JP6822537B2 - How to detect exosomes - Google Patents

Description

The present invention relates to a method for detecting exosomes secreted by various cells.

It is widely practiced to quantitatively analyze the effects of disease detection and treatment by detecting and analyzing a specific antigen (or antibody) associated with a disease as a biomarker.
In recent years, tiny membrane vesicles called exosomes are expected as new biomarkers.

Exosomes are contained in blood, lymph, saliva, urine, breast milk, semen, etc. Exosomes are generally spherical in liquid and are distributed around a diameter of about 100 nm. Exosomes are covered with a lipid bilayer membrane. Various substances such as proteins are present in the lipid bilayer membrane. In addition, various proteins and nucleic acids such as miRNA are contained inside the lipid bilayer membrane. In addition, exosomes are sometimes called microvesicles or extracellular vesicles, and there are a plurality of names.

Patent Document 1 describes a method for capturing and analyzing exosomes by using an immunological assay method by an enzyme-linked immunosorbent assay (ELISA).

International Publication No. 2009/09/386

In general, exosomes contain multiple different types of proteins. Among them, the protein to be recognized is a protein existing on the surface of the lipid bilayer, for example, a transmembrane protein, an adhesion molecule, a membrane transport protein, a membrane fusion protein, a sugar protein and the like. Hereinafter, these are simply collectively referred to as proteins. If multiple types of proteins can be identified for one exosome, selective detection of exosomes becomes possible. Therefore, it is expected that the disease specificity of detection can be enhanced and the accuracy and accuracy of diagnosis can be improved.

However, in the conventional exosome capture method as described in Patent Document 1, although it is possible to distinguish two types of proteins from one exosome, it is difficult to distinguish three types of proteins.

Therefore, it is necessary to capture and analyze exosomes a plurality of times using different antibodies, which complicates the process. Also in this case, it is difficult to distinguish whether or not three types of proteins are present in one exosome at the same time.

In view of these problems, an object of the present invention is to provide a method for detecting exosomes, which can improve disease specificity and improve the accuracy and accuracy of diagnosis as compared with the conventional case.

The present invention is a detection method for detecting whether or not an exosome having a first detection target substance, a second detection target substance, and a third detection target substance is present in the first sample solution. , The first detection target is obtained by mixing the first sample solution with the first buffer solution containing the first fine particles to which the first binding substance having the property of reacting with the first detection target substance is bound. The substance is reacted with the first binding substance to generate a mixed solution containing the first conjugate formed from the exosome having the first detection target substance and the first fine particles, and the produced mixed solution is produced. By substituting with the second buffer solution, a second sample solution containing an exosome having the first detection target substance was produced, and a second binding substance having the property of reacting with the second detection target substance was immobilized. A second sample solution is injected into the wells provided on the substrate, the exosomes of the first conjugate having the second detection target substance are reacted with the second binding substance, and then the second sample solution is released from the wells. By draining the sample solution and washing the inside of the well with the third buffer solution, the first binder is fixed on the substrate, and the third binding substance having the property of reacting with the third detection target substance is released. A fourth buffer containing the bound second microparticles is injected into the wells to react the exosomes of the first conjugate with the third detection target substance with the third binding substance to cause the first An analysis substrate on which an exosome having a detection target substance, a second detection target substance, and a third detection target substance is fixed is generated, and a detection signal is obtained from the reflected light when the surface of the analysis substrate is irradiated with laser light. By acquiring and determining that the acquired detection signal is a signal indicating the second fine particles, the exosome having the first detection target substance, the second detection target substance, and the third detection target substance is detected. Provided is a method for detecting an exosome.

According to the method for detecting exosomes of the present invention, it is possible to improve the disease specificity and improve the accuracy and accuracy of diagnosis as compared with the conventional method.

