JP7807502B2 - Exosome measurement method and exosome measurement kit - Google Patents

Description

The present invention relates to a method for measuring exosomes and a kit for measuring exosomes.

Exosomes are membrane vesicles with a diameter of approximately 40 to 150 nm that are produced by cells and released extracellularly. Exosomes are surrounded by a phospholipid bilayer membrane and contain a variety of cellular components, such as membrane proteins and microRNA (miRNA). Because different exosomes are released from cells depending on the cell type and state, exosomes are thought to mediate communication between cells and organs.

In recent years, exosomes secreted into bodily fluids such as blood and saliva have attracted attention as diagnostic markers for various diseases, including cancer and infectious diseases. For example, Patent Document 1 discloses an analytical method, analytical reagent, and analytical device for detecting exosomes, which serve as tumor markers. This method uses an antibody against an antigen contained in exosomes and an antibody against an antigen contained in the cells that secrete the exosomes, and detects exosomes based on the avidin-biotin interaction. Furthermore, Patent Document 2 discloses an exosome analysis device and exosome capture method in which antibodies against exosomes are immobilized in recesses in a base made of a synthetic resin or the like with an uneven structure, and exosomes are captured in the recesses.

International Publication No. 2013/094307 JP 2014-219384 A

As mentioned above, various methods for analyzing exosomes have been developed to date, but there is a need for methods that can measure exosomes with greater sensitivity and reproducibility.

The present invention aims to provide an exosome measurement method and exosome measurement kit that can measure the concentration of exosomes in a sample more sensitively and easily than conventional measurement methods.

A method for measuring exosomes according to one embodiment of the present invention includes the steps of: preparing a measurement chip including a metal film and a first binding substance immobilized on the metal film that binds to exosomes; providing a specimen containing exosomes on the metal film and allowing the exosomes contained in the specimen to bind to the first binding substance; labeling the exosomes with a fluorescent substance via a second binding substance that binds to the exosomes before or after binding to the first binding substance; and irradiating the metal film with excitation light so that surface plasmon resonance occurs in the metal film while the fluorescently labeled exosomes are bound to the first binding substance, and detecting the fluorescence emitted from the fluorescent substance, wherein one of the first binding substance and the second binding substance binds to a binding determinant known as an exosome marker, and the other binds to a binding determinant known as a marker for cells that secrete exosomes; and at least one of the first binding substance and the second binding substance is a lectin that can bind to a sugar chain on an exosome .

An exosome measurement kit according to one embodiment of the present invention comprises a measurement chip including a metal film and a first binding substance that binds to exosomes and is immobilized on the metal film; and a labeling reagent for labeling exosomes with a fluorescent substance, wherein the labeling reagent includes a second binding substance that binds to the exosomes.

The present invention makes it possible to measure the concentration of exosomes in a sample more sensitively and easily than conventional measurement methods.

FIG. 1A is a flowchart showing an example of the exosome measurement method according to the present embodiment. FIG. 1B is a flowchart showing another example of the exosome measurement method according to the present embodiment. FIG. 2A is a cross-sectional view illustrating the configuration of a measurement chip for PC-SPFS, and FIG. 2B is a cross-sectional view illustrating the configuration of a measurement chip for GC-SPFS. FIG. 3A is a schematic cross-sectional view showing an example of a measurement chip for PC-SPFS. FIG. 3B is a perspective schematic view showing another example of a measurement chip for PC-SPFS. FIG. 3C is a schematic cross-sectional view showing another example of a measurement chip for PC-SPFS. FIG. 4 is a calibration curve showing the relationship between the number of exosomes and the concentration. FIG. 5 is a standard curve of CD9-positive CD63-positive exosomes. FIG. 6 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S/N), and detection limit for CD9-positive CD63-positive exosomes. FIG. 7 is a calibration curve for CD9-positive, PSMA-positive exosomes. FIG. 8 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S/N), and detection limit for CD9-positive, PSMA-positive exosomes. FIG. 9 shows a calibration curve of CD9-positive CD63-positive exosomes derived from LNCap cells. FIG. 10 is a graph showing the relationship between irradiation energy, signal-to-noise ratio (S/N), and detection limit for CD9-positive CD63-positive exosomes derived from LNCap cells. FIG. 11 shows a calibration curve of CD9-positive exosomes bearing sugar chains derived from LNCap cells, using a lectin that recognizes sugar chains on exosomes. FIG. 12 shows a calibration curve of CD9-positive glycan-bearing exosomes derived from prostate cancer cells, using a lectin that recognizes glycans on exosomes.

Embodiments of the present invention will be described in detail below with reference to the drawings.

[Exosome measurement method]
Exosomes are secreted by both normal cells and disease-related cells such as cancer cells. Therefore, in order to use exosomes as disease markers, a method is needed that can distinguish and detect the large amount of exosomes derived from normal cells present in a sample (e.g., blood) from the smaller amount of exosomes derived from disease-related cells. The concentration of exosomes in serum is generally said to be about 1.0 × 10 ¹¹ cells/ml (1.0 × 10 ⁸ cells/μl). To detect disease-related (e.g., cancer) exosomes from this concentration, a method capable of detecting exosomes at 1.0 × 10 ⁶ cells/ml (1.0 × ^{10 3} cells/μl) or less is required.

In the exosome measurement method according to this embodiment, exosomes are measured using surface plasmon-field enhanced fluorescence spectroscopy (hereinafter also referred to as "SPFS") to improve measurement sensitivity. SPFS uses an electric field enhanced by surface plasmon resonance (hereinafter also referred to as "SPR") to excite fluorescent substances and cause them to emit fluorescence, allowing for more sensitive detection of targets (exosomes in this embodiment) than with general fluorescent immunoassays.

In addition, SPFS can also be used with whole blood as a sample, making it possible to detect disease-related exosomes easily and with minimal steps, without the need to purify exosomes in the blood.

Based on the above idea, the inventors conducted extensive research and discovered that exosomes can be measured with high sensitivity using SPFS, and completed the exosome measurement method according to the present embodiment.

The exosome measurement method according to this embodiment will now be described in detail. Figure 1A is a flowchart showing an example of the exosome measurement method according to this embodiment, which is a measurement method using a primary reaction and a secondary reaction. Figure 1B is a flowchart showing an example of the exosome measurement method according to this embodiment, which is a measurement method using a primary reaction, a secondary reaction, and a tertiary reaction.

