 μL of each sample, and the reaction was carried out at 30° C. to evaluate the AChE activity. In this case, the maximum concentration of AChE in the reaction solution was 50 mU / mL.

[0171] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 22 The time variation of AChE concentration and fluorescence intensity from the reporter molecule is shown. Figure 22 As shown, fluorescence intensity was found to vary with AChE concentration. Satisfactory detection was possible at concentrations corresponding to x1, x1 / 3, x1 / 9, and x1 / 27 of 50 mU / mL, 16.7 mU / mL, 5.56 mU / mL, and 1.85 mU / mL, respectively. Furthermore, the fluorescence intensity variation of the reporter molecule from the Amplite Fluorimetric Acetylcholinesterase Assay Kit was found to be greater than that from the SensoLyte 520 Acetylcholinesterase Activity Assay Kit.

[0172] Example 15 (AChE activity in extracellular vesicles derived from MDA-MB-231 or A549 was detected using the SensoLyte 520 Acetylcholinesterase Activity Assay Kit (Fluorimetric)) For each of the extracellular vesicles derived from MDA-MB-231 (50 μg protein in 50 μL of PBS) and the extracellular vesicles derived from A549 (50 μg protein in 50 μL of PBS) described in the "Materials, etc." section, 4 μL (equivalent to 4 μg for each of the extracellular vesicles derived from MDA-MB-231 and A549) was taken, and the AssayBuffer (Component D) included in the kit was added to the sample aliquots to provide 200 μL of each solution. The concentration of each solution of the extracellular vesicles derived from MDA-MB-231 and A549 was 20 μg / mL. In Example 8, the concentration of 20 μg / mL was set to x1, so the concentration of the solution was set to x1, and a 2-fold serial dilution was performed to prepare a standard sample with seven concentrations of each of the extracellular vesicles derived from MDA-MB-231 and A549. In addition, a sample not containing extracellular vesicles was similarly prepared and used as a sample for BG measurement. To detect AChE activity, 5 μL of acetylcholine solution (Component C) and 5 μL of AChE substrate solution (Component A) were added to 490 μL of Assay Buffer (Component D) to provide 500 μL of AChE detection solution. 50 μL of AChE test solution was added to 50 μL of each sample, and the reaction was performed at 30°C to evaluate AChE activity. In this case, the maximum concentration of extracellular vesicles in the reaction solution was 10 μg / mL for each of MDA-MB-231- and A549-derived extracellular vesicles.

[0173] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 23A The time changes of the concentration of extracellular vesicles derived from MDA-MB-231 and the fluorescence intensity from the reporter molecule are shown. Figure 23A As shown, the fluorescence intensity was found to vary with the concentration of extracellular vesicles. Figure 23B The time changes of A549-derived extracellular vesicle concentration and the fluorescence intensity from the reporter molecule are shown. Figure 23B As shown, it was found that the fluorescence intensity varied with the concentration of extracellular vesicles. From the comparison between extracellular vesicles derived from MDA-MB-231 and A549, it was found that the AChE activity of extracellular vesicles derived from A549 was strong. This is the same as the result of Example 12.

[0174] Example 16 (AChE activity in MDA-MB-231-derived extracellular vesicles was detected using the SensoLyte 520 Acetylcholinesterase Activity Assay Kit (Fluorimetric)) A 1 μL aliquot (equivalent to 1 μg) of the MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) from Example 1 was taken, and the Assay Buffer (Component D) used in Example 14 was added to the aliquot to provide a 250 μL solution. The concentration of this solution was 4 μg / mL. Since the concentration of 20 μg / mL in Example 8 was set to x1, the concentration of this solution was x1 / 5. In addition, in order to detect AChE activity, 2 μL acetylcholine solution (Component C) and the AChE substrate solution (Component A) of 2 μL are added in the Assay Buffer (Component D) of 96 μL to provide the AChE detection solution of 100 μL. The concentration of this solution is twice the concentration specified in the test kit. Similarly, 4 μL acetylcholine solution (Component C) and the AChE substrate solution (Component A) of 4 μL are added in the Assay Buffer (Component D) of 92 μL to provide the AChE detection solution (4 times of concentration) of 100 μL, and 6 μL acetylcholine solution (Component C) and the AChE substrate solution (Component A) of 6 μL are added in the Assay Buffer (Component D) of 88 μL to provide the AChE detection solution (6 times of concentration) of 100 μL. Therefore, three kinds of AChE detection solutions with three kinds of concentration were prepared. 50 μL of each AChE test solution was added to 50 μL of the sample, and the reaction was carried out at 30° C. to evaluate the AChE activity. In this case, the concentration of extracellular vesicles in the reaction solution was 2 μg / mL.

[0175] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 24 The time variation of fluorescence intensity due to the difference in reporter molecule concentration is shown. Figure 24 As shown in FIG, the fluorescence intensity was found to vary with the concentration of the reporter molecule. The variation in fluorescence intensity can be controlled by varying the concentration of the reporter molecule.

[0176] Example 17 (MMP14 activity or AChE activity in MDA-MB-231-derived extracellular vesicles was detected using Customized Product #1, Customized Product #2, Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence), or SensoLyte 520 Acetylcholinesterase Activity Assay Kit (Fluorimetric)) An 8 μL aliquot (equivalent to 8 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg protein in 50 μL PBS) was taken, and purified water was added to the aliquot to provide an 80 μL solution. The concentration of this solution was 100 μg / mL. In Example 8, the concentration of 20 μg / mL was set as x1, so the concentration of this solution was x5. Prepare the following three buffers for the respective activity assays. MMP14 buffer 50 mM Tris-Cl (pH: 7.6) 10mM CaCl2 0.005% Brij35 ·Amplite Fluorimetric Acetylcholinesterase Assay Kit(GreenFluorescence) Assay Buffer (Component B) Hereinafter referred to as "ALG buffer" ·SensoLyte 520Acetylcholinesterase Activity Assay Kit(Fluorimetric) Assay Buffer (Component D) Hereinafter referred to as "SL520 buffer"

[0177] For the detection of MMP14 activity, 1 μL of 1 mM custom product #1 was added to 100 μL of each of MMP14 buffer, ALG buffer, and SL520 buffer, thereby obtaining MMP14 detection solutions (1) each having a final concentration of 10 μM. For the detection of MMP14 activity, 1 μL of 1 mM custom product #2 was added to 100 μL of each of MMP14 buffer, ALG buffer, and SL520 buffer, thereby obtaining MMP14 detection solutions (2) each having a final concentration of 10 μM. For the detection of AChE activity (Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence)), 1 μL of acetylcholine stock solution (500-fold) and 2.5 μL of Thiolite Green stock solution (200-fold) were added to 500 μL of each of MMP14 buffer, ALG buffer, and SL520 buffer. Thus, an AChE detection solution (1) was obtained. For the detection of AChE activity (SensoLyte 520 Acetylcholinesterase Activity Assay Kit (Fluorimetric)), 1 μL of acetylcholine solution (component C) and 1 μL of AChE substrate solution (component A) were added to 100 μL of each of MMP14 buffer, ALG buffer, and SL520 buffer. Thus, an AChE detection solution (2) was obtained. 4 μL of the above sample was added to 100 μL of each of the above test solutions, reacted at 30°C, and evaluated for MMP14 and AChE activity. In this case, the extracellular vesicle concentration in each reaction solution was 4 μg / mL, corresponding to a final concentration x2 / 5 of that in Example 8. Furthermore, the same volume of sample was added as in the standard specification for each test solution. In this example, the volume of sample added was almost negligible, resulting in a reporter molecule concentration in each test solution approximately double that of the standard specification.

[0178] (Evaluation of MMP14 or AChE activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 25A The figure shows the temporal change of fluorescence intensity due to the difference in the measurement buffer when the MMP14 detection solution (1) was used. Figure 25A As shown, it was found that the fluorescence intensity varied depending on the difference in the buffer to be used. Although activity was confirmed in all cases, the MMP14 buffer, SL520 buffer, and ALG buffer were ranked in descending order of fluorescence intensity change. Figure 25B The figure shows the temporal change of fluorescence intensity due to the difference in the measurement buffer when the MMP14 detection solution (2) is used. Figure 25B As shown, it was found that the fluorescence intensity varied depending on the difference in the buffer to be used. Although activity was confirmed in all cases, MMP14 buffer, SL520 buffer, and ALG buffer were ranked in descending order of fluorescence intensity change, which was the same order as in the case of MMP14 detection solution (1). Figure 25C The figure shows the temporal change of fluorescence intensity due to the difference in the measurement buffer when the AChE detection solution (1) is used. Figure 25C As shown, it was found that the fluorescence intensity varied depending on the difference in the buffer to be used. Although activity was recognized in all cases, the SL520 buffer, MMP14 buffer, and ALG buffer were ranked in descending order of fluorescence intensity change. Figure 25D The figure shows the temporal change of fluorescence intensity due to the difference in the measurement buffer when the AChE detection solution (2) is used. Figure 25D As shown, it was found that the fluorescence intensity varied depending on the difference in the buffer to be used. Although activity was confirmed in all cases, the MMP14 buffer, SL520 buffer, and ALG buffer were ranked in descending order of fluorescence intensity change, which was the same order as in the case of MMP14 detection solutions (1) and (2). Therefore, when MMP14 activity and AChE activity are detected simultaneously, any buffer can be used. MMP14 buffers and SL520 buffers whose respective activities are widely improved by using various reporter molecules are preferred, and MMP14 buffers with the highest activity improvement are more preferred.

[0179] Example 18 (Hole Manufacturing) Figure 5A and Figure 5B The hole 204 shown is produced by a CYTOP application step, a photolithography step, and an etching / resist removal step. In the CYTOP applying step, a quartz substrate (synthetic quartz substrate, AQ grade, thickness: 1 mm, manufactured by AGC Inc.) was used as the lower substrate 201 and treated with a silane coupling agent (KBE-903, manufactured by Shin-Etsu Silicone), and then CYTOP (CTL-809A, manufactured by AGC Inc.) was applied. In the photolithography step, a positive photoresist (AZ P4903, AZ Electronic Materials) is applied. Next, a development process with an alkali is performed by UV exposure from above through a photomask having the desired pattern. Only in the portion irradiated with UV light by the development process, the photoresist is dissolved to expose the hydrophobic resin layer. In the etching / resist removal step, a portion of the resin layer is removed by etching using oxygen plasma via the dissolved photoresist portion to form a hydrophobic partition wall. Finally, the photoresist was dissolved with an organic solvent to form the desired holes 204. The holes 204 each had a diameter of 5 μm, a depth of 4 μm, and a volume of 78.5 fL, and had a pitch of 10 μm, and the number of holes was about 1,000,000.

[0180] (Fabrication of Hole Arrays) Figure 5A and Figure 5B The well array 200 shown is manufactured to include a lower substrate 201 having the wells 204 formed thereon, an upper substrate 202, an inlet portion (not shown), and an outlet portion (not shown). Polycarbonate (thickness: 1 mm) was used as the upper substrate 202, and the openings of the wells 204 and the top surfaces of the partition walls 203 were opposed to each other across a space 205. The distance from the top surface of each partition wall 203 to the upper substrate 202 in the space 205 was 250 μm.

[0181] (Preparation of Reaction Solution for Wells) A reaction solution for filling well 204 was prepared. 1 μL (equivalent to 1 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the following buffer (4) was added to the aliquot to provide a 150 μL solution. The concentration of this solution was 6.7 μg / mL. In Example 8, the concentration of 20 μg / mL was set to x1, so the concentration of this solution was x1 / 3. Buffer (4) 50 mM Tris-Cl (pH: 7.6) 10mM CaCl2 0.01% Brij35

[0182] Furthermore, in order to detect MMP14 activity, 1 mM custom compound #2 was diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM custom compound #2. 100 μL of MMP14 detection solution was added to 100 μL of sample to prepare a reaction solution. In this case, the concentration of extracellular vesicles in the reaction solution was 3.3 μg / mL, and the concentration of the reporter molecule in the reaction solution was 5 μM. 100 μL of the 200 μL reaction solution prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0183] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. Figure 26A The time-dependent changes in fluorescence intensity from the reporter molecule via MDA-MB-231-derived extracellular vesicles are shown. Figure 26A As shown, it was found that the fluorescence intensity increased with the reaction time, and the MMP14 activity could be detected.

[0184] (Fill the wells with the reaction solution) The reaction solution is injected from the injection port portion on the upper substrate 202, and the reaction solution is fed so that the hole 204 is covered. Next, the hole array 200 is allowed to stand under reduced pressure to degas the space 205 so as to fill the hole with the reaction solution. Thereafter, a hydrophobic solvent to be sealed is supplied to the space 205. Fluorinated oils AsahiKlin AE-3000 (manufactured by AGC Inc.) and Fomblin Y-25 (manufactured by Solvay SA) are used as hydrophobic solvents. Fluorinated oil Simoa (trademark) SR-X Sealing Oil: 102767 (manufactured by Quanterix Corporation) is further used as another hydrophobic solvent.