It is a flowchart for demonstrating the method of forming the conjugate of an exosome and a first fine particle. It is a schematic cross-sectional view of a sample solution containing an exosome and an exosome. It is a schematic diagram of a buffer solution containing magnetic fine particles and magnetic fine particles. It is a schematic cross-sectional view of the mixed solution of the sample solution containing an exosome and the buffer solution containing magnetic fine particles. It is sectional drawing which shows typically the state which the bonded body is magnetically collected. It is a schematic cross-sectional view of the sample liquid in which the conjugate is dispersed. It is a schematic diagram of an exosome capture unit. It is an enlarged perspective view of the well of the portion cut by BB of FIG. 7A. It is a flowchart for demonstrating the capture method of an exosome. It is a schematic cross-sectional view of the solution injected into a well. It is a schematic diagram of a buffer solution containing fine particles injected into a well, and fine particles. It is sectional drawing which shows typically the state which the antibody and the block layer are fixed in the track region. It is sectional drawing which shows typically the state which the exosome and the conjugate are fixed in the recess of the track region. It is an enlarged cross-sectional view which shows the dimensional shape of the convex part and the concave part of a track area. It is a schematic diagram which shows the state which the coupling body is fixed in the recess of the track area.

[Formation of conjugates of exosomes and first microparticles]
First, a method for forming a conjugate of an exosome and a first fine particle will be described with reference to FIGS. 1 to 6.

FIG. 1 is a flowchart for explaining a method of forming a bond between the exosome 10 and the magnetic fine particles 20. FIG. 2A is a schematic cross-sectional view of the sample solution 1 containing the exosome 10. FIG. 2B is a schematic cross-sectional view of the exosome 10. Although there are various particle diameters of exosome 10, in FIG. 2A and the like, the particle diameters of exosome 10 are all shown as the same.

In FIG. 1, in step S1, as shown in FIG. 2A, the operator prepares a sample solution 1 (first sample solution) containing the exosome 10 to be detected.

As shown in FIG. 2B, the exosome 10 is covered with a lipid bilayer membrane 11. There are a plurality of types of proteins such as transmembrane proteins in the lipid bilayer membrane 11. The number of proteins and the position on the lipid bilayer membrane 11 differ depending on the type of exosome and also differ depending on the individual. Recognition is performed using these surface molecules as antigens using an antigen-antibody reaction. Many papers have reported the existence of molecules such as CD63, CD9, Rab-5b, and various other proteins as surface molecules that are antigens. In FIG. 2B, symbols 12, 13 and 14 are attached to three types of proteins (detection target substances) which are antigens for identifying exosomes.

The average particle size Ra of exosome 10 is, for example, 100 nm.

The average particle size Ra of the exosome 10 means an average value obtained by measuring the particle size of the exosome 10 by an arbitrary measuring method. As the measurement method, for example, a wet measurement method for observing the exosome 10 in a solution using a nanoparticle tracking method, or a dry measurement method for observing the exosome 10 while maintaining its morphology with a transmission electron microscope. There are methods and so on.

In the latter measurement method, in order to enable dry observation while maintaining the morphology of exosome 10, a sample containing exosome 10 is treated according to the method of observing cells.

Specifically, the sample is fixed on a substrate, and the concentration of ethanol is increased step by step from low-concentration ethanol to 100% pure ethanol, and ethanol impregnation is repeated over several steps. As a result, the water content in the sample is replaced with ethanol and dehydrated.

The sample is then impregnated with a solution containing a synthetic resin soluble in ethanol to replace the ethanol with the synthetic resin. Then, the sample replaced with the synthetic resin is sliced and observed.

Further, by rapidly freezing the sample containing the exosome 10, it is possible to dehydrate the sample while maintaining the morphology of the exosome 10 so that it can be observed.

FIG. 3A is a schematic view of the buffer solution 2 containing the magnetic fine particles 20. FIG. 3B is a schematic view of the magnetic fine particles 20.

In step S2, the operator prepares a buffer solution 2 (first buffer solution) containing the magnetic fine particles 20 (first fine particles) as shown in FIG. 3A.

