Ultrasonic irradiation apparatus

Abstract

To provide an ultrasonic irradiation device capable of improving uniformity when detecting an object with multiple channels.SOLUTION: The ultrasonic irradiation apparatus 10 includes an ultrasonic generator 2, a sample plate 4, and a rotor blade 5. The ultrasonic generator 2 generates ultrasonic waves and irradiates the inside of the container 1 with the generated ultrasonic waves. The sample plate 4 has a plurality of wells 44 arranged axisymmetrically, and is fixed to the rotary shaft 3. An object to be irradiated with ultrasonic waves is placed in each of the plurality of wells 44. The rotary blade 5 is fixed to the rotary shaft 3 on the lower side of the sample plate 4, receives ultrasonic waves, is self-rotated by the sound pressure of the ultrasonic waves, and rotates the sample plate 4 via the rotary shaft 3 so that the plurality of wells 44 are axially symmetrically rotated.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an ultrasonic irradiation device. [Background technology]

[0002] Conventionally, an amyloid assay device described in Patent Document 1 is known. The amyloid assay device described in Patent Document 1 includes an ultrasonic irradiation device and a plate reader. A microplate is mounted on the ultrasonic irradiation device. The microplate has a plurality of wells arranged in 12 rows and 8 columns. Each of the plurality of wells contains a protein solution.

[0003] The ultrasonic irradiation device irradiates the protein solution placed in each well with ultrasonic waves. When a supersaturated solution of amyloid-causing proteins is subjected to ultrasonic irradiation, amyloid is produced.

[0004] When amyloid is induced from a protein, the amyloid emits fluorescence by binding to an amyloid-specific fluorescent dye, and the presence of the amyloid-specific fluorescent dye in multiple wells of the microplate where the amyloid is induced causes fluorescence, allowing a plate reader to detect the amyloid in the solution by detecting this fluorescence.

[0005] In this way, the amyloid assay device described in Patent Document 1 promotes amyloid formation by irradiating a protein solution placed in each well with ultrasound, and detects amyloid formed by the ultrasound irradiation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2012 / 017739 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Masatomo So, Hisashi Yagi, Kazumasa Sakurai, Hirotsugu Ogi, Hironobu Naiki and Yuji Goto, "Ultrasonication-Dependence Acceleration of Amyloid Fibril Formation," J. Mol. Boil. (2011)412, 568-577. Summary of the Invention [Problem to be solved by the invention]

[0008] However, when performing multi-channel detection in which a supersaturated solution of protein is placed in multiple wells and amyloid formed by ultrasonic irradiation is detected, the following problem arises.

[0009] It is extremely difficult to make the ultrasonic intensity introduced into each channel the same, resulting in large variations between channels. To reduce this variation between channels, attempts have been made to irradiate ultrasonic waves while rotating a container containing many samples in water. However, this method has the drawback of being incompletely uniform, and the entire device becomes large in size because the container is forcibly rotated using a stage or other device.

[0010] Furthermore, in a fluorescence spectrophotometer, it is effective to introduce a stirrer to stir the solution, but there is a problem in that it is difficult to introduce a stirrer when the amount of solution is very small.

[0011] Therefore, according to an embodiment of the present invention, an ultrasonic irradiation device capable of improving uniformity when detecting an object through multiple channels is provided. [Means for solving the problem]

[0012] (Configuration 1) According to an embodiment of the present invention, an ultrasonic irradiation device includes a sample plate, a rotation device, and an ultrasonic generator. The sample plate has a plurality of wells arranged axially symmetrically. The rotation device rotates the sample plate so that the plurality of wells rotate axially symmetrically. The ultrasonic generator generates ultrasonic waves and irradiates the generated ultrasonic waves to a solution introduced into each of the plurality of wells while the sample plate is rotating.

[0013] (Configuration 2) In configuration 1, the rotating device rotates the sample plate by the sound pressure of ultrasonic waves generated by the ultrasonic generator.

[0014] (Configuration 3) In the second configuration, the rotating device is arranged on the bottom surface of the sample plate along the circumferential direction of the sample plate and comprises a plurality of blades that rotate the sample plate by the sound pressure of ultrasonic waves.

[0015] (Configuration 4) In the second aspect, the ultrasonic irradiation device further includes a rotating shaft to which the sample plate is fixed. The rotating device is fixed to the rotating shaft and includes a rotor that rotates by itself in response to the sound pressure of the ultrasonic waves, thereby rotating the sample plate via the rotating shaft.

[0016] (Configuration 5) In configuration 4, the rotating shaft, sample plate, and rotor are immersed in a medium consisting of a fluid.

[0017] (Configuration 6) In the fourth or fifth configuration, the ultrasonic irradiation device further includes a seal. The seal closes the openings of the wells and is thinner than the thickness of the wells. Ultrasonic waves are irradiated to the solution in the wells through the seal.

[0018] (Configuration 7) In any of configurations 4 to 6, the rotor blade has an inverted truncated cone shape and includes a plurality of blades arranged circumferentially around the inclined surface of the inverted truncated cone, each of the plurality of blades having a spiral shape extending in the length direction of the rotor shaft.

[0019] (Configuration 8) In any one of configurations 1 to 7, the sample plate has a plurality of through-holes for allowing bubbles generated in a fluid medium when the medium is irradiated with ultrasonic waves to escape into the atmosphere.

[0020] (Configuration 9) In configuration 8, the plurality of through-holes are arranged axially symmetrically on the sample plate.

[0021] (Configuration 10) In any of configurations 1 to 9, each of the plurality of wells has a cylindrical shape with a predetermined curvature at the intersection of the side surface and the bottom surface.

[0022] (Configuration 11) In any one of the configurations 1 to 10, the solution comprises a solution containing a protein and a fluorescent molecule that emits light when adsorbed to the protein aggregates that have been aggregated by ultrasonic irradiation.

[0023] (Configuration 12) In the eleventh aspect, the ultrasound irradiation device further includes a light irradiation device and a light detection device. The light irradiation device irradiates the solution with excitation light having a predetermined wavelength. The light detection device detects fluorescence emitted by the fluorescent molecules when the excitation light is irradiated by the light irradiation device.

[0024] (Configuration 13) In configuration 12, the sample plate is made of a disc-shaped transparent body, and the photodetector is disposed on the outer periphery of the sample plate at a position facing the wells of the sample plate.

[0025] (Configuration 14) In configuration 13, the sample plate is placed in a container having a transparent window facing the wells of the sample plate.

[0026] (Configuration 15) In any one of configurations 1 to 14, the ultrasonic irradiation device further includes a circulation control unit. The circulation control unit controls the temperature of the fluid medium to a predetermined temperature, degasses the medium, and circulates the degassed medium, controlled to the predetermined temperature, within a container in which the sample plate is placed. [Effects of the Invention]

[0027] This improves uniformity when detecting an object across multiple channels. [Brief explanation of the drawings]

[0028] [Figure 1] 1 is a schematic diagram of an ultrasonic irradiation device according to an embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram of the ultrasonic generator shown in FIG. [Figure 3] FIG. 2 is a perspective view of the sample plate shown in FIG. [Figure 4] 1 is a cross-sectional view of the seal 8, the main body 41, the wells 44 and the wall member 45 in the region of two adjacent wells 44, 44. FIG. [Figure 5] FIG. 2 is a perspective view of the rotor shown in FIG. 1. [Figure 6] 1 is a diagram for explaining a method for detecting fluorescence emitted from a plurality of solutions placed in a plurality of wells. FIG. 2 is a diagram showing the relationship between light intensity and time. [Figure 7] FIG. 10 is a diagram showing the relationship between light intensity and time. [Figure 8] FIG. 10 is a schematic diagram of another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 9] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 10] FIG. 10 is a perspective view of the sample plate shown in FIG. [Figure 11]FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 12] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 13] 10 is a cross-sectional view of the seals 8 and 9, the main body 41, the wall member 45, and the cylindrical member 54 in the region of two adjacent cylindrical members 54, 54 of the sample plate 4B. [Figure 14] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 15] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 16] FIG. 16 is a perspective view of the sample plate 4 as seen from the ultrasonic generator 2 side in FIG. [Figure 17] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 18] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 19] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 20] FIG. 20 is a schematic diagram of the circulation control unit 15 shown in FIG. [Figure 21] FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. [Figure 22] FIG. 10 is a diagram showing the relationship between ultrasonic amplitude and time. [Figure 23] 4 is a flowchart for explaining the operation of the ultrasonic irradiation device according to the embodiment of the present invention. [Figure 24] FIG. 1 is a schematic diagram of the apparatus used to form amyloid fibrils described in Non-Patent Document 1. [Figure 25] FIG. 1 is a graph showing the time dependence of normalized fluorescence intensity of Thioflavin T in the present invention. [Figure 26] FIG. 10 is a diagram showing the time dependence of fluorescence intensity in a conventional example. [Figure 27] FIG. 10 is another diagram showing the time dependence of normalized fluorescence intensity in the present invention. [Figure 28] FIG. 1 is a conceptual diagram showing the time dependence of fluorescence intensity in high-risk individuals and healthy individuals. DETAILED DESCRIPTION OF THE INVENTION

[0029] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals and description thereof will not be repeated.

[0030] Fig. 1 is a schematic diagram of an ultrasonic irradiation device according to an embodiment of the present invention. Referring to Fig. 1, an ultrasonic irradiation device 10 according to the embodiment of the present invention includes a container 1, an ultrasonic generator 2, a rotating shaft 3, a sample plate 4, a rotor 5, a light irradiation device 6, a light detection device 7, and a seal 8.

[0031] The container 1 has a hollow cylindrical shape. A medium, such as water, is placed inside the container 1. The container 1 is made of stainless steel, glass, acrylic, or the like.

[0032] The ultrasonic generator 2 is placed in contact with the bottom surface 1A of the container 1. The ultrasonic generator 2 generates ultrasonic waves having a frequency of, for example, 30 kHz, and irradiates the generated ultrasonic waves from the bottom surface 1A of the container 1 into the interior of the container 1.

[0033] One end of the rotating shaft 3 is rotatably attached to the bottom member 11 of the container 1. In this case, one end of the rotating shaft 3 is attached to the bottom member 11 of the container 1 via a bearing or the like (not shown). This allows the rotating shaft 3 to rotate with low resistance.

[0034] The sample plate 4 has a circular planar shape and is fixed to the rotating shaft 3 so that its central axis coincides with the center of the rotating shaft 3. In this case, the sample plate 4 is fixed to the other end of the rotating shaft 3 so that the sticker 8 affixed to the upper surface of the sample plate 4 is positioned above the medium (water) in the container 1. The sample plate 4 has a plurality of wells 44. Each of the plurality of wells 44 is filled with, for example, a solution containing a protein and fluorescent molecules that adsorb to protein aggregates and emit light.

[0035] In an embodiment of the present invention, the protein is, for example, amyloid β, which causes Alzheimer's disease, or α-synuclein, which causes Parkinson's disease. When the protein is amyloid β or α-synuclein, the fluorescent molecule is thioflavin T. Thioflavin T adsorbs to aggregates of amyloid β or α-synuclein and emits fluorescence.

[0036] The rotor 5 is fixed to the rotor shaft 3 below the sample plate 4 (on the side of the bottom member 11 of the container 1).

[0037] The light irradiation device 6 is placed, for example, above the sample plate 4 and irradiates the solution placed in each well 44 of the sample plate 4 through the seal 8 with excitation light having a wavelength of, for example, 450 nm.

[0038] The photodetector 7 is placed, for example, above the wells 44 of the sample plate 4, and detects fluorescence emitted by fluorescent molecules in the solution through the seal 8. The fluorescence has a wavelength of, for example, 487 nm.