(Preparation of measurement chip)
First, a measurement chip containing a metal film and a first binding substance that binds to exosomes is prepared (step S10). In SPFS, SPR is generated by coupling evanescent waves generated when the metal film is irradiated with light (excitation light in this embodiment) with surface plasmons. Known techniques for generating SPR include placing a prism on one side of the metal film (Kretschmann configuration) and forming a diffraction grating on the metal film. SPFS employing the former technique is called prism coupling (PC)-SPFS, and SPFS employing the latter technique is called grating coupling (GC)-SPFS. The exosome measurement method according to this embodiment may employ either PC-SPFS or GC-SPFS.

As mentioned above, the metal film generates SPR when irradiated with excitation light. There are no particular limitations on the type of metal that makes up the metal film, as long as it is a metal that can generate SPR. Examples of metals that make up the metal film include gold, silver, copper, aluminum, and alloys of these.

The first binding substance is capable of binding to exosomes and is immobilized on a metal membrane to capture exosomes in a sample. Typically, the first binding substance is uniformly immobilized in a predetermined region (reaction field) on the metal membrane. The type of first binding substance immobilized on the metal membrane is not particularly limited, as long as it is capable of binding to exosomes, i.e., binds to the first binding determinant possessed by exosomes. Examples of binding substances include antibodies capable of binding to exosomes, nucleic acids capable of binding to exosomes, lipids capable of binding to exosomes, and proteins other than antibodies capable of binding to exosomes, such as lectins capable of binding to glycans on exosomes. Here, the binding determinant possessed by exosomes refers to a portion of the exosome that the binding substance recognizes when binding to the exosome. For example, if the binding substance is an antibody, the binding determinant is the antigenic determinant (epitope) of the antigen. If the binding substance is a ligand, the binding determinant is the binding site of the corresponding receptor.

The first binding determinant on exosomes to which the first binding substance binds is not particularly limited, but includes binding determinants known as exosome markers and binding determinants known as markers of cells that secrete exosomes. Binding determinants known as exosome markers include CD9, CD63, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, and LAMP1. Antibodies against these binding determinants are commercially available, and such antibodies can be used as the first binding substance. When a substance (e.g., an antibody) that reacts with the above-mentioned substance is used as the first binding substance that binds to a binding determinant known as an exosome marker, it binds only to exosomes and is therefore effective for detecting exosomes in a sample.

Binding determinants known as markers for cells that secrete exosomes include caveolin-1, PSMA, EpCAM, Glypican-1, Survivin, CD91, Tspan8, CD147, EGFR, HER2, CD44, galactin, and integrin. Antibodies against these binding determinants are also commercially available, and these antibodies can be used as the first binding substance. When a binding substance (e.g., an antibody) that binds to a binding determinant known as a marker for cells that secrete exosomes is used as the first binding substance, it binds only to specific cells and exosomes secreted therefrom, and is therefore effective for detecting exosomes derived from specific cells in a sample.

When the first binding substance is an antibody, the antibody may be a monoclonal antibody, a polyclonal antibody, or an antibody fragment. Furthermore, the type of first binding substance immobilized on the metal film may be one type, or two or more types. For example, when the first binding substance immobilized on the metal film is an antibody, the antibody may be one or two or more types of monoclonal or polyclonal antibodies.

It is preferable to select the first binding substance taking into consideration its combination with the second binding substance, which will be explained in relation to the fluorescent label. Preferred combinations of the first binding substance and the second binding substance will be described later.

The method for immobilizing the binding substance is not particularly limited. For example, a self-assembled monolayer (hereinafter referred to as "SAM") or polymer film to which a binding substance (e.g., an antibody) is bound may be formed on a metal film. Examples of SAMs include films formed with substituted aliphatic thiols such as HOOC-(CH ₂ ) ₁₁ -SH. Examples of materials that can constitute the polymer film include polyethylene glycol and MPC polymer. Alternatively, a polymer having a reactive group (or a functional group that can be converted into a reactive group) that can bind to a binding substance (e.g., an anti-exosome antibody) may be immobilized on a metal film, and the binding substance (e.g., an antibody) may be bound to this polymer.

The measurement chip is preferably a structure with each piece measuring a few mm to a few cm in length, but may also be a smaller or larger structure that does not fall within the category of a "chip."

Figure 2A is a cross-sectional schematic diagram illustrating the configuration of a measurement chip for PC-SPFS, and Figure 2B is a cross-sectional schematic diagram illustrating the configuration of a measurement chip for GC-SPFS. For ease of explanation, the sizes and shapes of the components in these figures are not accurate. These figures also show an example in which an anti-exosome antibody that recognizes an antigen on exosomes is used as the first binding substance.

As shown in Figure 2A, the measurement chip 100 for PC-SPFS has a prism 110, a metal film 120, and an anti-exosome antibody (first binding substance) 130 (layer) that recognizes an antigen on exosomes. The prism 110 is made of a dielectric material that is transparent to the excitation light L1, and has an incident surface 111 onto which the excitation light L1 is incident, a film-forming surface 112 from which the excitation light L1 is reflected, and an exit surface 113 from which the reflected light L2 is emitted. The shape of the prism 110 is not particularly limited. In the example shown in Figure 2A, the prism 110 is a cylinder with a trapezoidal base. The surface corresponding to one base of the trapezoid is the film-forming surface 112, the surface corresponding to one leg is the incident surface 111, and the surface corresponding to the other leg is the exit surface 113. Examples of materials for the prism 110 include resin and glass. The material of the prism 110 is preferably a resin with a refractive index of 1.4 to 1.6 for the excitation light and low birefringence. The metal film 120 is disposed on the film formation surface 112 of the prism 110. There are no particular limitations on the method for forming the metal film 120. Examples of methods for forming the metal film 120 include sputtering, vapor deposition, and plating. There are no particular limitations on the thickness of the metal film 120, but it is preferably in the range of 30 to 70 nm.

As shown in Figure 2A, when excitation light L1 is irradiated onto metal film 120 via prism 110 so that SPR occurs in metal film 120, an electric field enhanced by SPR is generated near metal film 120. At this time, if exosomes 140 that have reacted with second binding substance 131 labeled with fluorescent substance 150 are bound to anti-exosome antibodies (first binding substances) 130 that recognize antigens on exosomes on metal film 120, fluorescent substance 150 is excited by the enhanced electric field and emits fluorescence L3.