As shown in FIG. 3B, the magnetic fine particles 20 are made of a synthetic resin such as polystyrene, which is formed in a substantially spherical shape. The magnetic fine particles 20 contain a magnetic material 21 such as iron oxide. An antibody 22 (first binding substance) that specifically binds to the antigen 12 (first detection target substance) of the exosome 10 is immobilized on the surface of the magnetic fine particles 20. The particle diameter Rb of the magnetic fine particles 20 will be described later.

FIG. 4 is a schematic cross-sectional view of a mixed solution 4 of a sample solution 1 containing an exosome 10 and a buffer solution 2 containing magnetic fine particles 20.

In step S3, the operator injects the sample solution 1 and the buffer solution 2 into a container 3 such as a microtube or a column and mixes them, as shown in FIG. Incubation treatment is performed to allow the antigen-antibody reaction to proceed for an appropriate period of time.

By the incubation treatment, a conjugate 5 (first conjugate) of the exosome 10 and the magnetic fine particles 20 in which the antigen 12 and the antibody 22 are specifically bound is formed in the mixed solution 4. Note that some sample solutions may contain exosomes that do not have antigen 12. Exosomes that do not have the antigen 12 are dispersed in the mixed solution 4 in a state where they do not bind to the magnetic fine particles 20.

FIG. 5 is a cross-sectional view schematically showing a state in which the conjugate 5 is magnetically collected.

In step S4, the operator magnetically collects the conjugate 5 using a magnet 6 or the like, as shown in FIG. For example, by bringing the magnet 6 close to the side surface 3a of the container 3, the conjugate 5 can be magnetically collected on the side surface 3a near the magnet 6. Therefore, the conjugate 5 can be separated from the mixed solution 4 by step S4.

The method for separating the conjugate 5 is not limited to magnetic collection. For example, it is also possible to use charged particles instead of the magnetic fine particles 20. Exosomes in which charged particles are fixed and exosomes in which charged particles are not fixed are separated from each other by utilizing the fact that the amount of charge is different and the movement in an electric field is different. For example, by using a cell sorter based on the principle of flow cytometry, it becomes possible to separate the conjugate 5 from the mixed solution 4.

When the magnetic fine particles 20 are very small, the magnetization induced by the external magnetic field is small. Therefore, it may be difficult to efficiently magnetically collect the conjugate 5 with a general permanent magnet. In that case, magnetic collection by the high-gradient magnetic separation method is preferable.

In step S5, the operator replaces the mixed solution 4 in the container 3 with a buffer solution. It is preferable to replace the inside of the container 3 with a buffer solution a plurality of times. Exosomes that do not have the antigen 12 dispersed in the mixed solution 4 are removed by being replaced with a buffer solution.

In step S6, the operator separates the magnet 6 from the container 3 and redistributes the conjugate 5 separated by magnetic collection in the buffer. At that time, a better dispersion state can be realized by performing a dispersion treatment such as applying ultrasonic waves to the container 3.

FIG. 6 is a schematic cross-sectional view of the sample liquid 7 in which the conjugate 5 is dispersed.

As shown in FIG. 6, the resuspension in which the conjugate 5 is dispersed in the buffer solution by the dispersion treatment in step S6 is designated as the sample solution 7 (second sample solution).

Next, a method for capturing exosomes will be described with reference to FIGS. 7 to 13.

[Exosome capture unit]
First, the configuration of an exosome capture unit for capturing exosomes will be described with reference to FIGS. 7 and 8.

FIG. 7A is a schematic top view of the exosome capture unit 30. FIG. 7B is a schematic cross-sectional view taken along the line AA of FIG. 7A. FIG. 7C is a schematic cross-sectional view for explaining that the cartridge 32 is removable from the substrate 31. FIG. 8 is a partially enlarged perspective view cut at BB of FIG. 7A.

As shown in FIG. 7A, the exosome capture unit 30 includes a substrate 31 and a cartridge 32.