[0039] The seal 8 is attached to the upper surface of the sample plate 4 so as to cover the multiple wells 44. The seal 8 is made of, for example, a Titer Stick HC Film 547-KTS series seal from Watson Corporation. The thickness of the seal 8 is, for example, 90 μm.

[0040] When the ultrasonic generator 2 generates ultrasonic waves, the generated ultrasonic waves are irradiated onto the medium (water) in the container 1 from below the container 1.

[0041] The ultrasonic waves propagate through the medium (water) and are irradiated onto the impeller 5 and the solution in each well 44. When the impeller 5 is irradiated with ultrasonic waves, as will be described later, the sound pressure of the ultrasonic waves causes the impeller 5 to rotate (self-rotate) in the circumferential direction of the impeller 5, thereby rotating the rotating shaft 3. As a result, the sample plate 4 rotates in the circumferential direction.

[0042] When the sample plate 4 rotates, the multiple wells 44 rotate axially symmetrically around the circumference of the sample plate 4. Then, while the multiple wells 44 are rotating, the light irradiation device 6 irradiates the solution in each well 44 with excitation light through the seal 8, and the light detection device 7 detects the fluorescence emitted by fluorescent molecules in the solution through the seal 8.

[0043] Fig. 2 is a schematic diagram of the ultrasonic generator 2 shown in Fig. 1. Referring to Fig. 2, the ultrasonic generator 2 includes a horn 21, a piezoelectric element 22, electrodes 23 and 24, and a voltage generator 25.

[0044] Horn 21 has the shape of an inverted truncated cone and is made of, for example, stainless steel. A truncated cone is a cone with a circular base. In other words, a truncated cone is a three-dimensional figure obtained by cutting a cone with a plane parallel to the base, excluding the small cone portion. In a truncated cone, the diameter of the circle on the top surface is smaller than the diameter of the circle on the bottom surface. In horn 21, the diameter of top surface 21A is larger than the diameter of bottom surface 21B, so horn 21 has the shape of an inverted truncated cone. Horn 21 is positioned so that top surface 21A is in contact with bottom surface 1A of container 1.

[0045] The piezoelectric element 22 is made of titanium oxide, barium oxide, etc. The electrode 23 is disposed on one surface of the piezoelectric element 22, and the electrode 24 is disposed on the other surface of the piezoelectric element 22.

[0046] The voltage generator 25 is electrically connected to the electrodes 23 and 24. The voltage generator 25 generates an impulse voltage (amplitude: for example, 300 V) and applies the generated impulse voltage to the electrodes 23 and 24.

[0047] When an impulse voltage is applied to electrodes 23 and 24, piezoelectric element 22 vibrates by contracting and expanding, generating ultrasonic waves. The ultrasonic waves generated by piezoelectric element 22 propagate through horn 21 and are irradiated onto the medium (water) in container 1 from upper surface 21A of horn 21.

[0048] Fig. 3 is a perspective view of the sample plate 4 shown in Fig. 1. Note that the seal 8 is omitted in Fig. 3.

[0049] 3(a) shows a perspective view of the sample plate 4 seen from the light irradiation device 6 side in FIG. 1, and FIG. 3(b) shows a perspective view of the sample plate 4 seen from the ultrasonic generator 2 side in FIG.

[0050] Referring to FIG. 3, the sample plate 4 includes a main body 41, a through-hole 42, a plurality of through-holes 43, a plurality of wells 44, and a plurality of wall members 45.

[0051] The main body 41 has a disk shape with a diameter of, for example, 60 mm. The through-hole 42 has a circular planar shape with a predetermined radius from the center of the disk-shaped main body 41, and penetrates the main body 41 in the thickness direction. The through-hole 42 is a hole into which the rotating shaft 3 is inserted. The through-hole 42 has a diameter of, for example, 4 mm.

[0052] The plurality of through holes 43 penetrate the main body portion 41 in the thickness direction and are arranged radially around the through hole 42. More specifically, the plurality of through holes 43 are arranged axially symmetrically on the main body portion 41 with the through hole 42 as the center, between the through hole 42 and the plurality of wells 44. That is, the plurality of through holes 43 are arranged at equal distances from the through hole 42, and are arranged on the main body portion 41 such that the angle formed by two lines connecting two adjacent through holes 43 in the circumferential direction of the main body portion 41 to the center of the through hole 42 is θ (constant). θ is, for example, 20°. Each of the plurality of through holes 43 has a planar shape whose width in the circumferential direction of the main body portion 41 gradually increases from the through hole 42 toward the outer periphery of the main body portion 41.

[0053] 3, the plurality of through-holes 43 includes, for example, 18 through-holes 43, and the plurality of wells 44 includes, for example, 18 wells 44. In this manner, the number of wells 44 is equal to the number of through-holes 43. Note that the number of wells 44 may be different from the number of through-holes 43.

[0054] The multiple wells 44 are arranged in the main body 41 at predetermined intervals around the circumference of the multiple through holes 43, along a circle CIR1 whose center is the through hole 42. That is, the multiple wells 44 are arranged axially symmetrically in the main body 41 with the through hole 42 as the center. In this case, each well 44 is provided in the main body 41 so that the circumferential center of the through hole 43 and the center of the well 44 are located on a straight line L1 that indicates one radius of the main body 41. As a result, the multiple (=18) wells 44 are provided corresponding to the multiple (=18) through holes 43, respectively. Furthermore, each of the multiple wells 44 protrudes downward from the main body 41 perpendicular to the plane of the circular shape of the main body 41.

[0055] The distance between each of the plurality of wells 44 and the center of the through-hole 42 is, for example, 25 mm. Each of the plurality of wells 44 has a diameter of, for example, 5 mm. Each of the plurality of wells 44 has a depth of, for example, 8 mm.

[0056] Each of the plurality of wall members 45 is disposed between two wells 44 adjacent to each other in the circumferential direction of the main body portion 41. Each of the plurality of wall members 45 has a height smaller than the depth of the well 44.

[0057] The sample plate 4 is made of, for example, polypropylene (transparent material) and is produced using a mold.

[0058] The term "axial symmetry" refers to a state in which, when viewed from the central axis, the shape and physical quantity do not change in the circumferential direction, but change depending only on the distance from the central axis.

[0059] Also, although the seal 8 is not shown in Figure 3, the seal 8 is attached to the main body 41 and the multiple wells 44 along the circumferential direction of the main body 41 so as to cover the multiple openings 44A of the multiple wells 44.

[0060] FIG. 4 is a cross-sectional view of the seal 8, the body 41, the wells 44 and the wall member 45 in the region of two adjacent wells 44, 44.

[0061] 4, well 44 protrudes downward from main body 41. Well 44 has opening 44A on the main body 41 side, and protrusion 44B provided in a circular shape around opening 44A.

[0062] The inner diameter of protrusion 44B is the same as the diameter of opening 44A. That is, protrusion 44B is arranged along the inner surface of well 44. The surface on which protrusion 44B is arranged is the surface that coincides with upper surface 41A of main body 41.

[0063] The seal 8 is attached onto the main body 41 and the protruding portion 44B so as to close the opening 44A of the well 44.

[0064] By providing the protrusion 44B, it becomes easier to attach a seal that covers the well 44 after the solution containing the protein and the fluorescent molecule has been placed in the well 44.

[0065] The height of the protrusion 44B is, for example, 0.3 mm, and the width of the protrusion 44B is, for example, 0.5 mm.

[0066] Furthermore, the corners where the bottom surface 44C and side surface 44D of the well 44 intersect do not have a right-angle cross-sectional shape, but rather have a cross-sectional shape with a predetermined curvature R. The curvature R is, for example, 0.5 mm. This prevents proteins from aggregating at the corners of the well 44 and causing the protein aggregates to become non-uniform when ultrasound is irradiated to the solution.

[0067] Furthermore, the bottom surface 44C of each well 44 has a flat portion FT having an area corresponding to a diameter equal to 50% or more of the outer diameter of the well 44. The bottom surface 44C has the flat portion FT, which makes it easier for ultrasonic energy to enter the well 44. In each well 44, the thickness of the well 44 at the bottom surface 44C is, for example, 0.5 mm.

[0068] 3(a), when the sample plate 4 is placed in the container 1, the bottom surface (the surface opposite to the top surface 41A) of the main body 41 is disposed so as to be in contact with the medium (water) in the container 1. As a result, the multiple wells 44 protrude downward from the main body 41, and a wall member 45 is provided between two adjacent wells 44 in the circumferential direction of the main body 41. Therefore, when the sample plate 4 is placed in the container 1, the area surrounded by the main body 41, the multiple wells 44, and the multiple wall members 45 is filled with the medium (water).

[0069] In this state, when ultrasound is irradiated onto the medium (water) from the bottom surface 1A side of the container 1, bubbles are generated in the medium (water) inside the container 1, and the generated bubbles also enter the area surrounded by the main body 41, the multiple wells 44, and the multiple wall members 45.

[0070] If the sample plate 4 does not have multiple through holes 43, air bubbles will accumulate unevenly between the medium (water) and the main body 41 of the sample plate 4 in the area surrounded by the main body 41, the multiple wells 44, and the multiple wall members 45, making it difficult to maintain the multiple wells 44 of the sample plate 4 in a completely axially symmetric state.

[0071] However, when the sample plate 4 is provided with a plurality of through-holes 43, even if bubbles are generated between the medium (water) and the main body 41 of the sample plate 4 in the area surrounded by the main body 41, the plurality of wells 44, and the plurality of wall members 45, the generated bubbles can be released into the atmosphere through the plurality of through-holes 43. As a result, the influence of the bubbles can be eliminated and the plurality of wells 44 of the sample plate 4 can be continuously rotated axially symmetrically.

[0072] Fig. 5 is a perspective view of the rotor 5 shown in Fig. 1. Fig. 5 shows a perspective view of the rotor 5 as seen from the ultrasonic wave generator 2 side in Fig. 1.

[0073] Referring to Figure 5, rotor 5 has a through hole 51, a plurality of blades 52, and a plurality of recesses 53. Rotor 5 has a truncated cone shape. Through hole 51 penetrates rotor 5 in the thickness direction. Rotor shaft 3 is inserted into through hole 51. Rotor 5 has, for example, a bottom surface 5A having a diameter of approximately 3 cm and a top surface 5B having a diameter of approximately 2.5 cm.

[0074] Rotor 5 has a structure in which a plurality of blades 52 and a plurality of recesses 53 are arranged in the circumferential direction of the inclined surface between top surface 5B and bottom surface 5A so that blades 52 and recesses 53 alternate.

[0075] Each of the plurality of blades 52 has a spiral shape in the direction from the top surface 5B to the bottom surface 5A, and has a shape in which the circumferential width increases from the top surface 5B to the bottom surface 5A. Each of the plurality of recesses 53 has a spiral shape in the direction from the top surface 5B to the bottom surface 5A, and has a shape in which the circumferential width decreases from the top surface 5B to the bottom surface 5A.

[0076] The rotor 5 is made of, for example, acrylic resin and is produced using a mold.

[0077] In the ultrasonic irradiation device 10, when ultrasonic waves are irradiated onto the rotor 5, the blades 52 receive the sound pressure of the ultrasonic waves, so that torque is generated in the rotor 5, causing the rotor 5 to rotate in the circumferential direction (self-rotating).

[0078] When the rotor 5 rotates in the circumferential direction (self-rotating), the rotary shaft 3 also rotates in the circumferential direction, and as a result, the multiple wells 44 of the sample plate 4 rotate axially symmetrically in the circumferential direction of the sample plate 4.