As shown in Figure 2B, the measurement chip 200 for GC-SPFS has a metal film 210 on which a diffraction grating 211 is formed and a layer of anti-exosome antibodies (first binding substances) 130 that recognize antigens on exosomes. The method for forming the metal film 210 is not particularly limited. Examples of methods for forming the metal film 210 include sputtering, vapor deposition, and plating. The thickness of the metal film 210 is not particularly limited, but is preferably within the range of 30 to 500 nm. The shape of the diffraction grating 211 is not particularly limited as long as it can generate evanescent waves. For example, the diffraction grating 211 may be a one-dimensional diffraction grating or a two-dimensional diffraction grating. For example, in a one-dimensional diffraction grating, multiple parallel convex portions are formed at predetermined intervals on the surface of the metal film 210. In a two-dimensional diffraction grating, convex portions of a predetermined shape are periodically arranged on the surface of the metal film 210. Examples of convex arrangements include a square lattice and a triangular (hexagonal) lattice. Examples of the cross-sectional shape of the diffraction grating 211 include a rectangular wave shape, a sine wave shape, a sawtooth shape, etc. The method for forming the diffraction grating 211 is not particularly limited. For example, a metal film 210 may be formed on a flat substrate (not shown), and then an uneven shape may be imparted to the metal film 210. Alternatively, the metal film 210 may be formed on a substrate (not shown) that has already been imparted with an uneven shape. Either method can form the metal film 210 including the diffraction grating 211.

As shown in Figure 2B, when excitation light L1 is irradiated onto the metal film 210 (diffraction grating 211) so that SPR occurs in the metal film 210 (diffraction grating 211), an electric field enhanced by SPR is generated near the metal film 210 (diffraction grating 211). At this time, if exosomes 140 that have reacted with second binding substance 131 labeled with fluorescent substance 150 are bound to anti-exosome antibodies (first binding substances) 130 that recognize antigens on exosomes on the metal film 210 (diffraction grating 211), the fluorescent substance 150 is excited by the enhanced electric field and emits fluorescence L3.

Figures 3A to 3C are cross-sectional schematic diagrams showing an example of a measurement chip for PC-SPFS. As shown in Figure 3A, the measurement chip 300 has a prism 110 having an incident surface 111, a deposition surface 112, and an exit surface 113, a metal film 120 formed on the deposition surface 112 of the prism 110, and a channel cover 310 disposed on the deposition surface 112 of the prism 110 or the metal film 120. In Figure 3, the incident surface 111 and the exit surface 113 are located in front of and behind the page, respectively. The measurement chip 300 also has a channel 320, a liquid injection section 330 connected to one end of the channel 320, and a reservoir section 340 connected to the other end of the channel 320. In this embodiment, the channel lid 310 is adhered to the metal film 120 (or prism 110) via an adhesive layer 350 such as double-sided tape, and the adhesive layer 350 also plays a role in defining the side shape of the channel 320. Although not shown in FIG. 3 , an anti-exosome antibody (first binding substance) 130 that recognizes an antigen on an exosome is immobilized in a partial region (reaction field) of the metal film 120 exposed within the channel 320. The liquid injection section 330 is closed by a liquid injection section covering film 331, and the storage section 340 is closed by a storage section covering film 341. The storage section covering film 341 has an air vent 342.

The flow channel lid 310 is formed from a material that is transparent to the fluorescence L3. However, as long as this does not interfere with the extraction of the fluorescence L3, a portion of the flow channel lid 310 may be formed from a material that is opaque to the fluorescence L3. Examples of materials that are transparent to the fluorescence L3 include resin. The flow channel lid 310 may be joined to the metal film 120 (or prism 110) by laser welding, ultrasonic welding, or pressure bonding using a clamp member, without using the adhesive layer 350. In this case, the side shape of the flow channel 320 is determined by the flow channel lid 310.

A pipette tip is inserted into the liquid injection section 330. At this time, the opening of the liquid injection section 330 (the through-hole provided in the liquid injection section covering film 331) comes into contact with the outer periphery of the pipette tip without any gaps. Therefore, liquid can be introduced into the flow path 320 by injecting it from the pipette tip into the liquid injection section 330, and the liquid in the flow path 320 can be removed by sucking it into the pipette tip. Furthermore, by alternately injecting and sucking the liquid, it is possible to transport the liquid back and forth within the flow path 320.

When an amount of liquid introduced from the liquid injector 330 into the flow channel 320 exceeds the volume of the flow channel 320, the liquid flows into the reservoir 340 from the flow channel 320. Liquid also flows into the reservoir 340 when the liquid is pumped back and forth within the flow channel 320. The liquid that flows into the reservoir 340 is agitated within the reservoir 340. Agitation of the liquid within the reservoir 340 makes the concentrations of components (e.g., exosomes, cleaning components, etc.) in the liquid (such as the specimen or cleaning solution) passing through the flow channel 320 uniform, making it easier for various reactions to occur within the flow channel 320 and improving the cleaning effect.

Figures 3B and 3C are schematic diagrams showing an example of a well-shaped measurement chip. Figure 3B is a schematic diagram of a well-shaped measurement chip 400 with a reaction detection unit on the bottom surface (see, for example, International Publication No. WO 2012/157403). In this chip, the dielectric member 412 is a hexahedron with a roughly trapezoidal cross section (truncated square pyramid), and the well member 418 is configured as a rectangle to match the shape of the dielectric member 412. Then, with a first binding substance that binds to the exosomes to be detected immobilized in the ligand immobilization region 416 on the metal thin film 414 of the sensor structure 22, a sample solution containing exosomes is supplied into the through-hole 420, and the sensor structure 422 is agitated.

Figure 3C also shows a well in which the reaction detection unit is located on the sidewall surface (see, for example, International Publication No. WO 2018/021238). The detection chip 500 has a well body 510 and a sidewall member 520. The well body 510 has a storage unit (well) 511 inside. The storage unit 511 is a bottomed recess configured to store liquid and is open to the outside through a first opening 512 at the top and a second opening 513 at the side. The sidewall member 520 has a prism 521 as an optical element, a metal film 525, and a reaction field 526. The prism 521 is an optical element made of a dielectric material that is transparent to excitation light and has an incident surface (not shown), a reflecting surface 523, and an exit surface (not shown). The prism 521 also functions as a sidewall that constitutes the storage unit 511. There is a capture region on the metal film 525, which is an area where a first binding substance for capturing exosomes in a sample is immobilized.

The type of sample is not particularly limited as long as it contains exosomes. Examples of samples include body fluids such as blood (serum, plasma, whole blood), urine, sweat, saliva, breast milk, semen, lymph, cerebrospinal fluid, and tears, as well as dilutions of these body fluids with physiological saline or buffer solutions. In the exosome detection method according to this embodiment, whole blood can also be used as the sample because exosomes are detected using SPFS. Therefore, from the standpoint of ease of acquisition, serum, plasma, whole blood, or dilutions of these are preferred samples. These samples may contain microvesicles, impurities, and the like in addition to exosomes. In the present invention, samples can be used directly for measurement without removal by centrifugation or filtering. However, there is no problem with using a purified liquid after centrifugation or filtering to remove or precipitate microvesicles and impurities for measurement. Furthermore, the method for removing microvesicles, impurities, and the like is not limited to these.