[0185] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed with a fluorescence microscope (BZ-X800 (manufactured by Keyence Corporation)). Fluorescence from each fluorescent substance was observed under the following conditions to acquire a fluorescent image. Custom Item #2 GFP: OP-87763 (manufactured by Keyence Corporation) Model: OP-87763 Excitation wavelength: 470 / 40nm Absorption wavelength: 525 / 50nm Dichroic wavelength: 495nm

[0186] 26B and Figure 26C Fluorescence microscopy images of the pores are shown. Figure 26B : is a fluorescence microscope image of a well sealed with AsahiKlin AE-3000 (manufactured by AGC Inc.) and Fomblin Y-25 (manufactured by Solvay SA) acting as a hydrophobic solvent after 3 hours of reaction. This image contains the fluorescence of 5-FAM derived from Customized Product #2 after release quenching and the background fluorescence of 5-FAM derived from Customized Product #2 under quenching. In addition, Figure 26CThis is a fluorescence microscope image of a well sealed with Simoa SR-X Sealing Oil (manufactured by Quanterix Corporation) acting as a hydrophobic solvent after 3 hours of reaction. Similar to the above, this image includes fluorescence from 5-FAM derived from Custom #2 after release quenching and background fluorescence from 5-FAM derived from Custom #2 under quenching. like Figure 26B and Figure 26C As shown, wells where fluorescence from 5-FAM derived from Customized Compound #2 after quenching and release was observed, and wells where only background fluorescence from 5-FAM derived from Customized Compound #2 under quenching was observed, can be identified. Therefore, it can be recognized that the wells are filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of Customized Compound #2 by MMP14 in MDA-MB-231-derived extracellular vesicles is taking place in the microcompartment.

[0187] Next, the fluorescence microscope image of the above embodiment after 3 hours of reaction was subjected to predetermined image processing to provide Figure 26D and Figure 26E In this case, Figure 26D is a histogram obtained by analyzing fluorescence microscope images of pores sealed with AsahiKlin AE-3000 (manufactured by AGC Inc.) and Fomblin Y-25 (manufactured by Solvay SA) serving as a hydrophobic solvent, and Figure 26E Figure 1 is a histogram obtained by analyzing fluorescence microscopy images of wells sealed with Simoa SR-X Sealing Oil. Figure 26D and Figure 26E The histograms shown in each show the number of wells, and each show the fluorescence intensity included in each portion of the fluorescence intensity obtained by dividing the fluorescence intensity into intervals of a constant value.

[0188] like Figure 26D and Figure 26EAs shown, each histogram is divided into three regions A, B, and C according to fluorescence intensity. Among them, region A with the lowest fluorescence intensity is attributed to the background fluorescence of the reporter molecule. In addition, the wells falling into the region B category are identified as positive wells B, and the wells falling into the region C category are identified as positive wells C. When the fluorescence intensities of positive wells B and positive wells C are compared with each other, the fluorescence intensity of positive wells C is stronger, so it is inferred that most positive wells B are filled with one extracellular vesicle per well, while most positive wells C are filled with multiple extracellular vesicles per well. In the present invention, it is conceived to detect the activity of "multiple" MMP14 molecules in "one" extracellular vesicle, and it is speculated that in addition to fluctuations in reaction efficiency, due to fluctuations in, for example, the size of the extracellular vesicles and the molecular density of MMP14 in each extracellular vesicle, no well-defined integer multiple peaks are observed.

[0189] Furthermore, when the concentration of extracellular vesicles having MMP14 activity is calculated from the total number of wells and the number of positive wells identified from each histogram by using the above-mentioned formula (1), the concentration can be estimated as follows.

[0190] [Table 2] Table 2

[0191] In a system using AE-3000 and Y25 as oil, λ is 0.235 and the volume of the microcompartment is 78.5 fL, so the extracellular vesicle concentration can be calculated to be 3.0 × 10 9 / mL. The concentration of the reaction solution was 3.3 μg protein / mL, so the extracellular vesicle concentration was 9.1×10 8 Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg of protein in 50 μL of PBS) contains 9.1×10 8 extracellular vesicles, in which MMP14 activity can be detected using custom product #2. Similarly, in the system using Simoa as the oil, λ is 0.188 and the volume of the microcompartment is 78.5 fL, so the extracellular vesicle concentration can be calculated to be 2.4 × 10 9 / mL. The concentration of the reaction solution was 3.3 μg protein / mL, so the extracellular vesicle concentration was 7.3×10 8 Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg of protein in 50 μL of PBS) contains 7.3×10 8 extracellular vesicles, in which MMP14 activity can be detected using custom product #2. Therefore, it was demonstrated that digital detection of extracellular vesicles is possible by utilizing MMP14 activity.

[0192] Example 19 (holes and hole patterns) Simoa Discs (16 discs): 100001 (manufactured by Quanterix Corporation) were used as the well array. The specifications are as follows. Pore ​​diameter: 4.25 μm Hole depth: 3.25 μm Pore ​​capacity: 50fL Number of wells in array: 239,000 Array range: 3mm×4mm

[0193] (Preparation of Reaction Solution for Wells) A reaction solution for filling the well 204 was prepared. 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the buffer (4) used in Example 18 was added to the aliquoted sample to provide a 200 μL solution. The concentration of the solution was 20 μg / mL. In Example 8, the concentration of 20 μg / mL was set to x1, so the concentration of the solution was also x1. The concentration of the solution was set to x1, and a 10-fold serial dilution was performed to prepare a standard sample with five concentrations. In addition, a sample without extracellular vesicles was similarly prepared and used as a sample for BG measurement.

[0194] Furthermore, to detect MMP14 activity, 1 mM custom product #2 and 1 mM Alexa Fluor 647 (manufactured by Molecular Probes), which served as a standard fluorescent substance, were diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM custom product #2 and 1 μM Alexa Fluor 647. 100 μL of MMP14 detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in the reaction solution was 10 μg / mL, and the concentrations of the reporter molecule and standard fluorescent substance in each reaction solution were 5 μM and 0.5 μM, respectively. 100 μL of each of the 200 μL reaction solutions prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0195] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 27The temporal changes in fluorescence intensity from Custom #2 by extracellular vesicles derived from MDA-MB-231 are shown. Figure 27 As shown, it was found that the fluorescence intensity increased with the reaction time, and MMP14 activity was detectable at concentrations of x1 and x1 / 10, but not at concentrations of x1 / 100 or lower.

[0196] (Fill the wells with the reaction solution) The reaction solution was injected from the injection port on upper substrate 202, and the solution was fed so that the wells 204 were covered. Next, the well array 200 was allowed to stand under reduced pressure to degas spaces 205, thereby filling the wells with the reaction solution. Thereafter, a hydrophobic solvent to be sealed was supplied to spaces 205. Fluorinated oil SR-X sealing oil was used as the hydrophobic solvent.

[0197] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the following conditions to acquire a fluorescent image. Reporter molecule (custom compound #2) GFP: OP-87763 (manufactured by Keyence Corporation) Model: OP-87763 Excitation wavelength: 470 / 40nm Absorption wavelength: 525 / 50nm Dichroic wavelength: 495nm

[0198] In this example, Customized #2 was used as a reporter molecule. In subsequent examples, when the same conditions as those in Example 19 are described, reporter molecules such as Customized #1, SenosoLyte, and Amplite were measured under the same conditions as described above, unless otherwise specified. Alexa Fluor 647 Cy5: OP-87766 (manufactured by Keyence Corporation) Model: OP-87766 Excitation wavelength: 620 / 60nm Absorption wavelength: 700 / 75nm Dichroic wavelength: 660nm

[0199] Figures 28A to 28F as well as Figures 28G to 28L Fluorescence microscopy images of the wells are shown. Figures 28A to 28FThe fluorescence microscope image containing the fluorescence of 5-FAM derived from Customized Product #2 and the background fluorescence of Customized Product #2 after 3 hours of reaction is shown. Figures 28G to 28L Each shows a fluorescence microscope image of fluorescence of a standard fluorescent substance after 3 hours of reaction.

[0200] Similarly, Figures 29A to 29F and Figures 29G to 29L Fluorescence microscopy images of the wells are shown. Figures 29A to 29F Each shows a fluorescence microscope image containing both the fluorescence of 5-FAM derived from Customized Product #2 and the background fluorescence of Customized Product #2 after 7 hours of reaction. Figures 29G to 29L Each shows a fluorescence microscope image containing fluorescence of a standard fluorescent substance after 7 hours of reaction.

[0201] like Figures 28G to 28L and Figures 29G to 29L As shown in FIG, the wells can be identified based on the fluorescence of the standard fluorescent substance. Figures 28A to 28E as well as Figures 29A to 29E As shown, fluorescence of the standard fluorescent substance was also observed in the wells where 5-FAM fluorescence was observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of customized substance #2 by MMP14 in MDA-MB-231-derived extracellular vesicles was effective in the microchamber.

[0202] Next, the fluorescence microscope image of the above embodiment after 3 hours of reaction was subjected to predetermined image processing to provide Figures 30A to 30E Similarly, the histograms shown in FIG. Figures 31A to 31E These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value.

[0203] The total number of wells identified from each histogram after 3 and 7 hours of reaction and the expected value (λ) of the number of positive wells relative to the concentration of the extracellular vesicles tested calculated using the above formula (1) are shown. In this case, in the sample without extracellular vesicles, no positive wells were observed at any reaction time. Figure 32 The coordinate diagram in the low positive ratio region is shown, where the separation between positive and negative is more satisfactory. The power approximation can be performed satisfactorily. The extracellular vesicle concentration can be accurately quantified. In this case, assuming that there are no artifacts in the extracellular vesicle concentration, such as aggregation of extracellular vesicles in the higher concentration region, it is believed that the approximation with an exponent close to 1 after a reaction time of 7 hours is more accurate.

[0204] As described in this embodiment Figure 27 As shown, only MMP14 activity at extracellular vesicle concentrations above x1 / 10 (corresponding to 1 μg / mL) could be detected using a 96-well plate, but MMP14 activity at extracellular vesicle concentrations above x1 / 10,000 (corresponding to 1 ng / mL) could be detected in a microchamber. Thus, the use of microchambers was found to increase detection sensitivity by approximately three orders of magnitude.

[0205] [Table 3] Table 3

[0206] From the approximation after 3 h of reaction time, λ at x1 is 0.6727, and the volume of the microcompartment is 50 fL, the extracellular vesicle concentration can be calculated to be 1.3 × 10 10 / mL. The concentration of the reaction solution is 10 μg protein / mL, so the extracellular vesicle concentration is 1.3×10 9 Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 1.3×10 9 extracellular vesicles, in which MMP14 activity can be detected using custom product #2. Similarly, from the approximation after 7 h of reaction time, λ at x1 is 0.8756, and the extracellular vesicle concentration can be calculated to be 1.8 × 10 10 Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 1.8×10 9 extracellular vesicles, in which MMP14 activity can be detected using custom product #2. Thus, it was demonstrated that digital detection of extracellular vesicles is possible by exploiting MMP14 activity even in different well arrays.

[0207] Example 20 (holes and hole patterns) A well array of Simoa Discs (manufactured by Quanterix Corporation) was used. (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the hole 204. Take 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add buffer (4) to the sample to provide a 200 μL solution. The concentration of the solution is 20 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is also x1. The concentration of the solution is set to x1, and a 10-fold serial dilution is performed to prepare a standard sample with five concentrations. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement.

[0208] In addition, to detect MMP14 activity, 1 mM custom product #1 and 1 mM Alexa Fluor 647 (which served as a standard fluorescent substance) were diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM custom product #1 and 5 μM Alexa Fluor 647. 100 μL of MMP14 detection solution was added to 100 μL of each sample to prepare reaction solutions for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 10 μg / mL, and the concentrations of the reporter molecule and standard fluorescent substance in each reaction solution were 5 μM and 2.5 μM, respectively. 100 μL of each of the 200 μL reaction solutions prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0209] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 33 The temporal changes in fluorescence intensity from Custom #1 by extracellular vesicles derived from MDA-MB-231 are shown. Figure 33 As shown, it was found that the fluorescence intensity increased with the reaction time, and MMP14 activity was detectable at concentrations of x1 and x1 / 10, but not at concentrations of x1 / 100 or lower.