[0079] As described above, "axial symmetry" means that when viewed from the central axis, the shape and physical quantities do not change in the circumferential direction, but change only depending on the distance from the central axis. Since the multiple wells 44 are arranged at equal distances from the center (through-hole 42) of the sample plate 4, in the rotating sample plate 4, the multiple physical quantities of the multiple solutions placed in the multiple wells 44 will be the same insofar as this is due to the structure of the sample plate 4.

[0080] As a result, if there are differences in protein aggregates among the multiple solutions placed in the multiple wells 44, it is believed that this is due to the proteins themselves, not the structure of the sample plate 4.

[0081] Therefore, the ultrasonic irradiation device 10 has a structure that can uniformly obtain protein aggregates in a plurality of solutions placed in a plurality of wells 44 .

[0082] In the ultrasonic irradiation device 10, the multiple wells 44 rotate axially symmetrically around the through-hole 42, so that the solution contained in each well 44 can be stirred without providing a stirrer to stir the solution contained in each well 44.

[0083] As a result, the ultrasonic irradiation device 10 can be made smaller than conventional devices.

[0084] As shown in FIG. 1, the rotor 5 is fixed to the rotating shaft 3 so that the bottom surface 5A is positioned above the top surface 5B, and therefore, in the ultrasonic irradiation device 10, the rotor 5 has the shape of an inverted truncated cone.

[0085] As shown in Figure 1, the ultrasound irradiated into the container 1 propagates through the medium (water) and is irradiated onto the rotor 5, and also propagates through the medium (water) along the inverted truncated cone shape of the rotor shaft 5 and is irradiated onto the solution in each well 44.

[0086] Therefore, the impeller 5 is preferably fixed to the rotating shaft 3 at a position where the ultrasonic waves propagating through the medium (water) along the inverted truncated cone shape of the impeller 5 are irradiated onto the solution in each well 44. That is, the impeller 5 has an inverted truncated cone shape for guiding the ultrasonic waves to the multiple wells 44, and has a structure in which the multiple blades 52 are arranged along the circumferential direction of the inclined surface of the inverted truncated cone, and is fixed to the rotating shaft 3 at a position where the ultrasonic waves are induced into the multiple wells 44. This allows the ultrasonic waves to be efficiently irradiated onto the solution in each well 44.

[0087] FIG. 6 is a diagram for explaining a method for detecting fluorescence emitted from a plurality of solutions placed in a plurality of wells 44. FIG. 7 is a diagram showing the relationship between light intensity and time. In FIG. 7, the vertical axis represents light intensity or fluorescence intensity, and the horizontal axis represents time. In FIG. 7, the time t1 to t 72 represents the time during which the fluorescence intensity of the fluorescence emitted from the solution begins to increase.

[0088] Referring to Figure 6, when detecting fluorescence emitted from multiple solutions placed in multiple wells 44, a reflective sheet 50 is attached to the upper surface of the main body 41 of the sample plate 4 between two circumferentially adjacent wells 44.

[0089] Then, while the multiple wells 44 of the sample plate 4 are rotating axially symmetrically, the light irradiation device 6 sequentially irradiates the multiple wells 44 and the multiple reflective sheets 50 with excitation light. When the light irradiation device 6 irradiates the reflective sheet 50 with excitation light, the light detection device 7 detects the light reflected from the reflective sheet 50, and when the light irradiation device 6 irradiates the wells 44 with excitation light, the light detection device 7 detects the fluorescence emitted from the solution in the wells 44. The intensity of the reflected light is much greater than the intensity of the fluorescence.

[0090] When reflected light and fluorescent light are detected while rotating the multiple wells 44 of the sample plate 4 axially symmetrically, the relationship between light intensity and time shown in FIG. 7(a) is obtained.

[0091] 7, it is assumed that excitation light is first irradiated onto the reflective sheet 50 (50a) shown in Fig. 6 while the multiple wells 44 of the sample plate 4 are rotating clockwise, and the sample plate 4 rotates twice. The rotation speed of the sample plate 4 is, for example, 2 Hz.

[0092] Referring to (a) of Figure 7, peaks P1_1 to P18_1 and P1_2 to P18_2 are peaks of light reflected by the reflecting sheet 50, and fluorescent signals SP1_1 to SP18_1 and SP1_2 to SP18_2 are fluorescent signals emitted by fluorescent molecules in the solution in the 18 wells 44.

[0093] When the sample plate 4 makes one rotation the first time, peaks P1_1 to P18_1 and fluorescent signals SP1_1 to SP18_1 are detected, and when the sample plate 4 makes one rotation the second time, peaks P1_2 to P18_2 and fluorescent signals SP1_2 to SP18_2 are detected.

[0094] The peak P1_1 is the peak of the light reflected by the reflecting sheet 50 (50a) shown in FIG. 6, and the fluorescent signal SP1_1 is the fluorescent signal emitted by the fluorescent molecules in the solution in the well 44 (44a) shown in FIG.

[0095] 6. Furthermore, peak P2_1 is the peak of light reflected by the reflective sheet 50 (50b) shown in FIG. 6, and fluorescent signal SP2_1 is the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44b) shown in FIG.

[0096] Furthermore, the peak P3_1 is the peak of the light reflected by the reflecting sheet 50 (50c) shown in FIG.

[0097] Similarly, peak P17_1 is the peak of reflected light by the reflective sheet 50 (50q) shown in Figure 6, and fluorescent signal SP17_1 is the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44q) shown in Figure 6.

[0098] 6. Furthermore, peak P18_1 is the peak of light reflected by the reflective sheet 50 (50r) shown in FIG. 6, and fluorescent signal SP18_1 is the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44r) shown in FIG. 6.

[0099] Furthermore, peak P1_2 is the peak of light reflected by the reflective sheet 50 (50a) shown in FIG. 6, and fluorescent signal SP2_1 is a fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44a) shown in FIG.

[0100] Furthermore, peak P2_2 is the peak of light reflected by the reflective sheet 50 (50b) shown in Figure 6, and fluorescent signal SP2_2 is a fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44b) shown in Figure 6.

[0101] Furthermore, peak P3_2 is the peak of the light reflected by the reflective sheet 50 (50c) shown in FIG.

[0102] Similarly, peak P17_2 is the peak of reflected light by the reflective sheet 50 (50q) shown in Figure 6, and fluorescent signal SP17_2 is the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44q) shown in Figure 6.

[0103] Furthermore, peak P18_2 is the peak of light reflected by the reflective sheet 50 (50r) shown in Figure 6, and fluorescent signal SP18_2 is a fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44r) shown in Figure 6.

[0104] When the peaks P1_1 to P18_1, P1_2 to P18_2 and the fluorescent signals SP1_1 to SP18_1, SP1_2 to SP18_2 are detected, the fluorescent signal SP1_1 and times t1 and t2, and the fluorescent signal SP1_2 and time t 37 ,t 38 are extracted from FIG. 7(a) to create the time dependence of the fluorescent signals SP1_1 and SP1_2 (see FIG. 7(b)).

[0105] In addition, the fluorescence signal SP2_1 and times t3 and t4, and the fluorescence signal SP2_2 and time t 39 ,t 40 and are extracted from FIG. 7(a) to create the time dependence of the fluorescent signals SP2_1 and SP2_2 (see FIG. 7(c)).

[0106] Similarly, the fluorescence signal SP18_1 and the time t 35 ,t 36 , the fluorescence signal SP18_2 and time t 71 ,t 72 and SP18_2 are extracted from FIG. 7(a) to generate the time dependence of the fluorescent signals SP18_1 and SP18_2 (see FIG. 7(d)).

[0107] The rotation speed of the sample plate 4 is 2 Hz, so it rotates twice per second. As a result, the time from when the fluorescent signal SP1_1 is detected until when the fluorescent signal SP1_2 is detected is 0.5 seconds. This time (0.5 seconds) corresponds to the time between time t2 and time t 37 It corresponds to the time between

[0108] On the other hand, the time from when ultrasound begins to be irradiated onto the solution in each well 44, when protein aggregates form, and when the fluorescent molecules begin to emit fluorescence due to the protein aggregates, until the fluorescence intensity reaches its maximum, is typically 10 minutes or more.

[0109] Therefore, in FIG. 7(b), the fluorescence intensity of the fluorescence signal SP1_1 at time t2 and the fluorescence intensity of the fluorescence signal SP1_2 at time t 37 Based on the fluorescence intensity at time t and the fluorescence intensity at time t, the fluorescence intensity is calculated as a function of time. 37 The fluorescence intensity at time t increases gradually until it reaches time t 37 It is believed that there is no problem in interpolating the fluorescence intensity between

[0110] So, time t2 and time t 37 The fluorescence intensity between these values ​​is interpolated to obtain the time dependence of the fluorescence intensity emitted from the fluorescent molecules in the solution in the well 44 (44a).

[0111] Similarly, for the fluorescent light signals SP2_1 and SP2_2 shown in FIG. 7(c), the time t4 and the time t 39 The fluorescence intensity between is interpolated to obtain the time dependence of the fluorescence intensity emitted from the fluorescent molecules in the solution in the well 44 (44b).

[0112] Similarly, the fluorescence signals SP18_1 and SP18_2 shown in FIG. 7(d) are also measured at time t 36 and time t 71 The fluorescence intensity between is interpolated to obtain the time dependence of the fluorescence intensity emitted from the fluorescent molecules in the solution in the well 44 (44r).

[0113] Therefore, by determining the reflecting sheet 50 (50a) onto which the light irradiation device 6 first irradiates excitation light and the rotation direction of the sample plate 4, it is possible to determine that the fluorescent signals SP1_1, SP1_2 detected after the peaks P1_1, P1_2 of the reflected light from the reflecting sheet 50 (50a) are the fluorescent signals of the fluorescent light emitted from the solution in the well 44 (44a) adjacent to the reflecting sheet 50 (50a) in the counterclockwise direction, and thereafter, the fluorescent signals of the fluorescent light emitted from the solution in each well 44 can be identified in a similar manner. As a result, the fluorescent signals of the fluorescent light emitted from the multiple solutions in the multiple wells 44 can be identified, and multiple protein aggregates in the multiple solutions in the multiple wells 44 can be detected.

[0114] It should be noted that only one reflection sheet 50 (50a) may be provided in the ultrasound irradiation device 10. In this case, after one peak such as peaks P1_1, P2_1 shown in Fig. 7 is observed, whose light intensity is greater than the fluorescence intensity of the fluorescence signal, 18 fluorescence signals such as fluorescence signals SP1_1, SP2_1 shown in Fig. 7 are observed, and therefore the fluorescence from the 18 solutions in the 18 wells 44 can be identified.

[0115] Furthermore, since it may take more than 10 hours for the protein aggregation reaction to be completed, it is not necessary to measure fluorescence for every rotation. For example, the fluorescence intensity of each well 44 for 10 rotations may be averaged every 10 minutes (the measurement time in this case is approximately 5 seconds), and the value may be recorded every 10 minutes.

[0116] Furthermore, the solutions contained in the wells 44 may be the same as or different from one another.

[0117] 8 is a schematic diagram of another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10A shown in FIG.

[0118] 8, an ultrasonic irradiation device 10A is the same as the ultrasonic irradiation device 10 shown in FIG. 1 except that the rotation axis 3 of the ultrasonic irradiation device 10 shown in FIG.

[0119] The rotating shaft 3A has a length shorter than the depth of the container 1. In the ultrasonic irradiation device 10A, the rotor 5 is fixed to one end of the rotating shaft 3A below the sample plate 4 (the end on the bottom surface 1A side of the container 1).

[0120] As a result, in the ultrasonic irradiation device 10A, the rotating shaft 3A, the sample plate 4, and the rotor 5 float so that the upper surface of the sample plate 4 is positioned above the medium (water).