The specimen may also be an exosome extract prepared from a body fluid. By extracting exosomes from a body fluid and using them as a specimen, it becomes possible to detect and quantify even trace amounts of exosomes, which are difficult to detect in the body fluid itself. There are no particular limitations on the method for extracting exosomes, and any conventionally known method for extracting exosomes can be used. For example, exosomes can be extracted from body fluids by ultracentrifugation, immunoprecipitation, or polymer precipitation.

A surfactant may also be added to the sample. Adding a surfactant prevents exosome aggregation and reduces background noise and variability in measurement values. Surfactants widely used in the biological and medical fields are preferred, including Tween 20, sodium deoxycholate, and Triton-X100. Surfactants do not destroy exosomes, but they can be used at a concentration that prevents their aggregation. For example, Tween 20 can be used at a concentration of 0.001% to 5%, preferably 0.005% to 1%, and more preferably 0.010% to 0.05%. Sodium deoxycholate can be used at a concentration of 0.001% to 0.025%, preferably 0.002% to 0.020%, and more preferably 0.003% to 0.010%. In addition, Triton-X100 can be used at a concentration of 0.001% to 0.010%, preferably 0.002% to 0.007%, and more preferably 0.003% to 0.005%.

(first order reaction)
Next, a sample is provided on the metal film of the measurement chip, and exosomes contained in the sample are allowed to bind to the first binding substance immobilized on the metal film (primary reaction; step S20). The method for providing the sample is not particularly limited. For example, the sample may be provided on the metal film using a pipette with a pipette tip attached to the end. While there is no particular limitation on the reaction time of the primary reaction, a longer reaction time is preferable from the viewpoint of increasing the reaction efficiency between the exosomes and the first binding substance, and is typically 5 to 180 minutes, preferably 60 to 150 minutes, and more preferably 100 to 120 minutes. After the primary reaction is completed, the surface of the metal film is typically washed with a buffer solution or the like to remove components not bound to the first binding substance (washing; step S21).
After cleaning, the optical blank can be measured (measuring the optical blank; step S2).

(Second-order reaction)
Next, a labeling reagent is provided on the metal film of the measurement chip, and exosomes bound to the binding substance are labeled with a fluorescent substance via a second binding substance that binds to the exosomes. The type of labeling reagent is not particularly limited as long as the fluorescently labeled second binding substance can react with exosomes bound to the first binding substance. For example, the labeling reagent is a fluorescently labeled second binding substance that binds to a second binding determinant on the exosomes (secondary reaction + fluorescent labeling; step S30 in Figure 1A). Alternatively, the labeling reagent may include both a second binding substance that binds to a second binding determinant on the exosomes and a fluorescently labeled third binding substance that binds to the second binding substance bound to the exosomes (secondary reaction; step S30 and fluorescent labeling (tertiary reaction); step S35 in Figure 1B).

The method for providing the labeling reagent is not particularly limited. For example, the labeling reagent may be provided on the metal film using a pipette equipped with a pipette tip. Typically, after the second and/or third reactions are completed, the surface of the metal film is washed with a buffer solution or the like to remove the second binding substance (or third binding substance) that is not bound to exosomes (washing; steps S31 and S36).

The type of second binding substance contained in the labeling reagent is not particularly limited as long as it can bind to exosomes. Examples of second binding substances include antibodies capable of binding to exosomes, nucleic acids capable of binding to exosomes, lipids capable of binding to exosomes, and proteins other than antibodies capable of binding to exosomes, such as lectins capable of binding to glycans on exosomes.

The second binding determinant on exosomes to which the second binding substance binds is not particularly limited, but includes binding determinants known as exosome markers and binding determinants known as markers of cells that secrete exosomes. Binding determinants known as exosome markers include CD9, CD63, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, and LAMP1. Antibodies against these binding determinants are commercially available, and such antibodies can be used as the second binding substance. When a substance (e.g., an antibody) that reacts with the above-mentioned substances is used as the second binding substance that binds to a binding determinant known as an exosome marker, it will bind only to exosomes, and is therefore effective for detecting only exosomes, even if other entities (e.g., cells) are bound to the metal film.

Binding determinants known as markers of exosome-secreting cells include caveolin-1, PSMA, EpCAM, Glypican-1, Survivin, CD91, Tspan8, CD147, EGFR, HER2, CD44, galactin, and integrin. Antibodies against these binding determinants are also commercially available, and these antibodies can be used as the second binding substance. When a binding substance (e.g., an antibody) that binds to a binding determinant known as a marker of exosome-secreting cells is used as the second binding substance, it binds only to specific cells and exosomes secreted therefrom, and is therefore effective in detecting only exosomes derived from specific cells from among the various exosomes bound to the metal film.

When the second binding substance is an antibody, the antibody may be a monoclonal antibody, a polyclonal antibody, or an antibody fragment. The labeling reagent may contain one or more types of second binding substances. For example, the anti-exosome antibody labeled with a fluorescent substance is one or more types of anti-exosome monoclonal antibodies or anti-exosome polyclonal antibodies. In this case, the anti-exosome monoclonal antibody and anti-exosome polyclonal antibody labeled with a fluorescent substance are preferably different from the one or more types of anti-exosome monoclonal antibodies immobilized on the metal film.

The type of second binding substance contained in the labeling reagent may be the same as or different from the type of first binding substance immobilized on the metal film. For example, both the first binding substance and the second binding substance may be antibodies, or one may be an antibody and the other a protein other than an antibody.

The first binding substance immobilized on the metal film and the second binding substance contained in the labeling reagent each bind to a binding determinant possessed by exosomes, but it is preferable that the first binding determinant bound by the first binding substance is different from the second binding determinant bound by the second binding substance. By having the first binding substance and the second binding substance bind to different binding determinants rather than competing for the same binding determinant, it becomes possible to more reliably immobilize exosomes on the metal film and label them with the labeling substance. Furthermore, by having the first binding determinant bound by the first binding substance and the second binding determinant bound by the second binding substance differ, it is possible to further narrow down the exosomes to be measured based on binding of the second binding substance from among the exosomes detected from a sample based on binding of the first binding substance.

For the first binding substance and the second binding substance, at least one of the first binding determinant and the second binding determinant is preferably a binding determinant known as an exosome marker. By using a binding substance that binds to a binding determinant known as an exosome marker, it is possible to detect only exosomes from substances in a sample. Furthermore, it is preferable that at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for cells that secrete exosomes. By using a binding substance that binds to a binding determinant known as a marker for cells that secrete exosomes, it is possible to detect exosomes derived from specific cells. In the present invention, it is more preferable that one of the first binding substance and the second binding substance binds to a binding determinant known as a marker for exosomes, and the other binds to a binding determinant known as a marker for cells that secrete exosomes. By using a combination of these two binding substances, it is possible to specifically detect exosomes secreted by specific cells. For example, cancer cells are known to secrete exosomes that are different from those of normal cells. Therefore, by detecting exosomes derived from cancer cells in body fluids, exosomes can be used as cancer markers.