[0210] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0211] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Figures 34A to 34F and Figures 34G to 34L Fluorescence microscopy images of the wells are shown. Figures 34A to 34F The fluorescence microscope images each contain fluorescence of 5-FAM derived from Customized Product #1 and background fluorescence of Customized Product #1 after 5 hours of reaction. Figures 34G to 34L Fluorescence microscope images each containing fluorescence of a standard fluorescent substance after 5 hours of reaction are shown. like Figures 34G to 34L As shown in FIG, the wells can be identified based on the fluorescence of the standard fluorescent substance. Figures 34A to 34E As shown in FIG, it was found that in the wells where 5-FAM fluorescence was observed, fluorescence of the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of the customized substance #1 by MMP14 in the extracellular vesicles derived from MDA-MB-231 was effective in the microcompartment. In this case, Figure 34F Fluorescence of 5-FAM was also observed in the absence of extracellular vesicles, thus revealing the occurrence of false positive results, however, the frequency was extremely low.

[0212] Next, the fluorescence microscope image of the above embodiment after 5 hours of reaction was subjected to predetermined image processing to provide Figures 35A to 35F These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 5 hours of reaction are shown, along with the expected value (λ) calculated using the above formula (1) relative to the concentration of the extracellular vesicles tested. In this case, the number of false-positive wells almost equal to x1 / 10,000 was also observed in the sample not containing extracellular vesicles, and thus it was found that detection at x1 / 10,000 was difficult. The expected value (λ) obtained by subtracting the number of positive wells when the extracellular vesicle concentration was zero is also shown. In addition, Figure 36 A graph showing the expected value λ versus the extracellular vesicle concentration is shown. The power approximation can be performed satisfactorily. The extracellular vesicle concentration can be accurately quantified. In this case, assuming that there are no disturbances in the extracellular vesicle concentration, such as aggregation of extracellular vesicles in higher concentration regions, an approximation in which the exponent of the expected value (λ) obtained by subtracting the number of positive wells when the extracellular vesicle concentration is zero is closer to 1 is considered to be more accurate.

[0213] As described in this embodiment Figure 33As shown, MMP14 activity can only be detected in a 96-well plate at extracellular vesicle concentrations of x1 / 10 (equivalent to 1 μg / mL) or higher, but MMP14 activity can be detected in a microchamber at extracellular vesicle concentrations of at least x1 / 10,000 (equivalent to 10 ng / mL). Thus, the use of microchambers was found to improve detection sensitivity by at least two orders of magnitude. Furthermore, the detection sensitivity of Customized Product #2 in Example 19 in the low-concentration range was found to be approximately one order of magnitude higher than that of Customized Product #1.

[0214] [Table 4] Table 4

[0215] In the approximation without BG correction, λ at x1 is 1.03 and the volume of the microcompartment is 50 fL, so the extracellular vesicle concentration can be calculated to be 2.1 × 10 10 / mL. The concentration of the reaction solution is 10 μg protein / mL, so the extracellular vesicle concentration is 2.1×10 9 / μg protein. Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 2.1×10 9 extracellular vesicles, in which MMP14 activity can be detected using custom product #1. Similarly, in the approximation with BG correction, λ at x1 is 1.07, and the extracellular vesicle concentration can be calculated to be 2.1 × 10 10 / mL, similar to the approximation without correction. Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 2.1×10 9 extracellular vesicles, in which MMP14 activity can be detected using custom product #1. Thus, it was demonstrated that digital detection of extracellular vesicles is possible by utilizing MMP14 activity even in the presence of different reporter molecules.

[0216] Example 21 (holes and hole patterns) Using the hole array of Simoa Discs. (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the hole 204. Take 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from A549 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add buffer (4) to the sample to provide a 200 μL solution. The concentration of the solution is 20 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is also x1. The concentration of the solution is set to x1, and a 10-fold serial dilution is performed to prepare a standard sample with five concentrations. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement. In addition, to detect MMP14 activity, 1 mM custom product #1 and 1 mM Alexa Fluor 647 (which served as a standard fluorescent substance) were diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM custom product #1 and 5 μM Alexa Fluor 647. 100 μL of MMP14 detection solution was added to 100 μL of each sample to prepare reaction solutions for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 10 μg / mL, and the concentrations of the reporter molecule and standard fluorescent substance in each reaction solution were 5 μM and 2.5 μM, respectively. 100 μL of each of the 200 μL reaction solutions prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0217] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 37 The temporal changes in fluorescence intensity from Custom #1 by A549-derived extracellular vesicles are shown. Figure 37 As shown, it was found that the fluorescence intensity increased with the reaction time, and MMP14 activity was detectable at a concentration of x1, but not at a concentration of x1 / 10 or lower.

[0218] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0219] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although illustration is omitted, a fluorescence microscope image containing both fluorescence derived from 5-FAM of Customized Product #1 and background fluorescence of Customized Product #1 after 5 hours of reaction, and a fluorescence microscope image containing fluorescence of a standard fluorescent substance after 5 hours of reaction were acquired. These wells can be identified based on the fluorescence of a standard fluorescent substance. Furthermore, it was found that in wells where 5-FAM fluorescence was observed, fluorescence from the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of customized product #1 by MMP14 in A549-derived extracellular vesicles was effective in the microcompartment. In this case, 5-FAM fluorescence was also observed in the fluorescence microscopy images of wells without extracellular vesicles, indicating that false positive results occurred, although the frequency was extremely low.

[0220] Next, the fluorescence microscope image of the above embodiment after 5 hours of reaction was subjected to predetermined image processing to provide Figures 38A to 38F These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 5 hours of reaction are shown, along with the expected value (λ) calculated using the above formula (1) relative to the concentration of the extracellular vesicles tested. In this case, substantially the same number of false positive wells as at x1 / 10,000 and x1 / 1,000 were observed in the sample not containing extracellular vesicles, and thus it was found that detection at x1 / 10,000 and x1 / 1,000 was difficult. The expected value (λ) obtained by subtracting the number of positive wells when the extracellular vesicle concentration was zero is also shown. In addition, Figure 39 A graph showing the expected value λ versus the extracellular vesicle concentration is shown. The power approximation can be performed satisfactorily. The extracellular vesicle concentration can be accurately quantified. In this case, assuming that there are no disturbances in the extracellular vesicle concentration, such as aggregation of extracellular vesicles in higher concentration regions, an approximation in which the exponent of the expected value (λ) obtained by subtracting the number of positive wells when the extracellular vesicle concentration is zero is closer to 1 is considered to be more accurate.

[0221] As described in this example Figure 37As shown, MMP14 activity can only be detected in a 96-well plate at extracellular vesicle concentrations of x1 (equivalent to 10 μg / mL) or higher, but can be detected in a microchamber at extracellular vesicle concentrations of at least x1 / 100 (equivalent to 100 ng / mL). Therefore, the use of microchambers was found to improve detection sensitivity by at least two orders of magnitude. Furthermore, the detection sensitivity of Customized Product #2 in Example 19 in the low-concentration range was found to be approximately one order of magnitude higher than that of Customized Product #1.

[0222] [Table 5] Table 5

[0223] In the approximation without BG correction, λ at x1 is 0.102 and the volume of the microcompartment is 50 fL, so the extracellular vesicle concentration can be calculated to be 2.0 × 10 9 / mL. The concentration of the reaction solution is 10 μg protein / mL, so the extracellular vesicle concentration is 2.0×10 8 Therefore, it is estimated that 1 μL of A549-derived extracellular vesicles (50 μg of protein in 50 μL of PBS) contains 2.0×10 8 extracellular vesicles, in which MMP14 activity can be detected using custom product #1. Similarly, in the BG-corrected approximation, λ at x1 is 0.120, and the extracellular vesicle concentration can be calculated to be 2.4 × 10 9 / mL, similar to the approximation without correction. Therefore, it is estimated that 1 μL of A549-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 2.4×10 8 extracellular vesicles, in which MMP14 activity can be detected using custom product #1. Therefore, it was demonstrated that digital detection of extracellular vesicles is possible even in cell-derived extracellular vesicles by utilizing MMP14 activity.

[0224] Example 22 (holes and hole patterns) Using the hole array of Simoa Discs. (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the hole 204. Take 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add buffer (2) to the sample to provide a 200 μL solution. The concentration of the solution is 20 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is also x1. The concentration of the solution is set to x1, and a 3-fold serial dilution is performed to prepare a standard sample with three concentrations. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement. In addition, to detect MMP14 activity, 1 mM Customized #1 or Customized #2 and 1 mM Alexa Fluor 647 (which serves as a standard fluorescent substance) were diluted with buffer (2) to prepare an MMP14 detection solution containing 10 μM Customized #1 or Customized #2 and 5 μM Alexa Fluor 647. 100 μL of MMP14 detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 10 μg / mL, and the concentrations of the reporter molecule and standard fluorescent substance in each reaction solution were 5 μM and 2.5 μM, respectively. 100 μL of each 200 μL reaction solution prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0225] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. The reaction conditions in this example are exactly the same as those in Example 8, and the MMP14 activity can be identified in exactly the same manner as in Example 8.

[0226] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0227] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not described, fluorescence microscope images were acquired after 1 hour and 2 hours of reaction, each containing both fluorescence from 5-FAM derived from Customized Substance #1 and background fluorescence from Customized Substance #1. Furthermore, fluorescence microscope images were acquired after 1 hour and 2 hours of reaction, each containing fluorescence from a standard fluorescent substance. Although not illustrated, fluorescence microscope images were similarly acquired after one hour of reaction and two hours of reaction, each containing both fluorescence derived from 5-FAM of Customized Substance #2 and background fluorescence of Customized Substance #2. Furthermore, fluorescence microscope images were acquired after one hour of reaction and two hours of reaction, each containing fluorescence from a standard fluorescent substance.

[0228] These wells can be identified based on the fluorescence of a standard fluorescent substance. Furthermore, it was found that in wells where 5-FAM fluorescence was observed, fluorescence of the standard fluorescent substance was also observed. Therefore, it was possible to confirm that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of Customized Product #2 by MMP14 in the extracellular vesicles derived from MDA-MB-231 was effective in the microcompartment. In this case, 5-FAM fluorescence was also observed in the fluorescence microscopy images of wells without extracellular vesicles, indicating that false positive results occurred in the system of Customized Product #1, but at an extremely low frequency. Meanwhile, no false positive wells were observed in the system of Customized Product #2. It was found that Customized Product #2 was more suitable when detecting lower concentrations of extracellular vesicles.

[0229] Next, the fluorescence microscope image is processed to provide Figures 40A to 40D The histogram shown is within 1 hour of reaction with Custom #1. Figures 40E to 40H The histogram shown is within 2 hours of reaction with Custom #1. Figures 41A to 41D The histograms shown are within 1 hour of reaction with Custom #2 and Figures 41E to 41H The histograms shown are those obtained after 2 hours of reaction with Custom # 2. These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 1 hour and after 2 hours of reaction with each reporter molecule are shown, relative to the expected value (λ) of the concentration of the extracellular vesicles tested calculated by using the above formula (1). In addition, Figure 42A shows a plot of the expected value λ versus the extracellular vesicle concentration of Custom #1, and Figure 42BA graph showing the expected value λ versus the extracellular vesicle concentration for Customized Product #2. The power approximation was performed satisfactorily. The extracellular vesicle concentration was accurately quantified. The results using a 96-well plate are reflected, and nearly identical detection results were obtained after 1 hour and 2 hours using the more active Customized Product #1. Furthermore, a greater number of positive wells were detected after 2 hours using Customized Product #2. Customized Product #1 is preferred when rapid results are required. As described above, false positive results were observed with Customized Product #1, but their frequency was extremely low. Therefore, Customized Product #2 is preferred when accurate detection in a lower concentration range is required, and the use of a reporter molecule is recommended, taking into account its characteristics. The expected values ​​of λ at x1 after 1 hour for Custom #1 and Custom #2 are 0.661 and 0.5098, respectively, with slight differences. However, the expected values ​​of λ at x1 after 2 hours for Custom #1 and Custom #2 are 0.7145 and 0.7649, respectively, which are almost the same.

[0230] Customization #1 System [Table 6] Table 6

[0231] Customization #2 System [Table 7] Table 7

[0232] Example 23 (holes and hole patterns) Using the hole array of Simoa Discs. (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling hole 204. Take 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg protein in 50 μL of PBS), and add the Assay buffer (component D) included in the SensoLyte 520MMP-14Assay Kit (Fluorimetric) to the aliquot to provide a 200 μL solution. The concentration of the solution is 20 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is also x1. The concentration of the solution is set to x1, and a 2-fold serial dilution is performed to prepare a standard sample with four concentrations. In addition, a sample without MMP14 is similarly prepared and used as a sample for BG measurement. In addition, to detect MMP14 activity, 4.95 mL of Assay Buffer (Component D) was added to 50 μL of MMP-14 Substrate (Component A) included in the kit to provide 5 mL of MMP14 detection solution. 500 μL of this was aliquoted, and 2.5 μL of 1 mM Alexa Fluor 647 (which served as a standard fluorescent substance) was added to the aliquot to prepare an MMP14 detection solution, which further contained 5 μM Alexa Fluor 647. 100 μL of MMP14 detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 10 μg / mL, and the concentration of the standard fluorescent substance in each reaction solution was 2.5 μM. 100 μL of each 200 μL reaction solution prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0233] (Evaluation of MMP14 activity using a 96-well plate) Evaluation was performed in the same manner as in Example 1. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 43 The time-dependent changes in fluorescence intensity from the reporter molecule via extracellular vesicles derived from MDA-MB-231 are shown. Figure 43 As shown, the fluorescence intensity was found to increase with the reaction time, and the MMP14 activity could be detected.