[0121] In the ultrasonic irradiation device 10A, when ultrasonic waves generated by the ultrasonic generator 2 are irradiated into the container 1, torque is generated in the rotor 5 by the sound pressure of the ultrasonic waves, and the floating rotor shaft 3A, sample plate 4, and rotor 5 rotate integrally in the circumferential direction of the sample plate 4. As a result, the multiple wells 44 of the sample plate 4 rotate axially symmetrically.

[0122] In addition, the ultrasound irradiated into the container 1 propagates through the medium (water) and is irradiated to the solution in each well 44 of the sample plate 4 while the floating rotating shaft 3A, sample plate 4, and rotor 5 rotate integrally in the circumferential direction of the sample plate 4.

[0123] The light irradiating device 6 irradiates the solution in each well 44 with excitation light through the seal 8 , and the light detecting device 7 detects fluorescence from the solution in each well 44 through the seal 8 .

[0124] Therefore, in the ultrasonic irradiation device 10A, the multiple wells 44 of the sample plate 4 can be rotated axially symmetrically, and the fluorescence from the solution in each well 44 can be detected to detect multiple protein aggregates contained in the multiple solutions in the multiple wells 44.

[0125] In the ultrasonic irradiation device 10A as well, the rotor 5 is preferably fixed to the rotary shaft 3A at a position where the ultrasonic waves propagating through the medium (water) along the inverted truncated cone shape of the rotor 5 are irradiated onto the solution in each well 44. In this case, the rotary shaft 3A has a length that allows the ultrasonic waves propagating through the medium (water) along the inverted truncated cone shape of the rotor 5 to be irradiated onto the solution in each well 44. This allows the ultrasonic waves to be efficiently irradiated onto the solution in each well 44.

[0126] Other explanations regarding the ultrasonic irradiation device 10A are the same as those regarding the ultrasonic irradiation device 10.

[0127] 9 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10B shown in FIG.

[0128] Referring to FIG. 9, the ultrasonic irradiation device 10B is the same as the ultrasonic irradiation device 10 shown in FIG. 1 except that the rotor 5 is removed and the sample plate 4 is replaced with a sample plate 4A.

[0129] The sample plate 4A is the same as the sample plate 4 except that a plurality of blades 46 are added to the sample plate 4.

[0130] The blades 46 are arranged on the bottom surface of the sample plate 4A (the surface opposite to the surface to which the sticker 8 is attached). The sample plate 4A is fixed to the rotation shaft 3 at the same position as the sample plate 4.

[0131] Fig. 10 is a perspective view of the sample plate 4A shown in Fig. 9. Fig. 10 shows a perspective view of the sample plate 4A seen from the ultrasonic generator 2 side in Fig. 9.

[0132] Referring to FIG. 10, the blades 46 are arranged axially symmetrically in the circumferential direction of the sample plate 4A with the through-hole 42 of the sample plate 4A as the center.

[0133] Each of the plurality of blades 46 has, for example, a rectangular parallelepiped shape. The plurality of blades 46 are arranged so that the rectangular side surface of each blade 46 is perpendicular to the circumferential direction of the main body 41. The plurality of blades 46 are also arranged so that two through holes 43 and two wells 44 are present in the region where two adjacent blades 46 are arranged in the circumferential direction of the main body 41.

[0134] In addition, the multiple blades 46 may be arranged so that one through hole 43 and one well 44 are present in the area where two adjacent blades 46 are arranged in the circumferential direction of the main body 41, or so that three through holes 43 and three wells 44 are present in the area where two adjacent blades 46 are arranged in the circumferential direction of the main body 41.

[0135] Furthermore, if the number of through-holes 43 and wells 44 present in the region where two adjacent blades 46 are arranged in the circumferential direction of the main body 41 increases, the number of blades 46 decreases.

[0136] Therefore, in the ultrasonic irradiation device 10B, it is sufficient that the blades 46 are arranged axially symmetrically in the number sufficient to generate a torque that allows the sample plate 4A to rotate in the circumferential direction around the through-hole 42.

[0137] In the ultrasonic irradiation device 10B, the blades 46 are arranged so that the rectangular side surfaces of the blades 46 are perpendicular to the circumferential direction of the main body 41. Therefore, when ultrasonic waves generated by the ultrasonic generator 2 are irradiated into the container 1, the sound pressure of the ultrasonic waves applied to the rectangular side surfaces of the blades 46 generates torque in the circumferential direction of the main body 41. As a result, the multiple wells 44 of the sample plate 4A rotate axially symmetrically around the through-holes 42.

[0138] Moreover, the ultrasonic waves irradiated into the container 1 propagate through the medium (water) and are irradiated onto the solution in each well 44 of the sample plate 4A.

[0139] The light irradiating device 6 irradiates the solution in each well 44 with excitation light through the seal 8 , and the light detecting device 7 detects fluorescence from the solution in each well 44 through the seal 8 .

[0140] Therefore, in the ultrasonic irradiation device 10B, the multiple wells 44 of the sample plate 4A can be rotated axially symmetrically, and the fluorescence from the solution in each well 44 can be detected to detect multiple protein aggregates contained in the multiple solutions in the multiple wells 44.

[0141] In the ultrasonic irradiation device 10B, the rotating shaft 3 may be replaced with the rotating shaft 3A shown in Fig. 8, and the sample plate 4A may be fixed to the rotating shaft 3A. In this case, the rotating shaft 3A and the sample plate 4A rotate together in the circumferential direction due to the sound pressure of the ultrasonic waves while suspended in the medium (water). As a result, the multiple wells 44 of the sample plate 4A rotate axially symmetrically in the circumferential direction around the through-hole 42.

[0142] In the ultrasonic irradiation device 10B, each of the plurality of blades 46 may have a spiral shape along the direction from the through-hole 42 toward the well 44.

[0143] Furthermore, similar to FIG. 5, the bottom surface of the sample plate 41 may be engraved in a spiral shape, so that the bottom surface of the sample plate 41 itself serves as a blade.

[0144] Other explanations regarding the ultrasonic irradiation device 10B are the same as those regarding the ultrasonic irradiation devices 10 and 10A.

[0145] 11 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10C shown in FIG.

[0146] Referring to Figure 11, the ultrasonic irradiation device 10C is the same as the ultrasonic irradiation device 10 except that the sample plate 4 and the seal 8 of the ultrasonic irradiation device 10 shown in Figure 1 are arranged so that the seal 8 is positioned below the sample plate 4.

[0147] In the ultrasonic irradiation device 10C, ultrasonic waves propagating through the medium (water) are irradiated onto the solution in each well 44 via the seal 8.

[0148] In addition, the light irradiation device 6 irradiates excitation light onto the solution in each well 44 through the bottom surface 44C of each well 44, and the light detection device 7 detects fluorescence from the solution in each well 44 through the bottom surface 44C of each well 44.

[0149] As described above, the thickness of the seal 8 is 90 μm, and the thickness at the bottom surface 44C of the well 44 is 0.5 mm. In the ultrasonic irradiation device 10C, ultrasonic waves propagating through the medium (water) are irradiated onto the solution in the well 44 via the seal 8. In this case, the ultrasonic waves have a wavelength of, for example, 2 cm, and are therefore irradiated onto the solution in the well 44 without being reflected much by the seal 8, which is 90 μm thick.

[0150] As a result, the ultrasonic waves can be applied to the solution in the well 44 more efficiently by applying the ultrasonic waves through the seal 8 that is thinner than the thickness of the bottom surface 44C of the well 44.

[0151] Therefore, the time required for protein aggregates to form in the solution can be shortened, and protein aggregates can be formed with good uniformity in the multiple solutions in the multiple wells 44. Then, the photodetector 7 can detect the protein aggregates formed with good uniformity in the multiple wells 44.

[0152] The ultrasonic irradiation device 10C may be an ultrasonic irradiation device 10A to which a modification from the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10C (a modification in which the sample plate 4 and the seal 8 are positioned so that the seal 8 is positioned below the sample plate 4) has been applied.

[0153] Other explanations regarding the ultrasonic irradiation device 10C are the same as those regarding the ultrasonic irradiation devices 10 and 10A.

[0154] 12 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10D shown in FIG.

[0155] Referring to FIG. 12, an ultrasonic irradiation device 10D is the same as the ultrasonic irradiation device 10 except that the sample plate 4 of the ultrasonic irradiation device 10 shown in FIG. 1 is replaced with a sample plate 4B and a seal 9 is added.

[0156] The sample plate 4B is the same as the sample plate 4, except that the wells 44 of the sample plate 4 are replaced with cylindrical members 54.

[0157] The seal 8 is attached to the upper surface of the sample plate 4B so as to close the upper opening of the cylindrical member 54, and the seal 9 is attached to the side (or bottom) of the cylindrical member 54 so as to close the lower opening of the cylindrical member 54. The seal 9 is made of the same seal as the seal 8 described above.

[0158] In the ultrasonic irradiation device 10D, the solution is placed in a cylindrical member 54 that is sealed by seals 8 and 9.

[0159] FIG. 13 is a cross-sectional view of the seals 8 and 9, the main body 41, the wall member 45, and the cylindrical member 54 in the region of two adjacent cylindrical members 54, 54 of the sample plate 4B.

[0160] 13(a), tubular member 54 has a shape that protrudes downward from main body 41. Tube member 54 has opening 54A on the main body 41 side and protrusion 54C that is circularly provided around opening 54A. Tube member 54 also has opening 54B on the side opposite main body 41. The diameter of opening 54B is the same as the diameter of opening 54A.

[0161] The inner diameter of the protrusion 54C is the same as the diameter of the opening 54A. That is, the protrusion 54C is arranged along the inner surface of the tubular member 54. The surface on which the protrusion 54C is arranged is the surface that coincides with the upper surface 41A of the main body 41.

[0162] The seal 8 is attached onto the main body 41 and the protruding portion 54C so as to close the opening 54A of the cylindrical member 54.

[0163] By providing the protrusion 54C, it becomes easier to attach the seal 8 that covers the tubular member 54.

[0164] The height of the protrusion 54C is the same as the height of the protrusion 44B described above, and the width of the protrusion 54C is the same as the width of the protrusion 44B described above.

[0165] The seal 9 (9a) is attached to the side surface 54D of the cylindrical member 54 so as to close the opening 54B. The seal 9 (9a) is attached to the side surface 54D of the cylindrical member 54, and since the side surface 54D protrudes downward from the main body portion 41, the seal 9 (9a) that closes the opening 54B can be easily attached.

[0166] 13(b), the cylindrical member 54 has an opening 54A and a protruding portion 54C on the side of the main body portion 41, similar to that shown in FIG. 13(a). The seal 8 is then affixed to the main body portion 41 and the protruding portion 54C, as described in FIG. 13(a).

[0167] Furthermore, cylindrical member 54 has a side surface 54D, a bottom surface 54E, and an opening 54F on the side opposite main body 41. Bottom surface 54E consists of the portion of bottom surface 44C shown in FIG. 4 excluding flat portion FT having an area equivalent to a diameter of 50% or more of the outer diameter of well 44 (portion having curvature R).

[0168] The seal 9 (9b) is affixed to the bottom surface 54E of the cylindrical member 54 so as to close the opening 54F. The seal 9 (9b) is affixed to the bottom surface 54E, which has a curvature R, and the bottom surface 54E is located at a position that protrudes downward from the main body 41, so that the seal 9 (9b) that closes the opening 54B can be easily affixed. Furthermore, since the cylindrical member 54 has the bottom surface 54E, it is possible to prevent proteins from aggregating at the corners of the cylindrical member 54 that are blocked by the seal 9 (9b) and causing the protein aggregates to become non-uniform when ultrasound is irradiated to the solution.