The substrate 31 has a disk shape equivalent to that of an optical disc such as a Blu-ray disc (BD), a DVD, or a compact disc (CD). The substrate 31 is made of, for example, a resin material such as a polycarbonate resin or a cycloolefin polymer generally used for optical discs.

As shown in FIG. 8, a track region 35 in which convex portions 33 and concave portions 34 are alternately arranged in the radial direction is formed on the surface of the substrate 31. The convex portion 33 and the concave portion 34 are formed in a spiral shape from the inner peripheral portion to the outer peripheral portion. The convex portion 33 corresponds to a land of an optical disc. The recess 34 corresponds to the groove of the optical disc.

As shown in FIG. 7A, the cartridge 32 has a ring shape. The cartridge 32 is formed with a plurality of cylindrical through holes 36 in the circumferential direction.

As shown in FIGS. 7B and 8, the exosome capture unit 30 has a plurality of wells 37 formed by a through hole 36 of the cartridge 32 and a track region 35 of the substrate 31. That is, the inner peripheral surface of the through hole 36 constitutes the inner peripheral surface of the well 37, and the track region 35 of the substrate 31 constitutes the bottom surface of the well 37. The well 37 is a container for storing the sample liquid 7 and the like. Further, it is convenient to insert a packing made of an elastically deformable material such as silicon rubber between the through hole 36 and the substrate 31 because the possibility of the solution leaking can be reduced.

As shown in FIG. 7C, the cartridge 32 is removable from the substrate 31. The detection of exosomes after exosome capture is performed by removing the cartridge 32 from the substrate 31 and using the substrate 31 alone.

[Exosome capture]
Next, a capture method for capturing the exosome 10 using the exosome capture unit 30 will be described with reference to FIGS. 9 to 15.

FIG. 9 is a flowchart for explaining a method for capturing exosome 10.

FIG. 10A is a schematic cross-sectional view of the buffer solution 40 containing the antibody 43 injected into the well 37. FIG. 10B is a schematic cross-sectional view of the sample liquid 7 containing the conjugate 5 injected into the well 37. In addition, in FIG. 10A and FIG. 10B, the substrate 31 and the cartridge 32 are shown as a simplified and integrated structure.

FIG. 11A is a schematic view of the buffer solution 8 containing the fine particles 50 injected into the wells 37. FIG. 11B is a schematic view of the fine particles 50.

FIG. 12A is a cross-sectional view schematically showing a state in which the antibody 43 is immobilized on the track region 35. FIG. 12B is a cross-sectional view schematically showing a state in which the block layer 41 is formed in the track region 35.

In addition, in FIG. 12A and FIG. 12B, the antibody 43 is shown to be fixed in an orientation substantially perpendicular to the substrate 31. However, in reality, the orientation in which the antibody 43 is immobilized differs depending on the fixation method. For example, when the antibody 43 is fixed using a hydrophobic bond, the antibody 43 has various orientations and is fixed to the substrate.

FIG. 13A is a cross-sectional view schematically showing a state in which the coupling body 5 is captured in the recess 34 of the track region 35. FIG. 13B is a cross-sectional view schematically showing a state in which the conjugate 9 in which the magnetic fine particles 20 and the fine particles 50 are bound to the exosome 10 is captured in the recess 34 of the track region 35.

For the sake of clarity, the exosomes 10 shown in FIGS. 13 (a) and 13 (b) are schematically shown in a simplified form of the exosomes 10 shown in FIG. 2 (b).

In FIG. 9, in step S11, as shown in FIG. 10 (a), the operator attaches antibody 43 (second binding substance) that specifically binds to antigen 13 (second detection target substance) of exosome 10. The buffer solution 40 containing the above is injected into the well 37 of the exosome capture unit 30.

The operator incubates the exosome capture unit 30 for a suitable time and at a suitable temperature. For example, it is allowed to incubate at 4 ° C. overnight, which is used for general immunological measurement. As a result, the antibody 43 is fixed in the track region 35 on the substrate 31.

In step S12, the operator discharges the buffer solution 40 from the well 37 and cleans the well 37 with the buffer solution. The antibody 43 that was not immobilized in the track region 35 is removed by this washing.