[0234] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0235] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not illustrated, a fluorescence microscope image containing fluorescence from 5-FAM of the SensoLyte reporter molecule and background fluorescence of the SensoLyte reporter molecule was acquired after 1 hour of reaction. In addition, a fluorescence microscope image containing fluorescence from a standard fluorescent substance was acquired after 1 hour of reaction. These wells can be identified based on the fluorescence of a standard fluorescent substance. Furthermore, it was found that in wells where 5-FAM fluorescence was observed, fluorescence from the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of the SensoLyte reporter molecule by MMP14 in the MDA-MB-231-derived extracellular vesicles was effective in the microcompartment. In this case, no 5-FAM fluorescence was observed in the fluorescence microscopy images of wells without extracellular vesicles.

[0236] Next, the above fluorescence microscope image is processed to provide Figures 44A to 44E The histograms shown are obtained after 1 hour of reaction with the SensoLyte reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 1 hour of reaction are shown, relative to the expected value (λ) of the extracellular vesicle concentration tested calculated using the above formula (1). Figure 45 A plot showing the expected value λ versus the extracellular vesicle concentration. The power approximation performs satisfactorily, enabling accurate quantification of the extracellular vesicle concentration. By line approximation, the expected value λ for SensoLyte at x1 is 0.469, while the expected values ​​λ for Customized Compound #1 and Customized Compound #2 in Example 22 at the same reaction time are 0.661 and 0.5098, respectively, which are slightly lower values. This is believed to be affected by the MMP14 detection sensitivity of the SensoLyte reporter molecule, which is slightly lower than that of Example 4. Figure 12 Those values ​​for Custom #1 and Custom #2 shown in . Thus, it was demonstrated that digital detection of extracellular vesicles is possible by utilizing MMP14 activity even in the presence of different reporter molecules.

[0237] SensoLyte reporter system [Table 8] Table 8

[0238] Example 24 (holes and hole patterns) Using the hole array of Simoa Discs. (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. Take 2 μL (equivalent to 2 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add the buffer (4) used in Example 18 to the aliquot to provide a 200 μL solution. The concentration of the solution is 10 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is x1 / 2. The solution is subjected to a 10-fold serial dilution to prepare a standard sample with three concentrations. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement.

[0239] In addition, to detect AChE activity, 1 μL of acetylcholine stock solution (500 times) and 2.5 μL of Thiolite Green stock solution (200 times) included in the Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence) and 5 μL of 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were added to 1000 μL of buffer (4) to provide 1008.5 μL of AChE detection solution. The concentration of this solution was 1 / 2 of the concentration specified in the kit. 100 μL of AChE detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 5 μg / mL, the concentration of the reporter molecule in each reaction solution was half the concentration specified in the kit, and the concentration of the standard fluorescent substance in each reaction solution was 2.5 μM. 100 μL of each of the 200 μL reaction solutions prepared for each well was used to measure AChE activity using a 96-well plate.

[0240] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1, except that the reaction temperature was set to 30° C. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 46 The time-dependent changes in fluorescence intensity from the reporter molecule via extracellular vesicles derived from MDA-MB-231 are shown. Figure 46 As shown, it was found that the fluorescence intensity increased with the reaction time, and the AChE activity was detected. In this example, the expression "x1" represents a half of the concentration represented by the expression "x1" in Example 8.

[0241] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0242] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Figures 47A to 47F Fluorescence microscopy images of the wells are shown. Figures 47A to 47C The fluorescence microscope images shown each contain both the fluorescence derived from the Amplite reporter molecule and the Amplite reporter molecule background fluorescence after 3 hours of reaction. Figures 47D to 47F The fluorescence microscope images shown each contain fluorescence of a standard fluorescent substance after a reaction for 3 hours. like Figures 47D to 47F As shown in FIG, the wells can be identified based on the fluorescence of the standard fluorescent substance. Figures 47A to 47C As shown, fluorescence of the standard fluorescent substance was also observed in the wells where fluorescence of the Amplite reporter was observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter, and that the reaction of the Amplite reporter with AChE in the MDA-MB-231-derived extracellular vesicles was proceeding within the microchamber.

[0243] Next, the above fluorescence microscope image is processed to provide Figures 48A to 48C The histograms shown are for a 3-hour reaction with the Amplite reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells identified from each histogram after 3 hours of reaction and the expected value (λ) of the number of positive wells relative to the concentration of the extracellular vesicles tested calculated using the above formula (1) are shown. Figure 49 A plot showing the expected value λ versus the extracellular vesicle concentration. The power approximation performs satisfactorily, enabling accurate quantification of the extracellular vesicle concentration.

[0244] Amplite Reporter System [Table 9] Table 9

[0245] exist Figure 49In the approximation, λ at x1 is 0.0386 and the volume of the microcompartment is 50 fL, so the concentration of extracellular vesicles that can be detected based on AChE activity using the Amplite reporter molecule can be calculated to be 7.7 × 10 8 / mL. The concentration of the reaction solution is 5 μg protein / mL, so the extracellular vesicle concentration is 1.5×10 8 Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 1.5×10 8 extracellular vesicles, in which AChE activity can be detected using the Amplite reporter molecule. It was demonstrated that in addition to the digital detection of extracellular vesicles by using the MMP14 activity described in the previous examples, the digital detection of extracellular vesicles can also be performed by using the AChE activity.

[0246] Example 25 (holes and hole patterns) Using the hole array of Simoa Discs. (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. Take 2 μL (equivalent to 2 μg) of the A549-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add buffer (4) to the aliquot to provide a 200 μL solution. The concentration of the solution is 10 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is x1 / 2. The solution is subjected to a 10-fold serial dilution to prepare a standard sample with three concentrations. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement.

[0247] In addition, to detect AChE activity, 1 μL of acetylcholine stock solution (500 times) and 2.5 μL of Thiolite Green stock solution (200 times) included in the Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence) and 5 μL of 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were added to 1000 μL of buffer (4) to provide 1008.5 μL of AChE detection solution. The concentration of this solution was 1 / 2 of the concentration specified in the kit. 100 μL of AChE detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 5 μg / mL, the concentration of the reporter molecule in each reaction solution was half the concentration specified in the kit, and the concentration of the standard fluorescent substance in each reaction solution was 2.5 μM. 100 μL of each of the 200 μL reaction solutions prepared for each well was used to measure AChE activity using a 96-well plate.

[0248] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1, except that the reaction temperature was set to 23° C. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 50 The figure shows the temporal changes in the fluorescence intensity of the reporter molecule by A549-derived extracellular vesicles. Figure 50 As shown in Figure 1, it was found that the fluorescence intensity increased with the reaction time, and AChE activity was detected at concentrations of x1 and x1 / 10, but not at a concentration of x1 / 100. In this example, the expression "x1" represents half the concentration represented by the expression "x1" in Example 8.

[0249] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0250] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 23° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not illustrated, a fluorescence microscope image containing both fluorescence derived from the Amplite reporter molecule and background fluorescence of the Amplite reporter molecule was acquired after 1.5 hours of reaction. In addition, a fluorescence microscope image containing fluorescence of the standard fluorescent substance was acquired after 1.5 hours of reaction. These wells can be identified based on the fluorescence of a standard fluorescent substance. Furthermore, it was found that in wells where fluorescence of the Amplite reporter molecule was observed, fluorescence of the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the reaction of the Amplite reporter molecule, which was carried out by AChE in A549-derived extracellular vesicles, was taking place within the microcompartment.

[0251] Next, the above fluorescence microscope image is processed to provide Figures 51A to 51D The histograms shown are those obtained after 1.5 hours of reaction with the Amplite reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 1.5 hours of reaction are shown, relative to the expected value (λ) of the extracellular vesicle concentration tested, calculated using the above formula (1). Figure 52 A plot showing the expected value λ versus the extracellular vesicle concentration. The power approximation performs satisfactorily, enabling accurate quantification of the extracellular vesicle concentration.

[0252] Amplite Reporter System [Table 10] Table 10

[0253] exist Figure 52 In the approximation, λ at x1 is 0.0436 and the volume of the microcompartment is 50 fL, so the concentration of extracellular vesicles that can be detected based on AChE activity using the Amplite reporter molecule can be calculated to be 8.7 × 10 8 / mL. The concentration of the reaction solution is 5 μg protein / mL, so the extracellular vesicle concentration is 1.7×10 8 Therefore, it is estimated that 1 μL of A549-derived extracellular vesicles (50 μg of protein in 50 μL of PBS) contains 1.7×10 8 extracellular vesicles, in which AChE activity can be detected using the Amplite reporter molecule. This indicates that, in addition to the digital detection of MDA-MB-231-derived extracellular vesicles described in the previous examples, digital detection of A549-derived extracellular vesicles can also be performed using AChE activity. Furthermore, even after a reaction at room temperature for 1.5 hours, sufficient detection was possible.

[0254] Example 26 (holes and hole patterns) Using the hole array of Simoa Discs.

[0255] (Preparation of Reaction Solution for Wells) A reaction solution for filling the well 204 was prepared. 2 μL (equivalent to 2 μg) of the A549-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the buffer (4) used in Example 18 was added to the aliquoted sample to provide a 200 μL solution. The concentration of the solution was 10 μg / mL. In Example 8, the concentration of 10 μg / mL was set to x1, so the concentration of the solution was x1 / 2. The solution was subjected to a 10-fold serial dilution to prepare a standard sample having three concentrations. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. In addition, to detect AChE activity, 10 μL of acetylcholine stock solution (component C) and 10 μL of AChE substrate solution (component A) included in the SensoLyte 520 Acetylcholinesterase Activity Assay Kit and 2.5 μL of 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were added to 480 μL of buffer (4) to obtain 502.5 μL of AChE detection solution. The concentration of this solution was x2 of the concentration specified in the kit. 100 μL of AChE detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 5 μg / mL, the concentration of the reporter molecule in each reaction solution was x2 the concentration specified in the kit, and the concentration of the standard fluorescent substance in each reaction solution was 2.5 μM. 100 μL of each of the 200 μL reaction solutions prepared for each well was used for AChE activity measurement using a 96-well plate.

[0256] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1, except that the reaction temperature was set to 23° C. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 53 The figure shows the temporal changes in the fluorescence intensity of the reporter molecule by A549-derived extracellular vesicles. Figure 53 As shown in Figure 1, it was found that the fluorescence intensity increased with the reaction time, and AChE activity was detected at a concentration of x1, but not at concentrations of x1 / 10 and x1 / 100. In this example, the expression "x1" represents half the concentration represented by the expression "x1" in Example 8.

[0257] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0258] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 23° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not illustrated, a fluorescence microscope image containing both fluorescence derived from the SensoLyte reporter molecule and background fluorescence of the SensoLyte reporter molecule was acquired after 5 hours of reaction. Furthermore, a fluorescence microscope image containing fluorescence of the standard fluorescent substance was acquired after 5 hours of reaction. These wells can be identified based on the fluorescence of the standard fluorescent substance. Furthermore, it was found that in wells where fluorescence of the SensoLyte reporter molecule was observed, fluorescence of the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the reaction of the SensoLyte reporter molecule caused by AChE in the A549-derived extracellular vesicles was occurring within the microcompartment.

[0259] Next, the fluorescence microscope image is processed to provide Figures 54A to 54D The histograms shown are obtained after 5 hours of reaction with the SensoLyte reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 5 hours of reaction are shown, relative to the expected value (λ) of the extracellular vesicle concentration tested, calculated using the above formula (1). Figure 55 A plot showing the expected value λ versus the extracellular vesicle concentration. The power approximation performs satisfactorily, enabling accurate quantification of the extracellular vesicle concentration.