[0169] In the ultrasonic irradiation device 10D, the sample plate 4B shown in FIG. 13(a) or the sample plate 4B shown in FIG. 13(b) is used.

[0170] When the sample plate 4B shown in (a) of Figure 13 is used, after sealing the opening 54A with the seal 8, a solution containing the protein and fluorescent molecules may be poured into the tubular member 54 through the opening 54B, and then the opening 54B may be sealed with the seal 9 (9a). Alternatively, after sealing the opening 54B with the seal 9 (9a), a solution containing the protein and fluorescent molecules may be poured into the tubular member 54 through the opening 54A, and then the opening 54A may be sealed with the seal 8.

[0171] Furthermore, when the sample plate 4B shown in (b) of Figure 13 is used, after sealing the opening 54A with the seal 8, a solution containing the protein and fluorescent molecules may be poured into the tubular member 54 through the opening 54F, and then the opening 54F may be sealed with the seal 9 (9b), or after sealing the opening 54F with the seal 9 (9b), a solution containing the protein and fluorescent molecules may be poured into the tubular member 54 through the opening 54A, and then the opening 54A may be sealed with the seal 8.

[0172] Alternatively, a simple disk-shaped (e.g., 60 mm diameter, 8 mm thick) plate made of a transparent or translucent resin material (e.g., acrylic resin or polypropylene) with many through-holes (e.g., 5 mm inner diameter) may be used, with stickers 8 and 9 attached to both sides. In this case, too, sticker 8 may be attached first, the solution may be poured in, and then sticker 9 may be attached, or vice versa.

[0173] In the ultrasonic irradiation device 10D, ultrasonic waves propagating through the medium (water) are irradiated onto the rotor 5, causing the rotor shaft 3, the sample plate 4B to which the seals 8 and 9 are attached, and the rotor 5 to rotate in the circumferential direction of the sample plate 4B. In addition, ultrasonic waves propagating through the medium (water) are irradiated via the seal 9 to the solution in the cylindrical member 54 sealed by the seals 8 and 9.

[0174] Furthermore, the light irradiating device 6 irradiates the solution in the cylindrical member 54 with excitation light through the seal 8, and the light detecting device 7 detects the fluorescence from the solution in the cylindrical member 54 through the seal 8.

[0175] As a result, the ultrasonic waves can be applied to the solution inside the cylindrical member more efficiently by applying the ultrasonic waves through the seal 9, which is thinner than the thickness of the side surface 54D (or bottom surface 54E) of the cylindrical member 54.

[0176] Therefore, the time required for protein aggregates to form in the solution can be shortened, and protein aggregates can be formed uniformly in the multiple solutions in the multiple tubular members 54.

[0177] Then, the photodetector 7 can detect highly uniform protein aggregates formed in the multiple solutions in the multiple cylindrical members 54.

[0178] The ultrasonic irradiation device 10D may be obtained by applying a modification from the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10D (changing the sample plate 4 to the sample plate 4B and adding a seal 9) to either of the ultrasonic irradiation devices 10A and 10B.

[0179] The change from ultrasonic irradiation device 10 to ultrasonic irradiation device 10D (the change from sample plate 4 to sample plate 4B and the addition of seal 9) essentially changes well 44 to cylindrical member 54 and seals openings 54A, 54B (or openings 54A, 54F) on both sides of cylindrical member 54 with seals 8, 9, respectively. Therefore, even if multiple blades 46 are arranged on the inner periphery of multiple wells 44 in ultrasonic irradiation device 10B, the change from ultrasonic irradiation device 10 to ultrasonic irradiation device 10D (the change from sample plate 4 to sample plate 4B and the addition of seal 9) can be applied to ultrasonic irradiation device 10B.

[0180] In addition, when the change from ultrasonic irradiation device 10 to ultrasonic irradiation device 10D (change from sample plate 4 to sample plate 4B and add sticker 9) is applied to ultrasonic irradiation device 10C, the only difference is that the positions of stickers 8 and 9 are reversed from the positions of stickers 8 and 9 in ultrasonic irradiation device 10D, so the change from ultrasonic irradiation device 10 to ultrasonic irradiation device 10D (change from sample plate 4 to sample plate 4B and add sticker 9) applied to ultrasonic irradiation device 10C is essentially the same as ultrasonic irradiation device 10D.

[0181] Other explanations regarding the ultrasonic irradiation device 10D are the same as those regarding the ultrasonic irradiation devices 10, 10A, and 10B.

[0182] 14 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10E shown in FIG.

[0183] Referring to Figure 14, the ultrasonic irradiation device 10E is the same as the ultrasonic irradiation device 10C shown in Figure 11, except that the sample plate 4 with the sticker 8 attached and the rotor 5 are fixed at different positions on the rotating shaft 3 so that they are immersed in the medium (water).

[0184] In the ultrasonic irradiation device 10E, the sample plate 4 with the seal 8 attached and the rotor 5 are fixed to the rotating shaft 3 so that they are immersed in the medium (water), for example, by the following method.

[0185] In the ultrasonic irradiation device 10C, the rotor 5 is fixed to the rotary shaft 3 at a position closer to the bottom surface 1A of the container 1 than the position where the rotor 5 is fixed to the rotary shaft 3. Then, after a solution containing proteins and fluorescent molecules is poured into the multiple wells 44, the multiple wells 44 are sealed with a seal 8, and the sample plate 4 with the seal 8 attached is fixed to the rotary shaft 3 so that the seal 8 is positioned on the rotor 5 side. After that, a medium (water) is poured into the container 1 so that the sample plate 4 with the seal 8 attached and the rotor 5 are immersed.

[0186] The method for fixing the sample plate 4 with the seal 8 attached and the rotor 5 to the rotor shaft 3 so that the rotor 5 is immersed may be other than the above method.

[0187] The ultrasonic generator 2 generates ultrasonic waves and irradiates the generated ultrasonic waves onto the medium (water) from the bottom surface 1A of the container 1. The ultrasonic waves propagating through the medium (water) are irradiated onto the rotor 5, causing the rotor shaft 3, the sample plate 4 to which the seal 8 is affixed, and the rotor 5 to rotate in the circumferential direction of the sample plate 4. The ultrasonic waves propagating through the medium (water) are irradiated onto the solution in each well 44 via the seal 8.

[0188] Furthermore, in the ultrasound irradiation device 10E, the light irradiation device 6 irradiates the solution in each well 44 with excitation light via the medium (water) and the bottom surface 44C of the well 44 of the sample plate 4, and the light detection device 7 detects fluorescence from the solution in each well 44 via the medium (water) and the bottom surface 44C of the well 44 of the sample plate 4. Alternatively, the container 1 may be made of a transparent material such as acrylic resin, and the light irradiation device 6 and the light detection device 7 may be disposed on the side surface to measure fluorescence from the side of the sample plate 4.

[0189] In the ultrasonic irradiation device 10E, each well 44 is positioned closer to the bottom surface 1A of the container 1 than the ultrasonic irradiation device 10C, and ultrasonic waves are irradiated to the solution through a seal 8 that is thinner than the bottom surface 44C of the well 44, so that stronger ultrasonic waves can be irradiated to the solution than in the ultrasonic irradiation device 10C.

[0190] Therefore, the time required for protein aggregates to form in the solution can be shortened, and protein aggregates can be formed uniformly in the multiple solutions in the multiple wells 44.

[0191] Then, the photodetector 7 can detect highly uniform protein aggregates formed in the multiple solutions in the multiple wells 44.

[0192] The ultrasonic irradiation device 10E may be any one of the ultrasonic irradiation devices 10, 10A, 10B, and 10D, in which a modification from the ultrasonic irradiation device 10C to the ultrasonic irradiation device 10E (a modification in which the sample plate 4 with the sticker 8 attached and the fixed positions of the sample plate 4 and the rotor 5 on the rotating shaft 3 are changed so that the rotor 5 is immersed in the medium (water)) has been applied.

[0193] When the ultrasonic irradiation device 10C is changed to the ultrasonic irradiation device 10E and applied to the ultrasonic irradiation device 10A, the rotating shaft 3A, the sample plate 4 with the seal 8 attached, and the rotor 5 in the ultrasonic irradiation device 10A are immersed in the medium (water), and the total weight of the rotating shaft 3A, the sample plate 4, and the rotor 5 is adjusted so that they float.

[0194] Other explanations regarding the ultrasonic irradiation device 10E are the same as those regarding the ultrasonic irradiation devices 10, 10A, 10B, and 10D.

[0195] 15 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10F shown in FIG.

[0196] Referring to Figure 15, the ultrasonic irradiation device 10F is configured by integrating a sample plate 4 and a rotor 5 to which a seal 8A is attached instead of the seal 8 of the ultrasonic irradiation device 10C shown in Figure 11, and the integrated sample plate 4 and rotor 5 are immersed in a medium (water), but is otherwise the same as the ultrasonic irradiation device 10C.

[0197] The rotor 5 is bonded to the upper surface 41A of the main body 41 of the sample plate 4 so that the bottom surface 5A of the rotor 5 is in contact with the upper surface 41A of the main body 41 of the sample plate 4. The integrated sample plate 4 and rotor 5 are then fixed to the rotating shaft 3 so that the rotor 5 has the shape of an inverted truncated cone at a position where it is immersed in the medium (water).

[0198] The seal 8A is attached to the upper surface 41A of the main body 41 of the sample plate 4 in an area not in contact with the bottom surface 5A of the rotor 5 so as to cover the multiple wells 44. The seal 8A is made of the same material as the seal 8.

[0199] FIG. 16 is a perspective view of the sample plate 4 seen from the ultrasonic generator 2 side in FIG.

[0200] 16, in the sample plate 4, the plurality of through-holes 43 are arranged axially symmetrically within a region surrounded by a circle CIR2 with the through-hole 42 at the center. In the ultrasonic irradiation device 10F, the bottom surface 5A of the rotor 5 has a diameter smaller than the diameter of the circle CIR2. The rotor 5 is bonded to the main body 41 of the sample plate 4 so that the bottom surface 5A contacts the top surface 41A of the main body 41 of the sample plate 4 within the region surrounded by the circle CIR2.

[0201] In this way, the bottom surface 5A of the rotor 5 has a diameter smaller than the diameter of the circle CIR2, and the seal 8A is affixed to the upper surface 41A of the main body 41 of the sample plate 4 so as to block the multiple wells 44, so that a portion of the outer periphery of the multiple through holes 43 is not blocked by the bottom surface 5A of the rotor 5 and the seal 8A.

[0202] As a result, bubbles generated in the medium (water) below the sample plate 4 move to the upper end side of the container 1 through the plurality of through-holes 43 of the sample plate 4 and are released into the atmosphere.

[0203] Therefore, even in the ultrasonic irradiation device 10F having an integrated sample plate 4 and rotor 5, the multiple wells 44 of the sample plate 4 rotate axially symmetrically around the through-hole 42, eliminating the influence of bubbles generated in the medium (water).

[0204] In the ultrasonic irradiation device 10F, after a solution containing proteins and fluorescent molecules is placed in the multiple wells 44 of the integrated sample plate 4 and rotor 5, the multiple wells 44 are sealed with seals 8A, and the integrated sample plate 4 and rotor 5 are fixed to the rotary shaft 3 at a position where they are immersed in the medium (water). Then, the medium (water) is poured into the container 1 so that the integrated sample plate 4 and rotor 5 are immersed.

[0205] The ultrasonic generator 2 generates ultrasonic waves and irradiates the generated ultrasonic waves from the bottom surface 1A of the container 1 to the medium (water). The ultrasonic waves propagating through the medium (water) are irradiated to the rotor 5, causing the rotor shaft 3 and the integrated sample plate 4 and rotor 5 to rotate in the circumferential direction of the sample plate 4. The ultrasonic waves propagating through the medium (water) are irradiated to the solution in each well 44 via the seal 8A.