The third binding substance contained in the labeling reagent is not particularly limited as long as it can be labeled with a fluorescent substance and can bind to the second binding substance. Examples of third binding substances include antibodies, nucleic acids, lipids, and proteins other than antibodies (e.g., lectins) that can bind to the antibodies, nucleic acids, lipids, and proteins other than antibodies used as the second binding substance. For example, using a combination of second and third binding substances such that multiple third binding substances bind to one second binding substance allows more fluorescent substance to bind to exosomes than when the second binding substance is directly fluorescently labeled, thereby improving measurement sensitivity. Furthermore, exosomes can be labeled using a third binding substance even in cases where it is difficult to directly label the second binding substance with a fluorescent substance, or where fluorescent labeling changes, reduces, or eliminates the binding ability of the second binding substance to exosomes.

The type of fluorescent substance used to label the second or third binding substance is not particularly limited as long as it is compatible with SPFS. Examples of fluorescent substances include cyanine dyes, Alexa Fluor® dyes from Thermo Scientific, and CF dyes from Biotium. Among commercially available fluorescent dyes, Alexa Fluor dyes and CF dyes have high quantum efficiency for the wavelength of excitation light used in SPFS. CF dyes also exhibit little bleaching during fluorescence detection, allowing for stable fluorescence detection. The method for labeling the binding substance with a fluorescent substance is not particularly limited and can be appropriately selected from known methods. For example, the fluorescent substance can be bound to an amino group or sulfhydryl group of the binding substance (e.g., an anti-exosome antibody).

In the above explanation, exosomes are bound to a first binding substance immobilized on a metal film, and then the exosomes are labeled with a fluorescent substance using a labeling reagent. However, exosomes may also be labeled with a fluorescent substance using a labeling reagent before binding to the first binding substance immobilized on the metal film. In this case, the sample and labeling reagent (second binding substance) can be mixed before providing the sample on the metal film. Alternatively, a fluorescently labeled third binding substance can be added to the mixture of the sample and labeling reagent (second binding substance). Furthermore, the step of binding exosomes to a binding substance immobilized on a metal film and the step of labeling the exosomes with a fluorescent substance can be performed simultaneously. In this case, the sample and labeling reagent (second binding substance, or second and third binding substances) can be provided on the metal film simultaneously.

(Fluorescence measurement)
Next, fluorescence indicating the amount of exosomes is measured by SPFS (step S40). Specifically, while fluorescently labeled exosomes are bound to the binding substance immobilized on the metal film, excitation light is irradiated onto the metal film to generate SPR in the metal film, and the fluorescence emitted from the fluorescent substance is measured. Typically, a signal value correlating with the amount of exosomes is calculated by subtracting a previously measured optical blank value from the measured fluorescence value. If necessary, the signal value may be converted to the amount (number/ml) or concentration (μg/ml) of exosomes using a previously prepared calibration curve.

For example, a calibration curve for exosomes can be obtained by preparing samples at different dilutions using commercially available exosomes, determining the number and concentration of exosomes in each sample, determining the signal value using the method of the present invention, and plotting the signal value against the number and concentration of exosomes. Alternatively, the number and concentration of exosomes in a sample can be measured separately to obtain a calibration curve (Figure 4) in which the number of exosomes is plotted against the exosome concentration. Using such a calibration curve, the amount of exosomes counted as a number can be converted to a concentration (μg/ml). This conversion also enables comparison with other companies' devices that detect exosome amount as a concentration (μg/ml).

The method for measuring the number of exosomes is not particularly limited, but for example, the qNano/Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.) can be used. The method for measuring the concentration of exosomes is also not particularly limited, but for example, protein quantification (BCA method, manufactured by ThermoFischer) can be used.

As shown in Figure 2A, when using a measurement chip 100 for PC-SPFS, excitation light L1 is irradiated onto the metal film 120 via the prism 110. This causes SPR in the metal film 120, and the fluorescent substance 150 present near the metal film 120 is excited by the enhanced electric field and emits fluorescence L3. The angle of incidence of the excitation light L1 on the metal film 120 is set so that SPR occurs in the metal film 120, and is preferably the resonance angle or enhancement angle. Here, "resonance angle" refers to the angle of incidence at which the amount of reflected light L2 is minimized when the angle of incidence of the excitation light L1 on the metal film 120 is scanned. Furthermore, "enhancement angle" refers to the angle of incidence at which the amount of scattered light (plasmon scattered light) of the same wavelength as the excitation light L1 emitted above the metal film 120 (the opposite side of the prism 110) is maximized when the angle of incidence of the excitation light L1 on the metal film 120 is scanned.

As shown in Figure 2B, when using a measurement chip 200 for GC-SPFS, excitation light L1 is directly irradiated onto the metal film 210 (diffraction grating 211). This causes SPR in the metal film 210 (diffraction grating 211), and the fluorescent substance 150 present near the metal film 210 (diffraction grating 211) is excited by the enhanced electric field and emits fluorescence L3. The angle of incidence of excitation light L1 with respect to the metal film 210 is set so that SPR occurs in the metal film 210, but it is preferable that the angle be such that the intensity of the enhanced electric field formed by SPR is strongest. The optimal angle of incidence of excitation light L1 is set appropriately depending on the pitch of the diffraction grating 211, the wavelength of excitation light L1, the type of metal constituting the metal film 210, etc.

The type of excitation light is not particularly limited, but is typically laser light. For example, the excitation light is laser light emitted from a laser light source with an output of 10 μW to 30 mW. The irradiation energy of the excitation light is 7.5 μW/ ^mm² to 30 mW/ ^mm² , preferably 8.5 μW/ ^mm² to 10 mW/ ^mm² , and more preferably 9.5 μW/ ^mm² to 5 mW/ ^mm² . By setting the irradiation energy of the excitation light to 30 mW/ ^mm² or less, the fluorescence intensity is increased, the signal-to-noise ratio (S/N) is increased, and smaller amounts of exosomes can be detected, thereby improving the detection limit. The wavelength of the excitation light is appropriately set depending on the excitation wavelength of the fluorescent substance used. Furthermore, if the light intensity exceeds 30 mW/ ^mm² , dissociation of the antigen-antibody reaction due to the heat of irradiation progresses, resulting in a poor signal-to-noise ratio.

The fluorescence detector is preferably installed in the direction where the fluorescence intensity is highest relative to the measurement chip. For example, as shown in Figure 2A, when using a measurement chip 100 for PC-SPFS, the direction where the fluorescence intensity L3 is highest is the normal direction of the metal film 120, so the detector is installed directly above the measurement chip. On the other hand, as shown in Figure 2B, when using a measurement chip 200 for GC-SPFS, the direction where the fluorescence intensity L3 is highest is a direction that is somewhat inclined relative to the normal line of the metal film 120, so the detector is installed in a position that is not directly above the measurement chip. The detector may be, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

The above procedure allows the concentration of exosomes contained in the sample to be measured.

[Exosome measurement kit]
The exosome measurement kit according to this embodiment is a set comprising the above-described measurement chip and the above-described labeling reagent (comprising a fluorescent substance, a second binding substance, and optionally a third binding substance). Preparing the above-described measurement chip and labeling reagent in advance in this manner allows users (e.g., medical professionals) to more easily perform the above-described exosome measurement method. The measurement kit may further comprise a surfactant. The use of a surfactant can reduce background noise and variability in measurement values.

[effect]
As described above, according to the exosome measurement method or measurement kit of the present embodiment, exosomes in a sample can be measured with high sensitivity and ease using SPFS.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Experiment 1: Measurement of exosomes from the blood of healthy subjects using antibodies against two types of exosomes A measurement chip 300 with the configuration shown in Figure 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance to a specific region (reaction area) of the metal film 120 (thin gold film) exposed within the flow channel 320.

Standard exosomes (lyophilized product) derived from healthy individuals purchased from Cosmo Bio Co., Ltd. were hydrated and used as specimen samples. Dilutions were made in a 10-fold dilution series using PBS containing 1% BSA.
The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

The number of exosomes in each sample was measured at different dilution rates. Measurements were performed using a qNano/Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.).

Furthermore, the signal value correlating with the amount of exosomes was measured for the same sample as above. The sample (either diluted blood) was introduced into the flow channel 320 from the liquid injection unit 330 using a pipette tip, and the liquid was pumped back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection unit 330, the inside of the flow channel 320 was washed once with a cleaning solution. Next, a labeled reagent (an anti-CD63 monoclonal antibody labeled with an Alexa Fluor dye) was introduced into the flow channel 320 from the liquid inlet 330 and sent back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. The labeled reagent in the flow channel 320 was removed from the liquid inlet 330, and the flow channel 320 was washed once with a cleaning solution. Next, a measurement solution was introduced into the flow channel 320 from the liquid inlet 330. In this state, the fluorescence value was measured using SPFS. That is, excitation light (laser light) was irradiated onto the metal film 120 from the prism 110 side so that the angle of incidence of the excitation light with respect to the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the irradiation energy amount was 3.8 mW/ ^mm2 . The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate a signal value correlated with the amount of exosomes. The same measurement was performed six times for each sample.

The measured signal values were plotted against the number of exosomes (cells/μl) in the sample to obtain a calibration curve for CD9-positive CD63-positive exosomes (Figure 5).

As is clear from the plot in Figure 5, there was a correlation between the number of exosomes in the sample and the signal value, and the detection limit was low at 2.1 x ¹⁰² /µl.

Next, the signal value correlating with the amount of exosomes was calculated in the same manner as above, except that the output of the excitation light used for detection was changed. The output of the excitation light used for detection was 5 mW, 15 mW, or 30 mW, and the respective irradiation energy amounts were 3.8 mW/mm ² , 11.3 mW/mm ² , or 22.6 mW/mm ^2. For each irradiation energy amount, the detection limit when measuring exosomes from the sample was determined in the same manner as above.

In addition, the signal-to-noise ratio (S/N) for the above measurements was calculated as follows: The signal value (S) was calculated by subtracting the optical blank from the measurement signal in the presence of exosomes, and the noise value (N) was calculated by subtracting the optical blank from the measurement signal in the absence of exosomes. Next, the S/N ratio was calculated.

The calculated S/N and detection limit were plotted against the amount of irradiation energy, and a graph (Figure 6) was obtained showing the relationship between irradiation energy, S/N, and detection limit for CD9-positive CD63-positive exosomes derived from healthy individuals.

In the plots in Figure 6, when the irradiation energy of the excitation light used to measure exosomes from a sample was low, the S/N ratio increased (i.e., the noise to the signal decreased) and the detection limit was improved.

Experiment 2: Measurement of exosomes associated with prostate cancer using antibodies against exosome-secreting cells and antibodies against exosomes

A measurement chip 300 with the configuration shown in Figure 3 was prepared. An anti-PSMA monoclonal antibody was immobilized as a first binding substance in a specific region (reaction area) of the metal film 120 (thin gold film) exposed within the flow channel 320.

Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell line, purchased from Cosmo Bio Co., Ltd. were hydrated and used as specimen samples. Dilutions were made in a 10-fold dilution series using PBS containing 1% BSA.
The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

The number of LNCap cell-derived exosomes in the samples was measured. Measurements were performed using a qNano/Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.).

Furthermore, to create a calibration curve for CD9-positive PSMA-positive exosomes derived from LNCap cells using the same samples as above, signal values correlating with the amount of exosomes were measured for each sample at different dilutions. A sample (either diluted culture supernatant) was introduced into the flow channel 320 from the liquid injection unit 330 using a pipette tip, and the liquid was pumped back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, an anti-CD9 monoclonal antibody labeled with a labeling reagent (Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection unit 330, and the liquid was pumped back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. After removing the labeling reagent from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, the measurement solution was introduced into the flow channel 320 from the liquid injection unit 330. In this state, the fluorescence value was measured using SPFS. That is, excitation light (laser light) was irradiated onto the metal film 120 from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW/ ^mm2 . The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate a signal value correlated with the amount of exosomes. The same measurement was performed six times for each sample.

The measured signal values were plotted against the number of exosomes (cells/μl) in the sample to obtain a calibration curve for CD9-positive, PSMA-positive exosomes (Figure 7).

As is clear from the plot in Figure 7, the above method was able to detect exosomes (CD9-positive, PSMA-positive exosomes) derived from specific cells (prostate cancer cells). Furthermore, there was a correlation between the number of CD9-positive, PSMA-positive exosomes and the signal value, and the detection limit was low at 1.7 x ¹⁰² cells/µl.

Next, the signal value correlating with the amount of CD9-positive, PSMA-positive exosomes was calculated in the same manner as above, except that the output of the excitation light used for detection was changed. The output of the excitation light used for detection was 5 mW, 15 mW, or 30 mW, and the respective irradiation energy amounts were 3.8 mW/mm ² , 11.3 mW/mm ² , and 22.6 mW/mm ^2. For each irradiation energy amount, the detection limit when measuring exosomes from the sample was determined in the same manner as above.

In addition, the signal-to-noise ratio (S/N) was calculated for the above measurements in the same way as in Experiment 1.

The calculated S/N and detection limit were plotted against the amount of irradiation energy, and a graph (Figure 8) was obtained showing the relationship between irradiation energy, S/N, and detection limit for CD9-positive, PSMA-positive exosomes.

In the plot of Figure 8, when the irradiation energy of the excitation light used to measure CD9-positive, PSMA-positive exosomes was low, the S/N ratio increased (i.e., the noise to signal decreased), and the detection limit also improved.

Experiment 3: Measurement of exosomes derived from prostate cancer cells using two types of exosome antibodies

A measurement chip 300 with the configuration shown in Figure 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance in a specific region (reaction area) of the metal film 120 (thin gold film) exposed within the flow channel 320.

Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell line, purchased from Cosmo Bio Co., Ltd. were hydrated and used as specimen samples. Dilutions were made in a 10-fold dilution series using PBS containing 1% BSA.
The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

To create a calibration curve for CD9+CD63+ exosomes derived from LNCap cells, signal values correlating with the amount of exosomes were measured for each sample at different dilutions. A sample (either diluted culture supernatant) was introduced into the flow channel 320 from the liquid injection unit 330 using a pipette tip, and the liquid was pumped back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, a labeled reagent (an anti-CD63 monoclonal antibody labeled with Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection unit 330, and the liquid was pumped back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. After removing the labeled reagent from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, the measurement solution was introduced into the flow channel 320 from the liquid injection unit 330. In this state, the fluorescence value was measured using SPFS. That is, excitation light (laser light) was irradiated onto the metal film 120 from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW/ ^mm2 . The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate a signal value correlated with the amount of exosomes. The same measurement was performed six times for each sample.

The same samples as above were used to measure the number of LNCap cell-derived exosomes in the samples. The number of extracted exosomes was measured using a qNano/Nanoparticle Multi-Analyzer (Meiwafosis Co., Ltd.). The weight was determined by protein quantification (BCA method, ThermoFischer).

The measured signal values were plotted against the number of exosomes (cells/μl) in the sample to obtain a calibration curve for CD9-positive CD63-positive exosomes (Figure 9).

As is clear from the plots in Figure 9, the above method was able to detect CD9+CD63+ exosomes even in samples derived from specific cells (prostate cancer cells). Furthermore, there was a correlation between the number of CD9+CD63+ exosomes and the signal value, and the detection limit was low at 3.2 x ¹⁰⁰ cells/µL.

Next, the signal value correlating with the amount of CD9-positive CD63-positive exosomes was calculated in the same manner as above, except that the output of the excitation light used for detection was changed. The output of the excitation light used for detection was 5 mW, 15 mW, or 30 mW, and the respective irradiation energy amounts were 3.8 mW/mm ² , 11.3 mW/mm ² , and 22.6 mW/mm ^2. For each irradiation energy amount, the detection limit when measuring exosomes from the sample was determined in the same manner as above.

In addition, the signal-to-noise ratio (S/N) was calculated for the above measurements in the same way as in Experiment 1.

The calculated S/N and detection limit were plotted against the amount of irradiation energy, and a graph (Figure 10) was obtained showing the relationship between irradiation energy, S/N, and detection limit for CD9-positive CD63-positive exosomes.

In the plot of Figure 10, when the irradiation energy of the excitation light used to measure CD9-positive CD63-positive exosomes was low, the S/N ratio increased (i.e., the noise to the signal decreased), and the detection limit also improved.

Experiment 4: Measurement of CD9-positive glycan-bearing exosomes using an antibody against exosomes and WFA lectin, which recognizes glycans on exosomes.

A measurement chip 300 with the configuration shown in Figure 3 was prepared. An anti-CD9 monoclonal antibody was immobilized as a first binding substance in a specific region (reaction area) of the metal film 120 (thin gold film) exposed within the flow channel 320.

Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell line, purchased from Cosmo Bio Co., Ltd. were hydrated and used as specimen samples. Dilutions were made in a 10-fold dilution series using PBS containing 1% BSA.
The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

To create a calibration curve for CD9-positive glycan-bearing exosomes derived from LNCap cells, signal values correlating with the amount of exosomes were measured for each sample at different dilutions. A sample (either diluted culture supernatant) was introduced into the flow channel 320 from the liquid injection unit 330 using a pipette tip, and the liquid was pumped back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, a labeled reagent (WFA lectin labeled with Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection unit 330, and the liquid was pumped back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. After removing the labeled reagent from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, the measurement solution was introduced into the flow channel 320 from the liquid injection unit 330. In this state, the fluorescence value was measured using SPFS. That is, excitation light (laser light) was irradiated onto the metal film 120 from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW/ ^mm2 . The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate a signal value correlated with the amount of exosomes. The same measurement was performed six times for each sample.

The number of LNCap cell-derived exosomes in the same samples as above was measured. The number of extracted exosomes was measured using a qNano/Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.).

The measured signal values were plotted against the number of exosomes (cells/μl) in the sample to obtain a calibration curve for CD9-positive glycan-bearing exosomes (Figure 11).

As is clear from the plots in Figure 11, the above method was able to detect CD9-positive glycan-bearing exosomes even in samples derived from specific cells (prostate cancer cells). Furthermore, there was a correlation between the number of CD9-positive glycan-bearing exosomes and the signal value, and the detection limit was low at 9.9 x ¹⁰ cells/µl.

Experiment 5: Measurement of exosomes derived from prostate cancer cells using antibodies against exosome-secreting cells and lectins that recognize glycans on exosomes

A measurement chip 300 with the configuration shown in Figure 3 was prepared. An anti-PSMA monoclonal antibody was immobilized as a first binding substance in a specific region (reaction area) of the metal film 120 (thin gold film) exposed within the flow channel 320.

Standard exosomes (lyophilized product) derived from LNCap cells, a type of prostate cancer cell line, purchased from Cosmo Bio Co., Ltd. were hydrated and used as specimen samples. Dilutions were made in a 10-fold dilution series using PBS containing 1% BSA.
The sample dilution solution in the device contained a surfactant, and the concentration of the surfactant was 0.05%.

The number of LNCap cell-derived exosomes in the samples was measured. Measurements were performed using a qNano/Nanoparticle Multi-Analyzer (manufactured by Meiwafosis Co., Ltd.).

Furthermore, to create a calibration curve for PSMA-positive glycan-bearing exosomes derived from LNCap cells, the signal value correlating with the amount of exosomes was measured for each sample at different dilutions. The sample (either diluted culture supernatant) was introduced into the flow channel 320 from the liquid injection unit 330 using a pipette tip, and the liquid was pumped back and forth (primary reaction). The reaction time for the primary reaction was 100 minutes. After removing the sample from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, WFA lectin labeled with a labeling reagent (Alexa Fluor dye) was introduced into the flow channel 320 from the liquid injection unit 330, and the liquid was pumped back and forth (secondary reaction). The reaction time for the secondary reaction was 10 minutes. After removing the labeling reagent from the flow channel 320 from the liquid injection unit 330, the flow channel 320 was washed once with a cleaning solution. Next, the measurement solution was introduced into the flow channel 320 from the liquid injection unit 330. In this state, the fluorescence value was measured using SPFS. That is, excitation light (laser light) was irradiated onto the metal film 120 from the prism 110 side so that the angle of incidence of the excitation light on the metal film 120 was an enhancement angle, and the fluorescence emitted at that time was detected. The output of the excitation light used for detection was 5 mW, and the amount of irradiation energy was 3.8 mW/ ^mm2 . The optical blank value measured in advance was subtracted from the obtained fluorescence value to calculate a signal value correlated with the amount of exosomes. The same measurement was performed six times for each sample.

The measured signal values were plotted against the number of exosomes (cells/μl) in the sample to obtain a calibration curve for PSMA-positive glycan-bearing exosomes (Figure 12).

As is clear from the plot in Figure 12, the above method was able to detect exosomes (exosomes bearing PSMA-positive glycans) derived from specific cells (prostate cancer cells). Furthermore, there was a correlation between the number of PSMA-positive glycan-bearing exosomes and the signal value, and the detection limit was low at 2.5 x ¹⁰² /µL.

This application claims priority from Japanese Patent Application No. 2019-119824, filed June 27, 2019. The entire contents of that application are incorporated herein by reference.

By using the exosome measurement method or measurement kit according to this embodiment, exosomes can be measured with high sensitivity and ease. Therefore, the exosome detection method and measurement kit according to the present invention are useful, for example, in clinical testing.

100, 200, 300, 400, 500 Measurement chip 110 Prism 111 Incident surface 112 Film formation surface 113 Emitting surface 120 Metal film 130 Anti-exosome antibody (first binding substance)
131 Anti-exosome antibody (second binding substance)
140 Exosome 150 Fluorescent substance 210 Metal film 211 Diffraction grating 310 Channel cover 320 Channel 330 Liquid injection section 331 Liquid injection section covering film 340 Reservoir section 341 Reservoir section covering film 342 Vent 350 Adhesive layer 412 Dielectric member 414 Metal thin film 416 Ligand immobilization region 418 Well member 420 Through-hole 422 Sensor structure 510 Well body
511 Storage section
520 Side wall member
521 Prism
523 Reflective surface
525 Metal film
526 Reaction field
L1 Excitation light L2 Reflected light L3 Fluorescence

Claims (19)

Preparing a measurement chip including a metal film and a first binding substance that binds to exosomes and is immobilized on the metal film;
providing a specimen containing exosomes on the metal film and allowing the exosomes contained in the specimen to bind to the first binding substance;
labeling the exosomes with a fluorescent substance via a second binding substance that binds to the exosomes before or after binding to the first binding substance;
a step of irradiating the metal film with excitation light so that surface plasmon resonance occurs in the metal film while the exosomes labeled with the fluorescent substance are bound to the first binding substance, and detecting fluorescence emitted from the fluorescent substance;
Including,
one of the first binding substance and the second binding substance binds to a binding determinant known as a marker for exosomes, and the other binds to a binding determinant known as a marker for cells that secrete exosomes;
At least one of the first binding substance and the second binding substance is a lectin capable of binding to a sugar chain on an exosome.
Methods for measuring exosomes. The method for measuring exosomes according to claim 1, wherein the first binding substance binds to a first binding determinant carried by the exosome, and the second binding substance binds to a second binding determinant carried by the exosome, the first binding determinant and the second binding determinant being different. The method for measuring exosomes according to claim 2, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for the exosome. The method for measuring exosomes according to claim 2 or 3, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for cells that secrete the exosomes. The method for measuring exosomes according to any one of claims 1 to 4, wherein in the labeling step, the exosomes are labeled with a fluorescent substance using a third binding substance that binds to the second binding substance. The method for measuring exosomes according to any one of claims 1 to 5, wherein the irradiation energy of the excitation light is 7.5 μW/ ^mm² or more and ¹⁰ mW/mm² or less. The method for measuring exosomes according to any one of claims 1 to 6, wherein a surfactant is added to the sample containing exosomes before providing the sample on the metal film. The method for measuring exosomes according to claim 7, wherein the surfactant is at least one selected from the group consisting of Tween 20, sodium deoxycholate, and Triton-X100. The exosome measurement method according to any one of claims 1 to 8, wherein the sample is serum, plasma, whole blood, or a dilution thereof. The method for measuring exosomes according to any one of claims 1 to 8, wherein the sample is an exosome extract. the metal film is disposed on a prism;
The excitation light is irradiated onto the metal film through a prism.
The method for measuring exosomes according to any one of claims 1 to 10. the metal film includes a diffraction grating;
the first binding substance is immobilized on the diffraction grating;
The excitation light is irradiated onto the diffraction grating.
The method for measuring exosomes according to any one of claims 1 to 10. A measuring chip including a metal film and a first binding substance that binds to exosomes and is immobilized on the metal film;
A labeling reagent for labeling exosomes with a fluorescent substance;
Including,
the labeling reagent comprises a second binding substance that binds to the exosome;
one of the first binding substance and the second binding substance binds to a binding determinant known as a marker for exosomes, and the other binds to a binding determinant known as a marker for cells that secrete exosomes ;
At least one of the first binding substance and the second binding substance is a lectin capable of binding to a sugar chain on an exosome.
Exosome measurement kit. The exosome measurement kit according to claim 13, wherein the first binding substance binds to a first binding determinant carried by the exosome, and the second binding substance binds to a second binding determinant carried by the exosome, the first binding determinant and the second binding determinant being different. The exosome measurement kit according to claim 14, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for the exosome. The exosome measurement kit according to claim 14 or 15, wherein at least one of the first binding determinant and the second binding determinant is a binding determinant known as a marker for cells that secrete the exosomes. The exosome measurement kit according to any one of claims 13 to 16, wherein the labeling reagent further comprises a third binding substance that binds to the second binding substance. The exosome measurement kit according to any one of claims 13 to 17, further comprising a surfactant to be added to the sample. The exosome measurement kit according to claim 18, wherein the surfactant is at least one selected from the group consisting of Tween 20, sodium deoxycholate, and Triton-X100.