JP7773183B2 - Ultrasonic irradiation device - Google Patents

Description

This invention relates to an ultrasonic irradiation device.

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

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.

When amyloid is induced from a protein, the amyloid emits fluorescence by binding to an amyloid-specific fluorescent dye. The presence of the amyloid-specific fluorescent dye in multiple wells of a microplate where amyloid has been induced causes fluorescence, and a plate reader can detect this fluorescence to detect amyloid in solution.

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 then detects the amyloid formed by the ultrasound irradiation.

International Publication No. 2012/017739

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.

However, when performing multi-channel detection by placing a supersaturated protein solution in multiple wells and detecting amyloid formed by ultrasound irradiation, the following problems arise:

It is extremely difficult to ensure that the ultrasonic intensity introduced into each channel is 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 multiple samples in water, but this has the disadvantage of being incompletely uniform, and the structure requires forcibly rotating the container using a stage or other device, making the entire device large and complex.

In addition, while it is effective to introduce a stirrer to stir the solution in a fluorescence spectrophotometer, there is the problem that it is difficult to introduce a stirrer when the amount of solution is small.

Accordingly, an embodiment of the present invention provides an ultrasound irradiation device that can improve uniformity when detecting an object using multiple channels.

(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.

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

(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.

(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 includes a rotor fixed to the rotating shaft and configured to rotate the sample plate via the rotating shaft by self-rotating in response to the sound pressure of the ultrasonic waves.

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

(Configuration 6)
In the fourth or fifth aspect, 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.

(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 helical shape in the length direction of the rotor shaft.

(Configuration 8)
In any one of the configurations 1 to 7, 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.

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

(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.

(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.

(Configuration 12)
In the configuration 11, 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.

(Configuration 13)
In the 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.

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

(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.

Improves uniformity when detecting objects across multiple channels.

1 is a schematic diagram of an ultrasonic irradiation device according to an embodiment of the present invention. FIG. 2 is a schematic diagram of the ultrasonic generator shown in FIG. 1. FIG. 2 is a perspective view of the sample plate shown in FIG. 1 . 1 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. FIG. FIG. 2 is a perspective view of the rotor shown in FIG. 1 . 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. FIG. 10 is a diagram showing the relationship between light intensity and time. FIG. 10 is a schematic diagram of another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a perspective view of the sample plate shown in FIG. 9 . FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. 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. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. 16 is a perspective view of the sample plate 4 as seen from the ultrasonic generator 2 side in FIG. 15. FIG. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 20 is a schematic diagram of the circulation control unit 15 shown in FIG. 19 . FIG. 10 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. FIG. 10 is a diagram showing the relationship between ultrasonic amplitude and time. 4 is a flowchart for explaining the operation of the ultrasonic irradiation device according to the embodiment of the present invention. FIG. 1 is a schematic diagram of the apparatus used to form amyloid fibrils as described in Non-Patent Document 1. FIG. 1 is a graph showing the time dependence of normalized fluorescence intensity of Thioflavin T in the present invention. FIG. 10 is a diagram showing the time dependence of fluorescence intensity in a conventional example. FIG. 10 is another diagram showing the time dependence of normalized fluorescence intensity in the present invention. FIG. 1 is a conceptual diagram showing the time dependence of fluorescence intensity in high-risk individuals and healthy individuals.

Embodiments of the present invention will be described in detail with reference to the drawings. Note that identical or corresponding parts in the drawings will be designated by the same reference numerals, and their description will not be repeated.

Figure 1 is a schematic diagram of an ultrasonic irradiation device according to an embodiment of the present invention. Referring to Figure 1, the ultrasonic irradiation device 10 according to this 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.

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

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.

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.

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 seal 8 affixed to the top surface of the sample plate 4 is positioned above the medium (water) in the container 1. The sample plate 4 also has a plurality of wells 44. Each of the plurality of wells 44 is filled with, for example, a solution containing protein and fluorescent molecules that adsorb to protein aggregates and emit light.

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

The rotor 5 is fixed to the rotor shaft 3 below the sample plate 4 (towards the bottom member 11 of the container 1).

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

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

The seal 8 is attached to the top surface of the sample plate 4 so as to cover the multiple wells 44. The seal 8 is, for example, a WATSON Titer Stick HC Film 547-KTS series seal. The thickness of the seal 8 is, for example, 90 μm.

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

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

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

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

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 formed 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.

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

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.

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

Figure 3 is a perspective view of the sample plate 4 shown in Figure 1. Note that the seal 8 is omitted from Figure 3.

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

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.

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.

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

In FIG. 3, the plurality of through holes 43 consists of, for example, 18 through holes 43, and the plurality of wells 44 consists of, for example, 18 wells 44. In this way, 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.

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 centered on the through hole 42. That is, the multiple wells 44 are arranged axially symmetrically in the main body 41 with the through hole 42 at 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 circular plane of the main body 41.

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

Each of the multiple wall members 45 is disposed between two adjacent wells 44 in the circumferential direction of the main body 41. Each of the multiple wall members 45 has a height that is smaller than the depth of the well 44.

The sample plate 4 is made, for example, from polypropylene (transparent) and is produced using a mold.

Note that "axial symmetry" refers to a state in which, when viewed from the central axis, the shape and physical quantities do not change in the circumferential direction, but change depending only on the distance from the central axis.

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.

Figure 4 is a cross-sectional view of the seal 8, main body 41, well 44, and wall member 45 in the area of two adjacent wells 44, 44.

