JP2020020810A - Method for detecting exosome - Google Patents

Abstract

To provide a method for detecting exosomes, capable of enhancing disease specificity compared to conventional arts, and improving accuracy and probability of diagnosis.SOLUTION: A method for detecting exosomes having detection target substances 12 to 14 comprises: mixing, in a sample solution 1, a buffer solution 2 containing fine particles 12 to which a binding substance 22 that reacts with the detection target substance 12 is bound, to generate a sample solution 7 containing a conjugate 5 comprising exosomes and the fine particles 12 having the detection target substance 12; injecting the sample solution 7 on a substrate 31 on which a binding substance 43 that reacts with the detection target substance 13 is fixed; reacting the conjugate 5 having the detection target substance 13 with the binding substance 43 to fix the conjugate 5 on the substrate 31; injecting a buffer solution containing fine particles 50 to which a binding substance 54 that reacts with the detection target substance 14 is bound, to generate an analysis substrate on which exosomes having the detection target substances 12 to 14 are fixed; and determining that a detection signal obtained by irradiating a surface with laser light is a signal indicating the fine particles 50.SELECTED DRAWING: Figure 1

Description

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

It is widely practiced to detect and analyze a specific antigen (or antibody) associated with a disease as a biomarker to quantitatively analyze the effect of the discovery or treatment of a disease.
In recent years, small membrane vesicles called exosomes are expected as new biomarkers.

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

  Patent Literature 1 describes a method of capturing and analyzing exosomes by using an immunological assay based on an enzyme-linked immunosorbent assay (ELISA (Enzyme-Linked Immuno-Sorbent Assay)).

International Publication No. 2009/092386

  In general, exosomes contain a plurality of different types of proteins. Among them, proteins to be recognized are proteins existing on the surface of the lipid bilayer membrane, and include, for example, transmembrane proteins, adhesion molecules, membrane transport proteins, membrane fusion proteins, and glycoproteins. Hereinafter, these are simply referred to as proteins. If a plurality of types of proteins can be distinguished from one exosome, selective detection of exosomes becomes possible. Therefore, it is expected that the disease specificity of detection can be increased, and that the accuracy and precision of diagnosis can be improved.

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

  Therefore, it is necessary to perform exosome capture and analysis multiple 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 simultaneously present in one exosome.

  In view of such problems, an object of the present invention is to provide a method for detecting exosomes, which makes it possible to enhance disease specificity and improve the accuracy and precision of diagnosis as compared with the related art.

  The present invention relates to 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 a first sample solution, By mixing the first sample solution with a first buffer solution containing first fine particles bound to a first binding substance having a property of reacting with the first detection target substance, Reacting the substance with the first binding substance to generate a mixed liquid containing a first conjugate formed from exosomes having the first detection target substance and the first fine particles; By substituting with the second buffer solution, a second sample solution containing exosomes having the first target substance was generated, and the second binding substance having a property of reacting with the second target substance was fixed. Injecting a second sample solution into a well provided in the substrate, After reacting the exosome of the first conjugate having the target substance of No. 2 with the second conjugate, the second sample solution is discharged from the well, and the inside of the well is washed with a third buffer. Immobilizing the first conjugate on the substrate and injecting into the well a fourth buffer solution containing second fine particles to which a third binding substance having a property of reacting with the third substance to be detected is bound. Reacting the exosome of the first conjugate having the third substance to be detected with the third substance to form a first substance to be detected, a second substance to be detected, and a third substance to be detected An analysis substrate having exosomes immobilized thereon is generated, a detection signal is obtained from reflected light when the surface of the analysis substrate is irradiated with laser light, and the obtained detection signal is a signal indicating the second microparticle. By determining, the first detection target substance, the second detection target substance Out substance, and exosomes detection method for detecting a exosomes having a third detection target substance to provide.

  ADVANTAGE OF THE INVENTION According to the exosome detection method of this invention, it becomes possible to raise disease specificity conventionally and to improve the precision and accuracy of diagnosis.

