[go: up one dir, main page]

WO2019138865A1 - Dispositif de détection et procédé de détection - Google Patents

Dispositif de détection et procédé de détection Download PDF

Info

Publication number
WO2019138865A1
WO2019138865A1 PCT/JP2018/047599 JP2018047599W WO2019138865A1 WO 2019138865 A1 WO2019138865 A1 WO 2019138865A1 JP 2018047599 W JP2018047599 W JP 2018047599W WO 2019138865 A1 WO2019138865 A1 WO 2019138865A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
magnetic field
magnetic
detection
subject
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/JP2018/047599
Other languages
English (en)
Japanese (ja)
Inventor
健一 森脇
宇佐美　由久
西川　尚之
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.)
Fujifilm Corp
Original Assignee
Fujifilm Corp
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 Fujifilm Corp filed Critical Fujifilm Corp
Publication of WO2019138865A1 publication Critical patent/WO2019138865A1/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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to a detection apparatus and detection method for detecting bacteria or the like as a target substance.
  • the polymerase chain reaction is known as a highly sensitive detection method capable of detecting a small amount of virus or the like.
  • PCR polymerase chain reaction
  • the polymerase chain reaction method by amplifying only the nucleotide sequence to be tested by polymerase chain reaction, a virus to be tested can be amplified about one million times in 20 cycles. Therefore, highly sensitive detection of virus etc. is possible.
  • the polymerase chain reaction method has problems such as large influence of impurities, complicated pretreatment, and need of freezing storage.
  • a near field is formed on the detection plate described in Patent Document 1 and a conjugate containing the target substance (target substance) on the surface of the detection plate.
  • an optical detection method of detecting an object as an object and an optical signal.
  • the optical detection method described in Patent Document 1 generates, as an analyte, a conjugate in which a fluorescent particle (phosphor) and a magnetic particle are bound to a target substance using, for example, an antigen-antibody reaction.
  • the near field is generated on the front surface of the detection plate by the light irradiated from the back surface side under the total reflection condition.
  • the generated object is brought close to or separated from the near field (detection plate) by magnetic force, or the generated object is moved in parallel to the detection plate.
  • the target substance is detected by measuring the light amount fluctuation and the movement of light (bright spot) caused by the movement of the subject.
  • Patent Document 1 utilizes a near field.
  • the near field is formed near the pole of the detection surface.
  • Many foreign substances such as proteins are attached to the wall surface such as the detection surface, and further, fluorescent particles, magnetic particles and the like are nonspecifically adsorbed. These deposits inhibit smooth movement of the subject. Therefore, the movement of the subject due to the magnetic force becomes insufficient, and the detection sensitivity of the subject due to the movement decreases.
  • the subject also adheres to the near field or detection surface. Since the object attached to the detection surface can not move due to the magnetic force, the number of objects detected by the movement decreases with respect to the object actually present on the detection surface. As a result, the number of detection signals for noise decreases, and the signal-to-noise ratio decreases.
  • the object of the present invention is to solve such problems of the prior art, and in detection of a target substance using fluorescence and magnetic force, detection of the target substance with high S / N ratio and high sensitivity is enabled. Detection apparatus and detection method.
  • a detection device for detecting a combination of magnetic particles, a target substance and a fluorescent particle, or a target substance and a particle that is magnetic and emits fluorescence as an object
  • a cell containing a liquid containing a subject, an excitation light irradiating part for applying excitation light to the subject to cause fluorescence in the subject, an imaging part having a focal point in the cell for imaging the inside of the cell,
  • a magnetic field generating unit for moving the sample in a direction having an angle with respect to the optical axis of the imaging unit;
  • a detection apparatus characterized in that an intersection point of an optical axis of an excitation light irradiation unit and an optical axis of an imaging unit is located other than an inner wall surface of a cell.
  • [6] The detection device according to any one of [1] to [5], wherein the imaging unit is disposed at a position deviated from the optical path of the excitation light emitted by the excitation light irradiation unit.
  • [7] The detection according to any one of [1] to [6], wherein the optical axis of the imaging unit intersects a plane parallel to the vertical direction of the cell or a plane having an angle of 45 ° or less with respect to the vertical direction. apparatus.
  • the strength of the magnetic field generated by the magnetic field generation unit is 1 to 400 mT.
  • the present invention it is possible to detect a target substance with high sensitivity at a high S / N ratio by removing noise due to fluorescent particles etc. which are nonspecifically adsorbed to a cell or the like for detecting a target substance. is there.
  • FIG. 1 is a perspective view conceptually showing an example of a detection apparatus of the present invention.
  • FIG. 2 is a conceptual diagram for explaining the operation of the detection device shown in FIG.
  • FIG. 3 is a conceptual diagram for explaining the detection device shown in FIG.
  • FIG. 4 is a diagram conceptually showing an example of a system constituting the detection device of the present invention.
  • FIG. 5 is a diagram conceptually showing another example of the detection device of the present invention.
  • FIG. 6 is a diagram conceptually showing another example of the detection device of the present invention.
  • a numerical range represented using “to” means a range including the numerical values described before and after “to” as the lower limit value and the upper limit value.
  • FIG. 1 is a perspective view conceptually showing an example of a detection apparatus of the present invention which implements an example of the detection method of the present invention.
  • the detection device 10 includes a cell 12, an excitation light irradiation unit 14, an imaging unit 16, a first magnetic field generation unit 18, a second magnetic field generation unit 20, a third magnetic field generation unit 24 and a third And a fourth magnetic field generation unit 26.