In addition, step S11 and step S12 are steps necessary when an operator fixes an antibody 43. When the exosome capture unit 30 or the substrate 31 on which the antibody 43 is fixed in advance is used in a factory or the like, steps S11 and S12 can be omitted.

As shown in FIG. 12 (a), the antibody 43 is fixed to the convex portion 33 and the concave portion 34 constituting the track region 35 by a hydrophobic bond. The method for fixing antibody 43 is not limited to hydrophobic binding. The track region 35 may be appropriately chemically treated and the antibody 43 may be immobilized on the track region 35 by covalent bonding or the like. As a method for fixing the antibody 43 in the track region 35, a method generally used in immunology measurement methods can be used.

In step S13, the operator blocks the inside of the well 37 in order to prevent non-specific adsorption of the antigen other than the antigen-identifying portion of the antibody 43. Specifically, the operator injects skim milk diluted with buffer into the wells 37 and shakes the exosome capture unit 30 for an appropriate period of time in the same manner as in step S11.

Skim milk contains a protein that does not adhere to exosome 10, and is therefore suitable for blocking treatment. The substance used for the blocking treatment is not limited to skim milk as long as it has the same effect.

In step S14, the operator drains the buffer solution containing skim milk from the well 37 and cleans the well 37 with the buffer solution. The buffer solution used for washing may or may not contain skim milk. It is also possible to omit cleaning.

As shown in FIG. 12B, the block layer 41 is formed in the track region 35.

In step S15, the operator injects the sample solution 7 containing the conjugate 5 into the well 37 as shown in FIG. 10 (b), and similarly to step S11, the exosome capture unit 30 is appropriately used for an appropriate time. Incubate at a normal temperature. It may be shaken at the time of incubation. In step S15, the exosome capture unit 30 is shaken at 37 ° C. for, for example, about 2 hours.

As a result, the antigen 13 of the exosome 10 and the antibody 43 immobilized on the track region 35 are specifically bound by the antigen-antibody reaction. As shown in FIG. 13A, the coupling 5 is captured in the recess 34 of the track region 35. Note that some sample solutions may contain exosomes that do not have antigen 13. The exosome having no antigen 13 is dispersed as a conjugate 5 in the sample solution 7 in a state where it does not bind to the antibody 43 in the track region 35.

In step S16, the operator discharges the sample solution 7 from the well 37 and cleans the well 37 with a buffer solution. The conjugate 5 dispersed in the sample solution 7 and the conjugate 5 adhering to the track region 35 by non-specific adsorption that is not an antigen-antibody reaction are removed by this washing. That is, the exosomes that do not have the antigen 13 dispersed in the sample solution 7 are removed by washing with a buffer solution. Further, even when the magnetic fine particles 20 are contained in the sample liquid 7, the magnetic fine particles 20 are removed by this cleaning.

In step S17, as shown in FIG. 11A, the operator injects the buffer solution 8 (second buffer solution) containing the fine particles (second fine particles) 50 into the well 37, and similarly to step S11. , Exosome capture unit 30 is shaken for an appropriate period of time.

As shown in FIG. 11B, the fine particles 50 are formed of a synthetic resin such as polystyrene or glycidyl methacrylate formed in a substantially spherical shape. An antibody 54 (third binding substance) that specifically binds to the antigen 14 (third detection target substance) of the exosome 10 is immobilized on the surface of the fine particles 50. The particle size Rc of the fine particles 50 will be described later.

As a result, the antigen 14 of the exosome 10 and the antibody 54 of the fine particles 50 are specifically bound by the antigen-antibody reaction. As shown in FIG. 13B, the conjugate 9 in which the magnetic fine particles 20 and the fine particles 50 are bound to the exosome 10 is captured in the recess 34 of the track region 35. The exosome having no antigen 14 is captured in the recess 34 of the track region 35 without binding to the antibody 54 of the fine particles 50.