Referring to Figure 4, the well 44 protrudes downward from the main body 41. The well 44 has an opening 44A on the main body 41 side and a circular protrusion 44B around the opening 44A.

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

The seal 8 is affixed to the main body 41 and the protrusion 44B so as to cover the opening 44A of the well 44.

By providing the protrusion 44B, it becomes easier to attach a seal that covers the well 44 after a solution containing proteins and fluorescent molecules is placed in the well 44.

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.

Furthermore, the corner where the bottom surface 44C and side surface 44D of the well 44 intersect does not have a right-angle cross-sectional shape, but rather has 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 onto the solution.

Furthermore, the bottom surface 44C of each well 44 has a flat portion FT with an area corresponding to a diameter of 50% or more of the outer diameter of the well 44. The bottom surface 44C has a 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.

Referring again to FIG. 3(a), when the sample plate 4 is placed in the container 1, the bottom surface of the main body 41 (the surface opposite the top surface 41A) is positioned so that it comes into 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 circumferentially 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).

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

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, multiple wells 44, and multiple wall members 45, making it difficult to maintain complete axial symmetry among the multiple wells 44 of the sample plate 4.

However, if the sample plate 4 is provided with multiple 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, multiple wells 44, and multiple wall members 45, the generated bubbles can be released into the atmosphere through the multiple through-holes 43. As a result, the influence of the bubbles can be eliminated and the multiple wells 44 of the sample plate 4 can be continuously rotated axially symmetrically.

Figure 5 is a perspective view of the rotor 5 shown in Figure 1. Note that Figure 5 shows a perspective view of the rotor 5 as viewed from the ultrasonic generator 2 side in Figure 1.

Referring to Figure 5, the rotor 5 has a through-hole 51, multiple blades 52, and multiple recesses 53. The rotor 5 has a truncated cone shape. The through-hole 51 penetrates the rotor 5 in the thickness direction. The through-hole 51 is a hole into which the rotor shaft 3 is inserted. The rotor 5 has, for example, a bottom surface 5A with a diameter of approximately 3 cm and a top surface 5B with a diameter of approximately 2.5 cm.

The rotor 5 has a structure in which multiple blades 52 and multiple recesses 53 are arranged circumferentially on the inclined surface between the top surface 5B and the bottom surface 5A, with the blades 52 and recesses 53 alternating.

Each of the multiple wings 52 has a spiral shape extending from the top surface 5B to the bottom surface 5A, and its circumferential width increases from the top surface 5B to the bottom surface 5A. Each of the multiple recesses 53 has a spiral shape extending from the top surface 5B to the bottom surface 5A, and its circumferential width decreases from the top surface 5B to the bottom surface 5A.

The rotor blades 5 are made of, for example, acrylic resin and are manufactured using a mold.

In the ultrasonic irradiation device 10, when ultrasonic waves are irradiated onto the rotor 5, the multiple blades 52 are subjected to the sound pressure of the ultrasonic waves, generating torque in the rotor 5, causing the rotor 5 to rotate circumferentially (self-rotating).

When the rotor blades 5 rotate circumferentially (self-rotating), the rotating shaft 3 also rotates circumferentially, causing the multiple wells 44 of the sample plate 4 to rotate axially symmetrically in the circumferential direction of the sample plate 4.

As mentioned above, "axial symmetry" refers to a state in which, 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 positioned at equal distances from the center (through-holes 42) of the sample plate 4, the multiple physical quantities of the multiple solutions placed in the multiple wells 44 on the rotating sample plate 4 will be the same to the extent that this is due to the structure of the sample plate 4.

As a result, if differences in protein aggregates occur in the multiple solutions placed in the multiple wells 44, this is thought to be due to the proteins themselves, not the structure of the sample plate 4.

Therefore, the ultrasound irradiation device 10 has a structure that allows protein aggregates to be obtained uniformly in multiple solutions placed in multiple wells 44.

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

As a result, the ultrasound irradiation device 10 can be made smaller than conventional devices.

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

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.

Therefore, the impeller 5 is preferably fixed to the rotating shaft 3 at a position where ultrasound propagating through the medium (water) along the inverted truncated cone shape of the impeller 5 is irradiated onto the solution in each well 44. In other words, the impeller 5 has an inverted truncated cone shape for guiding ultrasound to multiple wells 44, and has a structure in which 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 ultrasound is induced into multiple wells 44. This allows ultrasound to be efficiently irradiated onto the solution in each well 44.

Fig. 6 is a diagram illustrating a method for detecting fluorescence emitted from multiple solutions placed in multiple 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, times _t1 to _t72 represent the time period during which the fluorescence intensity of the fluorescence emitted from the solutions begins to increase.

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

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, it 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.

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

In Figure 7, the multiple wells 44 of the sample plate 4 are rotating clockwise, and the excitation light is first irradiated onto the reflective sheet 50 (50a) shown in Figure 6, and the sample plate 4 rotates twice. The rotation speed of the sample plate 4 is, for example, 2 Hz.

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 reflective 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.

When the sample plate 4 rotates once 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 rotates once the second time, peaks P1_2 to P18_2 and fluorescent signals SP1_2 to SP18_2 are detected.

Peak P1_1 is the peak of light reflected by the reflective sheet 50 (50a) shown in Figure 6, and fluorescent signal SP1_1 is the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44a) shown in Figure 6.