  • the cell 12 is a container for containing a liquid containing a subject and is a cubic container having an opening (not shown) for introducing the liquid containing the subject.
  • the detection apparatus 10 uses as a subject a conjugate in which magnetic particles and fluorescent particles are bound or a conjugate in which magnetic and fluorescent particles are bound to a target substance such as a virus. It is something to detect.
  • a liquid containing a subject is contained in the cell 12. Then, the application of the magnetic field by the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 moves the subject inside the cell 12 and the excitation light irradiation unit 14 irradiates the inside of the cell 12 with excitation light. Then, the inside of the cell 12 is imaged by the imaging unit 16.
  • the target (target substance) in the liquid is detected depending on whether the moving bright spot is present in the image (moving image) captured by the imaging unit 16 of the detection device 10.
  • the presence or absence is detected, or the presence or absence and the amount of the subject in the liquid are measured.
  • a conjugate in which magnetic particles and fluorescent particles are bound to a target substance is used as an analyte.
  • the target substance (target substance) to be detected is not limited. Examples include viruses, bacteria, DNA, RNA, proteins, and contaminants.
  • the sample from which the target substance is collected there is no limitation on the sample from which the target substance is collected, and various types of substances considered to contain the target substance can be used.
  • body fluids such as blood and lymph, saliva, sweat, runny nose, tears, vomit, urine, feces, medicine, environmental water, clean water, sewage, wiping liquid, etc. are exemplified. These samples may be collected by known methods according to the samples.
  • a method of taking a sample by wiping the doorknob and the table or the like on the spot where food poisoning or the like has occurred with a swab or the like is exemplified.
  • Another example is a method of taking a sample by contacting a swab with vomit and urine and the like.
  • the saturation magnetization of the magnetic particles is not limited. Saturation magnetization of the magnetic particles is preferably 0.01 ⁇ 200A ⁇ m 2 / kg , more preferably in the range of 0.1 ⁇ 100A ⁇ m 2 / kg , 0.3 ⁇ 50A ⁇ m 2 / More preferably, it is kg. By setting the saturation magnetization amount of the magnetic particles to 0.01 A ⁇ m 2 / kg or more, the subject can be suitably moved by application of a magnetic field described later.
  • the subject is a combination of magnetic particles, a target substance and fluorescent particles. Further, by setting the saturation magnetization amount of the magnetic particles to 100 A ⁇ m 2 / kg or less, the subject is prevented from reaching the inner wall of the cell 12 due to the movement of the subject due to application of a magnetic field described later. The subject can be suitably moved in the liquid containing the subject.
  • the fluorescent particles are not limited, and various known substances used for detection of the target substance utilizing fluorescence can be used.
  • fluorescent dyes, quantum dots, rare earths, fluorescent pigments, biological fluorescent molecules and the like are exemplified.
  • Commercially available products are also available.
  • the fluorescent particles may be luminous particles. In this case, in a state in which the excitation light is temporarily blocked or extinguished, imaging in the cell 12 is continuously performed by the imaging unit 16 (imaging element 34) described later.
  • a luminous particle can also use a commercial item.
  • the target substance may emit fluorescence upon irradiation with excitation light. At this time, the target substance doubles as a fluorescent particle.
  • Physisorption is a method of binding a target substance to magnetic particles by using electrostatic bonding force such as hydrogen bonding. Physical adsorption is easy to carry out because processing of magnetic particles is unnecessary. On the other hand, in physical adsorption, the selectivity is low because magnetic particles and fluorescent particles do not specifically adsorb to the target substance. That is, in physical adsorption, magnetic particles and / or fluorescent particles may also bind to substances other than the target substance contained in the sample.
  • the antigen-antibody reaction utilizes specific binding with a target substance, and therefore has the advantage that magnetic particles and fluorescent particles can be selectively bound to the target substance. When using the antigen-antibody reaction, if the target substance is an antigen such as a virus, it is necessary to bind in advance an antibody against the virus as the target substance to the magnetic particle and / or the fluorescent particle. There is.
  • both the magnetic particle and the fluorescent particle are bound to the target substance
  • at least one of the bindings is preferably a specific binding to the target substance, as in an antigen-antibody reaction.
  • both magnetic and fluorescent particles are bound to the target substance, if both bindings are nonspecific, then both the magnetic and fluorescent particles bind to foreign substances other than the target substance. In this case, there arises a disadvantage that the target substance and the foreign matter can not be distinguished.
  • magnetic particles that emit fluorescence upon irradiation with excitation light and fluorescent particles that are magnetized can also be used.
  • the binding between the target substance and the particle is a specific binding with the target substance, such as an antigen-antibody reaction, of the magnetic particle that emits fluorescence upon irradiation with excitation light.
  • the magnetic particle that emits fluorescence upon irradiation of excitation light and the target substance is nonselective, the magnetic particle that emits fluorescence upon irradiation of excitation light is bound to a foreign substance other than the target substance. In this case, there arises a disadvantage that the target substance and the foreign matter can not be distinguished.
  • the cell 12 is a light transmitting container made of glass, resin, etc., and contains a liquid containing a subject.
  • a liquid containing a subject is also referred to as a “test liquid”.
  • a combination of magnetic particles, a target substance, and fluorescent particles is used as an object. Therefore, in the test liquid contained in the cell 12, in addition to the combined body of the magnetic particle which is the subject, the target substance and the fluorescent particle, the combined body of the magnetic particle and the target substance, the target substance and the fluorescent particle There may be a conjugate thereof, a target substance, magnetic particles, fluorescent particles and the like.