Therefore, in the recess 34 of the track region 35, the conjugate 9 to which the exosome 10 having all of the three types of detection target substances (proteins) 12, 13 and 14 for identifying the exosome 10 is bound, and the two types The exosome-bound conjugate 5 having the substances (proteins) 12 and 13 to be detected in the above is captured.

The fine particles 50 may include a magnetic material 21 in the same manner as the magnetic fine particles 20. By arranging the magnet on the back surface side of the exosome capture unit 30 in step S17, the fine particles 50 containing the magnetic material 21 can be quickly moved to the track region 35. As a result, the time of step S17 can be shortened.

In step S18, the operator discharges the buffer solution 8 from the well 37 and cleans the well 37 with the buffer solution. The fine particles 50 dispersed in the buffer solution 8 are removed by this washing.

For example, a laser beam is emitted from an externally arranged optical pickup toward the track region 35 in which the coupling 9 and the coupling 5 are captured, specifically, the recess 34. By analyzing the reflected light from the track region 35, only the conjugate 9 to which the fine particles 50 are bonded can be detected. Therefore, only exosomes 10 having all of the three types of detection target substances 12, 13, and 14 for identifying exosomes 10 can be detected.

Specifically, the optical pickup includes an objective lens for condensing the laser beam on the track region 35. By rotating the substrate 31 in the same manner as a general optical disc and moving the optical pickup in the radial direction of the substrate 31, the laser beam focused by the objective lens is scanned along the track (specifically, the recess 34). To do.

From the detection signal obtained by the reflected light from the track region 35, the fine particles 50 in the conjugate 9 captured in the recess 34 can be detected. Therefore, only the exosomes 10 constituting the conjugate 9 are detected by selecting only the signal from the conjugate 9 to which the fine particles 50 are bound from the detection signals obtained by the reflected light from the track region 35. Can be done.

That is, only the conjugate 9 can be detected by detecting the fine particles 50, and the exosome 10 in the conjugate 9 can be indirectly detected. In addition, the number of exosomes 10 can be indirectly measured by measuring the number of fine particles 50.

Here, the particle size of the magnetic fine particles 20 is set in a range in which the magnetic fine particles 20 have sufficient magnetization for magnetic separation and the change given to the detection signal is sufficiently small. The particle size of the magnetic fine particles 20 will be described later. As a result, the change in the detection signal appears only in the conjugate 9, not in the conjugate 5. That is, it is possible to detect only the conjugate 9 without performing complicated signal processing.

Of the three types of detection target substances 12, 13, and 14, depending on the sample solution, exosomes that do not have detection target substance 12, exosomes that do not have detection target substance 13, and exosomes that do not have detection target substance 14. May be included.

According to the method for capturing exosomes of the present embodiment, among the three types of substances 12, 13, and 14, exosomes that do not have the substance 12 to be detected are removed in step S5. For example, when the sample solution contains only exosomes having no substance 12 to be detected, all the exosomes are removed in step S5, so that only the exosomes having no substance 12 to be detected are contained in the sample solution. It can be identified that it is included.

According to the method for capturing exosomes of the present embodiment, exosomes lacking the substance to be detected 13 are removed in step S16. Exosomes that do not have the substance to be detected 14 are not detected because the fine particles 50 are not bound. That is, only exosomes 10 having three types of substances to be detected 12, 13, and 14 can be detected.

Therefore, according to the exosome capture method of the present embodiment, it is possible to identify three types of proteins existing in one exosome at a time. In addition, if the sample solution does not contain exosomes having all three types of substances to be detected, exosomes are not detected, so that the sample solution does not contain exosomes having all three types of substances to be detected. Can be identified. This makes it possible to improve the disease specificity and improve the accuracy and accuracy of diagnosis as compared with the conventional case.

The interrelationship between the exosome 10, the magnetic fine particles 20, the fine particles 50, and the track region 35 will be described with reference to FIGS. 14 and 15.