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

Furthermore, peak P3_1 is the peak of light reflected by the reflective sheet 50 (50c) shown in Figure 6.

Similarly, peak P17_1 is the peak of light reflected 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.

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

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

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 the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44b) shown in Figure 6.

Furthermore, peak P3_2 is the peak of light reflected by the reflective sheet 50 (50c) shown in Figure 6.

Similarly, peak P17_2 is the peak of light reflected 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.

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 the fluorescent signal emitted by fluorescent molecules in the solution in well 44 (44r) shown in Figure 6.

When peaks P1_1 to P18_1, P1_2 to P18_2 and fluorescence signals SP1_1 to SP18_1, SP1_2 to SP18_2 are detected, the fluorescence signal SP1_1 and times _t1 and _t2 , and the fluorescence signal SP1_2 and times _t37 and _t38 are extracted from Figure 7(a) to create the time dependence of the fluorescence signals SP1_1 and SP1_2 (see Figure 7(b)).

Furthermore, the fluorescence signal SP2_1 and times t ₃ and t ₄ , and the fluorescence signal SP2_2 and times t ₃₉ and t ₄₀ are extracted from FIG. 7( a ), and the time dependence of the fluorescence signals SP2_1 and SP2_2 is created (see FIG. 7( c )).

Similarly, the fluorescence signal SP18_1 and times t ₃₅ and t ₃₆ , and the fluorescence signal SP18_2 and times t ₇₁ and t ₇₂ are extracted from Figure 7(a) to create the time dependence of the fluorescence signals SP18_1 and SP18_2 (see Figure 7(d)).

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 to 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 _t37 in Figure 7(b).

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

Therefore, in FIG. 7(b), based on the fluorescence intensity of fluorescence signal SP1_1 at time _t2 and the fluorescence intensity of fluorescence signal SP1_2 at time _t37 , it is considered that there will be no problem if the fluorescence intensity between time _t2 and time _t37 is interpolated so that the fluorescence intensity gradually increases over time from the fluorescence intensity at time _t2 to the fluorescence intensity at time _t37 .

Therefore, the fluorescence intensity between time _t2 and time _t37 is interpolated to obtain the time dependence of the fluorescence intensity emitted from the fluorescent molecules in the solution in the well 44 (44a).

Similarly, for the fluorescent signals SP2_1 and SP2_2 shown in (c) of Figure 7, the fluorescent intensity between time _t4 and time _t39 is interpolated to obtain the time dependence of the fluorescent intensity emitted from the fluorescent molecules in the solution in well 44 (44b).

Similarly, for the fluorescent signals SP18_1 and SP18_2 shown in (d) of Figure 7, the fluorescent intensity between time _t36 and time _t71 is interpolated to obtain the time dependence of the fluorescent intensity emitted from the fluorescent molecules in the solution in well 44 (44r).

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

In addition, the ultrasound irradiation device 10 may be provided with only one reflective sheet 50 (50a). In this case, after a peak such as peaks P1_1, P2_1 shown in FIG. 7 is observed, where the 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.

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 can be averaged every 10 minutes (the measurement time in this case is approximately 5 seconds), and this value can be recorded every 10 minutes.

Furthermore, the multiple solutions placed in the multiple wells 44 may be the same as each other or different from each other.

Figure 8 is a schematic diagram of another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10A shown in Figure 8.

Referring to Figure 8, the ultrasonic irradiation device 10A is identical to the ultrasonic irradiation device 10 shown in Figure 1, except that the rotation axis 3 is replaced with a rotation axis 3A.

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 (the end on the bottom surface 1A side of the container 1) below the sample plate 4.

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

In the ultrasonic irradiation device 10A, when ultrasonic waves generated by the ultrasonic generator 2 are irradiated into the container 1, the sound pressure of the ultrasonic waves generates torque in the rotor 5, causing the floating rotor shaft 3A, sample plate 4, and rotor 5 to 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.

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

The light irradiation device 6 then irradiates the solution in each well 44 with excitation light through the seal 8, and the light detection device 7 detects fluorescence from the solution in each well 44 through the seal 8.

Therefore, even with the ultrasound 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 each of the multiple solutions in the multiple wells 44.

In the ultrasonic irradiation device 10A, 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.

The rest of the description of the ultrasonic irradiation device 10A is the same as the description of the ultrasonic irradiation device 10.

Figure 9 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10B shown in Figure 9.

Referring to Figure 9, the ultrasonic irradiation device 10B is identical to the ultrasonic irradiation device 10 shown in Figure 1 except that the rotor 5 is removed and the sample plate 4 is replaced with a sample plate 4A.

Sample plate 4A is identical to sample plate 4, except that it has multiple blades 46 added.

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 in the same position as the sample plate 4.

Figure 10 is a perspective view of the sample plate 4A shown in Figure 9. Note that Figure 10 shows a perspective view of the sample plate 4A as seen from the ultrasonic generator 2 side in Figure 9.

Referring to Figure 10, the multiple blades 46 are arranged axially symmetrically around the circumferential direction of the sample plate 4A, with the through-hole 42 of the sample plate 4A at the center.

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

The blades 46 may be arranged so that one through-hole 43 and one well 44 exist in the region 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 exist in the region where two adjacent blades 46 are arranged in the circumferential direction of the main body 41.

Furthermore, if the number of through holes 43 and wells 44 present in the area where two adjacent blades 46 are arranged in the circumferential direction of the main body 41 increases, the number of blades 46 will decrease.