  • the test liquid contained in the cell 12 contains the target substance in addition to the combination of the magnetic particle as the test object and the target substance. And magnetic particles may be present.
  • the test liquid contained in the cell 12 includes magnetic particles that emit fluorescence upon irradiation with excitation light, which is an analyte, and a target substance.
  • the cell 12 is a container for containing the test fluid.
  • the test liquid liquid containing an analyte specifically refers to a magnetic substance which is an analyte in a liquid containing a sample in which a target substance may be present, and magnetic particles and fluorescent particles. It is a liquid in which a combination of particles, a target substance and fluorescent particles is formed. That is, in the present invention, the test liquid is, in other words, a liquid in which a subject (target substance) may exist.
  • the sample is taken, for example, with a swab and supplied to the liquid contained in the cell 12.
  • the cell 12 is not limited, and various light transmitting containers that can be used for detection of a target substance by light can be used in medicine, biology and the like.
  • the cell 12 preferably has a transmittance of 50% or more of excitation light.
  • the inner wall of the cell 12 is preferably designed to be resistant to adhesion of the target substance, magnetic particles, fluorescent particles and various other particles.
  • treatment of a known commercially available blocking material is exemplified.
  • the cell 12 is, by way of example, a transparent cubic container having an opening (not shown) for introducing the test liquid, and the opening is directed upward Be done.
  • the excitation light irradiator 14 is for irradiating excitation light to the inside of the cell 12, that is, the test liquid contained in the cell 12 to cause the fluorescent particles to generate fluorescence.
  • the excitation light irradiation unit 14 has a light source 30 and a condensing optical system 32.
  • the light source 30 is for emitting excitation light for causing fluorescent particles to generate fluorescence.
  • the light source 30 is not limited, and various light sources capable of irradiating light including a component that excites fluorescent particles to generate fluorescence can be used.
  • Examples of the light source 30 include a light bulb such as a mercury lamp, a fluorescent lamp, a light emitting diode (LED), and a laser such as a semiconductor laser. Among them, LEDs and semiconductor lasers are suitably used.
  • the light source 30 preferably emits light of a wavelength range different from that of the fluorescence generated by the fluorescent particles as excitation light. As a result, it is possible to prevent the excitation light from becoming noise and lowering the detection sensitivity and accuracy of the object.
  • the condensing optical system 32 is provided as a preferable embodiment, and is for condensing the excitation light irradiated by the light source 30 and irradiating the inside of the cell 12.
  • the focusing optics 32 preferably have a focal point inside the cell 12. More preferably, the focusing optical system 32 has a focal point at a distance of 100 nm or more from the inner wall of the cell 12 and more preferably a focal point at a distance of 300 nm or more from the inner wall of the cell 12.
  • the spot diameter of the focal point can be appropriately selected according to the imaging unit 16, but 10 ⁇ m ⁇ to 10 mm ⁇ is preferable, and 100 ⁇ m ⁇ to 5 mm ⁇ is more preferable. As a result, noise due to the fluorescent particles nonspecifically adsorbed on the inner wall of the cell 12 is reduced, and detection of a highly sensitive subject with a higher S / N ratio becomes possible.
  • the condensing optical system 32 is not limited, and various known condensing optical systems such as a condensing optical system using one or more lenses can be used.
  • a condensing optical system 32 for example, a single-focus lens of fixed magnification in the range of 4 to 600 times is exemplified.
  • the condensing optical system 32 is not limited to the condensing optical system that condenses the light of the light source 30 so as to focus the light.
  • the condensing optical system 32 may be a collimating optical system that shapes the light from the light source 30 into parallel light. In this case, in FIG. 1, the excitation light emitted from the light collection optical system 32 becomes parallel light (substantially parallel light) without being collected.
  • the excitation light irradiator 14 preferably has a wavelength limiting filter between the light source 30 and the cell 12 for removing light other than the wavelength region of the excitation light from the light emitted by the light source 30.
  • a wavelength limiting filter known optical filters can be used depending on the wavelength region of the excitation light. By providing such a wavelength limiting filter, it is possible to suppress the incidence of light in an extra wavelength region that may become noise into the cell 12, and to obtain a high sensitivity S / N ratio high sensitivity object. Detection becomes possible.
  • the imaging unit 16 images the inside of the cell 12.
  • the imaging unit 16 includes an imaging element 34 and a focusing optical system 36.
  • the imaging device 34 is a known imaging device. Therefore, the imaging device 34 is not limited, and various imaging devices having sensitivity to light in this wavelength region can be used according to the wavelength region of the fluorescence generated by the fluorescent particles.
  • CMOS complementary metal oxide semiconductor
  • CCD charge-coupled device
  • the imaging device 34 a CMOS image sensor and a CCD image sensor are suitably used.
  • the condensing optical system 36 is an optical system which condenses the light path of the photometry by the imaging device 34 inside the cell 12 when viewed from the imaging device 34 side. That is, the focusing optical system 36 is a focusing optical system having a focal point inside the cell 12.
  • the focusing optical system 36 that is, the imaging unit 16 has a focal point inside the cell 12. More preferably, the focusing optical system 36 has a focal point at a distance of 100 nm or more from the inner wall of the cell 12 and more preferably a focal point at a distance of 300 nm or more from the inner wall of the cell 12.
  • the spot size of the focal point of the imaging unit 16 can be appropriately selected according to the imaging unit, but 10 ⁇ m square ( ⁇ m square) to 10 mm square (mm square) is preferable, and 100 ⁇ m square to 5 mm square is more preferable. As a result, noise due to the fluorescent particles nonspecifically adsorbed on the inner wall of the cell 12 is reduced, and detection of a highly sensitive subject with a higher S / N ratio becomes possible.