FIG. 14 is an enlarged cross-sectional view showing the dimensional shape of the convex portion 33 and the concave portion 34 of the track region 35. The depth of the concave portion 34 (height of the convex portion 33) is H, the width of the concave portion 34 is Wa, and the width of the convex portion 33 is Wb. The width Wa and the width Wb are the widths at the position of H / 2 indicated by the alternate long and short dash line.

FIG. 15A is a top view schematically showing a state in which the coupling 9 is fixed to the recess 34 of the track region 35. 15 (b) is a schematic cross-sectional view taken along the line CC of FIG. 15 (a). 15 (c) is a schematic cross-sectional view taken along the line DD of FIG. 15 (a).

As shown in FIGS. 15 (a) to 15 (c), the measurement accuracy of the exosome 10 is measured by arranging the exosomes 10 along the track direction and binding one fine particle 50 to one exosome 10 with a high probability. And measurement accuracy can be improved.

For example, as shown in the formula (1), the particle diameter Rb of the magnetic fine particles 20 is smaller than the spot diameter (k × λ) / NA of the laser beam focused on the recess 34, and the particle diameter Rc of the fine particles 50 is a spot. The diameter (k × λ) / NA or more is preferable.

Rb <(k × λ) / NA ≦ Rc… (1)

Note that λ is the central wavelength of the laser light emitted from the optical pickup and focused on the recess 34 by the objective lens. NA is the numerical aperture of the objective lens. k is a coefficient. k is, for example, 1/5.

By satisfying the formula (1), the optical pickup can accurately detect the fine particles 50 without being affected by the magnetic fine particles 20. Therefore, the measurement accuracy and measurement accuracy of the fine particles 50 can be improved. When λ = 405 nm, NA = 0.85, and k = 1/5, (k × λ) / NA = 95 nm, and the particle diameter Rb of the magnetic fine particles 20 is 95 nm or less, preferably about 50 nm. The particle diameter Rc of the fine particles 50 is, for example, 200 nm.

As shown in the formula (2), the width Wb of the convex portion 33 is preferably smaller than the average particle diameter Ra of the exosome 10.

Wb <Ra ... (2)

By satisfying the equation (2), the coupling body 5 is less likely to be located on the convex portion 33.

As shown in the formula (3), the width Wa of the recess 34 is larger than the sum of the average particle diameter Ra of the exosome 10 and the particle diameter Rb of the magnetic fine particles 20, and smaller than four times the average particle diameter Ra. Is preferable.

(Ra + Rb) <Wa <4 × Ra ... (3)

If (Ra + Rb) <Wa of the formula (3) is satisfied, the coupling body 5 can be captured in the recess 34.

As shown in FIG. 13A, the exosome 10 captured in the recess 34 is generally deformed from a spherical shape in a direction of expanding the contact area. As with the magnetic fine particles 20, when the fine particles 50 containing the magnetic material 21 are used, the deformation of the exosome 10 is promoted by the magnetic force by arranging the magnet on the back surface side of the exosome capture unit 30 in step S17.

Assuming that the spherical exosome 10 is transformed into an ellipsoid while maintaining its volume, if the diameter of the sphere changes by 50%, the diameter of the contact site with the recess 34, which is the long axis of the spheroid, increases by about 40%. .. Further, in reality, since the contact portion is deformed in a direction in which the area of the contact portion is larger than that of the spheroid shape, the diameter of the contact portion is increased by 50% or more, and in some cases 100% or more of the diameter of the original spherical shape. Sometimes.

From the above, it is preferable to satisfy Wa <4 × Ra of the formula (3).

Regarding Wa, as further shown in the formula (4), the width Wb of the convex portion 33 is preferably smaller than the particle diameter Rc of the fine particles 50. The width Wa of the recess 34 is preferably larger than the particle diameter Rc and smaller than twice the particle diameter Rc.

Wb <Rc <Wa <2 x Rc ... (4)

By satisfying Wb <Rc of the formula (4), the fine particles 50 are less likely to be located on the convex portion 33. By satisfying Rc <Wa of the formula (3), the fine particles 50 can enter the recess 34. By satisfying Wa <2 × Rc of the formula (4), it becomes difficult for two fine particles 50 to enter at the same time in the width direction of the recess 34, and the quantitative relationship between the exosome 10 and the fine particles 50 can be brought close to 1: 1.

As shown in the formula (5), the particle size Rc of the fine particles 50 is preferably larger than the average particle size Ra of the exosome 10.

Ra <Rc ... (5)

By satisfying the formula (5), it becomes difficult for a plurality of fine particles 50 to bind to the exosome 10 fixed to the recess 34, and the quantitative relationship between the exosome 10 and the fine particles 50 can be brought close to 1: 1. By satisfying the formula (5), the probability that the exosome 10 and the fine particles 50 react with each other increases, and as a result, the yield of the reaction can be improved.

As shown in the formula (6), the depth H of the recess 34 is preferably larger than 1/8 times the sum of the average particle diameter Ra of the exosome 10 and the particle diameter Rc of the fine particles 50.

(Ra + Rc) / 8 <H ... (6)

By satisfying the formula (6), the exosome 10 is easily trapped in the concave portion 34, non-specific adsorption of the fine particles 50 to the convex portion 33 is unlikely to occur, and the fine particles 50 are with the exosome 10 trapped in the concave portion 34. It becomes easier to combine.

It is more preferable that the depth H of the recess 34 satisfies the formula (7).

(Ra + Rc) / 6 <H ... (7)

It is preferable that all of the above equations (1) to (6) (or equation (7)) are satisfied, but it may be only partially satisfied.

The present invention is not limited to the embodiments described above, and various modifications can be made without departing from the gist of the present invention.

1 Sample solution (first sample solution)
2 Buffer solution (first buffer solution)
5 conjugate (first conjugate)
9 Combines (second combine)
10 Exosomes 12 Antigens (1st substance to be detected)
13 Antigen (second detection target substance)
14 Antigen (third substance to be detected)
20 Magnetic particles (first particles)
22 antibody (first binding substance)
31 Substrate 43 Antibody (second binding substance)
50 fine particles (second fine particles)
54 antibody (third binding substance)

Claims (2)

A detection method for detecting whether or not an exosome having a first detection target substance, a second detection target substance, and a third detection target substance is present in the first sample solution.
The first buffer solution containing the first fine particles to which the first binding substance having the property of reacting with the first detection target substance is bound to the first sample solution is mixed with the first sample solution. The detection target substance of No. 1 is reacted with the first binding substance to generate a mixed solution containing a first conjugate formed from the exosome having the first detection target substance and the first fine particles.
The generated said mixture to produce a second sample solution containing exosomes having the first detection target by replacing the second buffers, the property of reacting with the second detection target The second sample solution is injected into a well provided on a substrate on which the second binding substance having the substance is fixed, and the exosome of the first conjugate having the second detection target substance is transferred to the second sample solution. After reacting with the binding substance of the above, the second sample solution is discharged from the well, and the inside of the well is washed with a third buffer solution to fix the first binder on the substrate.
A fourth buffer solution containing a second fine particle to which a third binding substance having a property of reacting with the third detection target substance is bound is injected into the well, and the said having the third detection target substance. By reacting the exosome of the first conjugate with the third binding substance, the first detection target substance,
An analytical substrate on which an exosome having a second detection target substance and a third detection target substance was immobilized was generated.
A detection signal is acquired from the reflected light when the surface of the analysis substrate is irradiated with laser light.
By determining that the acquired detection signal is a signal indicating the second fine particles, an exosome having a first detection target substance, a second detection target substance, and a third detection target substance is detected. How to detect exosomes. When irradiating the laser beam, the laser beam is focused in the recess, the particle size of the first fine particles is smaller than the spot diameter of the focused laser light, and the particle size of the second fine particles is The method for detecting an exosome according to claim 1, wherein the spot diameter is equal to or larger than that of the spot diameter.

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