5 is a flowchart for explaining a method for forming a conjugate of exosomes and first fine particles. FIG. 2 is a schematic cross-sectional view of a sample solution containing exosomes and exosomes. It is a schematic diagram of a buffer solution containing magnetic fine particles and magnetic fine particles. FIG. 3 is a schematic cross-sectional view of a mixture of a sample solution containing exosomes and a buffer solution containing magnetic fine particles. It is sectional drawing which shows the state in which the coupling body was magnetically collected. FIG. 4 is a schematic cross-sectional view of a sample liquid in which a conjugate is dispersed. It is a schematic diagram of an exosome capture unit. FIG. 8 is an enlarged perspective view of a part of the well cut along the line BB in FIG. It is a flowchart for demonstrating the capture method of an exosome. FIG. 3 is a schematic sectional view of a solution injected into a well. FIG. 2 is a schematic diagram of a buffer solution containing fine particles injected into a well and fine particles. FIG. 4 is a cross-sectional view schematically showing a state in which an antibody and a block layer are fixed to a track region. FIG. 4 is a cross-sectional view schematically showing a state in which an exosome and a conjugate are fixed in a concave portion of a track region. FIG. 4 is an enlarged cross-sectional view showing a dimensional shape of a convex portion and a concave portion of a track area. FIG. 4 is a schematic diagram showing a state where the combined body is fixed to a concave portion of a track area.

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

  FIG. 1 is a flowchart for explaining a method for forming a conjugate of the exosome 10 and the magnetic fine particles 20. FIG. 2A is a schematic cross-sectional view of the sample solution 1 including the exosome 10. FIG. FIG. 2B is a schematic cross-sectional view of the exosome 10. In addition, although the particle diameter of the exosome 10 is various, the particle diameter of the exosome 10 is all the same in FIG.

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

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

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

  The average particle size Ra of the exosome 10 means an average value of values obtained by measuring the particle size of the exosome 10 by an arbitrary measuring method. Examples of the measurement method include a wet measurement method in which the exosome 10 is observed in a solution using a nanoparticle tracking method, and a dry measurement method in which the exosome 10 is observed while maintaining the form of the exosome 10 with a transmission electron microscope. There are methods.

  In the latter measurement method, in order to make it possible to observe the exosome 10 in a dry manner while maintaining the form of the exosome 10, a sample containing the exosome 10 is subjected to a treatment according to a method of observing cells.

  Specifically, the sample is immobilized on a substrate, and the ethanol concentration is increased every step from low-concentration ethanol to 100% -purity ethanol, and ethanol impregnation is repeated over several steps. Thereby, the water in the sample is replaced with ethanol and dehydrated.

  Next, the sample is impregnated with a solution containing a synthetic resin soluble in ethanol, thereby replacing ethanol with the synthetic resin. Then, the sample replaced with the synthetic resin is sliced and observed.

  In addition, by rapidly freezing the sample containing the exosome 10, it is possible to dehydrate the sample while maintaining the form of the exosome 10 to make the exosome 10 observable.

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

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

  As shown in FIG. 3B, the magnetic fine particles 20 are formed of a substantially spherical synthetic resin such as polystyrene. The magnetic fine particles 20 include a magnetic body 21 such as iron oxide. On the surface of the magnetic fine particles 20, an antibody 22 (first binding substance) that specifically binds to the antigen 12 (first detection target substance) of the exosome 10 is fixed. The particle size 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 exosomes 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. An incubation treatment for promoting the antigen-antibody reaction is performed for an appropriate 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 mixture 4. Note that, depending on the sample liquid, exosomes having no antigen 12 may be contained. Exosomes having no 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 where the coupling body 5 is magnetically collected.

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

  The method of separating the combined body 5 is not limited to magnetic collection. For example, it is also possible to use charged particles instead of the magnetic fine particles 20. The exosomes on which the charged particles are immobilized and the exosomes on which they are not immobilized are separated from each other by utilizing the fact that the exosomes have different amounts of electric charge and thus move differently in an electric field. For example, by using a cell sorter based on flow cytometry, the conjugate 5 can be separated 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 for a general permanent magnet to efficiently magnetically collect the combined body 5. In that case, magnetic collection by a high gradient magnetic separation method is preferable.

