[go: up one dir, main page]

WO2012017739A1 - Dispositif et procédé de dosage d'amyloïde - Google Patents

Dispositif et procédé de dosage d'amyloïde Download PDF

Info

Publication number
WO2012017739A1
WO2012017739A1 PCT/JP2011/063413 JP2011063413W WO2012017739A1 WO 2012017739 A1 WO2012017739 A1 WO 2012017739A1 JP 2011063413 W JP2011063413 W JP 2011063413W WO 2012017739 A1 WO2012017739 A1 WO 2012017739A1
Authority
WO
WIPO (PCT)
Prior art keywords
amyloid
microplate
ultrasonic
wells
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2011/063413
Other languages
English (en)
Japanese (ja)
Inventor
祐児 後藤
一正 櫻井
寿梓 八木
正智 宗
博次 荻
裕司 志鷹
幸吉 井戸
俊三 笠井
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CORONA ELECTRIC Co Ltd
ELEKON SCIENCE Co Ltd
Original Assignee
CORONA ELECTRIC Co Ltd
ELEKON SCIENCE Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CORONA ELECTRIC Co Ltd, ELEKON SCIENCE Co Ltd filed Critical CORONA ELECTRIC Co Ltd
Priority to JP2012527633A priority Critical patent/JP5841945B2/ja
Publication of WO2012017739A1 publication Critical patent/WO2012017739A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention relates to an amyloid assay device and an amyloid assay method.
  • Amyloidosis is known as a serious disease in an aging society. Amyloidosis is a general term for diseases in which abnormal protein aggregates called “amyloid fibrils”, which are aggregated with proteins and are not branched with a width of 10 nm, are deposited. Amyloidosis includes about 20 kinds of serious diseases including Alzheimer's disease, type II diabetes, prion disease, dialysis amyloidosis, AL amyloidosis, and Parkinson's disease. Although amyloid fibrils are thought to be a regular assembly of denatured proteins in the axial direction, there are many unclear points in the structure and formation mechanism.
  • amyloidosis In recent years, research on amyloidosis has been actively conducted, but it is difficult to predict the deposition of amyloid.
  • the formation of amyloid is a phenomenon similar to the crystal formation of a substance, and even if the causative substance exceeds a dangerous level, the energy barrier for amyloid formation is high, and thus the amyloid formation is often maintained in a supersaturated state. Under such circumstances, amyloid and oligomers thereof are not formed, amyloidosis does not develop, and the patient is in a latent state.
  • a major problem in research on amyloidosis is that it is difficult to predict the formation of associated abnormal aggregates such as amyloid and oligomers. Even in basic research in vitro, the reaction of the causative protein to form amyloid is slow, and in general, amyloid formation requires a long time of several days to several months.
  • Non-Patent Documents 1 and 2 describe that amyloid fibrils are efficiently induced by ultrasonic treatment.
  • Non-Patent Document 2 describes that fine and homogeneous amyloid particles are formed by antagonizing amyloid fibril formation induction and crushing by ultrasonic treatment.
  • amyloid assay could not be performed in a short time for each of a plurality of solutions containing proteins.
  • the present invention has been made in view of the above problems, and an object thereof is to provide an amyloid assay device and an amyloid assay method capable of performing an amyloid assay for each of a plurality of solutions containing proteins in a short time. It is in.
  • An amyloid assay device includes an ultrasonic irradiation unit that irradiates a solution containing a protein placed in each of a plurality of wells of a microplate with an ultrasonic wave, amyloid formed from the protein by the ultrasonic irradiation, and A fluorescence detection unit that detects fluorescence emitted by binding with an amyloid-specific fluorescent dye.
  • the ultrasonic wave irradiation unit irradiates the plurality of wells with ultrasonic waves nonuniformly.
  • the ultrasonic irradiation unit uniformly applies ultrasonic waves to the plurality of wells.
  • the amyloid assay device further includes a sound pressure measuring device that measures the sound pressure of the ultrasonic waves irradiated to each of the plurality of wells of the microplate by the ultrasonic irradiation unit.
  • the amyloid specific fluorescent dye comprises thioflavin T.
  • the ultrasonic irradiation unit includes a processing tank, an ultrasonic transducer, and a moving unit that moves the microplate.
  • the moving unit moves the microplate from the ultrasonic wave irradiation unit to the fluorescence detection unit.
  • the moving unit moves the microplate while the ultrasonic wave irradiation unit is irradiating the ultrasonic wave to the solution.
  • the amyloid assay method includes a step of irradiating a solution containing a protein placed in each of a plurality of wells of a microplate with an ultrasonic wave, and the amyloid formed from the protein and the amyloid-specific fluorescent dye bind to each other. And detecting the fluorescence emitted.
  • the plurality of wells are irradiated with ultrasonic waves non-uniformly.
  • the method further comprises preparing a microplate in which a solution containing a protein and an amyloid-specific fluorescent dye is placed in each of the plurality of wells, and in the step of preparing the microplate, the plurality of wells Put the same solution in each of the.
  • the plurality of wells are uniformly irradiated with ultrasonic waves.
  • the method further comprises preparing a microplate in which a solution containing a protein and an amyloid-specific fluorescent dye is placed in each of the plurality of wells, and in the step of preparing the microplate, the plurality of wells Put a different solution in each.
  • the amyloid assay method further includes a step of moving the microplate to a fluorescence detection section after detecting the fluorescence and before detecting the fluorescence.
  • the step of irradiating the ultrasonic wave moves the microplate while irradiating the ultrasonic wave to the solution.
  • amyloid assay can be performed in a short time for each of a plurality of solutions containing proteins.
  • (A) is a schematic diagram of an embodiment of an amyloid assay device according to the present invention, and (b) is a schematic diagram of a microplate used in the amyloid assay device shown in (a).
  • (A) is a schematic diagram of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment,
  • (b) is a schematic top view of the ultrasonic irradiation apparatus shown in (a),
  • (c) is ( It is typical sectional drawing of the ultrasonic irradiation apparatus shown to a).
  • (A) is a graph which shows the change of the size of the amyloid fiber by ultrasonic irradiation
  • (b) is a schematic diagram which shows an atomic force microscope (AFM) image when not irradiating an ultrasonic wave
  • (C) is a schematic diagram which shows an AFM image at the time of irradiating an ultrasonic wave with respect to the amyloid of (b). It is a schematic diagram of the plate reader in the amyloid assay device of this embodiment. It is a schematic diagram of an embodiment of an amyloid assay device according to the present invention.
  • (A) is a schematic diagram of the sound pressure measuring device used for the amyloid assay apparatus of this embodiment
  • (b) is a schematic diagram of the sensor part front-end
  • (A) to (d) are schematic diagrams showing the amount of amyloid when the ultrasonic irradiation time is 0 minutes, 90 minutes, 120 minutes, and 210 minutes, respectively.