[0206] Furthermore, in the ultrasound irradiation device 10F, the light irradiation device 6 irradiates excitation light onto the solution in each well 44 through the medium (water) and the bottom surface 44C of the well 44 of the sample plate 4, and the light detection device 7 detects fluorescence from the solution in each well 44 through the medium (water) and the bottom surface 44C of the well 44 of the sample plate 4.

[0207] In the ultrasonic irradiation device 10F, each well 44 is positioned closer to the bottom surface 1A of the container 1 than the ultrasonic irradiation device 10C, and ultrasonic waves are irradiated to the solution through a seal 8A that is thinner than the bottom surface 44C of the well 44, so that stronger ultrasonic waves can be irradiated to the solution than in the ultrasonic irradiation device 10C.

[0208] Therefore, the time required for protein aggregates to form in the solution can be shortened, and protein aggregates can be formed uniformly in the multiple solutions in the multiple wells 44.

[0209] Then, the photodetector 7 can detect highly uniform protein aggregates formed in the multiple solutions in the multiple wells 44.

[0210] The ultrasonic irradiation device 10F may be any one of the ultrasonic irradiation devices 10, 10A, and 10D, with the modification from the ultrasonic irradiation device 10C to the ultrasonic irradiation device 10E (the modification in which the sample plate 4 with the sticker 8A attached instead of the sticker 8 is integrated with the rotor 5, and the integrated sample plate 4 and rotor 5 are immersed in a medium (water)).

[0211] When the change from ultrasonic irradiation device 10C to ultrasonic irradiation device 10F is applied to ultrasonic irradiation device 10A, the rotating shaft 3A in ultrasonic irradiation device 10A and the integrated sample plate 4 and rotor 5 are immersed in the medium (water), and the total weight of the rotating shaft 3A, sample plate 4, and rotor 5 is adjusted so that they float.

[0212] Furthermore, when changing from the ultrasonic irradiation device 10C to the ultrasonic irradiation device 10F, one of the seals 8 and 9 is changed to the seal 8A. That is, in the integrated sample plate 4B and the rotor 5, the rotor 5 is adhered to the main body 41 of the sample plate 4B so that the bottom surface 5A of the rotor 5 contacts the surface of the main body 41 to which the seal 8 is attached or the surface of the main body 41 to which the seal 9 is attached.

[0213] Other explanations about the ultrasonic irradiation device 10F are the same as those about the ultrasonic irradiation devices 10, 10A, and 10D.

[0214] 17 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10G shown in FIG.

[0215] Referring to Figure 17, the ultrasonic irradiation device 10G is the same as the ultrasonic irradiation device 10 shown in Figure 1 except that a transparent window 12 is added and the position of the light detection device 7 is changed.

[0216] The transparent window 12 is disposed in the container 1 at a position facing the wells 44 of the sample plate 4. The transparent window 12 is made of a transparent material such as glass or acrylic.

[0217] In the ultrasonic irradiation device 10G, the photodetector 7 is placed at a position facing the transparent window 12 (at a position in the lateral direction of the container 1).

[0218] As described above, the sample plate 4 is made of a transparent body, and therefore, the fluorescence from the solution in each well 44 passes through the side wall of each well 44 and is emitted in the radial direction of the sample plate 4. As a result, the photodetector 7 detects the fluorescence through the transparent window 12 of the container 1.

[0219] When the solution in each well 44 is irradiated with ultrasound, bubbles are generated in the solution and move upward in the solution. As a result, when the fluorescence is detected by the photodetector 7 placed above the solution, noise may be generated.

[0220] However, in the ultrasound irradiation device 10G, the photodetector 7 detects fluorescence from the lateral direction of each well 44, so even if bubbles occur in the solution, the fluorescence can be detected while eliminating noise caused by the bubbles. As a result, multiple protein aggregates in multiple solutions in multiple wells 44 can be detected while eliminating noise caused by bubbles.

[0221] In the ultrasound irradiation device 10G, the container 1 may be made of a transparent material such as glass or acrylic, without providing the transparent window 12. By making the container 1 transparent, the light detection device 7 can detect fluorescence while removing noise caused by air bubbles, and as a result, multiple protein aggregates in multiple solutions in multiple wells 44 can be detected while removing noise caused by air bubbles. Furthermore, the light irradiation device 6 may be installed on the side of the container 1.

[0222] The ultrasonic irradiation device 10G may be any of the ultrasonic irradiation devices 10A, 10B, 10C, 10D, 10E, and 10F, with modifications from the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10G (modifications of adding a transparent window 12 and changing the position of the light detection device 7 to face the transparent window 12).

[0223] Other explanations regarding the ultrasonic irradiation device 10G are the same as those regarding the ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, and 10F.

[0224] 18 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10H shown in FIG.

[0225] Referring to FIG. 18, an ultrasonic irradiation device 10H is the same as the ultrasonic irradiation device 10, except that the container 1 of the ultrasonic irradiation device 10 is replaced with a cylindrical member 31 and the horn 21 of the ultrasonic generator 2 is used as the bottom member.

[0226] Cylindrical member 31 has a hollow cylindrical shape. Because horn 21 has an inverted truncated cone shape, the outer diameter of cylindrical member 31 is the same as the diameter of top surface 21A of horn 21. One axial end of cylindrical member 31 is bonded to top surface 21A of horn 21. As a result, cylindrical member 31 and horn 21 perform the same function as container 1 described above. In this way, horn 21 functions to propagate ultrasonic waves and to serve as the bottom member of the container.

[0227] In the ultrasonic irradiation device 10H, one end of the rotating shaft 3 is attached to the horn 21 via a bearing or the like (not shown). As a result, the rotating shaft 3 can rotate with low resistance.

[0228] In the ultrasonic irradiation device 10H, ultrasonic waves generated by the piezoelectric element 22 of the ultrasonic generator 2 propagate through the horn 21 and are directly irradiated from the horn 21 onto the medium (water).

[0229] Therefore, ultrasonic waves can be efficiently applied to the rotor 5 and the solution in each well 44 .

[0230] The ultrasonic irradiation device 10H may be a modification of the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10H (changing the container 1 to a cylindrical member 31 and changing the horn 21 of the ultrasonic generator 2 to a bottom member) applied to any of the ultrasonic irradiation devices 10A, 10B, 10C, 10D, 10E, 10F, and 10G.

[0231] Other explanations regarding the ultrasonic irradiation device 10H are the same as those regarding the ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, 10F, and 10G.

[0232] 19 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10I shown in FIG.

[0233] 19, an ultrasonic irradiation device 10I is the same as the ultrasonic irradiation device 10 shown in FIG. 1 except that pipes 13 and 14 and a circulation control unit 15 are added.

[0234] One end of the pipe 13 is fixed to the container 1 so as to penetrate the side wall 1B of the container 1 and communicate with the inside of the container 1, and the other end is connected to the circulation control unit 15.

[0235] One end of the pipe 14 is fixed to the container 1 so as to penetrate the side wall 1C of the container 1 and communicate with the inside of the container 1, and the other end is connected to the circulation control unit 15.

[0236] The inlet from the pipe 13 to the vessel 1 is provided below the outlet from the vessel 1 to the pipe 14.

[0237] Fig. 20 is a schematic diagram of the circulation control unit 15 shown in Fig. 19. Referring to Fig. 20, the circulation control unit 15 includes a buffer tank 151, a pump 152, a control unit 153, and a pipe 154. The buffer tank 151 is connected to the other end of the pipe 14 and to one end of the pipe 154. The pump 152 is connected to the other end of the pipe 154 and to the other end of the pipe 13.

[0238] Buffer tank 151 stores the medium (water) supplied from container 1 via pipe 14. Pump 152 sucks the medium (water) from buffer tank 151 via pipe 154 and supplies the sucked medium (water) into container 1 via pipe 13. Then, the medium (water) in buffer tank 151 is supplied by pump 152 into container 1 via pipe 13, and the medium (water) in container 1 is supplied into buffer tank 151 via pipe 14. Thus, pump 152 circulates the medium (water) between container 1 and buffer tank 151.

[0239] The control unit 153 controls the temperature of the medium (water) stored in the buffer tank 151 to a predetermined temperature (e.g., 37°C) and degasses the medium (water). This degassing removes dissolved gas from the medium (water) and prevents bubbles from forming in the medium (water) when ultrasonic waves are applied to the medium (water) in the container 1. As a result, the multiple wells 44 of the sample plate 4 can be stably rotated axially symmetrically. Furthermore, by controlling the temperature of the medium (water) to a predetermined temperature, the temperature of the solution in each well 44 can also be controlled to a predetermined temperature, eliminating the effects of temperature changes in the solution in each well 44 and allowing protein aggregates to form. As a result, variation in the protein aggregates formed in the multiple solutions in the multiple wells 44 can be further reduced.

[0240] In this way, the circulation control unit 15 circulates the medium (water) between the container 1 and the buffer tank 151 while controlling the temperature of the degassed medium (water) to a predetermined temperature.

[0241] The circulation control unit 15 may circulate a medium (water) that has only been degassed between the container 1 and the buffer tank 151, or may circulate a medium (water) that has only been controlled to a predetermined temperature between the container 1 and the buffer tank 151.

[0242] The ultrasonic irradiation device 10I may be configured by adding pipes 13 and 14 and a circulation control unit 15 to any one of the ultrasonic irradiation devices 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H.

[0243] Other explanations regarding the ultrasonic irradiation device 10I are the same as those regarding the ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H.

[0244] 21 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention, which may be an ultrasonic irradiation device 10J shown in FIG.

[0245] Referring to FIG. 21, an ultrasonic irradiation device 10J is the same as the ultrasonic irradiation device 10G shown in FIG. 17 except that the position of the light irradiation device 6 is changed.

[0246] In the ultrasound irradiation device 10J, the light irradiation device 6 is disposed at a position laterally of the container 1 so as to irradiate the solution in each well 44 with excitation light through the transparent window 12. As a result, in the ultrasound irradiation device 10J, the light irradiation device 6 irradiates the solution in each well 44 with excitation light through the transparent window 12, and the light detection device 7 detects the fluorescence emitted from the solution in each well 44 through the transparent window 12.

[0247] Therefore, even if bubbles are generated in each well 44, the influence of the generated bubbles can be eliminated, and the solution in each well 44 can be irradiated with excitation light and the fluorescence emitted from the solution in each well 44 can be detected.

[0248] In the above, it has been explained that the ultrasonic irradiation device 10G may be obtained by applying a modification from the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10G (modifications of adding a transparent window 12 and changing the position of the light detection device 7 to a position opposite the transparent window 12) to any of the ultrasonic irradiation devices 10A, 10B, 10C, 10D, 10E, and 10F.

[0249] The ultrasonic irradiation device 10J is the same as the ultrasonic irradiation device 10G except that the light irradiation device 6 is arranged at a different position so that excitation light is irradiated through the transparent window 12.

[0250] Therefore, the ultrasound irradiation device according to the embodiment of the present invention may be one in which the position of the photodetector 7 is changed so that fluorescence emitted from the solution in each well 44 is detected through the transparent window 12, or one in which the position of the light irradiation device 6 is changed so that excitation light is irradiated onto the solution in each well 44 through the transparent window 12, or one in which the position of the light irradiation device 6 is changed so that excitation light is irradiated onto the solution in each well 44 through the transparent window 12, and the position of the photodetector 7 is changed so that fluorescence emitted from the solution in each well 44 is detected through the transparent window 12. In the embodiment of the present invention, such changes may be applied to the ultrasound irradiation device 10I shown in FIG.