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

In the ultrasonic irradiation device 10B, each blade 46 is positioned so that the rectangular side of each blade 46 is 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, each blade 46 generates torque in the circumferential direction of the main body 41 due to the sound pressure of the ultrasonic waves applied to the rectangular side. As a result, the multiple wells 44 of the sample plate 4A rotate axially symmetrically around the through-hole 42.

Furthermore, the ultrasound irradiated into the container 1 propagates through the medium (water) and is irradiated onto the solution in each well 44 of the sample plate 4A.

The light irradiation device 6 then irradiates the solution in each well 44 with excitation light through the seal 8, and the light detection device 7 detects fluorescence from the solution in each well 44 through the seal 8.

Therefore, even in the ultrasound 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 each of the multiple solutions in the multiple wells 44.

In the ultrasonic irradiation device 10B, the rotating shaft 3 may be replaced with the rotating shaft 3A shown in Figure 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.

Furthermore, in the ultrasonic irradiation device 10B, each of the multiple blades 46 may have a spiral shape extending in the direction from the through-hole 42 toward the well 44.

Furthermore, similar to Figure 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 acts as a blade.

The rest of the description of the ultrasonic irradiation device 10B is the same as the description of the ultrasonic irradiation devices 10 and 10A.

Figure 11 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10C shown in Figure 11.

Referring to Figure 11, the ultrasonic irradiation device 10C is identical to the ultrasonic irradiation device 10 except that the sample plate 4 and seal 8 of the ultrasonic irradiation device 10 shown in Figure 1 are positioned so that the seal 8 is located below the sample plate 4.

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.

In addition, the light irradiation device 6 irradiates the solution in each well 44 with excitation light 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.

As described above, the thickness of the seal 8 is 90 μm, and the thickness of 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 irradiated onto the solution in the well 44 without being reflected much by the 90 μm thick seal 8.

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

This shortens the time it takes for protein aggregates to form in the solution, and allows protein aggregates to be formed with good uniformity in the multiple solutions in the multiple wells 44. The photodetector 7 can then detect the protein aggregates that have formed with good uniformity in the multiple wells 44.

The ultrasonic irradiation device 10C may be obtained by modifying the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10C (by positioning the sample plate 4 and seal 8 so that the seal 8 is positioned below the sample plate 4) and applying this to the ultrasonic irradiation device 10A.

The rest of the description of the ultrasonic irradiation device 10C is the same as the description of the ultrasonic irradiation devices 10 and 10A.

Figure 12 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10D shown in Figure 12.

Referring to Figure 12, the ultrasonic irradiation device 10D is identical to the ultrasonic irradiation device 10 except that the sample plate 4 of the ultrasonic irradiation device 10 shown in Figure 1 is replaced with sample plate 4B and a seal 9 is added.

Sample plate 4B is identical to sample plate 4, except that the well 44 of sample plate 4 is replaced with a cylindrical member 54.

Seal 8 is affixed to the top surface of sample plate 4B so as to close the upper opening of cylindrical member 54, and seal 9 is affixed to the side (or bottom) of cylindrical member 54 so as to close the lower opening of cylindrical member 54. Seal 9 is made of the same material as seal 8 described above.

In the ultrasonic irradiation device 10D, the solution is placed inside a cylindrical member 54 sealed by seals 8 and 9.

Figure 13 is a cross-sectional view of the seals 8, 9, main body 41, wall member 45, and tubular member 54 in the area of two adjacent tubular members 54, 54 of the sample plate 4B.

Referring to (a) of Figure 13, the tubular member 54 has a shape that protrudes downward from the main body 41. The tubular member 54 has an opening 54A on the side facing the main body 41, and a circular protrusion 54C surrounding the opening 54A. The tubular member 54 also has an opening 54B on the side opposite the main body 41. The diameter of the opening 54B is the same as the diameter of the opening 54A.

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.

The seal 8 is affixed to the main body 41 and the protruding portion 54C so as to close the opening 54A of the tubular member 54.

The protrusion 54C makes it easier to apply the seal 8 that covers the tubular member 54.

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

The seal 9 (9a) is affixed to the side surface 54D of the tubular member 54 so as to close the opening 54B. The seal 9 (9a) is affixed to the side surface 54D of the tubular member 54, and because the side surface 54D protrudes downward from the main body portion 41, the seal 9 (9a) that closes the opening 54B can be easily affixed.

Referring to FIG. 13(b), the tubular member 54 has an opening 54A and a protruding portion 54C on the main body portion 41 side, similar to 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).

The cylindrical member 54 also has a side surface 54D, a bottom surface 54E, and an opening 54F on the side opposite the main body 41. The bottom surface 54E consists of the bottom surface 44C shown in Figure 4 excluding the flat portion FT, which has an area equivalent to a diameter of at least 50% of the outer diameter of the well 44 (the portion with curvature R).

Seal 9 (9b) is affixed to the bottom surface 54E of the tubular member 54 so as to block the opening 54F. Seal 9 (9b) is affixed to the bottom surface 54E, which has a curvature R, and because the bottom surface 54E is located in a position that protrudes downward from the main body 41, seal 9 (9b) that blocks the opening 54B can be easily affixed. Furthermore, because the tubular member 54 has a bottom surface 54E, it is possible to prevent proteins from aggregating at the corners of the tubular member 54 blocked by seal 9 (9b) when ultrasound is irradiated to the solution, which could result in uneven protein aggregates.