  • (A) is a graph which shows the change of the amyloid formation degree according to the difference in protein concentration
  • (b) is a graph which shows the change of the amyloid formation degree in the physiological conditions similar to a patient internal environment.
  • (A) is a schematic diagram showing the sound pressure distribution of ultrasonic waves in each well
  • (b) is a schematic diagram showing the degree of amyloid formation
  • (c) is a graph showing the relationship between the sound pressure and the amount of amyloid. It is.
  • (A)-(d) is a schematic diagram for demonstrating determination of the risk of the onset of amyloidosis. It is a schematic diagram of the amyloid assay apparatus of this embodiment.
  • (A) is a typical top view of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment
  • (b) is a typical side view of (a).
  • It is a graph which shows the time change of the fluorescence intensity from the solution in each well in the plate reader at the time of using the amyloid assay apparatus of this embodiment.
  • It is a schematic diagram which shows the lag time of the solution in each well in the plate reader in the case shown in FIG.
  • (A) is a typical top view of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment
  • (b) is a typical side view of (a).
  • (A) is a typical top view of the ultrasonic irradiation apparatus in the amyloid assay apparatus of this embodiment
  • (b) is a typical side view of (a).
  • It is a schematic diagram of the amyloid assay apparatus of this embodiment.
  • It is a schematic diagram of the plate reader in the amyloid assay apparatus shown in FIG.
  • It is a schematic diagram which shows an example of the plate reader shown in FIG.
  • It is a graph which shows the time change of the fluorescence intensity from the solution in each well in the plate reader at the time of using the amyloid assay apparatus of this embodiment.
  • It is a schematic diagram which shows the lag time of the solution in each well in the plate reader in the case shown in FIG.
  • It is a graph which shows distribution of the lag time according to the presence or absence of the movement of the microplate at the time of ultrasonic irradiation.
  • FIG. 1A shows a schematic diagram of an amyloid assay device 100 of the present embodiment
  • FIG. 1B shows a schematic diagram of a microplate P used in the amyloid assay device 100.
  • the amyloid assay device 100 includes an ultrasonic irradiation device 10 and a plate reader 20.
  • a microplate P provided with a plurality of wells (holes or depressions) W is mounted.
  • 96 wells W are arranged in 12 rows and 8 columns.
  • Each of the plurality of wells W of the microplate P contains a protein solution.
  • the same solution may be put into the plurality of wells W of the microplate P, or different solutions may be put therein.
  • the same solution means that the solution component and the solution concentration are the same, and the different solution means that at least one of the solution component and the solution concentration is different.
  • the same solution includes body fluid (blood, etc.) of the same subject, and the different solution includes body fluid (blood, etc.) of a different subject.
  • the ultrasonic irradiation apparatus 10 irradiates the protein solution placed in each well W with ultrasonic waves.
  • amyloid is generated. This is presumably because local high temperature and high pressure are generated, the solution is disturbed, and supersaturation is disturbed.
  • an ultrasonic wave is irradiated to an amyloid fiber, the amyloid fiber is fragmented by impact.
  • the ultrasonic irradiation apparatus 10 may uniformly irradiate each of the samples placed in the plurality of wells W of the microplate P or may irradiate the ultrasonic waves nonuniformly. Good.
  • “irradiating ultrasonic waves to a plurality of samples non-uniformly” means “irradiating at least two samples with ultrasonic waves at different intensities (sound pressures)”
  • “Uniformly irradiating a plurality of samples” means “irradiating at least two samples with ultrasonic waves at the same intensity (sound pressure)”.
  • Such an ultrasonic irradiation device 10 can promote the formation of amyloid in each of the solutions placed in the plurality of wells W of the microplate P.
  • amyloid When amyloid is induced from protein, amyloid emits fluorescence by binding to amyloid-specific fluorescent dye.
  • the presence of the amyloid-specific fluorescent dye in the plurality of wells W of the microplate P in which amyloid is induced causes fluorescence to be emitted, and the plate reader 20 detects the amyloid in the solution by detecting this fluorescence. Can do.
  • FIG. 2 (a) shows a schematic front view of the ultrasonic irradiation device 10
  • FIG. 2 (b) shows a schematic top view of the ultrasonic irradiation device 10
  • FIG. 2 (c) shows the ultrasonic irradiation device. 10 is a schematic cross-sectional view.
  • the microplate P is mounted on the ultrasonic irradiation device 10.
  • the microplate P is preferably formed from a material with good ultrasonic efficiency.
  • the microplate P is formed from polystyrene.
  • Such a microplate P is formed from a mold.
  • a cap may be attached to the microplate P in case the solution contains an infectious substance.
  • the ultrasonic irradiation apparatus 10 functions as an “ultrasonic irradiation unit that irradiates ultrasonic waves to a solution containing a protein placed in each of a plurality of wells of a microplate”.
  • the ultrasonic irradiation apparatus 10 includes a processing tank 12 and an ultrasonic vibrator 14.
  • the treatment tank 12 has side plates 12a and 12b facing each other bent at 45 degrees inward at the center, side plates 12c and 12d facing each other communicating with the side plates 12a and 12b, and a bottom plate 12e connected to the side plates 12a to 12d. ing.
  • the treatment tank 12 is filled with water.
  • the ultrasonic waves generated in the ultrasonic transducer 14 are transmitted through water.
  • the ultrasonic transducers 14 are attached vertically to the inclined surfaces 12a2 and 12b2 of the side plates 12a and 12b, respectively.
  • a diaphragm 14a to which the ultrasonic transducer 14 is attached is disposed inside the inclined surfaces 12a2 and 12b2, and the ultrasonic waves intersect each other at a right angle so that the energy becomes stronger, and amyloid An effective crushing operation of the fiber is performed.
  • a microplate P is placed on top of the processing tank 12. The microplate P is fixed by a holder (not shown) with the tip of each well W immersed in water. This holder is arranged at the position where the ultrasonic wave is most easily received in the central portion of the treatment tank 12.
  • the ultrasonic wave passes through the vibration plate 14a and becomes strong and travels toward each well W of the microplate P.
  • the ultrasonic waves travel in a direction perpendicular to the respective inclined plates 14a, the ultrasonic waves intersect at right angles. Then, this intersection is set in advance near the central water surface of the treatment tank 12, and each well W of the microplate P is arranged in the vicinity thereof.
  • the ultrasonic wave passes through the water in the treatment tank 12 and reaches each well W of the microplate P, and further reaches the solution in each well W of the microplate P to induce and crush amyloid formation.
  • vibrator 14 and the inclination board 14a are arrange
  • the ultrasonic transducer 14 and the inclination plate 14a are not only the side plates 12a and 12b but also the side plate. You may attach to 12c, 12d.
  • the ultrasonic transducer 14 and the inclined plate 14a may be attached to the bottom plate 12e.
  • FIG. 3 (a) schematically shows the relationship between the size of amyloid fibrils and free energy.
  • FIG. 3B shows an AFM image of amyloid when no ultrasonic wave is irradiated
  • FIG. 3C shows an atomic force microscope image of amyloid when the ultrasonic wave is irradiated.
  • FIG. 4 shows a schematic diagram of the plate reader 20.
  • the plate reader 20 is also called a microplate reader.
  • the plate reader 20 functions as a “fluorescence detection unit that detects fluorescence emitted by the binding of amyloid formed from protein by irradiating ultrasonic waves and an amyloid-specific fluorescent dye”.
  • the microplate P is mounted on the plate reader 20 and fixed.
  • the microplate P is preferably the same as that mounted in the ultrasonic irradiation apparatus 10.
  • the cap When the cap is attached to the microplate P, it is preferable that the cap has high transparency.
  • the plate reader 20 may be mounted with a microplate P different from that mounted in the ultrasonic irradiation apparatus 10.
  • the plate reader 20 includes a light source 22 and a light receiving unit 24.
  • the light receiving unit 24 is a photomultiplier tube.
  • the optical fiber fb1 and fb2 are configured to be operable in a two-dimensional direction with respect to the main surface of the microplate P to which the tips are fixed.
  • the optical fibers fb1 and fb2 are moved while the microplate P is fixed to perform light irradiation and fluorescence detection.
  • the microplate P is moved without moving the light emitted toward the microplate P. It may be moved.
  • Fluorescence is generated from the solution in the well W by light irradiation.
  • the generated fluorescence is transmitted to the light receiving unit 24 via the optical fiber fb2, and the intensity thereof is measured.
  • the plate reader 20 can evaluate many samples.
  • the plate reader 20 selects a wavelength arbitrarily using a grating element.
  • the observer may move the microplate P.
  • the ultrasonic irradiation apparatus 10 and the plate reader 20 may be comprised integrally.
  • FIG. 5 shows an integrated amyloid assay device 100 ′ in which the ultrasonic irradiation device 10 and the plate reader 20 are integrated.
  • the microplate P is provided with optical fibers fb 1 and fb 2 in the upper part of the processing tank 12.
  • the amyloid assay device 100 and the integrated amyloid assay device 100 ′ further include a sound pressure measurement device that measures the sound pressure of the ultrasonic waves irradiated to the plurality of wells W of the microplate P by the ultrasonic irradiation device 10. Also good.
  • the sound pressure measuring device measures the sound pressure of the ultrasonic wave irradiated to each well W of the microplate P.
  • FIG. 6A shows the sound wave measuring device 30.
  • the sound pressure measuring device 30 includes a sensor unit 32, a voltage monitor unit 34, and a processing unit 36 that processes values monitored by the voltage monitor unit 34.
  • a processing unit 36 that processes values monitored by the voltage monitor unit 34.
  • sound pressure is measured in a state where the sensor unit 32 is infiltrated into the solution in the well W of the microplate P.
  • the sound pressure measurement and the fluorescence measurement may be performed separately or simultaneously.
  • the sensor unit 32 converts the sound pressure of the ultrasonic wave into a potential, and the voltage monitor unit 34 displays the potential waveform converted in the sensor unit 32.
  • the processing unit 36 the sound pressure, frequency, and harmonic component of the ultrasonic wave can be obtained.
  • the processing unit 36 may perform filtering or fast Fourier transform (FFT).
  • FIG. 6B shows an example of the tip of the sensor unit 32.
  • the sensor unit 32 includes a rod-shaped main body 32a and a piezoelectric element 32b provided at one end of the main body 32a.
  • the main body 32a is insulated at least on the surface.
  • the main body 32a is an alumina lot having a diameter of about 4 mm and a length of about 100 mm, for example.
  • the piezoelectric element 32b has a pair of metal electrodes 32b1 and 32b2 and a piezoelectric body 32b3 sandwiched between them.
  • Each of the metal electrodes 32b1 and 32b2 has a laminated structure of chromium and gold and has a thickness of 500 nm.
  • the piezoelectric body 32b3 is made of, for example, lead zirconate titanate (PZT), polyvinylidene fluoride (PVDF), quartz or langasite (La3Ga5SiO14).
  • PZT lead zirconate titanate
  • PVDF polyvinylidene fluoride
  • La3Ga5SiO14 langasite
  • the protective layer 32c is formed from, for example, an epoxy resin.
  • amyloid assay method [Amyloid assay method]
  • an embodiment of the amyloid assay method according to the present invention will be described with reference to FIG.
  • a microplate P in which a protein solution is placed in each of the plurality of wells W is prepared.
  • the solution also contains an amyloid specific fluorescent dye.
  • the amyloid specific fluorescent dye is Thioflavin T.
  • the ultrasonic wave is irradiated to the solution put in each of the plurality of wells W of the microplate P.
  • the sound pressure of the ultrasonic wave applied to each solution of the plurality of wells W may be uniform or non-uniform.
  • the fluorescence emitted by the binding of the amyloid derived from the protein and the amyloid-specific fluorescent dye is detected.
  • the fluorescence is detected by the plate reader 20.
  • the protein (amyloid) and the solution are discarded.
  • the amyloid assay device 100 and the integrated amyloid assay device 100 ' can be used for the study of abnormal protein aggregation. Alternatively, the amyloid assay device 100 and the integrated amyloid assay device 100 'are used to determine the risk of developing amyloidosis.
  • ⁇ 2 microglobulin that causes dialysis amyloidosis will be specifically described.
  • ⁇ 2 microglobulin is essentially a component of a histocompatibility antigen that is responsible for immune functions and an essential protein that is responsible for vital functions.
  • ⁇ 2 microglobulin is not degraded and the protein concentration in the blood increases. If this condition persists for many years, it is thought to cause amyloid deposition, but the detailed pathogenesis is unknown.
  • amyloid fibrils of ⁇ 2 microglobulin generally takes several days to one month when the test tube is left standing.
  • concentration of ⁇ 2 microglobulin for example, in vivo concentration: 0.05 mg / ml
  • formation of amyloid of ⁇ 2 microglobulin is performed using ultrasound.
  • the amyloid can be assayed in a short time (eg, hundreds of samples within minutes).
  • the sound pressure provided to the solution in each well W of the microplate P is, for example, 0 to 0.5 MPa, and the sound pressure value is adjusted. can do.
  • the sound pressure (average value ⁇ standard deviation) provided to the solution in each well W of the microplate P is 34600 ⁇ 17000 Pa
  • the frequency of the ultrasonic vibrator by the ultrasonic irradiation device 10 is 17.
  • the intensity of the ultrasonic transducer is 350 or 700 Watt.
  • the sound pressure provided to the solution in each well W, the vibration frequency of the ultrasonic vibrator by the ultrasonic irradiation device 10, and the strength of the ultrasonic vibrator are not limited to the above values.
  • the sound pressure provided to the solution in each well W, the frequency of the ultrasonic vibrator by the ultrasonic irradiation device 10 The value of the intensity of the ultrasonic transducer is arbitrary.
  • the amyloid-specific fluorescent dye is, for example, thioflavin T. In this case, when the plate reader 20 irradiates light having a wavelength of around 450 nm as excitation light, the plate reader 20 can detect fluorescence around 490 nm emitted from amyloid.
  • FIG. 8 shows a schematic diagram of the microplate P after the ultrasonic treatment.
  • an acidic condition of pH 2 and a protein concentration of 0.3 mg / ml were repeatedly subjected to ultrasonic irradiation for 1 minute and interruption for 9 minutes.
  • amyloid formation proceeded in the same manner.
  • FIG. 9 shows the change over time in the degree of amyloid formation.
  • group A which repeated ultrasonic irradiation for 1 minute and interruption for 9 minutes was compared with group B which repeated ultrasonic irradiation for 10 minutes and interruption for 5 minutes. did.
  • group B which repeated ultrasonic irradiation for 10 minutes and interruption for 5 minutes.
  • amyloid formation starts in about 90 minutes, whereas in the case of repeated irradiation with ultrasonic waves for 10 minutes and interruption for 5 minutes, it takes about 20 minutes.
  • Amyloid formation begins.
  • amyloid can be more efficiently formed by arbitrarily changing the ultrasonic treatment time.
  • the plurality of curves in each of the groups A and B show data of a plurality of wells having different sound pressures of ultrasonic waves irradiated at the same time.
  • One measurement shows that the experiment is highly reproducible.
  • the microplate P was allowed to stand without performing ultrasonic irradiation, but in this case, amyloid was not formed.
  • 10 (a) to 10 (d) are schematic diagrams showing the amount of amyloid when the ultrasonic irradiation time is 0 minutes, 90 minutes, 120 minutes, and 210 minutes, respectively. It is understood that the number of wells in which amyloid is formed increases with the irradiation time and the amount of amyloid increases in each well.
  • FIG. 11 (a) shows the change in the degree of amyloid formation according to the difference in protein concentration.
  • the solution is acidified, and ultrasonic irradiation for 1 minute and interruption for 9 minutes are repeated.
  • the protein concentration is 0.3 mg / ml, 0.2 mg / ml, 0.1 mg / ml, 0.05 mg / ml.
  • the degree of amyloid formation increases.
  • the protein concentration in the patient is approximately 0.05 mg / ml.
  • FIG. 11 (b) shows changes in the degree of amyloid formation under the same physiological conditions as the patient's internal environment.
  • the two graphs show data of two wells having a similar sound pressure level of the irradiated ultrasonic wave among the plurality of wells of the microplate P.
  • the protein concentration is 0.05 mg / ml and the solution is almost neutral.
  • FIG. 11B shows the reproducibility of the reaction can be observed and evaluated by using a plurality of wells W of the microplate P at the same time.
  • the amyloid assay device 100 and the integrated amyloid assay device 100 ′ can be suitably applied to the prevention and diagnosis of amyloidosis. Specifically, the risk level of developing amyloidosis can be determined using the amyloid assay device 100 or the integrated amyloid assay device 100 ′.
  • the ultrasonic irradiation apparatus 10 can provide a non-uniform or uniform sound pressure to the solution in the well W. For example, whether the sound pressure applied to the solution in the well W is nonuniform or uniform can be switched by an irradiation state switching unit provided in the ultrasonic irradiation apparatus 10.
  • the ultrasonic irradiation apparatus 10 When the ultrasonic irradiation apparatus 10 provides a non-uniform sound pressure to the solution of the well W, it is preferable to put the same sample (for example, blood of the same subject) into each well W of the microplate P. Or when the ultrasonic irradiation apparatus 10 provides a uniform sound pressure to the solution of the well W, it is preferable to put different samples (for example, blood of different subjects) in each well W of the microplate P.
  • FIG. 12A is a schematic diagram showing the sound pressure distribution of ultrasonic waves in each well
  • FIG. 12B is a schematic diagram showing the degree of amyloid formation.
  • 12 (a) and 12 (b) the vertical axes A to H correspond to the first to eighth columns of the microplate P
  • the horizontal axes 1 to 12 represent the first of the microplate P.
  • each well W of the microplate P contains the same sample (for example, blood of the same subject).
  • FIG. 12A is a schematic diagram showing the sound pressure distribution of ultrasonic waves in each well
  • FIG. 12B is a schematic diagram showing the degree of amyloid formation.
  • 12 (a) and 12 (b) the vertical axes A to H correspond to the first to eighth columns of the microplate P
  • the horizontal axes 1 to 12 represent the first of the microplate P.
  • each well W of the microplate P contains the same sample (for example, blood of the same subject).
  • FIG. 12C shows the relationship between the sound pressure and the amount of amyloid.
  • the sound pressure and the amount of amyloid have a substantially proportional relationship.
  • the risk level of developing amyloidosis can be specified by specifying the sound pressure at which a predetermined amount of amyloid is formed during a predetermined irradiation time.
  • the ultrasonic irradiation apparatus 10 provides a uniform sound pressure to the solution in the well W, it is preferable that a different sample is put in each well W of the microplate P. Specifically, blood or the like of different subjects is put in each well W of the microplate P.
  • FIG. 13 (a) shows different subjects. Blood or the like is collected from different subjects, and the solution is put into different wells W of the microplate P as shown in FIG. Thereafter, when each well W is irradiated with ultrasonic waves of uniform sound pressure, a predetermined amount of amyloid is formed in the solution of the subject having a relatively high risk of developing amyloidosis, as shown in FIG. A predetermined amount of amyloid is not formed in the solution of a subject with a relatively low risk of developing amyloidosis. For this reason, by setting the ultrasonic wave to be a threshold for the risk of developing amyloidosis, it is possible to determine whether the subject is positive or negative as shown in FIG.
  • the plate reader 20 measures the fluorescence emitted from the solution while the microplate P is in the processing tank 12.
  • the plate reader 20 may measure fluorescence emitted from the solution of the microplate P that has moved to the outside of the processing tank 12.
  • FIG. 14 shows a schematic diagram of an integrated amyloid assay device 100 ′.
  • the integrated amyloid assay device 100 ′ includes an ultrasonic irradiation device 10 and a plate reader 20.
  • MTP-810 or SH-9000 manufactured by Corona Electric Co., Ltd. is used as the plate reader 20.
  • a microplate having 96 wells W is used as the microplate P.
  • the microplate P may have 96 wells W of a half area black type.
  • the ultrasonic irradiation apparatus 10 includes a processing tank 12, an ultrasonic transducer 14, and a moving unit 16 that moves the microplate P in the processing tank 12.
  • a processing tank 12 For example, water is stored in the treatment tank 12.
  • the vibration plate 14 a is provided inside the treatment tank 12.
  • the moving unit 16 moves the microplate P from the inside of the processing tank 12 to the outside of the processing tank 12, and then the plate reader 20 moves the microplate P of the microplate P. Fluorescence emitted from the solution in the well W is detected.
  • the moving part 16 includes a support part 16t, an elevating part 16u attached to the support part 16t, and a fixed base 16v on which the microplate P is placed.
  • the fixed base 16v is attached to the elevating part 16u, and the elevating part 16u moves the fixed base 16v in the z direction. Accordingly, the microplate P moves from the inside of the processing tank 12 to the outside of the processing tank 12 or from the outside of the processing tank 12 into the processing tank 12.
  • the microplate P is moved from the inside of the processing tank 12 to the plate reader 20 (FIG. 14) outside the processing tank 12 by the elevating unit 16 u, and then the plate reader 20 is amyloid in the solution of the well W in the microplate P. Is detected.
  • amyloid fibrils are induced, amyloid emits fluorescence by binding to an amyloid-specific fluorescent dye, and amyloid can be detected by measuring this fluorescence.
  • the microplate P remains fixed.
  • ⁇ 2 microglobulin is used as the protein
  • thioflavin T is used as the amyloid-specific fluorescent dye.
  • 200 ⁇ L of 0.3 mg / ml ⁇ 2 microglobulin solution (containing 100 mM NaCl, 5 ⁇ M thioflavin T, pH 2.5) is placed in a microplate P having 96 wells W of the half area black type.
  • the microplate P is set to a temperature of 37 ° C.
  • the ultrasonic irradiation apparatus 10 repeats ultrasonic irradiation for 1 minute and interruption for 9 minutes.
  • FIG. 16 shows the change in fluorescence intensity over time when irradiated with ultrasonic waves.
  • attention is focused on the time from the start of the irradiation of ultrasonic waves to the start of the increase in fluorescence intensity.
  • the shortest time from the start of ultrasonic irradiation to the start of the increase in fluorescence intensity is about 50 minutes, and the fluorescence intensity from the solution in most wells starts increasing within 180 minutes.
  • the time from the start of ultrasonic irradiation to the start of increase in fluorescence intensity may be referred to as lag time.
  • FIG. 17 shows a schematic diagram of the microplate P showing the lag time.
  • the wells in the first row to the twelfth row are indicated as 1, 2, 3,... 12 respectively, and the wells in the first column to the eighth column are indicated as A, B,.
  • the lag time of the solution in most wells W near the center in the microplate P is about 100 minutes, and the lag time of the solution in the wells W near the end is about 120 minutes. is there.
  • the lag time of the solution in the well W at the end of the microplate P is longer than the lag time of the solution in the well W near the center.
  • the moving unit 16 moves the microplate P to the plate reader 20. It is not limited to. While the ultrasonic irradiation is performed, the moving unit 16 may move the microplate P. For example, the moving unit 16 may rotate the microplate P around a predetermined point, and thereby the microplate P may be rotated. In this case, the rotation speed of the microplate P is preferably in the range of 0.5 rpm to 11 rpm, and the rotation speed of the microplate P is more preferably about 6 rpm. Alternatively, the microplate P may move around by the moving unit 16 or may move in parallel. As described above, the moving unit 16 moves the microplate P in the processing tank 12, and thereby can irradiate the solution in each well W of the microplate P almost uniformly with ultrasonic waves.
  • the moving part 16 is attached so as to be movable along the slide part 16s1, a support part 16t1 attached so as to be movable along the slide part 16s1, a slide part 16s2 fixed to the support part 16t1, and a slide part 16s2. And a supporting portion 16t2.
  • the microplate P is placed on the tip of the support portion 16t2.
  • the slide portions 16s1 and 16s2 are referred to as the first slide portion 16s1 and the second slide portion 16s2
  • the support portions 16t1 and 16t2 are referred to as the first support portion 16t1 and the second support portion 16t2. is there.
  • the support portion 16t1 moves in the y direction along the slide portion 16s1, and accordingly, the microplate P also moves in the processing tank 12 in the y direction. Further, the support portion 16t2 moves in the x direction along the slide portion 16s2, and accordingly, the microplate P also moves in the processing tank 12 in the x direction. Thus, the moving unit 16 can move the microplate P in the processing tank 12 in the plane direction.
  • the moving unit 16 can move the microplate P along each of the x direction and the y direction, but the present invention is not limited to this.
  • the moving unit 16 may be capable of moving the microplate P along only one of the x direction and the y direction.
  • the first slide portion 16s1 and the first support portion 16t1 may be omitted from the ultrasonic irradiation apparatus 10 described with reference to FIG.
  • the 2nd slide part 16s2 and the 2nd support part 16t2 may be abbreviate
  • the moving unit 16 moves the microplate P in the plane direction in the processing bath 12, but the present invention is not limited to this.
  • the moving unit 16 may move the microplate P also in the vertical direction.
  • the moving unit 16 has the configuration shown in FIG. 18 except that the moving unit 16 further includes an elevating unit 16 u attached to the support unit 16 t 2 and a fixing base 16 v on which the microplate P is placed. It has the same configuration as that of the moving unit 16 described above with reference to the description, and redundant description is omitted to avoid redundancy.
  • the fixed base 16v is attached to the elevating part 16u, and the elevating part 16u moves the fixed base 16v in the z direction. Accordingly, the microplate P moves from the inside of the processing tank 12 to the outside of the processing tank 12 or from the outside of the processing tank 12 into the processing tank 12. Although detailed description is omitted here, the microplate P can be moved in three directions orthogonal to each other by the slide parts 16s1, 16s2 and the elevating part 16u. In the ultrasonic irradiation apparatus 10 shown in FIG. 19, the moving unit 16 moves the microplate P when irradiating ultrasonic waves, and can move the microplate P to the plate reader 20 after the ultrasonic irradiation. it can.
  • the integrated amyloid assay device 100 ′ includes an ultrasonic irradiation device 10 that irradiates a solution in the well W of the microplate P with ultrasonic waves, and a plate that detects fluorescence emitted from the amyloid in the solution of the well W in the microplate P. And a reader 20.
  • the ultrasonic irradiation apparatus 10 includes a circulation tank 12a used for circulating the liquid in the processing tank 12, a control unit 14s that controls the ultrasonic vibrator 14, And a drive unit 14t for driving the sound wave vibrator 14.
  • the number of ultrasonic transducers 14 is preferably as large as possible.
  • the sensor In order to measure the water temperature, when a sensor is attached to the treatment tank 12 to which the ultrasonic vibrator 14 is attached, the sensor may be damaged due to sound waves. For this reason, it is preferable to attach the sensor which measures water temperature to the circulation tank 12a, and to measure the temperature of the circulation tank 12a.
  • FIG. 21 shows a schematic diagram of the plate reader 20 in the amyloid assay device 100 or the integrated amyloid assay device 100 ′ of the present embodiment.
  • the plate reader 20 includes a light source 22, an input optical system Oa, an output optical system Ob, and a light receiving unit 24.
  • a light receiving unit 24 for example, a photomultiplier tube is used.
  • the light emitted from the light source 22 is applied to the solution in the well W of the microplate P through the input optical system Oa.
  • the fluorescence is detected by the light receiving unit 24 via the output optical system Ob.
  • FIG. 22 shows a schematic diagram of the plate reader 20.
  • the input optical system Oa includes lenses L1 to L4, slits S1 to S3, a color filter C1, gratings G1 and G2, and a half mirror H1.
  • the output optical system Ob includes slits S4 to S6, a color filter C2, gratings G3 and G4, and a half mirror H2.
  • the light emitted from the light source 22 is reflected by the grating G1 through the lenses L1, L2, the color filter C1, and the slit S1.
  • the f value of the lenses L1 and L2 is 60.
  • the color filter C1 blocks light having a wavelength other than the specific wavelength.
  • the reflected light is reflected by the grating G2 through the slit S2. Thereafter, the light passes through the slit S3, the half mirror H1, and the lenses L3 and L4, and finally irradiates the solution in each well of the microplate P via the half mirror H2.
  • the intensity of the light whose traveling direction is changed by the half mirror H1 may be detected by the photodetector PD. Based on this intensity, the intensity of light irradiated to the solution in each well of the microplate P can be easily adjusted.
  • ⁇ 2 microglobulin is used as the protein
  • thioflavin T is used as the amyloid-specific fluorescent dye.
  • 200 ⁇ L of 0.3 mg / ml ⁇ 2 microglobulin solution (containing 100 mM NaCl, 5 ⁇ M thioflavin T, pH 2.5) is placed in a microplate P having 96 wells W of the half area black type.
  • the microplate P is set to a temperature of 37 ° C.
  • the moving unit 16 moves the microplate P when irradiating ultrasonic waves.
  • the moving unit 16 rotates the microplate P.
  • the rotation speed of the microplate P is about 6 rpm.
  • the ultrasonic irradiation apparatus 10 repeats ultrasonic irradiation for 1 minute and interruption for 9 minutes.
  • FIG. 23 shows a temporal change in fluorescence intensity when the ultrasonic wave is irradiated while moving the microplate P.
  • the shortest lag time is about 50 minutes, and the longest is about 100 minutes.
  • FIG. 24 shows a schematic diagram of the microplate showing the lag time when the ultrasonic wave is irradiated while moving the microplate.
  • the wells in the first to twelfth rows are denoted as 1, 2, 3,..., 12, respectively, and the wells in the first to eighth columns are denoted as A, B,.
  • the lag time of the solution in most wells is about 70 to 80 minutes.
  • FIG. 16 and FIG. 17 mentioned above the time change of the fluorescence intensity at the time of similarly irradiating an ultrasonic wave in the state which fixed the microplate P, and the lag time of the MyProplate P are shown.
  • the average of the lag time is shortened and the variation of the lag time is suppressed. can do.
  • FIG. 25 shows the lag time distribution.
  • FIG. 25 shows the distribution according to the presence or absence of the movement of the microplate. The number of samples is 96.
  • Table 1 shows the average lag time and standard deviation according to the presence or absence of movement.
  • ⁇ 2 microglobulin causing dialysis amyloidosis is exemplified as a protein, but the present invention is not limited to this.
  • Other proteins may be used.
  • amyloid ⁇ peptide causing Alzheimer's disease
  • IAPP Islet amyloid polypeptide
  • type II diabetes causing type II diabetes
  • antibody L chain causing AL amyloidosis
  • ⁇ -synuclein causing Parkinson's disease Also good.
  • the formation of amyloid from the protein is promoted by ultrasonic waves, and the formed amyloid is detected.
  • amyloid fibrils formed by a peptide called IAPP are considered to cause type II diabetes.
  • type II diabetes the number of insulin receptors decreases or the function thereof decreases. If such a state continues, the islets of Langerhans ⁇ cells that synthesize insulin in the pancreas will also decrease and die.
  • IAPP synthesized in the pancreas together with insulin forms amyloid fibrils and deposits on the islets of Langerhans, thereby killing ⁇ -cells” is prominent.
  • Alzheimer's disease progresses when the neurons that make up the brain degenerate and die.
  • amyloid formation of amyloid ⁇ peptide called senile plaque occurred in the patient's brain, so it was thought that amyloid formation was the direct cause, but recently it is soluble and relatively small called oligomer
  • Abnormal aggregates have been shown to have strong cytotoxicity, which is considered a direct cause of neuronal cell death.
  • an oligomer assay can be performed by performing ELISA (Enzyme-Linked Immunosorbent Assay) using this oligomer and a specific antibody.
  • the ultrasonic irradiation device 10 and the plate reader 20 are used for the amyloid assay, but the present invention is not limited to this.
  • the object of formation and assay by the ultrasonic irradiation apparatus 10 and the plate reader 20 is not limited to amyloid, and may be any expression that is expressed by the ultrasonic irradiation apparatus 10 and can be read by the plate reader 20.
  • an amyloid assay can be performed in a short time for each of a plurality of solutions containing a protein.
  • the amyloid assay according to the invention is widely used not only in basic research but also in the areas of prevention and clinical medicine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention concerne un dispositif (100) de dosage d'amyloïde, qui comprend: une unité (10) d'émission ultrasonique qui émet des ultrasons vers des solutions contenant des protéines, ajoutées dans chacun d'une pluralité de puis d'une microplaque; et une unité (20) de détection de fluorescence qui détecte, au moyen de l'émission ultrasonique, la fluorescence libérée par les liaisons entre un colorant fluorescent spécifique d'amyloïde et les amyloïdes formés à partir de la protéine. L'unité (10) d'émission ultrasonique émet les ultrasons de manière régulière ou irrégulière vers la pluralité des puits. Une thioflavine (T) est avantageusement utilisée comme colorant fluorescent spécifique d'amyloïde.
PCT/JP2011/063413 2010-07-31 2011-06-10 Dispositif et procédé de dosage d'amyloïde Ceased WO2012017739A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2012527633A JP5841945B2 (ja) 2010-07-31 2011-06-10 アミロイドアッセイ装置およびアミロイドアッセイ方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010173225 2010-07-31
JP2010-173225 2010-07-31

Publications (1)

Publication Number Publication Date
WO2012017739A1 true WO2012017739A1 (fr) 2012-02-09

Family

ID=45559252

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/063413 Ceased WO2012017739A1 (fr) 2010-07-31 2011-06-10 Dispositif et procédé de dosage d'amyloïde

Country Status (2)

Country Link
JP (1) JP5841945B2 (fr)
WO (1) WO2012017739A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108061725A (zh) * 2017-02-16 2018-05-22 昆明医科大学 1,8-萘啶基荧光分子探针在氯胺酮娱乐性滥用神经机制研究及成瘾程度诊断中的应用
WO2019003601A1 (fr) * 2017-06-29 2019-01-03 国立大学法人大阪大学 Élément de génération d'ultrasons, dispositif d'émission d'ultrasons et dispositif d'observation de modification par ultrasons
CN113008850A (zh) * 2021-02-07 2021-06-22 首都医科大学宣武医院 诊断阿尔茨海默病的用途及诊断阿尔茨海默病的装置

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002540383A (ja) * 1999-01-25 2002-11-26 ミナーヴァ・バイオテクノロジーズ・コーポレーション 神経変性疾患における異常型タンパク質凝集の迅速かつ高感度の検出
JP2005060336A (ja) * 2003-08-19 2005-03-10 Japan Science & Technology Agency アミロイドβ蛋白質のアミロイド線維化を増幅するための試薬
JP2006090782A (ja) * 2004-09-22 2006-04-06 Fyuuensu:Kk 基板上に固定化した蛋白質の構造変化を検出する方法
WO2007063946A1 (fr) * 2005-11-30 2007-06-07 Fujifilm Ri Pharma Co., Ltd. Diagnostic et remede pour une maladie provoquee par l’aggregation et/ou le depot d’amyloide
JP2007536555A (ja) * 2004-05-10 2007-12-13 バイオクロミクス・アクチェボラーグ タンパク質のコンホメーション変化及び自己集合を決定するための方法
JP2008502880A (ja) * 2004-04-02 2008-01-31 メルク　エンド　カムパニー　インコーポレーテッド アミロイド前駆体タンパク質又はβアミロイド断片に結合する物質を検出する方法及び結合化合物
JP2008032554A (ja) * 2006-07-28 2008-02-14 Japan Science & Technology Agency βアミロイドオリゴマーを用いたバイオチップおよびアミロイドセンサーならびに生体試料中に存在するβアミロイドの検出方法
WO2009057577A1 (fr) * 2007-10-30 2009-05-07 Nihon Medi-Physics Co., Ltd. Utilisation d'un nouveau composé présentant une affinité pour les substances amyloïdes et procédé de production dudit composé

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001188070A (ja) * 1999-12-28 2001-07-10 Hitachi Ltd 自動分析装置及び自動分析方法
US20070077552A1 (en) * 2005-10-04 2007-04-05 Michael Hecht High throughput screen for inhibitors of polypeptide aggregation

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002540383A (ja) * 1999-01-25 2002-11-26 ミナーヴァ・バイオテクノロジーズ・コーポレーション 神経変性疾患における異常型タンパク質凝集の迅速かつ高感度の検出
JP2005060336A (ja) * 2003-08-19 2005-03-10 Japan Science & Technology Agency アミロイドβ蛋白質のアミロイド線維化を増幅するための試薬
JP2008502880A (ja) * 2004-04-02 2008-01-31 メルク　エンド　カムパニー　インコーポレーテッド アミロイド前駆体タンパク質又はβアミロイド断片に結合する物質を検出する方法及び結合化合物
JP2007536555A (ja) * 2004-05-10 2007-12-13 バイオクロミクス・アクチェボラーグ タンパク質のコンホメーション変化及び自己集合を決定するための方法
JP2006090782A (ja) * 2004-09-22 2006-04-06 Fyuuensu:Kk 基板上に固定化した蛋白質の構造変化を検出する方法
WO2007063946A1 (fr) * 2005-11-30 2007-06-07 Fujifilm Ri Pharma Co., Ltd. Diagnostic et remede pour une maladie provoquee par l’aggregation et/ou le depot d’amyloide
JP2008032554A (ja) * 2006-07-28 2008-02-14 Japan Science & Technology Agency βアミロイドオリゴマーを用いたバイオチップおよびアミロイドセンサーならびに生体試料中に存在するβアミロイドの検出方法
WO2009057577A1 (fr) * 2007-10-30 2009-05-07 Nihon Medi-Physics Co., Ltd. Utilisation d'un nouveau composé présentant une affinité pour les substances amyloïdes et procédé de production dudit composé

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIRONOBU NAIKI ET AL.: "First-Order Kinetic Model of Alzheimer's §-Amyloid Fibril Extension In Vitro", LABORATORY INVESTIGATION, vol. 74, no. 2, 1996, pages 374 - 383, XP009136410 *
OHHASHI YUMIKO ET AL.: "Ultrasonication-induced Amyloid Fibril Formation of beta2-Microglobulin", J BIOL CHEM, vol. 280, no. 38, 2005, pages 32843 - 32848 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108061725A (zh) * 2017-02-16 2018-05-22 昆明医科大学 1,8-萘啶基荧光分子探针在氯胺酮娱乐性滥用神经机制研究及成瘾程度诊断中的应用
WO2019003601A1 (fr) * 2017-06-29 2019-01-03 国立大学法人大阪大学 Élément de génération d'ultrasons, dispositif d'émission d'ultrasons et dispositif d'observation de modification par ultrasons
JPWO2019003601A1 (ja) * 2017-06-29 2020-08-06 国立大学法人大阪大学 超音波発生部材、超音波照射装置および超音波変性観察装置
US11623198B2 (en) 2017-06-29 2023-04-11 Osaka University Ultrasound generation member, ultrasound emission device, and ultrasound denaturation observation device
CN113008850A (zh) * 2021-02-07 2021-06-22 首都医科大学宣武医院 诊断阿尔茨海默病的用途及诊断阿尔茨海默病的装置

Also Published As

Publication number Publication date
JP5841945B2 (ja) 2016-01-13
JPWO2012017739A1 (ja) 2013-10-03

Similar Documents

Publication Publication Date Title
Lomont et al. Not all β-sheets are the same: amyloid infrared spectra, transition dipole strengths, and couplings investigated by 2D IR spectroscopy
Horrocks et al. Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-synuclein oligomers
Chou et al. Nanofluidic biosensing for β-amyloid detection using surface enhanced Raman spectroscopy
Devitt et al. Raman spectroscopy: an emerging tool in neurodegenerative disease research and diagnosis
Chen et al. Optical-resolution photoacoustic microscopy using transparent ultrasound transducer
Benilova et al. The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes
Xia et al. Regenerable and simultaneous surface plasmon resonance detection of aβ (1− 40) and aβ (1− 42) peptides in cerebrospinal fluids with signal amplification by streptavidin conjugated to an n-terminus-specific antibody
Singh et al. Ultrafast torsional dynamics of protein binding dye thioflavin-T in nanoconfined water pool
Gligorijevic et al. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria
Kabir et al. Biomolecular motor modulates mechanical property of microtubule
Heo et al. Identifying fibrillization state of Aβ protein via near-field THz conductance measurement
Natalello et al. Insoluble protein assemblies characterized by fourier transform infrared spectroscopy
Rioboó et al. Brillouin spectroscopy: from biomedical research to new generation pathology diagnosis
Luo et al. Optical imaging of beta-amyloid plaques in Alzheimer’s disease
JP2021104056A (ja) 生物学的実体におけるコンホメーションのハイスループット分析のためのシステムおよび方法
Chung et al. Intracellular Aβ42 aggregation leads to cellular thermogenesis
JP5841945B2 (ja) アミロイドアッセイ装置およびアミロイドアッセイ方法
Song et al. On-chip detection of the biomarkers for neurodegenerative diseases: technologies and prospects
CN110062603A (zh) 用于垂直横截面成像的多光子内窥显微镜
ES2834484T3 (es) Método para la preselección de fármacos para enfermedades de plegamiento incorrecto de proteínas
Moore et al. Proteomics analysis of amyloid and nonamyloid prion disease phenotypes reveals both common and divergent mechanisms of neuropathogenesis
Bodani et al. Antibody against small aggregated peptide specifically recognizes toxic Aβ-42 oligomers in Alzheimer’s disease
Zhou et al. Ultrastiff amyloid-fibril network of α-synuclein formed by surface seeding reaction confirmed by multichannel electrodeless quartz-crystal-microbalance biosensor
Juárez et al. Additional supra-self-assembly of human serum albumin under amyloid-like-forming solution conditions
Meyer et al. Ultrasensitive detection of Aβ42 seeds in cerebrospinal fluid with a nanopipette-based real-time fast amyloid seeding and translocation assay

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11814369

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2012527633

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11814369

Country of ref document: EP

Kind code of ref document: A1