[0260] SensoLyte reporter system [Table 11] Table 11

[0261] exist Figure 55 In the approximation, λ at x1 is 0.0184 and the volume of the microcompartment is 50 fL, so the concentration of extracellular vesicles that can be detected based on AChE activity using the SensoLyte reporter can be calculated to be 3.7 × 10 8 / mL. The concentration of the reaction solution is 5 μg protein / mL, so the extracellular vesicle concentration is 7.4×10 7 Therefore, it is estimated that 1 μL of A549-derived extracellular vesicles (50 μg of protein in 50 μL of PBS) contains 7.4×10 7 In Example 25, it was estimated that 1 μL of A549-derived extracellular vesicles contained 1.7×10 8 Extracellular vesicles in which AChE activity can be detected using the Amplite reporter molecule. However, the value of the number of extracellular vesicles in which AChE activity can be detected using the SensoLyte reporter molecule is about 40% of the number of extracellular vesicles in which AChE activity can be detected using the Amplite reporter molecule. The SNR between positive and negative wells for the Amplite reporter molecule is higher than the SNR between positive and negative wells for the SensoLyte reporter molecule, and positive and negative wells can be clearly separated. This is believed to contribute to the improvement of detection sensitivity. It was demonstrated that in addition to the digital detection of extracellular vesicles using the Amplite reporter described in the previous examples, digital detection of extracellular vesicles using the SensoLyte reporter can also be performed by using AChE activity.

[0262] Example 27 (holes and hole patterns) Using the hole array of Simoa Discs. (Preparation of Reaction Solution for Wells) A reaction solution for filling the well 204 was prepared. 2 μL (equivalent to 2 μg) of the A549-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and the buffer (4) used in Example 18 was added to the aliquoted sample to provide a 200 μL solution. The concentration of the solution was 10 μg / mL. In Example 8, the concentration of 20 μg / mL was set to x1, so the concentration of the solution was x1 / 2. The solution was subjected to a 10-fold serial dilution to prepare a standard sample having three concentrations. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP14 activity, 10 μL of 1 mM custom product #2 and 2.5 μL of 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were added to 490 μL of buffer (4), thereby providing 502.5 μL of MMP14 detection solution. 100 μL of MMP14 detection solution was added to 100 μL of each sample to prepare a reaction solution for each well. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 5 μg / mL, the concentration of the reporter molecule in each reaction solution was 10 μM, and the concentration of the standard fluorescent substance in each reaction solution was 2.5 μM. 100 μL of each of the 200 μL reaction solutions prepared for each well was used to measure MMP14 activity using a 96-well plate.

[0263] (AChE activity evaluation using 96-well plate) Evaluation was performed in the same manner as in Example 1, except that the reaction temperature was set to 23° C. ΔRFU was calculated by subtracting the measured value of the sample for BG measurement from the measured value of each sample. Figure 56 The figure shows the temporal changes in the fluorescence intensity of the reporter molecule by A549-derived extracellular vesicles. Figure 56 As shown in FIG. 1 , the fluorescence intensity increased with the reaction time, and MMP14 activity was detected at a concentration of x1, but not at concentrations of x1 / 10 and x1 / 100. In this example, the expression "x1" represents half the concentration represented by the expression "x1" in Example 8.

[0264] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0265] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 23° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not illustrated, a fluorescence microscope image containing both fluorescence from Customized Article #2 and background fluorescence of Customized Article #2 was acquired after 5 hours of reaction. In addition, a fluorescence microscope image containing fluorescence of a standard fluorescent substance was acquired after 5 hours of reaction. These wells can be identified based on the fluorescence of the standard fluorescent substance. Furthermore, it was found that fluorescence of the standard fluorescent substance was also observed in the wells where fluorescence of Customized Substance #2 was observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the reaction of Customized Substance #2 caused by MMP14 in A549-derived extracellular vesicles was proceeding within the microchamber.

[0266] Next, the fluorescence microscope image is processed to provide Figures 57A to 57D The histograms shown are those obtained after 5 hours of reaction with Custom # 2. These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The total number of wells and the number of positive wells identified from each histogram after 5 hours of reaction are shown, relative to the expected value (λ) of the extracellular vesicle concentration tested, calculated using the above formula (1). Figure 58 A plot showing the expected value λ versus the extracellular vesicle concentration. The power approximation performs satisfactorily, enabling accurate quantification of the extracellular vesicle concentration.

[0267] Customization #2 System [Table 12] Table 12

[0268] exist Figure 58 In the approximation of , λ at x1 is 0.2767 and the volume of the microcompartment is 50 fL, so the concentration of extracellular vesicles that can be detected by custom compound #2 based on MMP14 activity can be calculated to be 5.5×10 9 / mL. The concentration of the reaction solution is 5 μg protein / mL, so the extracellular vesicle concentration is 1.1×10 9 Therefore, it is estimated that 1 μL of A549-derived extracellular vesicles (50 μg of protein in 50 μL of PBS) contains 1.1×10 9 Extracellular vesicles, in which custom product #2 can be used to detect MMP14 activity.

[0269] Example 28 (Preparation of Antibody-Immobilized Particles) Homebrew 2.0 Multiplex Beads 647L1.5:103527 (manufactured by Quanterix Corporation) is used as magnetic particles. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (manufactured by Thermo Fisher Scientific Inc.) dissolved in the same Bead Conjugation Buffer is added to the magnetic particles to which the buffer is replaced with Bead Conjugation Buffer (manufactured by Quanterix Corporation), and the mixture is stirred at 4 ° C for 30 minutes, and then the magnetic particles are recovered with a magnet. The recovered magnetic particles are dispersed in Bead Conjugation Buffer, anti-CD9 antibody (ab58989, manufactured by Abcam plc.), anti-CD63 antibody (ab59479, manufactured by Abcam plc.) and anti-CD81 antibody (ab59477, manufactured by Abcam plc.) are added. Thereafter, the mixture is stirred at 4 ° C for 2 hours. Thereafter, the mixture was washed with Bead Washing Buffer (manufactured by Quanterix Corporation), and then Bead Blocking Buffer (manufactured by Quanterix Corporation) was added, and the mixture was stirred at room temperature for 45 minutes. The magnetic particles were recovered with a magnet, washed with Bead Washing Buffer, and then dispersed in Bead Diluent Buffer (manufactured by Quanterix Corporation) to provide an antibody-immobilized particle solution. The antibody-immobilized particle solution was stored at 4°C until use.

[0270] (holes and hole patterns) Using the hole array of Simoa Discs.

[0271] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. 2 μL (equivalent to 2 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and Sample Diluent Buffer (manufactured by Quanterix Corporation) was added to the aliquot to provide a 200 μL solution serving as a sample. The concentration of this solution was 10 μg / mL. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Each of the above samples was mixed with the following. Each sample prepared above: 100 μL The antibody-immobilized particles (2.0×10 7 Particles / mL): 25 μL The resulting mixed solution was shaken and reacted at 30°C for 30 minutes to form a particle complex of antibody-immobilized particles and extracellular vesicles. Thereafter, the complex was washed with a disk washer and then recovered with a magnet. To detect MMP14 activity, 1 mM custom compound #2 was diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM custom compound #2. 25 μL of the MMP14 detection solution was added to the recovered complex to provide a reaction solution for the wells.

[0272] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0273] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Figures 59A to 59D Shown are fluorescence microscope images of the wells taken after 5 hours of reaction. Figure 59A and Figure 59B The fluorescence microscope images each contain both the fluorescence from 5-FAM of the reporter molecule and the background fluorescence of the reporter molecule. Figure 59C and Figure 59D Shown are fluorescence microscopy images of the fluorescence of each containing Multiplex Beads 647. Figure 59C and Figure 59D , it is possible to identify pores filled with particles. In addition, from Figure 59A 5-FAM fluorescence was observed from the pores filled with particles. Therefore, it was confirmed that MDA-MB-231-derived extracellular vesicles were captured on the particles filling the microcompartments and that the reaction of Customized Substance #2 was activated by MMP14 in the extracellular vesicles.

[0274] Next, the fluorescence microscope image is processed to provide Figure 60A and Figure 60BThe histograms shown are those obtained after 5 hours of reaction with Custom # 2. These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. Shown are the number of wells filled with particles and the number of positive wells identified from each histogram after 5 hours of reaction, relative to the expected value (λ) of the concentration of the extracellular vesicles subjected to the test calculated by using the above formula (1).

[0275] Customization #2 System [Table 13] Table 13

[0276] The concentration of the sample was 10 μg / mL. 100 μL of sample was used, so 1 μg of protein was used in the reaction. 25 μL of the sample was used, with a concentration of 2.0 × 10 7 particles / mL, so the number of particles undergoing the reaction is 5.0×10 5 The expected value Δλ per particle (obtained by subtracting BG) was 0.159431, so the extracellular vesicle concentration was estimated to be 8.0×10 4 Extracellular vesicles / μg protein. Thus, extracellular vesicles are captured on particles bound to a capture antibody comprising a mixture of anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody, and the extracellular vesicles can be detected and quantified through their MMP14 activity.

[0277] Example 29 (Preparation of Antibody-Immobilized Particles) The antibody-immobilized particle solution was prepared in the same manner as in Example 28 and stored at 4°C until use.

[0278] (holes and hole patterns) Using the hole array of Simoa Discs.

[0279] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. 2 μL (equivalent to 2 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and Sample Diluent Buffer (manufactured by Quanterix Corporation) was added to the aliquot to provide a 200 μL solution serving as a sample. The concentration of this solution was 10 μg / mL. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Each of the above samples was mixed with the following. Each sample prepared above: 100 μL The antibody-immobilized particles (2.0×10 7 Particles / mL): 25 μL The resulting mixed solution was shaken and reacted at 30°C for 30 minutes to form a particle complex of antibody-immobilized particles and extracellular vesicles. Thereafter, the complex was washed with a disk washer and then recovered with a magnet. To detect AChE activity, 1 μL of acetylcholine stock solution (500x) and 2.5 μL of Thiolite Green stock solution (200x) included in the Amplite Fluorimetric Acetylcholinesterase Assay Kit (Green Fluorescence) were added to 1000 μL of buffer (4) to provide 1003.5 μL of AChE detection solution. The concentration of this solution was 1 / 2 of the concentration specified in the kit, but no equal amount of sample solution was added during the reaction, so the final concentration during the reaction was the concentration specified in the kit. 25 μL of AChE detection solution was added to the recovered complex to provide a reaction solution for the wells.

[0280] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0281] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Figures 61A to 61D Shown are fluorescence microscope images of the wells taken after 3 hours of reaction. Figure 61A and Figure 61B Each shows a fluorescence microscope image containing both fluorescence from the reporter molecule and background fluorescence from the reporter molecule. Figure 61C and Figure 61D Each shows a fluorescence microscope image containing the fluorescence of Multiplex Beads 647. Figure 61C and Figure 61D , it is possible to identify pores filled with particles. In addition, from Figure 61AFluorescence was observed from the pores filled with particles, confirming that MDA-MB-231-derived extracellular vesicles were captured on the particles filling the microcompartments and that the AChE activity of the extracellular vesicles was responsible for the response of the Amplite reporter molecule.

[0282] Next, the fluorescence microscope image is processed to provide Figure 62A and 62B The histograms shown are for a 3-hour reaction with the Amplite reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. Shown are the number of wells filled with particles and the number of positive wells identified from each histogram after 3 hours of reaction, relative to the expected value (λ) of the concentration of the extracellular vesicles subjected to the test calculated by using the above formula (1).

[0283] Amplite Reporter System [Table 14] Table 14

[0284] The concentration of the sample was 10 μg / mL. 100 μL of sample was used, so 1 μg of protein was used in the reaction. 25 μL of a concentration of 2.0 × 10 7 particles / mL, so the number of particles undergoing the reaction is 5.0×10 5 The expected value Δλ per particle (obtained by subtracting BG) was 0.059578, so the extracellular vesicle concentration was estimated to be 3.0 × 10 4 Extracellular vesicles / μg protein. Thus, extracellular vesicles are captured on particles bound to a capture antibody comprising a mixture of anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody, and the extracellular vesicles can be detected and quantified through their AChE activity.

[0285] Example 30 (Preparation of Antibody-Immobilized Particles) The antibody-immobilized particle solution was prepared in the same manner as in Example 28 and stored at 4°C until use.

[0286] (holes and hole patterns) Using the hole array of Simoa Discs.

[0287] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. 2 μL (equivalent to 2 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and Sample Diluent Buffer (manufactured by Quanterix Corporation) was added to the aliquot to provide a 200 μL solution serving as a sample. The concentration of this solution was 10 μg / mL. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Each of the above samples was mixed with the following. Each sample prepared above: 100 μL The antibody-immobilized particles (2.0×10 7 Particles / mL): 25 μL The resulting mixed solution was shaken and reacted at 30°C for 30 minutes to form a particle complex of antibody-immobilized particles and extracellular vesicles. Thereafter, the complex was washed with a disk washer and then recovered with a magnet. To detect AChE activity, 5 μL of acetylcholine solution (component C) and 5 μL of AChE substrate solution (component A) included in the SensoLyte 520 Acetylcholinesterase Activity Assay Kit were added to 490 μL of buffer (4) to provide 500 μL of AChE detection solution. The concentration of this solution was the concentration specified in the kit. No equal amount of sample solution was added during the reaction, so the final concentration during the reaction was x2 the concentration specified in the kit. 25 μL of AChE detection solution was added to the recovered complex to provide a reaction solution for the wells.