  • the condensing optical system 36 is not limited, and various known condensing optical systems such as a condensing optical system using one or more lenses can be used.
  • a condensing optical system 36 for example, a single-focus lens of fixed magnification in the range of 4 to 600 times is exemplified.
  • the focal point of the excitation light irradiator 14 (the condensing optical system 32) and the focal point of the imaging unit 16 (the condensing optical system 36) may or may not coincide.
  • the imaging unit 16 images a two-dimensional plane (focal plane) of a certain size at the focal position.
  • the focal point of the excitation light irradiation unit 14 and the focal point of the imaging unit 16 are slightly misaligned without being coincident with each other in order to suitably irradiate the excitation light to the two-dimensional plane. preferable.
  • the imaging unit 16 preferably includes an excitation light cut filter between the imaging element 34 and the cell 12 for removing light in the wavelength region of the excitation light.
  • the excitation light cut filter known optical filters can be used. By providing such an excitation light cut filter, it is possible to suppress the excitation light from being incident on the imaging element 34 and to become noise, and it is possible to detect an object with higher sensitivity.
  • the imaging part 16 (imaging element 34) is arrange
  • the detection apparatus 10 of this invention you may utilize various apparatuses which have an imaging function, such as a digital camera, a smart phone, a mobile telephone, etc. as the imaging part 16.
  • the detection device 10 may have a plurality of imaging units 16.
  • the intersection of the optical axis (optical path) of the excitation light irradiation unit 14 and the optical axis (optical path of the light to be measured) of the imaging unit 16 is other than the inner wall surface of the cell 12 Located (see FIG. 3). That is, in the present invention, the intersection of the optical axis of the excitation light irradiation unit 14 and the optical axis of the imaging unit 16 is located inside the cell 12.
  • the detection device 10 of the present invention removes noise caused by fluorescent particles and the like nonspecifically adsorbed on the inner wall surface of the cell 12 and has high sensitivity and high S / N ratio. It enables the subject (target substance). This point will be described in detail later.
  • the intersection point between the optical axis of the excitation light irradiation unit 14 and the optical axis of the imaging unit 16 is preferably located at a distance of 100 nm or more from the inner wall surface of the cell 12 inside the cell 12. It is more preferable to be located at a distance of 200 nm or more from the wall surface, and more preferable to be located at a distance of 300 nm or more from the inner wall surface of the cell 12. As a result, noise due to the fluorescent particles nonspecifically adsorbed on the inner wall of the cell 12 is reduced, and detection of a highly sensitive subject with a higher S / N ratio becomes possible.
  • the optical axis of the excitation light irradiator 14 is the optical axis of the light source 30 when the light collection optical system 32 is not provided, and when the light collection optical system 32 is provided, the light collection is performed. It is an optical axis of the optical system 32. Note that the optical axis of the light source 30 and the optical axis of the focusing optical system 32 generally coincide with each other. Further, in the present invention, the optical axis of the imaging unit 16 is the optical axis of the imaging device 34 when the condensing optical system 32 is not provided, and the condensing optical system is provided when the condensing optical system 32 is provided. It is an optical axis of the system 32. Note that the optical axis of the imaging device 34 and the optical axis of the focusing optical system 32 generally coincide with each other.
  • the imaging unit 16 perform imaging not from the bottom of the cell 12 but from the side surface of the cell 12 as shown in FIG. Specifically, the imaging unit 16 preferably performs imaging by causing the optical axis to intersect a plane parallel to the vertical direction of the cell 12. Alternatively, the imaging unit 16 preferably performs imaging by causing the optical axis to intersect a plane of the cell 12 having an angle of 45 ° or less, particularly 30 ° or less with respect to the vertical direction.
  • the first magnetic field generation unit 18, the second magnetic field generation unit 20, the third magnetic field generation unit 24, and the fourth magnetic field generation unit 26 apply (generate) a magnetic field in the cell 12 to move magnetic particles by magnetic force. It is a thing.
  • the first to fourth magnetic field generation units 18 to 26 are arranged such that one corresponds to one surface of the cubic cell 12 and surrounds the cell 12.
  • the first to fourth magnetic field generation units 18 to 26 are known magnetic field generation units using electromagnets, and include, as an example, an electromagnet, a power supply, and a switch. Ru.
  • the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 are arranged to move the subject in a direction having an angle with respect to the optical axis of the imaging unit 16. Specifically, it is preferable that the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 move the object so as to have an angle of less than 90 ° with respect to the detection surface of the imaging unit 16. Further, it is more preferable that the first to fourth magnetic field generation units 18 to 26 move the object in parallel with the detection surface of the imaging unit 16. In other words, the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 are configured such that the surface (and the extended surface) formed by the moving object is orthogonal to the optical axis of the imaging device 34 It is more preferable to move. Note that the surface formed by the moving subject is, in other words, an image drawn by the moving subject.
  • the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 have a plane formed by connecting the centers of the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 It is preferable to arrange so as to intersect the optical axis.
  • the plane formed by connecting the centers of the first to fourth magnetic field generating units 18 to 26 is orthogonal to the optical axis of the imaging unit 16. More preferably, they are arranged as In other words, the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 connect the centers of the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 with the detection surface of the imaging element 34. More preferably, they are arranged in parallel.