The ultrasonic irradiation device 10D uses the sample plate 4B shown in Figure 13(a) or the sample plate 4B shown in Figure 13(b).

When the sample plate 4B shown in Figure 13(a) is used, the opening 54A may be sealed with the seal 8, and then 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, the opening 54B may be sealed with the seal 9 (9a), and then 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.

Furthermore, when the sample plate 4B shown in FIG. 13(b) 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). Alternatively, 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.

Alternatively, stickers 8 and 9 may be attached to both sides of a simple disk-shaped (e.g., 60 mm diameter, 8 mm thickness) plate made of a transparent or translucent resin material (e.g., acrylic resin or polypropylene) with numerous through-holes (e.g., 5 mm inner diameter). 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.

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. Furthermore, ultrasonic waves propagating through the medium (water) are irradiated via the seal 9 to the solution inside the cylindrical member 54, which is sealed by the seals 8 and 9.

Furthermore, the light irradiation device 6 irradiates the solution in the tubular member 54 with excitation light through the seal 8, and the light detection device 7 detects fluorescence from the solution in the tubular member 54 through the seal 8.

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

This shortens the time it takes for protein aggregates to form in the solution, and allows protein aggregates to be formed uniformly in the multiple solutions within the multiple tubular members 54.

Then, the photodetector 7 can detect highly uniform protein aggregates formed in the multiple solutions in the multiple tubular members 54.

The ultrasonic irradiation device 10D may be obtained by modifying the ultrasonic irradiation device 10 to the ultrasonic irradiation device 10D (changing the sample plate 4 to the sample plate 4B and adding the seal 9) and applying this to either the ultrasonic irradiation device 10A or 10B.

The change from ultrasonic irradiation device 10 to ultrasonic irradiation device 10D (changing sample plate 4 to sample plate 4B and adding seal 9) essentially changes the well 44 to a cylindrical member 54 and seals the openings 54A, 54B (or openings 54A, 54F) on both sides of the 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 (changing sample plate 4 to sample plate 4B and adding seal 9) can be applied to ultrasonic irradiation device 10B.

Note that when ultrasonic irradiation device 10 is changed to ultrasonic irradiation device 10D (changing sample plate 4 to sample plate 4B and adding sticker 9) and 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. Therefore, when ultrasonic irradiation device 10 is changed to ultrasonic irradiation device 10D (changing sample plate 4 to sample plate 4B and adding sticker 9) and applied to ultrasonic irradiation device 10C, it is essentially the same as ultrasonic irradiation device 10D.

The rest of the description of the ultrasonic irradiation device 10D is the same as the description of the ultrasonic irradiation devices 10, 10A, and 10B.

Figure 14 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10E shown in Figure 14.

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 affixed thereto and the rotor 5 are fixed at different positions on the rotating shaft 3 so that they are immersed in the medium (water).

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.

In the ultrasonic irradiation device 10C, the rotor 5 is fixed to the rotor 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 rotor shaft 3. After a solution containing proteins and fluorescent molecules is poured into the multiple wells 44, the multiple wells 44 are sealed with seals 8, and the sample plate 4 with the seal 8 attached is fixed to the rotor shaft 3 so that the seal 8 is positioned on the rotor 5 side. A medium (water) is then poured into the container 1 so that the sample plate 4 with the seal 8 attached and the rotor 5 are immersed.

Note that methods other than those described above may be used to secure the sample plate 4 with the seal 8 attached and the rotor 5 to the rotor shaft 3 so that the sample plate 4 and rotor 5 are immersed.

The ultrasonic generator 2 then generates ultrasonic waves, which are irradiated 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, 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 also irradiated to the solution in each well 44 via the seal 8.

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 light detection device 7 may be positioned on the side to measure fluorescence from the side of the sample plate 4.

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

This shortens the time it takes for protein aggregates to form in the solution, and allows protein aggregates to be formed uniformly in multiple solutions within multiple wells 44.

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

The ultrasonic irradiation device 10E may be any of the ultrasonic irradiation devices 10, 10A, 10B, and 10D, modified from the ultrasonic irradiation device 10C to the ultrasonic irradiation device 10E (changing the fixed positions of the sample plate 4 and the rotor 5 on the rotation shaft 3 so that the sample plate 4 with the sticker 8 attached and the rotor 5 are immersed in the medium (water)).

When changing from ultrasonic irradiation device 10C to ultrasonic irradiation device 10E and applying it to ultrasonic irradiation device 10A, the rotating shaft 3A, sample plate 4 with sticker 8 attached, and rotor 5 of ultrasonic irradiation device 10A 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.

The rest of the description of the ultrasonic irradiation device 10E is the same as the description of the ultrasonic irradiation devices 10, 10A, 10B, and 10D.

Figure 15 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10F shown in Figure 15.

Referring to Figure 15, ultrasonic irradiation device 10F is identical to ultrasonic irradiation device 10C except that the sample plate 4 and rotor 5 are integrated together, with a seal 8A affixed instead of the seal 8 of ultrasonic irradiation device 10C shown in Figure 11, and the integrated sample plate 4 and rotor 5 are immersed in a medium (water).

The rotor 5 is adhered 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 contacts 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).

Seal 8A is affixed to the upper surface 41A of the main body 41 of the sample plate 4 in an area that does not contact the bottom surface 5A of the rotor 5, so as to cover multiple wells 44. Seal 8A is made of the same material as seal 8.

Figure 16 is a perspective view of the sample plate 4 seen from the ultrasonic generator 2 side in Figure 15.