  In step S5, the operator replaces the mixed solution 4 in the container 3 with a buffer. It is preferable to replace the inside of the container 3 with the buffer solution a plurality of times. The exosomes having no antigen 12 dispersed in the mixture 4 are removed by being replaced with a buffer.

  In step S6, the operator separates the magnet 6 from the container 3 and re-disperses the combined body 5 separated by magnetic collection in the buffer. At this time, if a dispersion treatment such as application of ultrasonic waves to the container 3 is performed, a better dispersion state can be realized.

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

  As shown in FIG. 6, the re-suspension in which the conjugate 5 is dispersed in the buffer solution by the dispersion process in step S6 is used as a sample solution 7 (second sample solution).

  Next, a method of capturing exosomes will be described with reference to FIGS.

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

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

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

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

  As shown in FIG. 8, on the surface of the substrate 31, a track region 35 in which convex portions 33 and concave portions 34 are alternately arranged in the radial direction is formed. 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 protrusion 33 corresponds to a land of the optical disk. The recess 34 corresponds to the groove of the optical disk.

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

  As shown in FIGS. 7B and 8, the exosome capturing unit 30 has a plurality of wells 37 formed by the through hole 36 of the cartridge 32 and the track area 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 area 35 of the substrate 31 constitutes the bottom surface of the well 37. The well 37 is a container for storing the sample solution 7 and the like. Further, if a packing made of a material that is elastically deformable, such as silicon rubber, is inserted between the through hole 36 and the substrate 31, the possibility of the solution leaking can be advantageously reduced.

  As shown in FIG. 7C, the cartridge 32 is detachable 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 capturing method for capturing the exosomes 10 using the exosome capturing unit 30 will be described with reference to FIGS.

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

  FIG. 10A is a schematic cross-sectional view of the buffer 40 containing the antibody 43 injected into the well 37. FIG. 10B is a schematic cross-sectional view of the sample solution 7 including the conjugate 5 injected into the well 37. Note that FIGS. 10A and 10B show a structure in which the substrate 31 and the cartridge 32 are simplified and integrated.

  FIG. 11A is a schematic diagram of the buffer 8 containing the fine particles 50 injected into the well 37. FIG. 11B is a schematic diagram of the fine particles 50.

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

  In FIGS. 12A and 12B, the antibody 43 is shown to be fixed in a direction substantially perpendicular to the substrate 31. However, actually, the direction in which the antibody 43 is fixed differs depending on the fixing method. For example, when the antibody 43 is immobilized using a hydrophobic bond, the antibody 43 is immobilized on the substrate in various directions.

  FIG. 13A is a cross-sectional view schematically showing a state in which the combined body 5 is captured in the concave portion 34 of the track area 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 respectively bonded to the exosome 10 is captured in the concave portion 34 of the track region 35.

  13 (a) and 13 (b), the exosome 10 shown in FIG. 2 (b) is schematically shown in a simplified manner.

  In FIG. 9, as shown in FIG. 10A, the operator at step S11, an antibody 43 (second binding substance) that specifically binds to the antigen 13 (second detection target substance) of the exosome 10 Is injected into the well 37 of the exosome capturing unit 30.

  The operator causes the exosome capturing unit 30 to incubate at an appropriate temperature for an appropriate time. For example, it is left to incubate at 4 ° C., which is used for general immunological measurement, for about overnight. Thus, the antibody 43 is fixed to the track region 35 on the substrate 31.

  In step S12, the operator discharges the buffer 40 from the well 37 and cleans the well 37 with the buffer. Note that the antibody 43 not fixed to the track region 35 is removed by this washing.

  Steps S11 and S12 are necessary steps when the operator fixes the antibody 43. When using the exosome capturing unit 30 or the substrate 31 to which the antibody 43 has been previously fixed in a factory or the like, steps S11 and S12 can be omitted.

  As shown in FIG. 12A, the antibody 43 is fixed to the convex portion 33 and the concave portion 34 constituting the track region 35 by hydrophobic bonding. The method for immobilizing the antibody 43 is not limited to hydrophobic bonding. The track region 35 may be subjected to an appropriate chemical treatment, and the antibody 43 may be fixed to the track region 35 using a covalent bond or the like. As a method for immobilizing the antibody 43 on the track region 35, a method generally used in an immunological assay can be used.