  • the centers of the first to fourth magnetic field generation units 18 to 26 refer to the optical axis direction of the imaging unit 16 and the imaging unit of the surface of the magnet (electromagnet or permanent magnet) of the magnetic field generation unit on the cell 12 side. 16 is the center in the direction orthogonal to the optical axis direction.
  • the magnetic field generating unit so that the moving object (magnetic particles) draws a two-dimensional image when viewed from the imaging unit.
  • the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 are configured such that the angle formed by the surface formed by the moving object and the detection surface of the imaging unit 16 is 45.degree. Or less. It is preferable to move the sample, more preferably to move the sample to 30 ° or less, and even more preferably to move the sample to 0 ° (that is, parallel). That is, in the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26, the plane formed by connecting the centers of the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 and the optical axis of the imaging unit 16 It is preferable that the angle formed be 135 ° or less, more preferably 120 ° or less, and still more preferably 90 ° (that is, orthogonal) .
  • the imaging unit 16 can two-dimensionally image the movement of the subject moving by the application of the magnetic field, and detect the subject (target substance) with higher sensitivity. It will be possible to
  • the magnetic field generator is not limited to one using an electromagnet.
  • the magnetic field generation unit may be configured by a permanent magnet, and a shielding means for the magnetic force that shields / passes the magnetic force from the permanent magnet to the cell 12.
  • the magnetic field strengths of the magnetic fields applied by the first to fourth magnetic field generation units 18 to 26 are not limited.
  • the magnetic field strengths of the magnetic fields applied by the first to fourth magnetic field generators 18 to 26 are preferably 1 to 400 mT, respectively.
  • the first to fourth magnetic field generators 18 to 26 The magnetic field strength of the applied magnetic field is more preferably 10 to 300 mT, and the magnetic field strength of the magnetic field applied by the first to fourth magnetic field generating units 18 to 26 is more preferably 20 to 200 mT.
  • the subject is a conjugate of magnetic particles, a target substance and fluorescent particles.
  • the magnetic field strength of the magnetic field applied by the first to fourth magnetic field generation units 18 to 26 is set to 400 mT or less, an object moving by application of the magnetic field may reach the inner wall of the cell 12. Therefore, the subject can be suitably moved in the test fluid.
  • the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 sequentially apply a magnetic field to combine a magnetic particle as an object, a target substance and a fluorescent particle.
  • the object (target substance) in the liquid to be tested is moved by moving the inside of the cell 12 by the excitation light irradiation unit 14 and imaging the inside of the cell 12 by the imaging unit 16. To detect.
  • FIG. 2 is a view of the detection device 10 as seen from the excitation light irradiation unit 14 and the imaging unit 16 side.
  • the present invention detects the subject T from the test liquid (a liquid containing the subject).
  • a liquid containing a sample that may contain the target substance, and magnetic particles and fluorescent particles is accommodated in the cell 12, and a combination of the target substance, which is the subject T, and the magnetic particles and the fluorescent particles.
  • the combined body of the magnetic particle and the target substance, and the target substance and the fluorescence Conjugates with particles, target substances, magnetic particles, fluorescent particles and the like may be present.
  • the excitation light irradiation unit 14 and the imaging unit 16 are omitted, and only the subject T is inside the cell 12. Is shown.
  • the test liquid When the test liquid is contained in the cell 12 and all of the first to fourth magnetic field generation units 18 to 26 are off, that is, the electromagnet is not driven, the object T, the magnetic particles, and the target substance In the cell 12 (liquid), a conjugate of the above, a conjugate of a target substance and a fluorescent substance, and the like are in a state of being randomly suspended.
  • the test object T, a combination of magnetic particles and a target substance, and magnetic particles move toward the first magnetic field generation unit 18 by magnetic force.
  • the subject T is a combination of magnetic particles, a target substance and fluorescent particles.
  • the conjugate of the target substance and the fluorescent particle, the target substance, and the fluorescent substance move due to Brownian movement or the like, since they do not have magnetic particles, they do not move due to magnetic force.
  • turning on the first magnetic field generation unit 18 means driving the electromagnet of the first magnetic field generation unit 18.
  • the other magnetic field generation units are also the same.
  • the first magnetic field generation unit 18 is turned off, and the second magnetic field generation unit 20 is turned on.
  • the analyte T, the combination of the magnetic particle and the target substance, and the magnetic particle move toward the second magnetic field generation unit 20 by the magnetic force, and the combination of the target substance and the fluorescent particle The target substance and the fluorescent substance do not move.
  • the second magnetic field generation unit 20 is turned off, and the third magnetic field generation unit 24 is turned on.
  • the analyte T, the combination of the magnetic particle and the target substance, and the magnetic particle move toward the third magnetic field generation unit 24 by the magnetic force, and the combination of the target substance and the fluorescent particle The target substance and the fluorescent substance do not move.
  • the third magnetic field generation unit 24 is turned off, and the fourth magnetic field generation unit 26 is turned on.
  • the analyte T, the combination of the magnetic particle and the target substance, and the magnetic particle move toward the fourth magnetic field generation unit 26 by the magnetic force, and the combination of the target substance and the fluorescent particle
  • the target substance and the fluorescent substance do not move.
  • the first magnetic field generation unit 18, the second magnetic field generation unit 20, the third magnetic field generation unit 24, and the fourth magnetic field generation unit 26 are sequentially turned on / off.
  • the object T, the conjugate of the magnetic particle and the target substance, and the magnetic particle move in a substantially square shape.