Referring to Figure 16, in the sample plate 4, the multiple through-holes 43 are arranged axially symmetrically within the area surrounded by the circle CIR2, with the through-hole 42 at the center. In the ultrasound 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 adhered 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 area surrounded by the circle CIR2.

In this way, the bottom surface 5A of the impeller 5 has a diameter smaller than the diameter of the circle CIR2, and the seal 8A is affixed to the top surface 41A of the main body 41 of the sample plate 4 so as to cover the multiple wells 44, so that part of the outer periphery of the multiple through-holes 43 is not covered by the bottom surface 5A of the impeller 5 and the seal 8A.

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

Therefore, even in the ultrasonic irradiation device 10F, which has 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 air bubbles generated in the medium (water).

In the ultrasonic irradiation device 10F, after a solution containing proteins and fluorescent molecules is placed into 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 rotor shaft 3 at a position where they are immersed in the medium (water). The medium (water) is then poured into the container 1 so that the integrated sample plate 4 and rotor 5 are immersed.

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 also irradiated to the solution in each well 44 via the seal 8A.

Furthermore, in the ultrasound irradiation device 10F, 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.

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

This shortens the time it takes for protein aggregates to form in the solution, and allows protein aggregates to be formed uniformly in multiple solutions within multiple wells 44.

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

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

When changing from ultrasonic irradiation device 10C to ultrasonic irradiation device 10F and applying it to ultrasonic irradiation device 10A, the rotating shaft 3A and the integrated sample plate 4 and rotor 5 in ultrasonic irradiation device 10A 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.

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

The rest of the description of the ultrasonic irradiation device 10F is the same as the description of the ultrasonic irradiation devices 10, 10A, and 10D.

Figure 17 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10G shown in Figure 17.

Referring to Figure 17, the ultrasound irradiation device 10G is the same as the ultrasound 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.

The transparent window 12 is placed in the container 1 at a position facing the well 44 of the sample plate 4. The transparent window 12 is made of a transparent material such as glass or acrylic.

In the ultrasonic irradiation device 10G, the light detection device 7 is positioned opposite the transparent window 12 (laterally of the container 1).

As described above, the sample plate 4 is made of a transparent material, so fluorescence from the solution in each well 44 passes through the sidewall of each well 44 and is emitted radially around the sample plate 4. As a result, the photodetector 7 detects the fluorescence through the transparent window 12 of the container 1.

When ultrasound is applied to the solution in each well 44, bubbles are generated in the solution, and these bubbles move upward in the solution. As a result, when fluorescence is detected by the photodetector 7 positioned above the solution, noise may be generated.

However, in the ultrasound irradiation device 10G, the photodetector 7 detects fluorescence from the side of each well 44, so even if air bubbles occur in the solution, the noise caused by the bubbles can be removed and the fluorescence can be detected. As a result, multiple protein aggregates in multiple solutions in multiple wells 44 can be detected with the noise caused by air bubbles removed.

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 eliminating noise caused by air bubbles, and as a result, multiple protein aggregates in multiple solutions in multiple wells 44 can be detected while eliminating noise caused by air bubbles. Furthermore, the light irradiation device 6 may be installed on the side of the container 1.

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

Other details regarding the ultrasonic irradiation device 10G are the same as those described for the ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, and 10F.

Figure 18 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10H shown in Figure 18.

Referring to Figure 18, the ultrasonic irradiation device 10H is identical to 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.

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

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.

In the ultrasonic irradiation device 10H, the 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).

This allows ultrasonic waves to be efficiently applied to the rotor 5 and the solution in each well 44.

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

The rest of the description of the ultrasonic irradiation device 10H is the same as the description of the ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, 10F, and 10G.

Figure 19 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10I shown in Figure 19.

Referring to Figure 19, the ultrasonic irradiation device 10I is the ultrasonic irradiation device 10 shown in Figure 1 with the addition of pipes 13 and 14 and a circulation control unit 15, but is otherwise the same as the ultrasonic irradiation device 10.

One end of the piping 13 is fixed to the container 1 so as to penetrate the side wall 1B of the container 1 and communicate with the interior of the container 1, and the other end is connected to the circulation control unit 15.

One end of the piping 14 is fixed to the container 1 so as to penetrate the side wall 1C of the container 1 and communicate with the interior of the container 1, and the other end is connected to the circulation control unit 15.

The inlet from pipe 13 to container 1 is located lower than the outlet from container 1 to pipe 14.

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

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, as the medium (water) in buffer tank 151 is supplied by pump 152 to container 1 via pipe 13, 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.

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 the generation of bubbles in the medium (water) when ultrasound is applied to the medium (water) in the container 1. As a result, the multiple wells 44 of the sample plate 4 can be rotated axially symmetrically in a stable manner. 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 between the multiple protein aggregates formed in the multiple solutions in the multiple wells 44 can be further reduced.

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

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.

Furthermore, the ultrasonic irradiation device 10I may be any one of the ultrasonic irradiation devices 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H to which pipes 13 and 14 and a circulation control unit 15 have been added.

The rest of the description of the ultrasonic irradiation device 10I is the same as the description of the ultrasonic irradiation devices 10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, and 10H.

Figure 21 is a schematic diagram of yet another ultrasonic irradiation device according to an embodiment of the present invention. The ultrasonic irradiation device according to an embodiment of the present invention may be ultrasonic irradiation device 10J shown in Figure 21.