  In step S13, the operator performs a blocking process on the inside of the well 37 in order to prevent the antigen from nonspecifically adsorbing to the portion other than the antigen identification portion of the antibody 43. Specifically, the operator injects the skim milk diluted with the buffer solution into the well 37, and shakes the exosome capturing unit 30 for an appropriate time as in step S11.

  Since skim milk contains a protein that does not adhere to exosome 10, it is suitable for a 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 discharges the buffer containing skim milk from the well 37 and cleans the well 37 with the buffer. The buffer used for washing may or may not contain skim milk. Further, the washing can be omitted.

  As shown in FIG. 12B, a block layer 41 is formed in the track area 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, sets the exosome capturing unit 30 for an appropriate time. Incubate at different temperatures. It may be shaken during the incubation. In step S15, the exosome capturing unit 30 is shaken at 37 ° C. for about 2 hours, for example.

  Thus, the antigen 13 of the exosome 10 and the antibody 43 fixed to the track region 35 are specifically bound by an antigen-antibody reaction. As shown in FIG. 13A, the combined body 5 is captured in the concave portion 34 of the track area 35. Note that, depending on the sample liquid, exosomes having no antigen 13 may be contained. The exosome without the antigen 13 is dispersed as the conjugate 5 in the sample solution 7 without being bound 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. The conjugate 5 dispersed in the sample solution 7 and the conjugate 5 attached to the track region 35 by non-specific adsorption other than the antigen-antibody reaction are removed by this washing. That is, exosomes having no antigen 13 dispersed in the sample solution 7 are removed by washing with a buffer. Further, even when the sample solution 7 contains the magnetic fine particles 20, the magnetic fine particles 20 are removed by this washing.

  In step S17, the operator injects a buffer solution 8 (second buffer solution) containing fine particles (second fine particles) 50 into the well 37 as shown in FIG. Then, the exosome capturing unit 30 is shaken for an appropriate 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 microparticle 50. The particle size Rc of the fine particles 50 will be described later.

  Thus, the antigen 14 of the exosome 10 and the antibody 54 of the microparticle 50 are specifically bound by an antigen-antibody reaction. As shown in FIG. 13B, the conjugate 9 in which the magnetic fine particles 20 and the fine particles 50 are respectively bonded to the exosome 10 is captured in the concave portions 34 of the track region 35. The exosome having no antigen 14 is captured in the concave portion 34 of the track region 35 without binding to the antibody 54 of the microparticle 50.

  Therefore, in the concave portion 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 two types of the conjugate 9 The conjugate 5 to which the exosomes having the detection target substances (proteins) 12 and 13 are bound is captured.

  The fine particles 50 may include the magnetic body 21 similarly to the magnetic fine particles 20. By arranging the magnet on the back surface side of the exosome capturing unit 30 in step S17, the fine particles 50 including the magnetic substance 21 can be quickly moved to the track area 35. As a result, step S17 can be shortened in time.

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

  For example, a laser beam is radiated from an optical pickup disposed outside toward a track region 35 where the coupling body 9 and the coupling body 5 are captured, specifically, a concave portion 34. By analyzing the reflected light from the track area 35, it is possible to detect only the combined body 9 to which the fine particles 50 are combined. Therefore, it is possible to detect only the exosome 10 having all of the three types of detection target substances 12, 13, and 14 for identifying the exosome 10.

  Specifically, the optical pickup includes an objective lens for condensing the laser light on the track area 35. By rotating the substrate 31 in the same manner as a general optical disk and moving the optical pickup in the radial direction of the substrate 31, the laser light condensed by the objective lens is scanned along the track (specifically, the concave portion 34). I do.

  From the detection signal obtained by the reflected light from the track region 35, the fine particles 50 in the combined body 9 captured in the concave portion 34 can be detected. Therefore, only the signal from the conjugate 9 to which the fine particles 50 are bound is selected from the detection signal obtained by the reflected light from the track region 35, thereby detecting only the exosome 10 constituting the conjugate 9 Can be.

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

  Here, the particle size of the magnetic fine particles 20 is set to a range that has sufficient magnetization to perform magnetic separation and that changes to the detection signal are sufficiently small. The particle diameter of the magnetic fine particles 20 will be described later. As a result, a change in the detection signal appears only in the conjugate 9 and does not occur in the conjugate 5. That is, it is possible to detect only the combination 9 without performing complicated signal processing.

  Depending on the sample liquid, among the three types of detection target substances 12, 13, and 14, exosomes without the detection target substance 12, exosomes without the detection target substance 13, and exosomes without the detection target substance 14 May be included.

  According to the exosome trapping method of the present embodiment, the exosome having no detection target substance 12 is removed in step S5 among the three types of detection target substances 12, 13, and 14. For example, when only exosomes without the target substance 12 are contained in the sample solution, all of the exosomes are removed in step S5, so only exosomes without the target substance 12 are contained in the sample solution. It can be specified that it is included.

  According to the exosome capturing method of the present embodiment, exosomes that do not have the detection target substance 13 are removed in step S16. Exosomes that do not have the detection target substance 14 are not detected because the fine particles 50 are not bound thereto. That is, it is possible to detect only the exosome 10 having the three types of detection target substances 12, 13, and 14.

  Therefore, according to the exosome capturing method of the present embodiment, it is possible to identify three types of proteins existing in one exosome at a time. Further, when the exosome having all three types of detection target substances is not contained in the sample liquid, exosomes are not detected, and thus the exosome having all three types of detection target substances is not contained in the sample liquid. Can be identified. This makes it possible to increase the disease specificity and improve the accuracy and accuracy of diagnosis as compared with the related art.

  The relationship 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.

  FIG. 14 is an enlarged cross-sectional view showing the dimensional shapes of the protrusions 33 and the recesses 34 of the track area 35. The depth of the recess 34 (the height of the projection 33) is H, the width of the recess 34 is Wa, and the width of the projection 33 is Wb. Note that the width Wa and the width Wb are the width at the position of H / 2 indicated by the dashed line.

  FIG. 15A is a top view schematically showing a state in which the coupling body 9 is fixed to the concave portion 34 of the track area 35. FIG. 15B is a schematic cross-sectional view taken along the line CC of FIG. FIG. 15C is a schematic cross-sectional view taken along a line DD in FIG.

  As shown in FIGS. 15A to 15C, the exosomes 10 are arranged in the track direction, and one microparticle 50 is bonded to one exosome 10 with a high probability, so that the measurement accuracy of the exosome 10 is improved. And measurement accuracy can be improved.

  For example, as shown in Expression (1), the particle diameter Rb of the magnetic fine particles 20 is smaller than the spot diameter (k × λ) / NA of the laser light focused on the concave portion 34, and the particle diameter Rc of the fine particles 50 is It is preferable that the diameter be equal to or larger than (k × λ) / NA.

  Rb <(k × λ) / NA ≦ Rc (1)

  Here, λ is the center wavelength of the laser light emitted from the optical pickup and focused on the concave portion 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 expression (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 projection 33 is preferably smaller than the average particle diameter Ra of the exosome 10.

  Wb <Ra (2)

  By satisfying the expression (2), it is difficult for the joint body 5 to be located on the protrusion 33.

  As shown in Expression (3), the width Wa of the concave portion 34 is larger than the sum of the average particle diameter Ra of the exosome 10 and the particle diameter Rb of the magnetic microparticles 20 and smaller than four times the average particle diameter Ra. Is preferred.

  (Ra + Rb) <Wa <4 × Ra (3)

  If (Ra + Rb) <Wa in the formula (3) is satisfied, the combined body 5 can be captured in the concave portion 34.

  As shown in FIG. 13A, the exosome 10 captured in the concave portion 34 generally deforms from a spherical shape in a direction to increase the contact area. In addition, similarly to the magnetic fine particles 20, when the fine particles 50 including the magnetic substance 21 are used, the deformation of the exosome 10 is promoted by the magnetic force by disposing the magnet on the back surface side of the exosome capturing unit 30 in step S17.

  Assuming that the spherical exosome 10 is deformed into an ellipsoid while maintaining its volume, when the diameter of the sphere changes by 50%, the diameter of the contact portion with the concave portion 34 which is the major axis of the spheroid increases by about 40%. . Further, in actuality, since the contact portion is deformed in a direction in which the area of the contact portion becomes larger than that of the spheroidal shape, the diameter of the contact portion increases by 50% or more, and sometimes 100% or more of the diameter of the original spherical shape. Sometimes.

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

  As for Wa, it is preferable that the width Wb of the convex portion 33 is smaller than the particle diameter Rc of the fine particles 50, as shown in Expression (4). The width Wa of the concave portion 34 is preferably larger than the particle diameter Rc and smaller than twice the value of the particle diameter Rc.

  Wb <Rc <Wa <2 × Rc (4)

  By satisfying Wb <Rc in the expression (4), the fine particles 50 are less likely to be located on the protrusions 33. By satisfying Rc <Wa in the equation (3), the fine particles 50 can enter the concave portions 34. By satisfying Wa <2 × Rc in the expression (4), it is difficult for two of the fine particles 50 to enter the width direction of the concave portion 34 at the same time, and the numerical relationship between the exosome 10 and the fine particles 50 can be made closer to one to one.

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

  Ra <Rc (5)

  By satisfying the expression (5), it becomes difficult for the plurality of microparticles 50 to bind to the exosome 10 fixed to the concave portion 34, and the number relationship between the exosome 10 and the microparticle 50 can be made closer to one to one. By satisfying the expression (5), the probability that the exosomes 10 and the fine particles 50 come into contact with each other is increased, and as a result, the reaction yield can be improved.

  As shown in Expression (6), it is preferable that the depth H of the concave portion 34 is larger than １ ／ 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 expression (6), the exosome 10 is easily trapped in the concave portion 34, and non-specific adsorption of the fine particles 50 to the convex portion 33 is less likely to occur. It becomes easier to combine.

  More preferably, the depth H of the recess 34 satisfies Expression (7).

  (Ra + Rc) / 6 <H (7)

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

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

1 sample liquid (first sample liquid)
2 Buffer (first buffer)
5. Conjugate (first conjugate)
9 Conjugate (second conjugate)
10 exosome 12 antigen (first detection target substance)
13 antigen (second detection target substance)
14 antigen (third substance to be detected)
20 Magnetic fine particles (first fine 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 a first sample solution,
The first sample solution is mixed with a first buffer solution containing first microparticles to which a first binding substance having a property of reacting with the first detection target substance is bound, whereby the first buffer solution is mixed. Reacting the detection target substance with the first binding substance to generate a mixed solution including a first conjugate formed from the exosome having the first detection target substance and the first microparticle;
By replacing the generated mixture with a second buffer, a second sample solution containing an exosome having the first target substance is generated, and has a property of reacting with the second target substance. The second sample solution is injected into a well provided on a substrate on which a second binding substance is immobilized, and the exosome of the first conjugate having the second substance to be detected is subjected to the second exosome. After reacting with the binding substance, the second sample solution is discharged from the well, and the inside of the well is washed with a third buffer solution, whereby the first binding body is fixed on the substrate,
Injecting, into the well, a fourth buffer solution including a second microparticle to which a third binding substance having a property of reacting with the third detection target substance is bound, and including the third detection target substance An analysis substrate on which an exosome of a first conjugate is reacted with the third binding substance to fix an exosome having a first detection target substance, a second detection target substance, and a third detection target substance Produces
Obtain a detection signal from reflected light when irradiating the surface of the analysis substrate with laser light,
By determining that the obtained detection signal is a signal indicating the second microparticle, an exosome having a first detection target substance, a second detection target substance, and a third detection target substance is detected. Exosome detection method.   When irradiating the laser light, the laser light is focused on the concave portion, and the particle diameter of the first fine particles is smaller than the spot diameter of the focused laser light, and the particle diameter of the second fine particles is small. 2. The method for detecting exosomes according to claim 1, wherein the diameter of the exosome is not less than the spot diameter.

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