  • the subject T or the like moves so as to draw a substantially square.
  • the subject T is a combination of magnetic particles, a target substance, and fluorescent particles.
  • the excitation light irradiation unit 14 when the inside of the cell 12 is irradiated with excitation light by the excitation light irradiation unit 14, the analyte T, the combination of the target substance and the fluorescent particle, and the fluorescent particle emit fluorescence.
  • the combination of the magnetic particle and the target substance, the magnetic particle, and the target substance do not undergo any change upon irradiation with the excitation light.
  • the subject T moves in a substantially rectangular shape by sequentially turning on / off the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26, but binding of the target substance to the fluorescent particle is performed.
  • the body and fluorescent particles do not move by magnetic force because they do not have magnetic particles.
  • the excitation light is irradiated, the subject T emits fluorescence, but in the same manner as the subject T, a combination of a magnetic particle and a target substance which moves in a substantially square shape, and the magnetic particle has fluorescent particles. It does not emit light because it is not. That is, in the state in which the first to fourth magnetic field generation units 18 to 26 are sequentially turned on / off and the excitation light is irradiated, it moves in a substantially square shape while emitting fluorescence. T only.
  • the excitation light is irradiated to the inside of the cell 12 while the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 are sequentially turned on / off, and the inside of the cell 12 is imaged by the imaging unit 16. If there is light (bright spot) that moves in a substantially rectangular shape in response to on / off of the first to fourth magnetic field generation units 18 to 26 in the captured image (during moving images), The presence of the subject T in the test solution, that is, the presence of the target substance in the sample can be detected.
  • the first magnetic field generation unit 18 to the first The light repeats a constant movement according to the on / off of the four magnetic field generation unit 26. Therefore, according to the present invention, even if the amount of the target substance in the sample is small, the target substance can be detected, and the presence or absence of the target substance in the sample can be detected.
  • the intersection of the optical axis of the excitation light irradiator 14 and the optical axis of the imaging unit 16 is located other than the inner wall surface of the cell 12. That is, in the present invention, the intersection of the optical axis of the excitation light irradiation unit 14 and the optical axis of the imaging unit 16 is located inside the cell 12. Further, in the present invention, the focal point of the imaging unit 16 is located inside the cell 12. Furthermore, in the present invention, by applying a magnetic field to the inside of the cell 12, the subject T is moved by the magnetic force inside the cell 12. According to the present invention, by having such a configuration, the subject is properly moved by the magnetic force in the test liquid, and the subject is detected inside the cell 12. By removing noise and the like due to the specifically adsorbed fluorescent particles, it is possible to detect an object with high sensitivity and high S / N ratio.
  • the detection surface of the cell 12 such as the detection surface
  • fluorescent particles, magnetic particles and the like are nonspecifically adsorbed.
  • These deposits inhibit smooth movement of the subject. Therefore, as in the near field, on the inner wall surface of the cell (in the vicinity of the inner wall surface), the movement of the subject due to the magnetic force becomes insufficient, and detection of the moving subject becomes difficult. That is, the detection sensitivity of the subject is reduced.
  • the object also adheres to the inner wall surface of the cell such as near field. Since the object attached to the detection surface can not move due to the magnetic force, the number of objects detected by the movement decreases with respect to the object actually present on the detection surface. Also in this point, the detection sensitivity of the subject is lowered. That is, the number of detection signals for noise is reduced, the S / N ratio is low, and the detection sensitivity is reduced.
  • the fluorescent substance is not always adsorbed specifically to the target substance, and many fluorescent particles adhere to the inner wall surface of the cell 12 by nonspecific adsorption. Therefore, when the inner wall surface of the cell 12 is irradiated with excitation light, the fluorescent particles nonspecifically adsorbed to this portion generate fluorescence.
  • the nonspecifically adsorbed fluorescent particles do not move even when magnetic force is applied.
  • an object is moved by a magnetic field using this, and nonspecifically adsorbed to the near field non-specifically due to the change of the light amount and the detection of the moved light (bright point). Differentiation between fluorescent particles and an object is performed, and noise due to fluorescent particles nonspecifically adsorbed in the near field is removed.
  • the unnecessary fluorescence due to the fluorescent particles that are nonspecifically adsorbed in the near field is detected by the imaging device, which again becomes noise and the S / N ratio decreases.
  • the intersection of the optical axis of the excitation light irradiator 14 and the optical axis of the imaging unit 16 is located other than the inner wall surface of the cell 12.
  • the focal point of the imaging unit 16 is located inside the cell 12. Therefore, in the detection device 10 of the present invention, the main detection area is not the inner wall surface of the cell 12, so there are few obstacles in the detection area that impede the movement of the subject, and the subject responds to the applied magnetic force. Move properly. Further, since the subject is not attached to any part, all the subjects present in the area can be moved by the magnetic force.
  • the subject is moved so as to draw a two-dimensional image, so that the subject can be prevented from contacting the inner wall of the cell 12. Therefore, according to the present invention, all the analytes present in the detection area move properly in accordance with the applied magnetic force, and therefore all the analytes present can be detected, so that the inside of the cell 12 can be detected like a near field.
  • the number of detections of the object is improved, that is, the S / N ratio is improved, and the object of high sensitivity (purpose Object) can be detected.