Referring to Figure 21, the ultrasound irradiation device 10J is identical to the ultrasound irradiation device 10G shown in Figure 17, except that the position of the light irradiation device 6 is changed.

In the ultrasound irradiation device 10J, the light irradiation device 6 is positioned 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.

Therefore, even if bubbles are generated within each well 44, the effects of the generated bubbles can be eliminated, allowing excitation light to be irradiated onto the solution in each well 44 and fluorescence emitted from the solution in each well 44 to be detected.

As explained above, the ultrasonic irradiation device 10G may be any of the ultrasonic irradiation devices 10A, 10B, 10C, 10D, 10E, and 10F, with the 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 face the transparent window 12).

The ultrasound irradiation device 10J is a device in which the position of the light irradiation device 6 of the ultrasound irradiation device 10G has been changed so that excitation light is irradiated through the transparent window 12.

Accordingly, in an ultrasound irradiation device according to an embodiment of the present invention, the position of the light detection device 7 may be changed so that fluorescence emitted from the solution in each well 44 is detected through the transparent window 12, or the position of the light irradiation device 6 may be changed so that excitation light is irradiated onto the solution in each well 44 through the transparent window 12, or the position of the light irradiation device 6 may be changed so that excitation light is irradiated onto the solution in each well 44 through the transparent window 12, and the position of the light detection device 7 may be changed so that fluorescence emitted from the solution in each well 44 is detected through the transparent window 12. In an embodiment of the present invention, such changes may be applied to the ultrasound irradiation device 10I shown in FIG. 19.

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 plurality of 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.

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 ultrasonic frequency that causes protein aggregation, and curve k2 shows the time dependence of the amplitude of ultrasonic waves with a lower frequency than curve k1. The frequency that causes protein aggregation is created from 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 ultrasonic frequency that causes protein aggregation, and curve k4 shows the time dependence of the ultrasonic amplitude that causes protein aggregation.

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 that has 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).

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).

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

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

In addition, instead of irradiating ultrasonic waves continuously, 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 burden on the ultrasonic generator 2.

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

Figure 23 is a flowchart illustrating the operation of an ultrasonic irradiation device according to an embodiment of the present invention.

In Figure 23, the operation of the ultrasound irradiation device will be described assuming that a solution is placed in the multiple wells 44 of the sample plate 4, 4A (or the multiple tubular members 54 of the sample plate 4B), a seal (at least one of seals 8, 8A, and 9) is affixed 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, and 9).

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

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

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

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

Then, the photodetector 7 detects multiple fluorescent signals emitted from each of the multiple solutions (step S6).

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 stores in advance a time T _AGG from when the ultrasonic irradiation of the solution begins until the protein in the solution begins to aggregate. When the elapsed time TELP from when the ultrasonic irradiation of the container 1 begins exceeds the time T _AGG , the _{ultrasonic generator} 2 determines to end the measurement, and when the elapsed time TELP is equal to or less than the time T _AGG , _{the ultrasonic generator} 2 determines not to end the measurement.

If it is determined in step S7 that measurement should not be terminated, the series of operations proceeds to step S1. Steps S1 to S7 are then repeatedly executed until it is determined in step S7 that measurement should be terminated.

Then, in step S7, if it is determined that measurement should be terminated, the ultrasonic generator 2 stops generating ultrasonic waves, and operation of the ultrasonic irradiation device ends.

The operation of the above-described ultrasound irradiation devices 10, 10A, 10C, 10E, 10F, 10G, 10H, and 10J is performed in accordance with steps S1 to S7 shown in Figure 23.

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

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

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

After steps S1 to S7 shown in Figure 23 are performed, the time dependence of the fluorescence intensity is obtained using the method described in Figure 7 based on the fluorescence signals detected by the photodetector 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.

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, to evaluate the uniformity of the multiple protein aggregates formed in each of the multiple solutions.

[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 apparatus used to form amyloid fibrils, as described in Non-Patent Document 1.

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

Each of the 96 wells 101 has a volume 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 protein concentration is 0.3 mg/mL, the NaCl concentration is 38 mM, and the pH of the solution is 2.5.

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. Ultrasound is then 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 protein (β2 microglobulin) aggregates in the solution in each well 101.

In this case, the ultrasonic frequency is 17 kHz to 20 kHz, the ultrasonic power is 350 W, and the measured temperature is 37°C.

Then, ultrasound was irradiated onto the solution in each well 101 by repeatedly irradiating it for 10 to 15 minutes and then stopping the irradiation for 5 minutes.

Furthermore, the fluorescence from the solution in each well 101 was detected to examine the time dependence of the fluorescence intensity 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.

The time dependence of fluorescence intensity described in Non-Patent Document 1 is considered the "conventional example."

Furthermore, the above-described ultrasonic irradiation device 10 was used to detect fluorescence from the solution in each well 44 and examine the time dependence of the fluorescence intensity. The time dependence of the fluorescence intensity detected using the ultrasonic irradiation device 10 is referred to as "the present invention."

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 the 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.

The opening 44A of each well 44 was then sealed with a seal 8. Ultrasonic waves were then 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 measured temperature was 37°C.

Then, ultrasonic waves were applied to the solution in each well 44 while rotating the sample plate 4 at a rotation speed of 2 Hz.

Figure 25 shows the time dependence of normalized fluorescence intensity of Thioflavin T in the present invention. Figure 26 shows the time dependence of fluorescence intensity in a conventional example.

In Figure 25, the vertical axis represents the normalized fluorescence intensity of thioflavin T, and the horizontal axis represents time.

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

25 , in the present invention, the multiple normalized fluorescence intensities of Thioflavin T begin to increase between time t _{R_1} and time t _{R_2} . Time t _{R_2} is shorter than one hour. In addition, in the time dependence of the multiple normalized fluorescence intensities 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%.

Referring to Figure 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 begins to increase is longer than 60 minutes (1 hour). Furthermore, in the time dependence of multiple fluorescence intensities, the coefficient of variation CV of the time until the fluorescence intensity begins to increase is 50%.

Referring to Figure 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 begin to increase is approximately 50 minutes or more. Furthermore, in the time dependence of multiple fluorescence intensities, the coefficient of variation CV of the time at which the fluorescence intensity begins to increase is 32%.

Therefore, the coefficient of variation CV (= 21%) in the present invention is less than half 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.

In conventional examples, when the container 110 (i.e., the sample plate 100) is rotated, the 96 wells 101 do not rotate axially symmetrically.

Therefore, as shown in the present invention, it has been experimentally demonstrated that by rotating the multiple wells 44 axially symmetrically, the time at which protein aggregates begin to form in the solution within 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.

In addition, in experiments according to the present invention, the ultrasound irradiation device 10C described above was used, but with a transparent window 12 provided in the container 1 and the position of the photodetector 7 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 through the transparent window 12 by the photodetector 7, and the time dependence of the normalized fluorescence intensity was obtained.

Then, we obtained the time dependence of normalized fluorescence intensity when α-synuclein aggregate seeds were added to the solution, and the time dependence of normalized fluorescence intensity when α-synuclein aggregate seeds were not added to the solution.

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

27 is a graph showing another time dependence of normalized fluorescence intensity according to the present invention. In FIG. 27, the vertical axis represents the normalized fluorescence intensity of Thioflavin T, and the horizontal axis represents time. Furthermore, curve group k _CG1 shows the time dependence of the normalized fluorescence intensity of Thioflavin T when no α-synuclein aggregation seeds are added, and curve group k _CG2 shows the time dependence of the normalized fluorescence intensity of Thioflavin T when α-synuclein aggregation seeds are added.

Referring to FIG. 27, when no α-synuclein aggregation seeds are added, the time it takes for protein (α-synuclein) aggregates to begin to form ranges from 0.5 hours to 2 hours (see curve family k _CG1 ).

On the other hand, when α-synuclein aggregation seeds are added, the time it takes for protein (α-synuclein) aggregates to begin to form is less than 0.5 hours (see curve group k _CG2 ).

Therefore, it has been experimentally clearly demonstrated that adding α-synuclein aggregation seeds can speed up the time it takes for protein (α-synuclein) aggregates to begin to form.

Furthermore, when no α-synuclein aggregation seeds are added, the time it takes for protein (α-synuclein) aggregates to begin to form ranges from 0.5 hours to 2 hours (see curve group k _CG1 ). Therefore, it has been experimentally demonstrated that the formation of protein (α-synuclein) aggregates can be accelerated by irradiating the solution with ultrasound through the seal 8.

As described above, it has been experimentally demonstrated that by irradiating a solution containing 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 than conventional methods, and with greater uniformity and at an accelerated rate.

α-Synuclein and β2-microglobulin are proteins found in the human body that cause Parkinson's disease and dialysis-related amyloidosis, respectively.

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

By using an ultrasound irradiation device according to an embodiment of the present invention to irradiate a solution containing alpha-synuclein with ultrasound and accelerate the formation of alpha-synuclein aggregates, it is 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 beta (the protein that causes Alzheimer's disease).

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. Fluorescence intensity also represents the amount of aggregates formed.

Referring to Figure 28, high-risk individuals who are at high risk of developing Parkinson's disease or Alzheimer's disease experience an increase in fluorescence intensity (amount of aggregates produced) in a shorter time than healthy individuals.

Therefore, by setting a threshold value th_RSK for determining that there is a high risk of developing Parkinson's disease or Alzheimer's disease in advance, and determining that there is a high risk of developing Parkinson's disease or Alzheimer's disease when the time _tINC at which the fluorescence intensity (amount of generated aggregates) begins to increase is equal to or less than the threshold value th_RSK, or determining that there is a low risk of developing Parkinson's disease or Alzheimer's disease when the time _tINC is longer than the threshold value th_RSK, it is possible to diagnose whether there is a high or low risk of developing Parkinson's disease or Alzheimer's disease in the future.

In the above-described embodiments, ultrasonic irradiation devices 10, 10A-10J that accelerate the formation of protein aggregates have been described. However, ultrasonic irradiation devices according to embodiments of the present invention are not limited to accelerated formation of protein aggregates and may be used for purposes other than the accelerated 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.

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.

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

In this embodiment of the present invention, the multiple tubular members 54 sealed with seals 8 and 9 constitute "multiple wells."

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

Furthermore, in this embodiment of the present invention, the cylindrical member 31 and the horn 21 constitute a "container."

The embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated 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.

This invention applies to ultrasound irradiation devices.

1 Container, 2 Ultrasonic wave generator, 3, 3A Rotating shaft, 4, 4A, 4B Sample plate, 5 Rotating blade, 6 Light irradiation device, 7 Light detection device, 8, 8A, 9 Seal, 10, 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H Ultrasonic wave 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 Cylinder 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 unit.

Claims (14)

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