[0288] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0289] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not illustrated, a fluorescence microscope image was obtained that included both fluorescence from the reporter molecule and background fluorescence from the reporter molecule. Furthermore, a fluorescence microscope image containing fluorescence from Multiplex Beads 647 was obtained. Pores filled with particles were identified. Furthermore, fluorescence was observed from pores filled with particles. Therefore, it was confirmed that extracellular vesicles derived from MDA-MB-231 were trapped on the particles filling the microcompartments, and that the reaction of the SensoLyte reporter molecule was activated by the AChE activity of the extracellular vesicles.

[0290] Next, the fluorescence microscope image is processed to provide Figure 63A and Figure 63B The histograms shown are obtained after 5 hours of reaction with the SensoLyte reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. Shown are the number of wells filled with particles and the number of positive wells identified from each histogram after 5 hours of reaction, relative to the expected value (λ) of the concentration of the extracellular vesicles subjected to the test calculated by using the above formula (1).

[0291] SensoLyte Molecular System [Table 15] Table 15 The concentration of the sample was 10 μg / mL. 100 μL of sample was used, so 1 μg of protein was used in the reaction. 25 μL of a concentration of 2.0 × 10 7 particles / mL, so the number of particles undergoing the reaction is 5.0×10 5 The expected value Δλ per particle (obtained by subtracting BG) was 0.04546, so the extracellular vesicle concentration was estimated to be 2.3 × 10 4 Extracellular vesicles / μg protein. Thus, extracellular vesicles are captured on particles bound to a capture antibody comprising a mixture of anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody, and the extracellular vesicles can be detected and quantified through their AChE activity.

[0292] Example 31 (Preparation of Antibody-Immobilized Particles) The antibody-immobilized particle solution was prepared in the same manner as in Example 28 and stored at 4°C until use.

[0293] (holes and hole patterns) Using the hole array of Simoa Discs.

[0294] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. 2 μL (equivalent to 2 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) was aliquoted, and Sample Diluent Buffer (manufactured by Quanterix Corporation) was added to the aliquot to provide a 200 μL solution serving as a sample. The concentration of this solution was 10 μg / mL. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Each of the above samples was mixed with the following. Each sample prepared above: 50 μL The antibody-immobilized particles (4.0×10 7 Particles / mL): 50 μL The resulting mixed solution was shaken and reacted at 30°C for 30 minutes to form a particle complex of antibody-immobilized particles and extracellular vesicles. Thereafter, the complex was washed with a disk washer and then recovered with a magnet. To detect MMP14 activity, 1 mM custom compound #2 was diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM custom compound #2. 100 μL of MMP14 detection solution was added to the recovered complex to provide a reaction solution for the wells, and the wells were filled with 25 μL of the reaction solution. The number of particles was increased fourfold, and the reaction was allowed to proceed. However, the number and concentration of particles used to fill the wells were ultimately the same as in the other examples.

[0295] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0296] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although not illustrated, a fluorescence microscope image was acquired that included both fluorescence from the reporter molecule 5-FAM and background fluorescence from the reporter molecule. Furthermore, a fluorescence microscope image was acquired that included fluorescence from Multiplex Beads 647. Particle-filled pores were identified. Furthermore, 5-FAM fluorescence was observed from the particle-filled pores. Thus, it was confirmed that MDA-MB-231-derived extracellular vesicles were trapped on the particles filling the microcompartments, and that the reaction of Customized Substance #2 was activated by MMP14 within the extracellular vesicles.

[0297] Next, the fluorescence microscope image is processed to provide Figure 64A and Figure 64B The histograms shown are those obtained after 5 hours of reaction with Custom # 2. These histograms each show the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. The number of wells filled with particles and the number of positive wells identified from each histogram after 5 hours of reaction are shown, and the expected value (λ) relative to the concentration of the extracellular vesicles tested is calculated using the above formula (1). Regarding BG, the value of Example 28 was used.

[0298] Customization #2 System [Table 16] Table 16

[0299] The concentration of the sample was 10 μg / mL, and 50 μL of sample was used, so 0.5 μg of protein was used in the reaction. The concentration of 50 μL was 4.0 × 10 7 particles / mL, so the number of particles undergoing the reaction is 2.0×10 6 The expected value Δλ for each particle (the value obtained by subtracting BG) was calculated from the sum of the number of particles and the number of positives in the two images to be 0.044354, so the extracellular vesicle concentration was estimated to be 1.8×10 5 Extracellular vesicles / μg protein. The extracellular vesicle concentration is greater than 8.0×10 4 Extracellular vesicles / μg protein, which is the value estimated in Example 28. This is probably because the capture efficiency of extracellular vesicles is improved by reacting with 4 times the number of particles at 5 times the particle concentration. Thus, extracellular vesicles are captured on particles bound to a capture antibody comprising a mixture of anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody, and the extracellular vesicles can be detected and quantified through their MMP14 activity.

[0300] Example 32 In this example, droplets were used as independent separation compartments.

[0301] (Preparation of Samples for Droplet Formation) The reaction solution for forming droplets was prepared as follows. 1.5 μL (corresponding to 1.5 μg) of MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg protein in 50 μL PBS) was aliquoted, and buffer (4) was added to the aliquot to provide a 300 μL solution. The concentration of this solution was 5 μg / mL. Furthermore, to detect MMP14 activity, 1.5 μL of 1 mM custom product #1 and 1.5 μL of 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were added to the above sample to provide an MMP14 detection solution containing custom product #1 and Alexa Fluor 647 at a final concentration of 5 μM.

[0302] (Preparation of Droplets) Droplets were prepared by pumping the reaction solution prepared above through an SPG emulsification membrane. The dispersed phase and the continuous phase are shown below. Dispersed phase: 80 μL of the reaction solution used to form droplets Continuous phase: 2.5 mL of aliphatic hydrocarbon (product name: Isopar L, manufactured by Exxon Mobil Corporation) in which a surfactant (product name: KF-6038, manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved at a concentration of 4% An SPG pumping connector (pore size: 10 μm, pore size: 5 μm, manufactured by SPG Technology Co., Ltd.) was used as an SPG emulsification membrane, and the number of pumping reciprocations was set to 10. Thus, droplets each having a diameter of about 1 μm to about 10 μm were obtained.

[0303] (Fluorescence microscopy observation) The emulsion prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 37° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Figures 65A to 65C Bright field and fluorescence microscopy images were acquired after 4 hours of reaction of a droplet generated using an SPG pumping linker (pore size: 10 μm). Figure 65A A bright field image of the droplet is shown. Figure 65BThe fluorescence microscope image includes both the fluorescence from the reporter molecule 5-FAM and the background fluorescence from the reporter molecule. Figure 65C Fluorescence microscope images showing fluorescence containing standard fluorescent substances. Figures 66A to 66C Bright field and fluorescence microscopy images were obtained after 4 hours of reaction of droplets generated using an SPG pumping linker (pore size: 5 μm). Figure 66A A bright field image of the droplet is shown. Figure 66B The fluorescence microscope image includes both the fluorescence from the reporter molecule 5-FAM and the background fluorescence from the reporter molecule. Figure 66C Fluorescence microscope images showing fluorescence containing standard fluorescent substances. Similar to Example 19, droplets can be identified based on the fluorescence of a standard fluorescent substance. It was found that in droplets where 5-FAM fluorescence was observed, fluorescence of the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of customized substance #1 by MMP14 in MDA-MB-231-derived extracellular vesicles was effective in the microcompartment.

[0304] Next, the image obtained using the SPG pumping linker having a pore size of 10 μm was analyzed in the same manner as in Example 19. The above fluorescence microscope image after 4 hours of reaction was subjected to predetermined image processing to provide the following image: Figure 67 The droplet size distribution is shown, and a histogram is obtained, which presents the number of droplets exhibiting corresponding fluorescence intensity included in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value in each particle size range. Furthermore, when the concentration of extracellular vesicles having MMP14 activity is calculated from the total number of droplets in each size range and the number of positive droplets identified from the histogram by using the above-mentioned formula (1), the concentration can be estimated as follows.

[0305] [Table 17] Table 17

[0306] From the above estimates, the mean and standard deviation of the extracellular vesicle concentration can be calculated to be 9.5 × 10 9 / mL and 7.0×10 8 / mL. The concentration of the reaction solution is 5.0 μg protein / mL, so the extracellular vesicle concentration is 1.9×10 9 Therefore, it is estimated that 1 μL of MDA-MB-231-derived extracellular vesicles (50 μg protein in 50 μL PBS) contains 1.9×10 9Extracellular vesicles, where MMP14 activity can be detected using reporter molecule custom compound #1. This result is similar to the 2.1×10 in the system in Example 20 where pores were used as microcompartments. 9 Thus, it was found that similar results were obtained in similar reaction systems, regardless of whether wells or droplets were used as microcompartments.

[0307] Example 33 (Preparation of Antibody-Immobilized Particles) The antibody-immobilized particle solution was prepared in the same manner as in Example 28 and stored at 4°C until use.

[0308] (Preparation of detection antibody in which the antibody is conjugated with biotin) Anti-CD9 antibody (ab58989), anti-CD63 antibody (ab59479), and anti-CD81 antibody (ab59477) were buffered with Biotinylation Reaction Buffer to provide an antibody solution. The biotinylation reagent NHS-PEG4-Biotin was added to the solution, the mixture was stirred, and then allowed to react at room temperature for 30 minutes. The resultant was then washed with Biotinylation Reaction Buffer and recovered with Biotinylation Reaction Buffer to provide a biotinylated detection antibody solution. The biotinylated detection antibody solution was stored at 4°C until use.

[0309] (holes and hole patterns) Using the hole array of Simoa Discs.

[0310] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling the well 204. Take 4 μL (equivalent to 4 μg) of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add Sample Diluent Buffer (manufactured by Quanterix Corporation) to the aliquot to provide a 400 μL solution to serve as a sample. The concentration of this solution is 10 μg / mL. This solution is subjected to a 3-fold serial dilution to prepare a standard sample with seven concentrations. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement.

[0311] Each of the above samples and antibody-immobilized particles were mixed as described below. Each sample prepared above: 100 μL The antibody-immobilized particles (2.0×107 Particles / mL): 25 μL The resulting mixed solution was shaken and reacted at 30° C. for 30 minutes to form a particle complex of antibody-immobilized particles and extracellular vesicles. Thereafter, the complex was washed with a disk washer and then recovered with a magnet.

[0312] The recovered composite particles were reacted with the following. Biotinylated detection antibody (0.3 μg / mL): 100 μL The resulting mixed solution was shaken and reacted at 30°C for 10 minutes to form sandwich-type particle complexes, in which the biotinylated detection antibody further bound to each particle complex of the antibody-immobilized particles and extracellular vesicles. The complexes were then washed using a disk washer and recovered using a magnet.

[0313] The recovered composite particles were reacted as follows. Streptavidin-conjugated β-galactosidase (SBG) (150 pM): 100 μL The resulting mixed solution was shaken and reacted at 30°C for 10 minutes to form a complex, in which the streptavidin-conjugated β-galactosidase further bound to the biotinylated detection antibody in each of the complexes. After washing in a disk washer, the complex was recovered using a magnet and used in subsequent reactions.

[0314] In addition, a reference evaluation was performed on some of the composites prepared above using Simoa SR-X. The evaluation using Simoa SR-X was performed according to the operating procedures of Simoa SR-X. To detect MMP14 activity, a 1 mM custom compound #2 and 100 μM resorufin β-D-galactopyranoside solution was diluted with buffer (4) to prepare an MMP14 and CD9 / CD63 / CD81 mixture detection solution (1) containing 10 μM custom compound #2 and 5 μM resorufin β-D-galactopyranoside. In addition, this solution was diluted 2-fold with buffer (4) to prepare a detection solution (2). Non-fluorescent resorufin β-D-galactopyranoside was degraded by β-galactosidase to produce fluorescent resorufin. In this case, the maximum wavelengths of excitation and fluorescence of resorufin were 570 nm and 585 nm, respectively. 50 μL of the detection solution (2) was added to the recovered complex to provide a reaction solution for the wells. Separately, 50 μL of the test solution (1) was added to 50 μL of each of the prepared standard samples and BG measurement samples at 7 concentrations, and the resulting solution was used when measuring MMP14 activity and β-galactosidase activity using a 96-well plate. Equal amounts of each sample and test solution (1) were added so that the final concentration of the reporter molecule was the same as that of the test solution (2).