  • the imaging unit 16 is nonspecific to the inner wall surface of the cell 12. It is possible to prevent the measurement of the fluorescence of the fluorescent particles adsorbed in a similar manner. That is, according to the present invention, noise due to the fluorescence generated by the fluorescent particles nonspecifically adsorbed on the inner wall surface of the cell 12 is removed, and a highly sensitive target substance having a high S / N ratio can be detected. become.
  • FIG. 4 conceptually shows an example of a detection system constituting the detection apparatus of the present invention which implements the detection method of the present invention.
  • the detection system shown in FIG. 4 uses a detection tube 40 comprising a cell 12.
  • the detection tube 40 has a filtration chamber 42 in communication with the cell 12, and further has a mixing chamber 46 in communication with the filtration chamber 42.
  • the filtration chamber 42 and the mixing chamber 46 are separated by the partition wall 48, and the filter 50 is provided between the filtration chamber 42 and the cell 12.
  • the partition wall 48 separating the filtration chamber 42 and the mixing chamber 46 is formed of a material such as aluminum foil that can be broken by the swab 52 or the like.
  • a sample that may contain the target substance is collected by the swab 52.
  • the mixing chamber 46 of the detection tube 40 contains a solvent capable of dissolving or dispersing the sample.
  • the solvent include phosphate buffer, tris buffer, acetate buffer, citrate buffer, tartrate buffer, water, water-based solvent, alcohol-based solvent and the like.
  • water it is preferable to use any of pure water, ion exchange water and distilled water.
  • the solution which contained the trace amount blocking agent in these solutions is also used.
  • the swab 52 from which the sample is collected is immersed in the solvent of the mixing chamber 46 and stirred to mix the sample with the solvent.
  • the partition wall 48 is broken by the swab 52, and the solvent mixed with the sample is supplied to the filtration chamber 42.
  • the filtration chamber 42 contains magnetic particles and fluorescent particles.
  • the magnetic particles and the fluorescent particles are mixed in the solvent containing the sample in the filtration chamber 42, an analyte in which the magnetic material, the target substance and the fluorescent material are combined is generated, and is stored in the cell 12.
  • the magnetic and fluorescent particles are not limited to the filtration chamber 42 and may be the mixing chamber 46 or the cell 12. Also, the magnetic particles and the fluorescent particles may be accommodated in different chambers (cells).
  • the liquid to be inspected falls from the filtration chamber 42 and is filtered by the filter 50 to remove large foreign matter and stored in the cell 12.
  • the first to fourth magnetic field generators 18 to 26 and The detection tube 40 is loaded in a detection unit having the excitation light irradiation unit 14 and the imaging unit 16 (not shown), and the object (target substance) is detected as described above.
  • the detection unit which comprises the detection system shown in FIG. 4, the excitation light irradiation part 14 and the imaging part 16 are arrange
  • the detection device 10 shown in FIG. 1 has four magnetic field generation units using electromagnets, the present invention is not limited thereto. That is, three magnetic field generators using electromagnets may be provided. At this time, the movement of the object or the like due to the magnetic force is substantially triangular. Or you may have five or more magnetic field generation parts. As described above, in the present invention, it is preferable to arrange the magnetic field generation unit so that the moving object (magnetic particles) draws a two-dimensional image when viewed from the imaging unit 16.
  • the movement of the subject moving by magnetic force that is, the figure drawn by the subject is not limited to a quadrilateral and a triangle, and is circular (substantially circular), elliptical (substantially elliptical), pentagon or more Various shapes are available, such as polygons (generally polygons) and irregular shapes.
  • the four first magnetic field generation units 18 to the fourth magnetic field generation unit 26 are fixed, but the present invention is not limited thereto.
  • the two magnetic field generation units of the first magnetic field generation unit 18 and the second magnetic field generation unit 20 are disposed so as to sandwich the cell 12 to form the first magnetic field generation unit 18.
  • the subject present in the test fluid may be moved. In this case, the subject moves in a circle.
  • only one first magnetic field generation unit 18 is provided, and the cell is rotated to move around the cell 12 with the first magnetic field generation unit turned on.
  • the subject present in the test fluid in 12 may be moved.
  • the magnetic field generation unit may use an electromagnet as in the first magnetic field generation unit 18, or may use a permanent magnet. Also in this case, the subject moves in a circular manner.
  • the movement mechanism of the magnetic field generation unit is not limited, and all known methods for moving various members can be used.
  • the preferable movement or the like of the magnetic field generation unit conforms to the configuration in which the above-mentioned magnetic field generation unit is sequentially turned on / off. That is, it is preferable that the magnetic field generation unit move the object so that the surface formed by the moving object intersects the optical axis of the imaging unit 16, and the object is perpendicular to the optical axis of the imaging unit 16 It is more preferable to move the
  • the moving mechanism of the magnetic field generation unit preferably moves the magnetic field generation unit so that the surface formed by the center of the moving magnet intersects the optical axis of the imaging unit 16 and is orthogonal to the optical axis of the imaging unit 16 It is more preferable to move the magnetic field generator so that
  • the number of movements of the object or the like by application of a magnetic field is not limited.
  • the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 shown in FIGS. 1 and 2 the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 are sequentially turned on / off.
  • the detection of the subject may be performed by repeating the process a plurality of times and drawing the substantially quadrilateral with the subject a plurality of times (a plurality of rounds).
  • the first magnetic field generation unit 18 to the fourth magnetic field generation unit 26 are sequentially turned on / off only once, and the detection of the object is performed by drawing a substantially rectangular shape by the object once. You may go.