[0251] In addition, the ultrasonic irradiation device according to the embodiment of the present invention may be one in which the rotor 5 of the ultrasonic irradiation device 10 described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the multiple blades 46 of the ultrasonic irradiation device 10B described above are removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10C described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10D described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10E described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10G described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10H described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10I described above is removed and the rotating shaft 3 is rotated by a motor, or one in which the rotor 5 of the ultrasonic irradiation device 10J described above is removed and the rotating shaft 3 is rotated by a motor.

[0252] Figure 22 is a diagram showing the relationship between ultrasonic amplitude and time. In Figure 22, the vertical axis represents ultrasonic amplitude, and the horizontal axis represents time. Furthermore, curve k1 shows the time dependence of the amplitude of a carrier wave having a higher frequency than the frequency of the ultrasonic wave that causes protein aggregation, and curve k2 shows the time dependence of the amplitude of ultrasonic waves having a lower frequency than curve k1. The frequency that causes protein aggregation is created by curves k1 and k2 (due to the so-called amplitude modulation relationship). Furthermore, curve k3 shows the time dependence of the amplitude of another carrier wave having a higher frequency than the frequency of the ultrasonic wave that causes protein aggregation, and curve k4 shows the time dependence of the amplitude of the ultrasonic wave that causes protein aggregation.

[0253] Referring to (a) of Figure 22, the ultrasound has an amplitude that varies according to the time dependence (see curve k1) of the amplitude of a carrier wave having a frequency (e.g., 150 kHz) higher than the frequency (approximately 30 kHz) that causes cavitation in the solution and promotes protein aggregation (see curve k1).

[0254] Referring to (b) of Figure 22, the ultrasound has an amplitude that varies according to the time dependence (see curve k3) of the amplitude of a carrier wave having a frequency (e.g., 150 kHz) higher than the frequency (approximately 30 kHz (see curve k4)) that causes cavitation in the solution and promotes protein aggregation (see curve k3).

[0255] In the above-mentioned ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J, the ultrasonic generator 2 may generate ultrasonic waves of 30 kHz and irradiate the container 1 (or a container consisting of the tubular member 31 and the horn 21) with ultrasonic waves whose amplitude is changed by the amplitude of a carrier wave having a higher frequency (see curve k1 in Figure 22), or may generate ultrasonic waves of 30 kHz and irradiate the container 1 (or a container consisting of the tubular member 31 and the horn 21) with ultrasonic waves whose amplitude is changed by the amplitude of a carrier wave having a higher frequency (see curve k3 in Figure 22).

[0256] By irradiating the container 1 with ultrasound whose amplitude is changed depending on the amplitude of a carrier wave having a frequency higher than the frequency for protein aggregation, the ultrasound whose amplitude changes depending on the amplitude of the carrier wave can propagate through the medium (water) at a shorter carrier wavelength, so that the ultrasound contributes to stirring the solution in the well 44 of the sample plate 4, 4A (or the tubular member 54 of the sample plate 4B), thereby reducing measurement variability.

[0257] It should be noted that instead of continuously irradiating ultrasonic waves, ultrasonic waves may be irradiated by repeating a cycle of, for example, 0.5 seconds of irradiation followed by a 0.5 second pause, thereby reducing the load on the ultrasonic wave generator 2.

[0258] Furthermore, for example, 30 kHz ultrasound may be applied for only the first 5 minutes to generate nuclei for protein aggregation, and thereafter, high-frequency (e.g., 150 kHz) ultrasound may be applied in cycles of 0.5 seconds followed by 0.5 seconds of rest, to simply stir the solution in the well 44 (or the tubular member 54 of the sample plate 4B).

[0259] FIG. 23 is a flowchart for explaining the operation of the ultrasonic irradiation device according to the embodiment of the present invention.

[0260] In Figure 23, the operation of the ultrasound irradiation device is described under the assumption that a solution is placed in multiple wells 44 of sample plate 4, 4A (or multiple tubular members 54 of sample plate 4B), a seal (at least one of seals 8, 8A, 9) is attached to the area where the multiple wells 44 (or multiple tubular members 54) are arranged, and the openings of the multiple wells 44 (or multiple tubular members 54) are blocked by the seal (at least one of seals 8, 8A, 9).

[0261] Referring to FIG. 23, when the operation of the ultrasonic wave irradiation device is started, the ultrasonic wave generator 2 generates ultrasonic waves (step S1), and irradiates the generated ultrasonic waves into the container 1 (step S2).

[0262] The ultrasonic waves irradiated into the container 1 propagate through the medium (water) and are irradiated onto the rotor 5 and the plurality of wells 44 of the sample plate 4 (step S3).

[0263] As a result, the rotor 5 rotates due to the sound pressure of the ultrasonic waves, and the multiple wells 44 of the sample plate 4 rotate axially symmetrically (step S4).

[0264] Then, the light irradiation device 6 irradiates the excitation light onto the solutions in the wells 44 of the sample plate 4 while the wells 44 are rotating axially symmetrically (step S5).

[0265] Thereafter, the photodetector 7 detects a plurality of fluorescent signals emitted from each of the plurality of solutions (step S6).

[0266] Subsequently, the ultrasonic generator 2 determines whether or not to end the measurement (step S7). More specifically, the ultrasonic generator 2 has a built-in timer and measures the time T from when the ultrasonic waves start to be irradiated onto the solution until the proteins in the solution start to aggregate. AGG is stored in advance, and the elapsed time T ELP is time T AGG When the elapsed time T ELP is time T AGG If it is equal to or less than this, it is determined that the measurement is not to be ended.

[0267] If it is determined in step S7 that the measurement should not be ended, the series of operations proceeds to step S1. Then, steps S1 to S7 are repeatedly executed until it is determined in step S7 that the measurement should be ended.

[0268] Then, in step S7, when it is determined that the measurement is to be ended, the ultrasonic wave generator 2 stops generating ultrasonic waves, and the operation of the ultrasonic wave irradiation device ends.

[0269] The operations of the above-described ultrasonic irradiation devices 10, 10A, 10C, 10E, 10F, 10G, 10H, and 10J are executed in accordance with steps S1 to S7 shown in FIG.

[0270] Furthermore, the operation of the ultrasonic irradiation device 10B described above is performed according to a flowchart in which step S4 of steps S1 to S7 shown in FIG. 23 is changed to "a step of rotating multiple blades 46 by the sound pressure of ultrasonic waves to rotate multiple wells 44 of the sample plate 4A axially symmetrically."

[0271] Furthermore, the operation of the above-described ultrasonic irradiation device 10D is performed according to a flowchart in which, among steps S1 to S7 shown in FIG. 23, the "multiple wells" in steps S3, S4, and S5 are replaced with "multiple cylindrical members 54 blocked by seals 8 and 9."

[0272] Furthermore, the operation of the above-described ultrasonic irradiation device 10I is performed according to steps S1 to S7 shown in FIG. 23 while a medium (water) whose temperature is controlled to a predetermined temperature by the circulation control unit 15 is circulated in the container 1.

[0273] Then, after steps S1 to S7 shown in FIG. 23 are performed, the time dependence of the fluorescence intensity is obtained based on the fluorescence signals detected by the photodetector 7 using the method described in FIG. 7, and an increase in the fluorescence intensity is detected based on the obtained time dependence of the fluorescence intensity to detect the formation of protein aggregates, for all of the multiple fluorescence signals.

[0274] This makes it possible to detect whether or not protein aggregates have formed in each of the multiple solutions, and if protein aggregates have formed in each of the multiple solutions, it is possible to evaluate the uniformity of the multiple protein aggregates formed in each of the multiple solutions.

[0275] [experiment] An experiment in which ultrasonic waves are used to accelerate the formation of protein aggregates will be described. Figure 24 is a schematic diagram of the device used to form amyloid fibrils, as described in Non-Patent Document 1.

[0276] Referring to FIG. 24, Non-Patent Document 1 uses a sample plate 100 having 96 wells 101 arranged in 8 rows and 12 columns.

[0277] Each of the 96 wells 101 has a capacity of 0.2 mL. A solution containing β2 microglobulin as a protein, thioflavin T as a fluorescent molecule, and sodium chloride (NaCl) is placed in each of the 96 wells 101. In this case, the concentration of the protein is 0.3 mg / mL, the concentration of NaCl is 38 mM, and the pH of the solution is 2.5.

[0278] The sample plate 100, with the solution placed in each of the 96 wells 101, is floated on the medium (water) in the container 110. Then, ultrasonic waves are irradiated from the bottom surface 110A of the container 110 onto the solution in each well 101 of the sample plate 100, excitation light 140 having a wavelength of 450 nm is irradiated onto the solution in each well 101, and fluorescence 150 having a wavelength of 490 nm emitted by thioflavin T in the solution in each well 101 is detected to detect aggregates of the protein (β2 microglobulin) in the solution in each well 101.

[0279] In this case, the frequency of the ultrasonic waves is 17 kHz to 20 kHz, and the power of the ultrasonic waves is 350 W. The measurement temperature is 37°C.

[0280] Then, ultrasonic waves were irradiated onto the solution in each well 101 by repeatedly irradiating the solution with ultrasonic waves for 10 to 15 minutes and then stopping the irradiation for 5 minutes.

[0281] In addition, the time dependence of the fluorescence intensity was examined by detecting fluorescence from the solution in each well 101 in two cases: when ultrasound was applied without rotating the container 110, and when ultrasound was applied while rotating the container 110 using a rotation stage (not shown). When rotating the container 110, the rotation speed of the container 110 was 6 rpm.

[0282] The time dependence of the fluorescence intensity described in Non-Patent Document 1 is taken as the "conventional example."

[0283] Furthermore, the time dependence of the fluorescence intensity was examined by detecting the fluorescence from the solution in each well 44 using the ultrasonic irradiation device 10. The time dependence of the fluorescence intensity detected using the ultrasonic irradiation device 10 is referred to as "the present invention."

[0284] In an experiment using the ultrasound irradiation device 10, a solution containing β2-microglobulin as a protein and thioflavin T as a fluorescent molecule was placed in each of 18 wells 44. 0.2 mL of the solution was placed in each well 44. The concentration of the solution was 0.3 mg / mL, and the pH of the solution was 2.5.

[0285] Then, the opening 44A of each well 44 was sealed with a seal 8. After that, ultrasonic waves were generated by the ultrasonic generator 2 and irradiated into the container 1. In this case, the ultrasonic frequency was 30 kHz and the ultrasonic power was 30 W. The measurement temperature was 37°C.

[0286] Then, the solution in each well 44 was irradiated with ultrasonic waves while rotating the sample plate 4 at a rotation speed of 2 Hz.

[0287] Fig. 25 is a graph showing the time dependence of normalized fluorescence intensity of Thioflavin T in the present invention, and Fig. 26 is a graph showing the time dependence of fluorescence intensity in a conventional example.

[0288] In FIG. 25, the vertical axis represents the normalized fluorescence intensity of Thioflavin T, and the horizontal axis represents time.

[0289] In Fig. 26, the vertical axis represents fluorescence intensity and the horizontal axis represents time. Fig. 26(a) shows the time dependence of fluorescence intensity when the container 110 (i.e., sample plate 100) is not rotated, and Fig. 26(b) shows the time dependence of fluorescence intensity when the container 110 (i.e., sample plate 100) is rotated.

[0290] Referring to FIG. 25, in the present invention, a plurality of normalized fluorescence intensities of Thioflavin T are measured at time t R_1 ~time t R_2 It starts to increase between time t R_2 In addition, in the time dependence of the normalized fluorescence intensity of Thioflavin T, the coefficient of variation (CV) of the time at which the normalized fluorescence intensity of Thioflavin T begins to increase is 21%.

[0291] 26(a), in the conventional example, when the container 110 (i.e., the sample plate 100) is not rotated, the time until the fluorescence intensity starts to increase is longer than 60 minutes (1 hour). In the time dependence of the multiple fluorescence intensities, the coefficient of variation CV of the time until the fluorescence intensity starts to increase is 50%.

[0292] 26(b), in the conventional example, when the container 110 (i.e., the sample plate 100) is rotated, the time it takes for the fluorescence intensity to start increasing is approximately 50 minutes or more. In the time dependence of the fluorescence intensity, the coefficient of variation (CV) of the time at which the fluorescence intensity starts to increase is 32%.

[0293] Therefore, the coefficient of variation CV (=21%) in the present invention is less than half of the coefficient of variation CV (=50%) in the conventional example when the container 110 (i.e., sample plate 100) is not rotated, and is approximately 0.66 times the coefficient of variation CV (=32%) in the conventional example when the container 110 (i.e., sample plate 100) is rotated.

[0294] In the conventional example, when the container 110 (that is, the sample plate 100) is rotated, the 96 wells 101 do not rotate axially symmetrically.

[0295] Therefore, as shown in the present invention, it has been experimentally shown that by rotating the multiple wells 44 axially symmetrically, the time at which protein aggregates begin to form in the solution in the multiple wells 44 can be made faster than in conventional examples, and the time at which protein aggregates begin to form can be made more uniform than in conventional examples.

[0296] Furthermore, in the experiments of the present invention, an ultrasound irradiation device was used in which a transparent window 12 was provided in the container 1 of the above-described ultrasound irradiation device 10C, and the position of the photodetector 7 was changed to face the transparent window 12 as shown in Figure 17. A solution containing alpha-synuclein (the protein that causes Parkinson's disease) as a protein, thioflavin T as a fluorescent molecule, and sodium chloride (NaCl) was placed into multiple wells 44 of a sample plate 4, and the fluorescence from the solution was detected by the photodetector 7 through the transparent window 12, and the time dependence of the normalized fluorescence intensity was obtained.

[0297] Then, the time dependence of the normalized fluorescence intensity when α-synuclein aggregate seeds were added to the solution and when no α-synuclein aggregate seeds were added to the solution were obtained.

[0298] In this case, the protein (α-synuclein) concentration was 0.5 mg / mL, the NaCl concentration was 100 mM, the pH of the solution was 7.4, the ultrasonic frequency was 27 kHz, and the ultrasonic power was about 30 W.

[0299] Fig. 27 is a graph showing the time dependence of another normalized fluorescence intensity in the present invention. In Fig. 27, the vertical axis represents the normalized fluorescence intensity of Thioflavin T, and the horizontal axis represents time. In addition, the group of curves k CG1 shows the time dependence of the normalized fluorescence intensity of Thioflavin T without adding α-synuclein aggregation seeds, and the curve family k CG2 shows the time dependence of the normalized fluorescence intensity of thioflavin T upon addition of α-synuclein aggregation seeds.

[0300] Referring to FIG. 27, when no aggregation seeds of α-synuclein are added, the time it takes for protein (α-synuclein) aggregates to begin to form ranges from 0.5 hours to 2 hours (curve group k CG1 reference).

[0301] On the other hand, when α-synuclein aggregation seeds are added, the time required for protein (α-synuclein) aggregates to begin to form is shorter than 0.5 hours (curve family k CG2 reference).

[0302] Therefore, it was clearly demonstrated experimentally that the time at which protein (α-synuclein) aggregates begin to form can be accelerated by adding α-synuclein aggregation seeds.

[0303] Furthermore, when no α-synuclein aggregation seeds are added, the time it takes for protein (α-synuclein) aggregates to begin to form is in the range of 0.5 to 2 hours (curve group k CG1 It has been experimentally demonstrated that the formation of protein (α-synuclein) aggregates can be accelerated by irradiating a solution with ultrasound through a seal 8.

[0304] As described above, it has been experimentally demonstrated that by irradiating a solution containing a protein (α-synuclein or β2-microglobulin) with ultrasound using an ultrasound irradiation device according to an embodiment of the present invention, aggregates of the protein (α-synuclein or β2-microglobulin) can be formed in a shorter time and with better uniformity than in conventional examples.

[0305] Alpha-synuclein and beta-2 microglobulin are proteins found in the human body that cause Parkinson's disease and dialysis-related amyloidosis, respectively.

[0306] Normally, alpha-synuclein aggregates in the human body over a long period of time, such as decades, and causes Parkinson's disease.

[0307] Therefore, by irradiating a solution containing α-synuclein with ultrasound using an ultrasound irradiation device according to an embodiment of the present invention and accelerating the formation of α-synuclein aggregates, it becomes possible to diagnose whether or not there is a risk of developing Parkinson's disease in the future. The same applies to other disease-causing proteins such as amyloid β (the protein that causes Alzheimer's disease).

[0308] Figure 28 is a conceptual diagram showing the time dependence of fluorescence intensity in high-risk individuals and healthy individuals. In Figure 28, the vertical axis represents fluorescence intensity, and the horizontal axis represents time. The fluorescence intensity represents the amount of aggregates formed.

[0309] Referring to FIG. 28, in high-risk individuals who are at high risk of developing Parkinson's disease or Alzheimer's disease, the fluorescence intensity (the amount of aggregates produced) increases in a shorter time than in healthy individuals.

[0310] Therefore, a threshold value th_RSK for determining whether a subject is at high risk of developing Parkinson's disease or Alzheimer's disease is set in advance, and the time t INC If th_RSK is equal to or less than the threshold value, the risk of developing Parkinson's disease or Alzheimer's disease is determined to be high, and INC When the time t_RSK is longer than the threshold th_RSK, the risk of developing Parkinson's disease or Alzheimer's disease is determined to be low, thereby making it possible to diagnose whether the risk of developing Parkinson's disease or Alzheimer's disease in the future is high or low.

[0311] In the above-described embodiments, ultrasonic irradiation devices 10, 10A to 10J for accelerating the formation of protein aggregates have been described, but the ultrasonic irradiation devices according to the embodiments of the present invention are not limited to accelerating the formation of protein aggregates and may be used for purposes other than accelerating the formation of protein aggregates. In general, any device may be used that irradiates ultrasonic waves onto objects within multiple wells 44 (or multiple tubular members 54 blocked with seals 8, 9) while rotating the multiple wells 44 (or multiple tubular members 54 blocked with seals 8, 9) axially symmetrically.

[0312] Therefore, the ultrasonic irradiation device according to the embodiment of the present invention is a sample plate having a plurality of wells arranged axially symmetrically; a rotating device that rotates the sample plate so that the plurality of wells rotate axially symmetrically; The sample plate may be provided with an ultrasonic generator that generates ultrasonic waves and irradiates the generated ultrasonic waves onto the solution introduced into each of the multiple wells while the sample plate is rotating.

[0313] Furthermore, in the above-described embodiment, the medium was described as water, but in the embodiment of the present invention, this is not limited to this, and the medium may be a fluid or a gas (e.g., air).

[0314] In this embodiment of the present invention, the plurality of cylindrical members 54 closed with the seals 8 and 9 constitute "plurality of wells."

[0315] In addition, in this embodiment of the present invention, the rotor 5 constitutes a "rotating device", and the plurality of blades 46 provided on the sample plate 4A constitute the "rotating device".

[0316] Furthermore, in this embodiment of the present invention, the cylindrical member 31 and the horn 21 form a "container."

[0317] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the description of the above embodiments, and is intended to include all modifications within the meaning and scope of the claims. [Industrial Applicability]

[0318] The present invention is applied to an ultrasonic irradiation device. [Explanation of symbols]

[0319] 1 container, 2 ultrasonic generator, 3, 3A rotating shaft, 4, 4A, 4B sample plate, 5 rotor, 6 light irradiation device, 7 light detection device, 8, 8A, 9 seal, 10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H ultrasonic irradiation device, 11 bottom member, 12 transparent window, 13, 14, 154 piping, 15 circulation control unit, 21 horn, 22 piezoelectric element, 23, 24 electrode, 25 voltage generator, 31, 54 cylindrical member, 41 main body, 42, 43, 51 through hole, 44 well, 44A, 54A, 54B, 54F opening, 44B, 54C protrusion, 45 wall member, 46 blade, 50 reflective sheet, 52 blade, 53 recess, 151 Buffer tank, 152 pump, 153 control section.

Claims

1. a sample plate having a plurality of wells arranged axially symmetrically; a rotating device that rotates the sample plate so that the plurality of wells rotate axially symmetrically; an ultrasonic generator that generates ultrasonic waves and irradiates the generated ultrasonic waves onto the solutions introduced into each of the plurality of wells while the sample plate is rotating; The rotating device is an ultrasonic irradiation device that rotates the sample plate by the sound pressure of the ultrasonic waves generated by the ultrasonic generator.

2. 2. The ultrasonic irradiation device according to claim 1, wherein the rotating device is composed of a plurality of blades arranged on the bottom surface of the sample plate along the circumferential direction of the sample plate and rotating the sample plate by the sound pressure of the ultrasonic waves.

3. Further comprising a rotation shaft to which the sample plate is fixed, 2. The ultrasonic irradiation device according to claim 1, wherein the rotating device comprises a rotor fixed to the rotating shaft and rotating by itself in response to the sound pressure of the ultrasonic waves, thereby rotating the sample plate via the rotating shaft.

4. The ultrasonic irradiation device according to claim 3 , wherein the rotating shaft, the sample plate, and the rotor are immersed in a medium made of a fluid.

5. a seal that closes the openings of the plurality of wells and is thinner than the thickness of the wells; 5. The ultrasonic irradiation device according to claim 3, wherein the ultrasonic waves are irradiated to the solution in the plurality of wells through the seal.

6. The rotor blade has an inverted truncated cone shape and includes a plurality of blades arranged in a circumferential direction of an inclined surface of the inverted truncated cone, The ultrasonic irradiation device according to claim 3 , wherein each of the plurality of blades has a spiral shape in the length direction of the rotation shaft.

7. 7. The ultrasonic irradiation device according to claim 1, wherein the sample plate has a plurality of through holes for allowing bubbles generated in a fluid medium by irradiating the medium with ultrasonic waves to escape into the atmosphere.

8. The ultrasonic irradiation device according to claim 7 , wherein the plurality of through-holes are arranged axially symmetrically on the sample plate.

9. The ultrasonic irradiation device according to claim 1 , wherein each of the plurality of wells has a cylindrical shape with a predetermined curvature at an intersection between a side surface and a bottom surface.

10. 10. The ultrasonic irradiation device according to claim 1, wherein the solution contains a protein and a fluorescent molecule that is adsorbed to the protein aggregates that have aggregated due to the ultrasonic irradiation and emits light.

11. a light irradiation device that irradiates the solution with excitation light having a predetermined wavelength; The ultrasound irradiation device according to claim 10 , further comprising a light detection device that detects fluorescence emitted by the fluorescent molecules when the excitation light is irradiated by the light irradiation device.

12. The sample plate is made of a transparent disc-shaped body, The ultrasonic irradiation device according to claim 11 , wherein the light detection device is disposed on the outer periphery of the sample plate at a position facing the well of the sample plate.

13. The sample plate is placed in a container; The ultrasonic irradiation device according to claim 12 , wherein the container has a transparent window at a position facing the well of the sample plate.

14. 14. The ultrasonic irradiation device according to claim 1, further comprising a circulation control unit that controls the temperature of a medium made of a fluid to a predetermined temperature, degasses the medium, and circulates the degassed medium controlled to the predetermined temperature within a container in which the sample plate is placed.

Images