[0315] (Evaluation of MMP14 and β-galactosidase activities in 96-well plates) Evaluation was performed in the same manner as in Example 1, except that the reaction temperature was set to 30°C. ΔRFU was calculated by subtracting the measured value of the BG measurement sample from the measured value of each sample. In this case, resorufin generated by β-galactosidase activity was simultaneously measured at an excitation wavelength of 545±20 nm and a fluorescence wavelength of 590±20 nm, and ΔRFU was similarly calculated. Figure 68A The figure shows the temporal changes in the fluorescence intensity of the MMP14 activity reporter molecule from MDA-MB-231-derived extracellular vesicles. Figure 68A As shown in FIG. 1 , the fluorescence intensity increased with the reaction time, and MMP14 activity was detected at a concentration of x1 / 9 or higher, but not at a concentration of x1 / 27 or lower. In this example, the expression "x1" represents half the concentration represented by the expression "x1" in Example 8. Figure 68B The figure shows the temporal change of the fluorescence intensity of the β-galactosidase activity reporter (resorufin β-D-galactopyranoside) from MDA-MB-231-derived extracellular vesicles. Figure 68B As shown, it was found that no activity could be detected at all. This system does not include a CD9 / CD63 / CD81 detection system, and therefore it was found that the intrinsic β-galactosidase activity of extracellular vesicles derived from MDA-MB-231 was equal to or less than the detection limit of the system, and it was also found that the system did not affect the evaluation of β-galactosidase activity used in the CD9 / CD63 / CD81 detection system.

[0316] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0317] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence derived from each fluorescent substance was observed to obtain a fluorescent image under the same conditions as in Example 19. In this case, fluorescence derived from resorufin was observed using a TRITC filter. Resorufin TRITC: OP-87764 (manufactured by Keyence) Model: OP-87764 Excitation wavelength: 545 / 25nm Absorption wavelength: 605 / 70nm Dichroic wavelength: 565nm

[0318] Figures 69A to 69C Each shows an example of a fluorescence microscope image of a well taken after 4 hours of reaction. These figures show images obtained from a standard sample with a concentration of x1 / 9. Figure 69A A fluorescence microscopy image is shown that contains both the fluorescence from 5-FAM of Custom #2 and the background fluorescence of the reporter molecule. Figure 69B The fluorescence microscopy image shown contains resorufin fluorescence derived from resorufin β-D-galactopyranoside and background fluorescence of the reporter molecule. Figure 69C The fluorescence microscope image of the fluorescence containing Multiplex Beads 647 is shown. Figure 69C Identify the pores filled with particles. Figure 69A and Figure 69B It was found that 5-FAM fluorescence and resorufin fluorescence were observed from the pores filled with particles. Therefore, it was confirmed that: extracellular vesicles derived from MDA-MB-231 were captured on the particles filling the microcompartments, and the reaction of customized product #2 was activated by MMP14 in the extracellular vesicles; and the biotinylated detection antibody bound to CD9 / CD63 / CD81 in the extracellular vesicles, and the reaction of the reporter molecule (resorufin β-D-galactopyranoside) was activated by streptavidin-coupled β-galactosidase further bound to biotin. Therefore, it was recognized that by using enzyme activity and surface antigens, which are biomarkers with different properties, extracellular vesicles captured on particles can be detected in a multiplexed manner. Next, the fluorescence microscope image is processed to provide Figure 70A (5-FAM) and Figure 70B (resorufin) are histograms of fluorescence derived from each reporter molecule. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. Similarly, for other dilution series of standard samples, fluorescence microscope images were analyzed to provide histograms. The number of particle-filled wells and the number of positive wells identified from each histogram after 4 hours of reaction are shown, along with the expected value (λ) relative to the concentration of the extracellular vesicles tested, calculated using the above formula (1). Also shown are the results obtained by evaluating similarly prepared samples using the Simoa SR-X ("Simoa" column).

[0319] [Table 18] Table 18

[0320] Figure 71A A coordinate graph of the expected value λ (Simoa, AEB) for each standard sample is shown. It was found that the expected value λ obtained by using CD9 / CD63 / CD81 and its approximate curve obtained from the fluorescence microscope image were in satisfactory agreement with those obtained using Simoa SR-X. In this case, for Simoa, a simulation correction based on the brightness value of the hole was performed in the high AEB region, so it was considered that a higher value was obtained, and it was found that the value obtained for λ or AEB less than 1 was extremely satisfactory to the value obtained from the fluorescence microscope image. In addition, it was found that the number of extracellular vesicles that can be detected by using MMP14 is about one order of magnitude smaller than the number of extracellular vesicles that can be detected by using CD9 / CD63 / CD81. The BG of the expected value λ in this system can be estimated to be about 0.01, so Figure 71B Five graphs showing expected values ​​on the higher concentration side. The number of extracellular vesicles detectable by MMP14 activity changed at an almost constant rate relative to the number of extracellular vesicles detectable by using CD9 / CD63 / CD81, indicating that the multiplex detection system effectively operates over a dynamic range of approximately two orders of magnitude. Furthermore, the multiplex detection system resulted in detection sensitivity approximately one order of magnitude higher than that of MMP14 activity using a 96-well plate. For example, it can be seen that 99.7% of extracellular vesicles detectable with MMP14 at a concentration [au] of x1 are present in pores (particles) containing extracellular vesicles detectable with at least one of CD9, CD63, and CD81, which are commonly used biomarkers for extracellular vesicles. It can be seen that this multiplex detection system is effectively used as a means of identifying co-localization of biomarkers.

[0321] The embodiments of the present invention can be implemented by a method executed by a computer of a system or device, by performing one or more functions of the above-mentioned embodiments with a computer of a system or device (e.g., an application-specific integrated circuit (ASIC)) configured to read and execute computer-executable instructions (e.g., one or more programs) recorded on a storage medium, and / or by reading and executing computer-executable instructions from a storage medium with a computer of a system or device including one or more circuits configured to perform one or more functions of the above-mentioned embodiments and for, for example, performing one or more functions of the above-mentioned embodiments, and / or by controlling one or more circuits to perform one or more functions of the above-mentioned embodiments. The computer may include one or more processors (e.g., a central processing unit (CPU) or a microprocessor (MPU)), and may include a network of a single computer or a single processor to read and execute computer-executable instructions. Computer-executable instructions may be provided to the computer, for example, from a network or a storage medium. The storage medium may include, for example, one or more of the following: a hard disk, a random access memory (RAM), a read-only memory (ROM), a storage device of a distributed computing system, an optical disk (e.g., a compact disk (CD), a digital versatile disk (DVD), or a Blu-ray disk (BD)), a flash memory device, and a memory card.

[0322] Example 34 (Detection of MMP activity in extracellular vesicles derived from MDA-MB-231 or A549 using reporter molecules SB2, SB6, SB7, SB9, SB10, or SB14) 4 μL of each of extracellular vesicles derived from MDA-MB-231 (50 μg of protein in 50 μL of PBS) and extracellular vesicles derived from A549 (50 μg of protein in 50 μL of PBS) was aliquoted (equivalent to 4 μg for each of the extracellular vesicles derived from MDA-MB-231 and A549), and buffer (4) was added to the aliquoted sample to provide 500 μL of each solution. The solution concentration was 8 μg / mL for each of the extracellular vesicles derived from MDA-MB-231 and A549. In Example 8, the concentration of 20 μg / mL was set to x1, so the concentration of the solution was x1 / 2.5 for each of the extracellular vesicles derived from MDA-MB-231 and A549. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP activity, 100 μM SB2, SB6, SB7, SB9, SB10, or SB14 of SensoLyte 520 MMP Substrate Sampler Kit (Fluorimetric) AS-71170 was diluted with buffer (4) to prepare a 10 μM MMP detection solution. 50 μL of the MMP detection solution was added to 50 μL of each sample, and the reaction was carried out at 30°C to evaluate the MMP activity. In this case, for each of the MDA-MB-231-derived and A549-derived extracellular vesicles, the concentration of the extracellular vesicles in each reaction solution was 4 μg / mL, and the concentration of the reporter molecule in the reaction solution was 5 μM.

[0323] (Evaluation of MMP activity using 96-well plate) Evaluation was performed in the same manner as in Example 1. Table 19 shows the change in fluorescence intensity of MDA-MB-231-derived extracellular vesicles after 1 hour by different reporter molecules. As shown in Table 19, it was found that MDA-MB-231-derived extracellular vesicles changed the fluorescence intensity.

[0324] [Table 19] Table 19 reporter molecules Fluorescence change (ΔRFU) after 1 hour of reaction at 30°C SB2 32,088 SB6 52,130 SB7 39,031 SB9 62,330 SB10 36,239 SB14 6,831

[0325] Table 20 shows the change in fluorescence intensity after 1 hour by different reporter molecules using A549-derived extracellular vesicles. As shown in Table 20, it was found that A549-derived extracellular vesicles changed the fluorescence intensity.

[0326] [Table 20] Table 20 reporter molecules Fluorescence change (ΔRFU) after 1 hour of reaction at 30°C SB2 1,154 SB6 2,043 SB7 1,228 SB9 1,775 SB10 1,220 SB14 801

[0327] From Tables 19 and 20, it was found that the change in fluorescence intensity by MDA-MB-231-derived extracellular vesicles was about 30 times that by A549-derived extracellular vesicles (in the case of SB14, slightly less than 10 times).

[0328] Example 35 (holes and hole patterns) Using the hole array of Simoa Discs.

[0329] (Preparation of Reaction Solution for Wells) A reaction solution for filling hole 204 is prepared. 12 μL (relative to 12 μg) of extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) are taken, and buffer (4) is added to the sample to provide 1200 μL of solution. The concentration of the solution is 10 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of the solution is x1 / 2. The solution is further diluted 2 times to prepare a standard sample with a concentration of x1 / 4, further diluted 3 times to prepare a standard sample with a concentration of x1 / 12, and further diluted 3 times to prepare a standard sample with a concentration of x1 / 36. Thus, standard samples with four concentrations are prepared. In addition, a sample without extracellular vesicles is similarly prepared and used as a sample for BG measurement.

[0330] In addition, to detect MMP activity, 100 μM SB2, SB6, SB7, SB9, SB10, or SB14 of SensoLyte 520 MMP Substrate Sampler Kit (Fluormetric) AS-71170 and 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were diluted with buffer (4) to prepare an MMP detection solution containing 10 μM SB2, SB6, SB7, SB9, SB10, or SB14 and 2 μM Alexa Fluor 647. 50 μL of each MMP detection solution was added to 50 μL of each sample to provide a reaction solution for the wells. In this case, the maximum concentration of extracellular vesicles in each reaction solution was 5 μg / mL, and the concentrations of the reporter molecule and the standard fluorescent substance in each reaction solution were 5 μM and 1 μM, respectively.

[0331] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0332] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although the description is omitted, a fluorescence microscope image containing fluorescence derived from 5-FAM of each of SB2, SB6, SB7, SB9, SB10 and SB14 and background fluorescence of each of SB2, SB6, SB7, SB9, SB10 and SB14 after 6 hours of reaction, as well as a fluorescence microscope image containing fluorescence of a standard fluorescent substance after 6 hours of reaction were obtained. These wells can be identified based on the fluorescence of a standard fluorescent substance. Furthermore, it was found that in wells where 5-FAM fluorescence was observed, fluorescence of the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of each of SB2, SB6, SB7, SB9, SB10, and SB14 by the MMP in the MDA-MB-231-derived extracellular vesicles was effective in the microcompartment. In this case, 5-FAM fluorescence was observed even in the fluorescence microscopy images of wells without extracellular vesicles, thus revealing the occurrence of false positive results, although the frequency was extremely low.

[0333] Next, although the description is omitted, the fluorescence microscope images of the above-described example after 6 hours of reaction were subjected to predetermined image processing to provide histograms. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. Table 21 shows the total number of wells and the number of positive wells identified from each histogram after 6 hours of reaction, calculated using the above formula (1) relative to the expected value (λ) of the extracellular vesicle concentration tested. The power approximation can be satisfactorily applied to the plot of the expected value λ versus the extracellular vesicle concentration. The extracellular vesicle concentration can be accurately quantified. The approximate equation and R 2 The results are shown in Table 22. In addition, the extracellular vesicle concentration at the x1 concentration calculated from λ and the microcompartment volume of 50 fL at the x1 concentration is also shown in Table 22.

[0334] [Table 21] Table 21

[0335] [Table 22] Table 22

[0336] Thus, it was demonstrated that digital detection of extracellular vesicles is possible by using MMP activity in different reporter molecules.

[0337] Example 36 (holes and hole patterns) Using the hole array of Simoa Discs.

[0338] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling well 204. Take 4 μL (equivalent to 4 μg) of the A549-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add buffer (4) to the aliquot to provide a 400 μL solution. The concentration of this solution is 10 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of this solution is x1 / 2.

[0339] In addition, in order to detect MMP activity, 100 μM SB2, SB6, SB7, SB9, SB10 or SB14 of SensoLyte 520 MMP Substrate Sampler Kit (Fluormetric) AS-71170 and 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were diluted with buffer (4) to prepare an MMP detection solution containing 10 μM SB2, SB6, SB7, SB9, SB10 or SB14 and 2 μM Alexa Fluor 647. 50 μL of each MMP detection solution was added to 50 μL of the sample to provide a reaction solution for the wells. In this case, the concentration of extracellular vesicles in each reaction solution was 5 μg / mL, and the concentrations of the reporter molecule and the standard fluorescent substance in each reaction solution were 5 μM and 1 μM, respectively.

[0340] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as a hydrophobic solvent, and the wells were filled with each reaction solution in the same manner as in Example 19.

[0341] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although the description is omitted, fluorescence microscope images containing both the fluorescence of 5-FAM derived from each of SB2, SB6, SB7, SB9, SB10, and SB14 and the background fluorescence of each of SB2, SB6, SB7, SB9, SB10, and SB14 were obtained after 4 hours and 24 hours of the reaction, and fluorescence microscope images containing the fluorescence of the standard fluorescent substance were obtained after 4 hours and 24 hours of the reaction. These wells can be identified based on the fluorescence of a standard fluorescent substance. Furthermore, it was found that in wells where 5-FAM fluorescence was observed, fluorescence from the standard fluorescent substance was also observed. Therefore, it was confirmed that the wells were filled with the sample and the reagent containing the reporter molecule, and that the cleavage reaction of SB2, SB6, SB7, SB9, SB10, or SB14 by MMPs in A549-derived extracellular vesicles was taking effect in the microcompartments.

[0342] Next, although the description is omitted, the fluorescence microscope images of the above-described example after 4 hours and 24 hours of reaction were subjected to predetermined image processing to provide histograms. Each of these histograms shows the number of wells exhibiting the corresponding fluorescence intensity in each fluorescence intensity interval obtained by dividing the fluorescence intensity into intervals of a constant value. Table 23 shows the total number of wells and the number of positive wells identified from each histogram after 4 and 24 hours of reaction, calculated using the above formula (1) relative to the expected value (λ) of the extracellular vesicle concentration tested. In addition, the extracellular vesicle concentration at the x1 concentration, calculated from the λ at the x1 concentration and the microcompartment volume of 50 fL, is also shown in Table 24.

[0343] [Table 23] Table 23

[0344] [Table 24] Table 24

[0345] Thus, it was demonstrated that digital detection of MMP activity can be performed by using extracellular vesicles derived from different cell lines and different reporter molecules.

[0346] Example 37 (MMP14 activity of MDA-MB-231-derived extracellular vesicles was detected using the following reporter molecules: Customized #11, Customized #12, Customized #13, Customized #14, Customized #15, or Customized #16) 4 μL of the extracellular vesicles derived from MDA-MB-231 described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS) (equivalent to 4 μg of extracellular vesicles derived from MDA-MB-231) was aliquoted, and buffer (4) was added to the aliquoted sample to provide a 400 μL solution. The concentration of the extracellular vesicles derived from MDA-MB-231 in the solution was 10 μg / mL. In Example 8, the concentration of 20 μg / mL was set to x1, so the concentration of the extracellular vesicles derived from MDA-MB-231 in the solution was x1 / 2. In addition, a sample free of extracellular vesicles was similarly prepared and used as a sample for BG measurement. Furthermore, to detect MMP14 activity, 1 mM Customized #11, Customized #12, Customized #13, Customized #14, Customized #15, or Customized #16 was diluted with buffer (4) to prepare a 10 μM MMP14 detection solution. 50 μL of the MMP14 detection solution was added to 50 μL of each sample, and the reaction was carried out at 30° C., and the MMP14 activity was evaluated. In this case, the concentration of MDA-MB-231-derived extracellular vesicles in each reaction solution was 5 μg / mL, and the concentration of the reporter molecule in each reaction solution was 5 μM.

[0347] (Evaluation of MMP activity using 96-well plate) Evaluation was performed in the same manner as in Example 1. Table 25 shows the change in fluorescence intensity after 2 hours from different reporter molecules by MDA-MB-231-derived extracellular vesicles. As shown in Table 25, it was found that MDA-MB-231-derived extracellular vesicles changed the fluorescence intensity.

[0348] [Table 25] Table 25 reporter molecules Fluorescence change (ΔRFU) after 2 hours of reaction at 30°C Custom Item #11 33,665 Custom Item #12 32,801 Custom Item #13 19,279 Custom Item #14 34,878 Custom Item #15 16,774 Custom Item #16 4,424

[0349] Example 38 (holes and hole patterns) Using the hole array of Simoa Discs.

[0350] (Preparation of Reaction Solution for Wells) Prepare a reaction solution for filling well 204. Take 4 μL (equivalent to 4 μg) of the MDA-MB-231-derived extracellular vesicles described in the "Materials, etc." section (50 μg of protein in 50 μL of PBS), and add buffer (4) to the aliquot to provide 800 μL of solution. The concentration of this solution is 5 μg / mL. In Example 8, the concentration of 20 μg / mL is set to x1, so the concentration of this solution is x1 / 4.

[0351] In addition, to detect MMP14 activity, 1 mM Customized #11, Customized #12, Customized #13, Customized #14, Customized #15, or Customized #16 and 1 mM Alexa Fluor 647 serving as a standard fluorescent substance were diluted with buffer (4) to prepare an MMP14 detection solution containing 10 μM Customized #11, Customized #12, Customized #13, Customized #14, Customized #15, or Customized #16 and 2 μM Alexa Fluor 647. 50 μL of the MMP14 detection solution was added to 50 μL of the sample to provide a reaction solution for the wells. In this case, the concentration of extracellular vesicles in the reaction solution was 2.5 μg / mL, and the concentrations of the reporter molecule and the standard fluorescent substance in the reaction solution were 5 μM and 1 μM, respectively.

[0352] (Fill the wells with the reaction solution) Fluorinated oil SR-X Sealing Oil was used as the hydrophobic solvent, and the wells were filled with the reaction solution in the same manner as in Example 19.

[0353] (Fluorescence microscopy observation) The well array prepared above was subjected to a reaction to promote the generation of fluorescence by incubation at 30° C. Thereafter, observation was performed using a fluorescence microscope (BZ-X800). Fluorescence originating from each fluorescent substance was observed under the same conditions as in Example 19 to acquire a fluorescent image. Although the description is omitted, fluorescence microscope images of both the fluorescence of 5-FAM derived from Customized #11, Customized #12, Customized #13, Customized #14, Customized #15, or Customized #16 and the background fluorescence of Customized #11, Customized #12, Customized...

Claims

1. A method for detecting extracellular vesicles having a target enzyme, the method comprising: a partitioning step of partitioning the extracellular vesicles and a reagent comprising a reporter molecule to be altered by a target enzyme to emit a signal into a plurality of independent separation compartments; a signal generating step of allowing the target enzyme and the reporter molecule to react with each other to generate a signal; a signal detection step of detecting the signal; as well as an identifying step comprising determining a signal strength of each of the independent separation compartments based on the detection result obtained in the signal detecting step, and identifying each of the independent separation compartments having a signal strength exceeding a predetermined threshold.

2. The method for detecting extracellular vesicles according to claim 1, wherein The signal detection step includes an image acquisition step of acquiring an image containing the individual separation compartments.

3. The method for detecting extracellular vesicles according to claim 2, wherein: The identifying step includes identifying each of the independent separation compartments having a signal intensity exceeding the predetermined threshold by processing the image acquired in the image acquiring step.

4. The method for detecting extracellular vesicles according to any one of claims 1 to 3, wherein The reporter molecule is labeled.

5. The method for detecting extracellular vesicles according to any one of claims 1 to 4, wherein The reporter molecule is labeled with a fluorescent substance.

6. The method for detecting extracellular vesicles according to any one of claims 1 to 5, wherein The reporter molecule is labeled with a quencher and a fluorescent substance.

7. The method for detecting extracellular vesicles according to any one of claims 1 to 6, wherein The reporter molecule is a molecule that reacts with any one selected from the group consisting of: a hydrolase; an oxidoreductase; a transferase; and an isomerase to emit a signal.

8. The method for detecting extracellular vesicles according to any one of claims 1 to 7, wherein The reporter molecule is a molecule that reacts with any one selected from the following to emit a signal: protease; esterase; glycosidase; oxidase; peroxidase; catalase; superoxide dismutase; acetylase and deacetylase; kinase; protein kinase; glycosyltransferase; cis-trans isomerase; racemase; and mutase.

9. The method for detecting extracellular vesicles according to any one of claims 1 to 8, wherein The reporter molecule is a molecule that reacts with a molecule belonging to the MMP family to emit a signal.

10. The method for detecting extracellular vesicles according to any one of claims 1 to 9, wherein The reporter molecule is a molecule that reacts with MMP14 to emit a signal.

11. The method for detecting extracellular vesicles according to any one of claims 1 to 8, wherein The reporter molecule is a molecule that reacts with AChE to emit a signal. 12 . The method for detecting extracellular vesicles according to claim 1 , further comprising the step of mixing the extracellular vesicles with a capture protein to be bound to the extracellular vesicles to form a complex.

13. The method for detecting extracellular vesicles according to claim 12, wherein: The capture protein will bind to the membrane protein of the extracellular vesicle.

14. The method for detecting extracellular vesicles according to claim 12 or 13, wherein: The capture protein is any one selected from the group consisting of anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-raft antibody, anti-ALIX antibody, anti-syndecan antibody, anti-TSG101 antibody, anti-HSP70 antibody, anti-HSP90 antibody, anti-SNARE antibody, anti-Rab antibody, anti-Annexin antibody, anti-MMP antibody, anti-ADAM antibody, anti-AChE antibody, anti-integrin antibody, anti-selectin antibody and anti-EpCAM antibody.

15. The method for detecting extracellular vesicles according to any one of claims 12 to 14, wherein The capture protein is labeled.

16. The method for detecting extracellular vesicles according to any one of claims 12 to 15, wherein The captured protein is labeled with particles.

17. The method for detecting extracellular vesicles according to claim 16, wherein: The particles have a particle size of 1 μm or more and 10 μm or less.

18. The method for detecting extracellular vesicles according to any one of claims 1 to 16, further comprising a recovery step, the recovery step comprising mixing the extracellular vesicles and a capture protein to be bound to the extracellular vesicles to form a complex, and recovering the formed complex. 19 . The method for detecting extracellular vesicles according to claim 18 , further comprising the step of mixing the complex with a second capture protein to be bound to the extracellular vesicles.

20. The method for detecting extracellular vesicles according to any one of claims 1 to 19, wherein The reagent contains two or more reporter molecules.

21. The method for detecting extracellular vesicles according to claim 20, wherein: The two or more reporter molecules each produce a different signal.

22. The method for detecting extracellular vesicles according to claim 21, wherein The signal detection step includes separately detecting the signal intensity of a signal emitted from each of the two or more reporter molecules.

23. The method for detecting extracellular vesicles according to any one of claims 1 to 22, wherein: The method further comprises the step of staining at least one of the membrane or protein of the extracellular vesicles.

24. The method for detecting extracellular vesicles according to any one of claims 1 to 23, wherein The identifying step includes identifying each of the independent separation compartments having a signal strength exceeding the predetermined threshold based on at least either a ratio or a difference between a signal strength of a reference compartment and a signal strength of each of the independent separation compartments.

25. The method for detecting extracellular vesicles according to claim 24, wherein: The reference compartment is a compartment that does not contain the extracellular vesicles and includes a sample containing a reporter molecule.

26. The method for detecting extracellular vesicles according to any one of claims 1 to 25, wherein Each of the independently separated compartments is a droplet.

27. The method for detecting extracellular vesicles according to any one of claims 1 to 26, wherein Each of the independently separated compartments is a well.

28. The method for detecting extracellular vesicles according to any one of claims 1 to 27, wherein Each of the independent separation chambers has a volume of 0.1 fL or more and 1400 fL or less.

29. A kit for detecting extracellular vesicles with a target enzyme in independently isolated compartments, the kit comprising: a reagent comprising a reporter molecule to be changed by the target enzyme to emit a signal; as well as A container for providing multiple independently separated compartments.

30. A device for detecting extracellular vesicles having a target enzyme, the device comprising: a dispensing unit configured to distribute the extracellular vesicles and a reagent comprising a reporter molecule to be changed by a target enzyme to emit a signal into a plurality of independent separation compartments; a signal generating unit configured to cause the target enzyme and the reporter molecule to react with each other to generate a signal; a signal detection unit configured to detect the signal; as well as An identification unit is configured to determine a signal strength of each of the independent separation compartments based on the detection result obtained by the signal detection unit, and identify each of the independent separation compartments having a signal strength exceeding a predetermined threshold. 31 . A program for causing a computer included in an extracellular vesicle detection device to execute the method for detecting extracellular vesicles according to any one of claims 1 to 28 , so that the extracellular vesicle detection device executes the method.

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