Landscapes

  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention aborde le problème de la fourniture d'un dispositif de détection et d'un procédé de détection grâce auxquels, lors de la détection d'un échantillon d'essai, ledit échantillon d'essai comprenant, par exemple, une substance cible combinée à une particule magnétique et à une particule fluorescente, la substance cible, par exemple un virus, peut être détectée avec une grande sensibilité. Pour résoudre ce problème, l'invention concerne un dispositif de détection qui comprend une cellule abritant un liquide contenant un échantillon d'essai, une partie d'émission de lumière d'excitation qui émet, en direction de l'échantillon d'essai, une lumière d'excitation de façon à générer une fluorescence, une partie d'imagerie focalisée sur l'intérieur de la cellule, et une partie de génération d'un champ magnétique qui va amener l'échantillon d'essai à se déplacer dans une direction présentant un certain angle par rapport à l'axe optique de la partie d'imagerie, le point d'intersection entre l'axe optique de la partie d'émission de lumière d'excitation et l'axe optique de la partie d'imagerie étant positionné ailleurs que sur la face interne de la paroi de la cellule.
PCT/JP2018/047599 2018-01-15 2018-12-25 Dispositif de détection et procédé de détection Ceased WO2019138865A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018004120 2018-01-15
JP2018-004120 2018-01-15

Publications (1)

Publication Number Publication Date
WO2019138865A1 true WO2019138865A1 (fr) 2019-07-18

Family

ID=67218282

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/047599 Ceased WO2019138865A1 (fr) 2018-01-15 2018-12-25 Dispositif de détection et procédé de détection

Country Status (1)

Country Link
WO (1) WO2019138865A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220099674A1 (en) * 2020-09-30 2022-03-31 The Cleveland Clinic Foundation Rapid test system for viral and bacterial infections

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053827A1 (en) * 2006-03-28 2009-02-26 Inverness Medical Switzerland Gmbh Assay device and method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053827A1 (en) * 2006-03-28 2009-02-26 Inverness Medical Switzerland Gmbh Assay device and method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOKURA, SUSUMU: "Study on the non-contact magnetic drive of magnetic fine particles in the fluid", ACADEMIC PAPER *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220099674A1 (en) * 2020-09-30 2022-03-31 The Cleveland Clinic Foundation Rapid test system for viral and bacterial infections
WO2022072391A1 (fr) 2020-09-30 2022-04-07 The Cleveland Clinic Foundation Système de test rapide pour infections virales et bactériennes
EP4221505A4 (fr) * 2020-09-30 2024-10-16 The Cleveland Clinic Foundation Système de test rapide pour infections virales et bactériennes

Similar Documents

Publication Publication Date Title
US7764821B2 (en) Methods and algorithms for cell enumeration in a low-cost cytometer
US20060129327A1 (en) Ultrasensitive sensor and rapid detection of analytes
US20060257941A1 (en) Integration of fluids and reagents into self-contained cartridges containing particle and membrane sensor elements
US20060257854A1 (en) Membrane assay system including preloaded particles
EP1723420A1 (fr) Systeme de dosage base sur des particules et des membranes
US20060257991A1 (en) Integration of fluids and reagents into self-contained cartridges containing particle-based sensor elements and membrane-based sensor elements
WO2005083423A2 (fr) Systeme et procede d'integration de fluides et de reactifs dans des cartouches autonomes contenant des detecteurs de membrane et de particules
CA2704216A1 (fr) Spr microfluidique hybride et dispositif d'imagerie moleculaire
CN109804235B (zh) 目标物质检测装置及目标物质检测方法
JP7028455B2 (ja) 標的物質検出装置及び標的物質検出方法
CN101432739A (zh) 超灵敏传感器和分析物的快速检测
WO2019138865A1 (fr) Dispositif de détection et procédé de détection
JP2020030136A (ja) 目的物質の検出方法
US12203851B2 (en) Method for detecting objective substance
JP7411219B2 (ja) 磁場と重力を用いる標的物質検出装置及び標的物質検出方法
JP4810639B2 (ja) 赤外蛍光粒子を用いた定量方法
JP2019124521A (ja) 検出装置および検出方法
US20120202189A1 (en) Rapid, semi-automated method to detect respiratory virus infected cells in direct specimens
JP2023025766A (ja) 標的物質の検出方法及び標的物質の検出キット
WO2019181400A1 (fr) Méthode de traitement d'échantillon et récipient pour traitement d'échantillon
JP2023025765A (ja) 標的物質の検出方法及び標的物質の検出キット
WO2019235270A1 (fr) Procédé de détection d'une substance cible et kit de détection d'une substance cible
JP2020060440A (ja) 目的物質検出装置及び目的物質検出方法

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: 18900194

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18900194

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP