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WO2019235270A1 - Procédé de détection d'une substance cible et kit de détection d'une substance cible - Google Patents

Procédé de détection d'une substance cible et kit de détection d'une substance cible Download PDF

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Publication number
WO2019235270A1
WO2019235270A1 PCT/JP2019/020765 JP2019020765W WO2019235270A1 WO 2019235270 A1 WO2019235270 A1 WO 2019235270A1 JP 2019020765 W JP2019020765 W JP 2019020765W WO 2019235270 A1 WO2019235270 A1 WO 2019235270A1
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WIPO (PCT)
Prior art keywords
liquid sample
substance
target substance
light
sample introduction
Prior art date
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Ceased
Application number
PCT/JP2019/020765
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English (en)
Japanese (ja)
Inventor
雅人 安浦
藤巻　真
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.)
National Institute of Advanced Industrial Science and Technology AIST
Original Assignee
National Institute of Advanced Industrial Science and Technology AIST
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Application filed by National Institute of Advanced Industrial Science and Technology AIST filed Critical National Institute of Advanced Industrial Science and Technology AIST
Priority to JP2020523639A priority Critical patent/JP7320845B2/ja
Publication of WO2019235270A1 publication Critical patent/WO2019235270A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • 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
    • 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

Definitions

  • the present invention relates to a target substance detection method for detecting a target substance by using a change in an optical signal when a target substance existing in a liquid sample is moved using a magnetic field, and the implementation of the target substance detection method.
  • the present invention relates to a target substance detection kit suitably used.
  • An external force assisted sensor for example, an external force assisted near-field illumination biosensor (External-Force-Assisted) that detects the target substance by comparatively observing a conjugate obtained by binding magnetic particles to the target substance before and after applying a magnetic field.
  • Near Field Illumination Biosensor (see Non-Patent Documents 1 and 2) is one of them, and an optical signal based on the target substance bound to the magnetic particles moves before and after application of the magnetic field, while detection is used for detection.
  • a conjugated conjugate is prepared. Specifically, first, an analyte liquid of the target substance, a first liquid containing a first binding substance that binds the magnetic particles and the magnetic particles to the target substance, the photoresponsive substance, and the light A second liquid containing a second binding substance that binds a responsive substance to the target substance is prepared.
  • the first binding substance and the second binding substance are selected from substances that can bind to the target substance.
  • the first binding substance and the second binding substance must also bind to the magnetic particles and the photoresponsive substance. Therefore, the first binding substance that binds to the magnetic particles and the second binding substance that binds to the photoresponsive substance are selected from different substances.
  • the first liquid is added to the analyte liquid and allowed to react for a predetermined time, and the magnetic particles are bound to the target substance via the first binding substance.
  • the second liquid is added to the analyte liquid and reacted for a predetermined time, the photoresponsive substance is bound to the target substance via the second binding substance, and the first binding to the target substance is performed.
  • the combined body in which the magnetic particles and the photoresponsive substance are bonded through the substance and the second binding substance is obtained.
  • the order of adding the first liquid and the second liquid is changed, the second liquid is added first, and the first liquid is added later, so that the first binding substance and the second liquid are added to the target substance.
  • the combined body in which the magnetic particles and the photoresponsive substance are bonded via the binding substance is obtained.
  • the order of adding the first liquid and the second liquid to the analyte liquid is such that the affinity (binding) with the target substance is between the selected first binding substance and the second binding substance.
  • the first liquid is added first when the affinity of the first binding substance is low, and when the affinity of the second binding substance is low, the first binding substance is low.
  • Add 2 liquids first that is, when a binding substance having a higher affinity with the target substance is added first, the binding site of the target substance is filled with the binding substance, and the affinity with the target substance added later is lower. Therefore, any one of the magnetic particles and the photoresponsive substance does not form a conjugate with the target substance and is excluded from the detection target. Such a procedure is cumbersome for the user and hinders efficient detection testing.
  • the detection target is removed from the detection target due to the difference in the procedure described above or the passage of time, the accuracy of the detection result is lowered and the detection result becomes unstable.
  • the present invention provides a target substance detection method and a target substance detection kit that can solve the above-mentioned problems in the prior art and can detect a target substance efficiently, highly accurately and stably using the external force support type sensor.
  • the purpose is to do.
  • Means for solving the above problems are as follows. That is, ⁇ 1> A liquid sample is introduced onto the front surface and transmitted through the light irradiated from the rear surface side or the front surface side as propagating light so that it can propagate to the surface side opposite to the side irradiated with the light.
  • a detection plate capable of generating near-field light on the surface by light that is introduced onto the surface and irradiated with total reflection conditions on the surface.
  • the liquid sample introduction plate is arranged and the liquid sample is held on the surface of the liquid sample introduction plate with respect to the liquid sample holding portion that can hold the liquid sample on the surface of the liquid sample introduction plate.
  • a first combined body moving step of moving in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the introduction plate and a direction moving away from the liquid sample introduction plate; and the liquid sample introduction plate The combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate is drawn onto the surface of the liquid sample introduction plate by applying a magnetic field from a magnetic field application unit disposed on the back side. In the state where the magnetic field is applied, the magnetic field application unit is moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate, and the coupling is performed by following the movement of the magnetic field application unit.
  • a combined body moving step which is one of the second combined body moving steps for moving the body, and the movement of the combined body in accordance with the combined body moving step of the optical signal based on the propagating light or the near-field light.
  • An optical signal detection step for detecting by a change in signal, wherein the liquid sample has a magnetic particle and a photoresponsive substance each forming a conjugate with a target substance, and a first that binds the magnetic particle to the target substance.
  • a binding substance and a second binding substance that binds the photoresponsive substance to the target substance, wherein the same binding substance is used for the first binding substance and the second binding substance.
  • a target substance detection method for detecting by a change in signal, wherein the liquid sample has a magnetic particle and a photoresponsive substance each forming a conjugate with a target substance, and a first that binds the magnetic particle to the target substance.
  • a binding substance and a second binding substance that binds the photoresponsive substance to the target substance, wherein the same binding substance is used for
  • the target substance detection method according to ⁇ 1>, wherein the target substance is a substance having a plurality of binding sites of the same type, and the binding substance is a substance that can specifically bind to the binding site.
  • the liquid sample is prepared by mixing one liquid detection liquid containing a magnetic particle and a photoresponsive substance each in a state of being bound to a binding substance with respect to an analyte liquid of a target substance. > To ⁇ 2>.
  • ⁇ 4> The target substance detection method according to any one of ⁇ 1> to ⁇ 3>, wherein the photoresponsive substance is irradiated with propagating light or near-field light to generate scattered light.
  • the target substance detection method according to any one of ⁇ 1> to ⁇ 3>, wherein the photoresponsive substance generates fluorescence upon irradiation with propagating light or near-field light.
  • the optical signal detection step is a step of detecting a signal change of an optical signal based on propagating light
  • the photoresponsive substance includes a light absorbing substance that generates light absorption when irradiated with the propagating light.
  • ⁇ 7> The target substance detection method according to any one of ⁇ 1> to ⁇ 6>, wherein the magnetic particles are spherical particles having a diameter of 5 nm to 6,500 nm.
  • the target substance detection method according to any one of ⁇ 1> to ⁇ 7>, wherein the photoresponsive substance is a spherical particle having a diameter of 50 nm to 6,500 nm.
  • the combined body moving step is the first combined body moving step, after the liquid sample introduction and holding step and before the combined body moving step, all or all of the combined bodies in the liquid sample are applied by applying an attracting magnetic field.
  • Feature target substance detection kit Magnetic particles that form a conjugate with a target substance, a photoresponsive substance that forms the conjugate with the target substance, a first binding substance that binds the magnetic particles to the target substance, and the photoresponse And a second binding substance that binds the target substance to the target substance, and the first binding substance and the second binding substance have a single liquid detection solution composed of the same binding substance.
  • a target substance detection method and a target substance detection kit that can solve the above-described problems in the prior art and can detect a target substance efficiently, highly accurately, and stably using the external force support type sensor. Can be provided.
  • Via binding substance B 1, B 2 to the target substance T magnetic particles M and photoresponsive material O is an explanatory diagram showing a conjugate bound a total of three. It is explanatory drawing of the target substance detection apparatus.
  • FIG. 7 is a cross-sectional view taken along line AA in FIG.
  • FIG. 9 is a cross-sectional view taken along line AA in FIG. It is explanatory drawing of 1 A of target substance detection apparatuses. It is explanatory drawing of the target substance detection apparatus 1B.
  • FIG. (2) which shows typically a mode on the surface of a liquid sample introduction board in an observation field of view observed with an imaging device after a combined body movement process.
  • FIG. 13 is a cross-sectional view taken along line AA in FIG. 1 is an explanatory diagram of a target substance detection device 10.
  • FIG. It is a figure (3) which shows typically the mode on the surface of the liquid sample introduction board in the observation visual field observed with an imaging device after a combined body movement process.
  • FIG. 16 is a cross-sectional view taken along line AA in FIG. It is explanatory drawing of the target substance detection apparatus 20.
  • FIG. It is explanatory drawing of 20 A of target substance detection apparatuses. It is explanatory drawing of the target substance detection apparatus 30.
  • FIG. It is explanatory drawing of the target substance detection apparatus.
  • FIG. 1 shows the mode on the said surface of a liquid sample introduction plate after a conjugate
  • FIG. It is a figure (2) which shows the mode on the said surface of a liquid sample introduction plate before a coupling body movement process. It is a figure (2) which shows the mode on the said surface of a liquid sample introduction plate after a conjugate
  • FIG. It is a figure which shows the image after 7 second passage from the said one time point in the said moving image of the 1st detection by the target substance detection method based on Example 1.
  • FIG. 1 It is a figure which shows the image in one time in the moving image of the 2nd detection by the target substance detection method which concerns on Example 1.
  • FIG. It is a figure which shows the image after 5.3 second progress from the said one time point in the said moving image of the 2nd detection by the target substance detection method which concerns on Example 1.
  • FIG. It is a figure which shows the image in the one time point in the moving image of the 1st detection by the target substance detection method which concerns on the comparative example 1.
  • FIG. It is a figure which shows the image after 6.3 second progress from the said one time point in the said moving image of the 1st detection by the target substance detection method which concerns on the comparative example 1.
  • FIG. 1 It is a figure which shows the image in the one time point in the moving image of the 2nd detection by the target substance detection method which concerns on the comparative example 1.
  • FIG. It is a figure which shows the image after 2.7 second progress from the said one time point in the said moving image of the 2nd detection by the target substance detection method which concerns on the comparative example 1.
  • the target substance detection method according to the present invention is an external force assisted sensor that detects a target substance by comparatively observing a conjugate obtained by binding magnetic particles to the target substance before and after applying a magnetic field, for example, external force assisted near field illumination. It can be carried out using a biosensor (External-Force-Assisted Near Field Illumination Biosensor).
  • a biosensor External-Force-Assisted Near Field Illumination Biosensor
  • the external force support type sensor will be described in detail.
  • the external force support type sensor will be described as a “target substance detection device”.
  • the target substance detection device includes a liquid sample holding unit, a light irradiation unit, a magnetic field application unit, and an optical signal detection unit, and includes other units as necessary.
  • the liquid sample holding part is a part in which a liquid sample introduction plate is arranged and a liquid sample is held on the surface of the liquid sample introduction plate.
  • the liquid sample introduction plate propagates to the surface side opposite to the side irradiated with the light using the transmitted light of the light irradiated from the back side or the surface side as the propagation light while the liquid sample is introduced onto the surface.
  • a translucent plate that is made possible, a reflective plate that is capable of propagating the reflected light of the light irradiated from the surface side as the propagating light as the propagating light, and the liquid sample.
  • the propagation light is generally light that does not include near-field light that exhibits abrupt attenuation at a position that is separated from the generation source by a distance of several hundred nm to several ⁇ m. It means that near-field light is not included, and means light that does not show abrupt attenuation at a position separated from the surface of the liquid sample introduction plate by a distance of several hundred nm to several ⁇ m.
  • the near-field light means light that shows abrupt attenuation at a position separated from the surface of the liquid sample introduction plate by a distance of several hundred nm to several ⁇ m.
  • the light-transmitting plate is not particularly limited and can be appropriately selected depending on the purpose.
  • a known translucent plate can be used.
  • the reflecting plate is not particularly limited and may be appropriately selected depending on the purpose.
  • a known plate such as a glass plate, a plastic plate, or a metal plate used for an observation stage of a known episcopic microscope is used.
  • a reflector can be used.
  • the introduction plate is not particularly limited and may be appropriately selected depending on the purpose.
  • the introduction plate includes the light transmission plate and the reflection plate, and is a known plate for introducing other liquid samples.
  • a shaped member can be used.
  • the detection plate is not particularly limited and may be appropriately selected depending on the purpose.
  • a known detection plate such as a known surface plasmon resonance sensor or a known waveguide mode sensor is used. be able to.
  • the said surface is surface-treated with the adsorption inhibitor which suppresses adsorption
  • the adsorption inhibitor which suppresses adsorption
  • the target substance is the protein
  • a known blocking method that suppresses adsorption of the protein can be selected as the surface treatment method.
  • the blocking method is not particularly limited, and examples thereof include a method using polyethylene glycol, a method using ethanolamine, a method using skim milk, and a method using a silane coupling agent.
  • maintenance part there is no restriction
  • the plate-like translucent member and the liquid sample introduction plate may be sandwiched, and the liquid layer of the liquid sample may be held on the surface of the liquid sample introduction plate.
  • maintenance part it can also comprise with the bowl-shaped liquid cell in which a bottom face is comprised with the said liquid sample introduction plate.
  • the liquid sample holding unit may be divided into a plurality of regions by dividing a plurality of regions on the surface of one liquid sample introduction plate.
  • a flow path capable of feeding liquid is formed between the outside and the space on the surface of the liquid sample introduction plate as the liquid sample holding portion. That is, according to the target substance detection device, since the target substance can be detected ignoring the presence of impurities adsorbed on the liquid sample introduction plate, the cleaning process of the liquid sample introduction plate is sequentially performed. Therefore, when the flow path is formed in the liquid sample holder, the liquid sample is simply replaced through introduction and discharge of the liquid sample through the flow path. Detection can be advanced, and the detection operation can be made more efficient.
  • the “cleaning process” means a process of removing the contaminants adsorbed on the surface of the liquid sample introduction plate by a physical polishing process, a peeling process using chemicals, or a dissolving process. , It does not include a process of rinsing with water when changing the liquid sample.
  • the light irradiation unit is formed of any one of a back surface side light irradiation unit, a front surface side light irradiation unit, a side surface side light irradiation unit, and a total reflection light irradiation unit.
  • the back surface side light irradiating unit can irradiate the light from the back surface of the liquid sample introducing plate when the liquid sample introducing plate is formed of the light transmitting plate.
  • the back surface side light irradiating unit can irradiate the light from the back surface of the liquid sample introducing plate when the liquid sample introducing plate is formed of the light transmitting plate.
  • the said back surface side light irradiation part there is no restriction
  • the surface-side light irradiating unit can irradiate the light from the surface side of the liquid sample introducing plate when the liquid sample introducing plate is formed of either the light transmitting plate or the reflecting plate.
  • the said surface side light irradiation part there is no restriction
  • the side-side light irradiating unit is configured so that the liquid sample is introduced from the side surface side of the liquid sample introduction plate with respect to the liquid sample held on the liquid sample introduction plate when the liquid sample introduction plate is formed by the introduction plate.
  • the light can be irradiated in a direction parallel to the in-plane direction of the surface of the sample introduction plate.
  • the total reflection light irradiating unit can irradiate the light on the surface under total reflection conditions when the liquid sample introduction plate is formed by the detection plate.
  • the total reflection on the surface is not particularly limited in the incident direction of the light as long as the total reflection condition can be satisfied on the surface of the detection plate.
  • the light can be introduced into the waveguide structure from the surface side through the prism, and the total reflection condition on the surface of the detection plate can be satisfied by utilizing the total reflection in the waveguide structure.
  • the prism may be formed as a partial structure of the detection plate.
  • the light irradiation unit can be configured in the same manner.
  • the back side light irradiation part the surface side light irradiation part, the side surface side light irradiation part, and the total reflection light irradiation part
  • a known light emitting device such as a lamp, an LED device, or a laser light irradiation device can be used.
  • the back side light irradiation unit, the front side light irradiation unit, and the total reflection light irradiation unit are not particularly limited with respect to optical elements other than the light source, and a known optical microscope, a known surface plasmon resonance sensor, A known optical element used in a known waveguide mode sensor can be appropriately adopted depending on the purpose.
  • the magnetic field application unit is formed by one of a first magnetic field application unit and a second magnetic field application unit. Any one of the first magnetic field application unit and the second magnetic field application unit has a role of moving the conjugate introduced on the surface of the liquid sample introduction unit, and the target substance. In the detection apparatus, the movement of the conjugate is used for detection of the target substance.
  • the first magnetic field application unit is disposed on the surface side or the side surface side of the liquid sample introduction plate and magnetically applies the combination in the liquid sample introduced onto the surface of the liquid sample introduction plate. Is a member that is moved in either a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate or a direction away from the liquid sample introduction plate.
  • the first magnetic field application unit is not particularly limited as long as it is such a member, and can be appropriately selected according to the purpose.
  • the first magnetic field application unit can be configured using a known electromagnet and permanent magnet.
  • the permanent magnet In the case of using the permanent magnet, for example, the permanent magnet is held on a moving member, and the magnetic field by the permanent magnet is in the proximity state where the magnetic field by the permanent magnet extends on the surface of the liquid sample introduction plate. It is possible to control the movement between the liquid sample introduction plate and the separated state that does not reach the surface, and to turn on and off the application state of the magnetic field on the surface of the liquid sample introduction plate. Further, for example, a known magnetic shield member is controlled to be opened and closed in an open state in which the magnetic field is applied to the surface of the liquid sample introduction plate and a shield state in which the magnetic field is not applied to the surface of the liquid sample introduction plate. The application state of the magnetic field to the surface of the liquid sample introduction plate can be turned on and off.
  • the first magnetic field application unit is not particularly limited, but has a through-hole, an incomplete ring shape such as a U shape, or a plurality of members arranged in an annular or incomplete ring shape. It is preferable that it is the structure comprised.
  • the first magnetic field application unit is formed in this way, the surface side of the liquid sample introduction plate through the through hole or the inside of the ring or the incomplete ring when the surface side light irradiation unit is used.
  • the surface of the liquid sample introduction plate in any case of the front side light irradiation unit, the back side light irradiation unit, the side surface side light irradiation unit, and the total reflection light irradiation unit can be detected by the optical signal detection unit through the inside of the through hole or the annular member.
  • the members arranged in an annular shape are not particularly limited as long as they do not obstruct the light irradiation or the optical path of the optical signal, and may be those that can individually control the application state of the magnetic field.
  • the second magnetic field application unit is arranged on the back side of the liquid sample introduction plate and applies the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate by applying a magnetic field.
  • the liquid sample introduction plate can be drawn toward the surface side and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate with the magnetic field applied. It is a member.
  • the second magnetic field application unit is not particularly limited as long as it is such a member, and can be appropriately selected according to the purpose.
  • the second magnetic field application unit can be configured using a known electromagnet and permanent magnet.
  • the electromagnet or the permanent magnet is held on a slide member, and the surface side light irradiation unit, the back side light irradiation unit, the side surface side light irradiation unit, or the total reflection light irradiation unit of the liquid sample introduction plate is used.
  • An initial state in which the electromagnet or the permanent magnet is positioned in the vicinity of a region (detection region) irradiated with the light from the light irradiation unit, and a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate It can comprise by carrying out movement control between the states which moved the said electromagnet or the said permanent magnet toward the direction which has.
  • the second magnetic field application unit is not particularly limited, but has a through-hole formed therein, an incomplete ring shape such as a U shape, or a plurality of members arranged in a ring shape or an incomplete ring shape. It is preferable that it is the structure comprised.
  • the members arranged in an annular shape are not particularly limited as long as they do not obstruct the light irradiation or the optical path of the optical signal, and may be those that can individually control the application state of the magnetic field.
  • the optical signal detection unit is arranged on the front surface side, the back surface side, or the side surface side of the liquid sample introduction plate and applies the magnetic field by the first magnetic field application unit and the second magnetic field application unit.
  • the movement of the combined body accompanying the movement can be detected by a signal change of an optical signal based on the propagating light or the near-field light.
  • the optical signal detector is not particularly limited and may be appropriately selected depending on the purpose.
  • a known optical element such as a photodiode or a photomultiplier tube or a known optical element such as an objective lens may be used. Can be configured.
  • the optical signal detection unit is not particularly limited, but it is preferable that the state of the detection region on the surface of the liquid sample introduction plate can be acquired as a two-dimensional image. If the two-dimensional image can be acquired, position information and size information of the optical signal in the two-dimensional image appearing as a light spot or a dark spot can be easily acquired, and the two-dimensional image before and after the combined body is moved.
  • the optical signal is information related to the combined body, or the combined body such as scratches on the surface of the liquid sample introduction plate, the foreign matter, fluctuation of the light source output, etc. It is possible to clearly distinguish whether the information is not involved.
  • an imaging device may be selected as the optical signal detection unit. Furthermore, when the two-dimensional image is continuously captured and observed as a moving image, it is possible to more clearly identify the movement of the optical signal in the two-dimensional image that appears as a light spot or a dark spot. .
  • the imaging device there is no restriction
  • the optical signal detection unit is out of the imageable range and the near-field light generation region (several hundred nm from the surface of the liquid sample introduction plate).
  • a method of performing detection after arranging the conjugate once on the surface of the liquid sample introduction plate or in the vicinity of the surface. preferable.
  • Examples of detecting the target substance include detection of the presence or absence of the target substance, detection of the amount of the target substance (quantitative measurement), real-time observation of the presence state of the target substance, and the like.
  • the detection in the optical signal detection unit accompanying the movement of the combined body by the magnetic field application unit will be described.
  • the optical signal based on the propagating light detected by the optical signal detection unit includes an upper part of the surface of the liquid sample introduction plate in the same manner as the optical signal acquired by the known transmission microscope or the epi-illumination microscope.
  • An optical signal 2 an optical signal 3 that can be distinguished from the optical signal 1, and generated on the surface of the liquid sample introduction plate.
  • the optical signal also includes a noise signal caused by fluctuations in the light source output. If the optical signals 2 to 4 and the noise signal cannot be distinguished except for the optical signal 1 processed as a background signal, the detection accuracy is lowered. However, in the target substance detection device, the combined body is moved based on the magnetic field applying section formed by the first magnetic field applying section and the second magnetic field applying section of the combined body, Since it is detected as a signal change of the optical signal based on the propagating light, the optical signal 2, the optical signals 3 and 4, and the noise signal can be clearly distinguished. That is, the optical signals 3 and 4 and the noise signal are optical signals that do not change before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit.
  • the optical signal 2 includes scattered light, reflected light, phase difference, transmitted light based on differential interference, fluorescence of the photoresponsive substance, phosphorescence, etc. emitted when the conjugate is irradiated with the propagating light.
  • each of the liquid sample holding unit, the light irradiation unit, and the optical signal detection unit may be a known phase contrast microscope
  • the optical system in the differential interference microscope is configured.
  • Examples of the change of the optical signal 2 include intensity increase / decrease, phase change, position shift, defocus, and appearance / disappearance.
  • the optical signal based on the near-field light detected by the optical signal detection unit includes, in the liquid sample, the same as the optical signal acquired by a known surface plasmon resonance sensor or a known waveguide mode sensor.
  • the optical signal also includes a noise signal caused by fluctuations in the light source output.
  • the target substance detection device when the near-field light is used, similarly to the case where the propagation light is used, if the optical signals 5 to 7 and the noise signal cannot be distinguished, the detection sensitivity is lowered.
  • the combined body is moved based on the magnetic field applying section formed by the first magnetic field applying section and the second magnetic field applying section of the combined body, and the change is moved to the Since it is detected as a signal change of the optical signal based on near-field light, the optical signal 5, the optical signals 6 and 7, and the noise signal can be clearly distinguished.
  • the optical signals 6 and 7 and the noise signal are optical signals that do not change before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit
  • the optical signal 5 Is an optical signal that changes before and after the application of the magnetic field by the first magnetic field application unit and before and after the movement of the second magnetic field application unit because it is caused by the combined body including the magnetic particles.
  • optical signal 5 which is focused as the changing optical signal
  • various aspects can be taken according to the type of the photoresponsive substance and the type of the optical system of the target substance detection device. That is, examples of the optical signal 5 include scattered light emitted when the conjugate is irradiated with the near-field light, light emission such as fluorescence, and an optical signal based on light absorption of the conjugate.
  • increase / decrease in intensity, position movement, and appearance / disappearance can be mentioned.
  • the other part is not particularly limited and may be appropriately selected depending on the purpose.
  • a third magnetic field application part a known transmission microscope, a known episcopic microscope, a known total reflection microscope, An arbitrary portion used for a known surface plasmon resonance sensor, a known waveguide mode sensor, or the like can be given.
  • the third magnetic field application unit is further disposed on the back side of the liquid sample introduction plate and the liquid sample introduction plate when the magnetic field application unit is formed by the first magnetic field application unit. It is a portion that can draw the combined body in the introduced liquid sample onto the surface of the liquid sample introduction plate by applying a magnetic field.
  • the magnetic field application unit When the magnetic field application unit is formed by the second magnetic field application unit, the combined body in the liquid sample is attracted onto the surface of the liquid sample introduction plate by the application of the magnetic field. Therefore, the detection of the optical signal by the optical signal detection unit is performed by focusing on the surface of the liquid sample introduction plate or in the vicinity thereof, thereby detecting the movement state of the combined body drawn on the surface. be able to.
  • the optical signal detection unit performs the detection of the optical signal by focusing on the surface of the liquid sample introduction plate or the vicinity thereof. For example, immediately after the liquid sample is introduced into the liquid sample introduction plate, the conjugate does not necessarily attract the surface of the liquid sample introduction plate. It is in a state of floating in the liquid layer.
  • the combined body in the floating state exists outside the imageable range where the optical signal can be detected by the optical signal detection unit or outside the near-field light generation region, the combined body is not detected. Become. Therefore, when the optical signal is detected by the optical signal detection unit while focusing on the surface of the liquid sample introduction plate or the vicinity thereof, the liquid sample is introduced into the liquid sample introduction plate, and then the coupling is performed. It is necessary to wait for the body to gravity settle on the surface of the liquid sample introduction plate, and it takes time to prepare for detection. In particular, when the specific gravity of the conjugate is small, a longer time is required. Therefore, by applying the magnetic field by the third magnetic field applying unit, the conjugate floating in the liquid layer of the liquid sample is drawn to the surface side of the liquid sample introduction plate, thereby shortening the detection preparation time. And more efficient detection can be performed.
  • said 3rd magnetic field application part can select suitably, For example, it can comprise using a well-known electromagnet and a permanent magnet.
  • the third magnetic field application unit is configured to prevent the combination from being moved by the first magnetic field application unit after the combination is attracted to the surface side of the liquid sample introduction plate. Therefore, it is necessary to adjust the strength to weaken the application state of the magnetic field that attracts and to turn on / off control.
  • the permanent magnet when the permanent magnet is used, for example, the permanent magnet is held on a moving member, and the magnetic field generated by the permanent magnet extends into the liquid layer of the liquid sample and the magnetic field generated by the permanent magnet is the liquid.
  • the intensity adjustment or the on-off control of the magnetic field application state can be performed through excitation and demagnetization of the electromagnet.
  • the application state of the magnetic field can be turned on and off.
  • the third magnetic field application unit is not particularly limited, but has a through-hole formed therein, an incomplete ring shape such as a U shape, or a plurality of members arranged in a ring shape or an incomplete ring shape. It is preferable that it is the structure comprised.
  • an incomplete ring shape such as a U shape
  • a plurality of members arranged in a ring shape or an incomplete ring shape It is preferable that it is the structure comprised.
  • the members arranged in an annular shape are not particularly limited as long as they do not obstruct the light irradiation or the optical path of the optical signal, and may be those that can individually control the application state of the magnetic field.
  • the conjugate can be concentrated in the detection region on the surface of the liquid sample introduction plate, and the target substance can be detected with higher accuracy. It can be carried out.
  • the target substance detection method of the present invention includes a liquid sample introduction and holding step, a light irradiation step, a conjugate transfer step, and an optical signal detection step, and includes other steps as necessary.
  • liquid sample introduction and holding process In the liquid sample introduction and holding step, the liquid sample is introduced onto the front surface and transmitted light of light irradiated from the rear surface side or the front surface side is used as propagation light on the surface opposite to the side irradiated with the light.
  • a translucent plate capable of propagating, a reflective plate capable of propagating reflected light of light irradiated from the surface side as the propagating light as the propagating light, and the liquid sample. Can introduce near-field light on the surface by the introduction plate introduced on the surface, and the liquid sample introduced on the surface and light irradiated on the surface under total reflection conditions.
  • the liquid sample introduction plate is arranged with respect to a liquid sample holding portion in which a liquid sample introduction plate formed by any of the detection plates is arranged and the liquid sample can be held on the surface of the liquid sample introduction plate The liquid on the surface of A step of the introduced retaining fee.
  • the liquid sample includes a magnetic particle and a photoresponsive substance that form a conjugate with a target substance, a first binding substance that binds the magnetic particle to the target substance, and the photoresponsive substance as the target substance. And a second binding substance to be bound.
  • Examples of the target substance include various proteins including virus-like particles such as DNA, RNA, virus, capsid protein, extracellular vesicles such as exosomes, microvesicles, and large oncosomes, fungi, and contaminants.
  • Examples of the target substance test liquid for verifying the presence of the target substance include, for example, blood, saliva, urine, liquid chemicals, environmental water, water and sewage, beverages, food homogenizing solutions, wipes, powders, etc. And a solution obtained by dissolving the solid sample in a solvent such as water, and a gas phase concentrated liquid obtained by collecting gas and fine particles in the gas phase.
  • the role of the magnetic particles is to cause movement associated with application of the magnetic field in a state where a conjugate with the target substance is formed.
  • the magnetic particle is not particularly limited as long as it plays the above role, and can be appropriately selected according to the purpose, and known magnetic beads or the like can be used.
  • the role of the photoresponsive substance is to generate the optical signal based on the propagating light or the near-field light in a state where a conjugate with the target substance is formed.
  • the photoresponsive substance is not particularly limited as long as it plays the above role and can be appropriately selected according to the purpose, and generates scattered light upon irradiation with the propagating light or the near-field light.
  • a light scattering material, a fluorescent material that generates fluorescence, or a light absorbing material that generates light absorption can be used.
  • resin particles such as polystyrene beads prepared so as to have the respective properties, metal nanoparticles such as gold nanoparticles, silver nanoparticles, gold nanorods, and gold nanostars are known.
  • the particles can be used.
  • known fluorescent materials such as fluorescent dyes, quantum dots, fluorescent beads, beads with quantum dots, and fluorescent dyes can be used.
  • light responsiveness refers to the property of generating an optical signal that can be detected in the optical signal detection step in response to irradiation with the propagating light or the near-field light.
  • the first binding substance and the second binding substance have a role of binding the magnetic particles and the photoresponsive substance to the target substance, and are in a state of being combined with the magnetic particles and the photoresponsive substance And binding to the target substance to form a conjugate in which the magnetic particles and the photoresponsive substance are bound to the target substance via the first binding substance and the second binding substance.
  • FIG. 1 is an explanatory diagram for explaining the situation when the first binding substance B 1 and the second binding substance B 2 having a lower affinity than the first binding substance B 1 are used. These are explanatory drawings explaining the situation in the case of using the first binding substance B 1 and the second binding substance B 2 having a higher affinity than the first binding substance B 1 , and FIG. If the binding substance B 1 and the second coupling between the substance B 2 are identical binding agent used is an explanatory diagram for explaining the status of.
  • the magnetic particles M in a state where the first binding substance B 1 is bound to the analyte liquid of the target substance T and the photoresponsive substance O in a state where the second binding substance B 2 is bound Think about the situation when you add it. Different binding substances are used for the first binding substance B 1 and the second binding substance B 2, and the second binding substance B 2 has a lower affinity for the target substance T than the first binding substance B 1. Then, as shown in FIG. 1, the first binding substance B 1 having high affinity preferentially binds to the target substance T, and the target substance T and the magnetic particles M via the first binding substance B 1 However, there is a situation in which the binding between the target substance T and the photoresponsive substance O via the second binding substance B2 cannot be obtained. In this case, since the optical signal based on the photoresponsive substance O cannot be detected, the target substance T is excluded from the detection target.
  • the second binding substance B 2 has more affinity for the target substance T than the first binding substance B 1.
  • the second binding substance B 2 having a high affinity preferentially binds to the target substance T, and the target substance T and the light via the second binding substance B 2 are assumed.
  • a bond with the responsive substance O is obtained, a situation occurs in which a bond between the target substance T and the magnetic particles M via the first binding substance B1 cannot be obtained.
  • the conjugate of the target substance T and the photoresponsive substance O cannot be moved before and after the application of the magnetic field, and the optical signal based on the photoresponsive substance O cannot be distinguished from the noise signal. It is out of detection.
  • the binding substance is not particularly limited and can be appropriately selected according to the type of the target substance and the detection material.
  • Various substances that give a bond by a known antigen-antibody reaction, binding by an aptamer, DNA hybridization, biotin-avidin bond, chelate bond, amino bond and the like can be used.
  • Specific examples include monoclonal antibodies, aptamers, DNA probes, RNA probes, peptides, protein A, protein G, avidin and derivatives thereof (streptavidin, neutravidin, etc.), biotin and the like.
  • polyclonal antibodies which are aggregates of different types of antibodies that can bind to a certain antigen, cannot guarantee to give the same binding substance to each of the magnetic particles and the photoresponsive substance, It is not preferable as the binding substance.
  • the target substance detection method since the same binding substance is used for the first binding substance and the second binding substance, the target of the type having a plurality of binding sites of the same type It is particularly suitable for the detection of substances (for example, virus-like particles such as viruses and capsid proteins, and extracellular vesicles such as exosomes, microvesicles, and large oncosomes). It is preferable that the substance be capable of binding to.
  • substances for example, virus-like particles such as viruses and capsid proteins, and extracellular vesicles such as exosomes, microvesicles, and large oncosomes.
  • the magnetic particles and the photoresponsive substance will be further described.
  • the conjugate can be a detection target.
  • the size of the magnetic particles and the photoresponsive substance is excessive with respect to the size of the target substance, and only two of the magnetic particles and the photoresponsive substance can bind to the target substance. In the situation, both of the two particles that bind to the target substance become the magnetic particles or the photoresponsive substance, increasing the risk of being removed from the detection target.
  • the upper limit of the size of the magnetic particles and the photoresponsive substance is set so that three or more of the magnetic particles and the photoresponsive substance can be bonded to one target substance, It is preferable to give an opportunity for any one of the three or more magnetic particles and the photoresponsive substance to be bonded to a different type from the remaining particles (see FIG. 4).
  • FIG. 4 is an explanatory diagram showing a combined body in which a total of three magnetic particles M and photoresponsive substances O are bonded to the target substance T via the binding substances B 1 and B 2 .
  • the size of the target substance that is required to be detected using the target substance detection device (the external force assisting sensor) is generally about 1 nm to 1,000 nm.
  • the upper limit of the diameter is 6,500 nm. And is more preferably 1,000 nm.
  • the lower limit of the diameter of the magnetic particle if the magnetic particle is too small, the magnetization value of the magnetic particle becomes small, and it may be difficult to receive a sufficient force to move the optical signal. For this reason, the lower limit of the diameter of the magnetic particles is preferably 5 nm. When the diameter of the magnetic particles is 5 nm or more, detection can be performed using a magnetic field generated by a general permanent magnet.
  • the magnetic particles are preferably spherical particles having a diameter of 5 nm to 6,500 nm, and more preferably spherical particles having a diameter of 5 nm to 1,000 nm.
  • the lower limit of the diameter of the photoresponsive substance when the optical signal is generated using the scattered light, if the photoresponsive substance is too small, it cannot be detected beyond the detection limit of the optical system that performs the detection. There is a fear. Therefore, the lower limit of the diameter of the photoresponsive substance is preferably 50 nm. When the diameter of the photoresponsive substance is 50 nm or more, detection can be performed using a general light source in the visible light wavelength region (400 nm to 700 nm).
  • the photoresponsive substance is preferably a spherical particle having a diameter of 50 nm to 6,500 nm, and a spherical particle having a diameter of 50 nm to 1,000 nm when the optical signal is generated using the scattered light. It is more preferable.
  • the spherical particles include not only true spheres but also irregular spherical particles such as elliptical spheres, and the diameter of the irregular spherical particles corresponds to the maximum diameter of the particles.
  • the magnetic particles and the light with respect to the content of the target substance In the scene of preparing the liquid sample, from the viewpoint of binding three or more of the magnetic particles and the photoresponsive substance to one target substance, the magnetic particles and the light with respect to the content of the target substance. It is preferable to carry out the preparation so that the total content of responsive substances is 3 times or more. In particular, the greater the total content of the magnetic particles and the photoresponsive substance, the greater the number of bindings to the target substance, so that the target substance can be detected with high accuracy. Therefore, the magnetic particles and the photoresponsive substance may be contained in a content greater than or equal to the content that causes saturation in the binding with the target material relative to the content of the target material. In this case, the number of the target substances present in the liquid sample is unknown at the stage before detection, but an excessive amount may be introduced in view of the number of target substances assumed from a rule of thumb.
  • the method for preparing the liquid sample is not particularly limited and may be appropriately selected depending on the purpose. For example, (1) after holding the analyte liquid of the target substance in the liquid sample holder, A method in which the magnetic particles bound to the first binding substance and the photoresponsive substance bound to the second binding substance are added to the sample liquid and mixed; (2) to the liquid sample holder Prior to introduction, the analyte liquid of the target substance is held in the liquid sample holder, and then the magnetic particles combined with the first binding substance and the light combined with the second binding substance Examples thereof include a method in which a responsive substance is added to the sample liquid and mixed, and a (pre-mixing method) method.
  • the magnetic particles in the mixing container and the combined body containing the magnetic particles are collected by the magnet through the mixing container, so that they do not flow down by the magnet.
  • the magnetic particles in the mixing container and the combined body containing the magnetic particles are collected by the magnet through the mixing container, so that they do not flow down by the magnet.
  • the magnetic particles in the mixing container and the combined body containing the magnetic particles are collected by the magnet through the mixing container, so that they do not flow down by the magnet.
  • contamination of the liquid sample introduced into the liquid sample holding unit can be suppressed and the introduced into the liquid sample holding unit In a liquid sample, the conjugate can be concentrated. As a result, it is possible to carry out detection with higher accuracy than when the method (1) is applied.
  • the method of adding the magnetic particles bound to the first binding substance and the photoresponsive substance bound to the second binding substance to the analyte liquid there is no particular limitation on the method of adding the magnetic particles bound to the first binding substance and the photoresponsive substance bound to the second binding substance to the analyte liquid, depending on the purpose.
  • (3) the liquid containing the magnetic particles combined with the first binding substance and the second binding substance combined with the analyte liquid A method of adding two liquids with a liquid containing a photoresponsive substance (two liquid method), and (4) the magnetic particles bound to the first binding substance and the second binding to the analyte liquid.
  • a method of adding one liquid detection solution containing the photoresponsive substance bound to the substance can be mentioned.
  • each of the target substance in the state of being bound to the binding substance with respect to the analyte liquid by preparing and storing the detection liquid in advance, each of the target substance in the state of being bound to the binding substance with respect to the analyte liquid.
  • the liquid sample can be prepared simply by mixing one detection liquid containing the magnetic particles and the photoresponsive substance, and pretreatment before detection can be efficiently performed.
  • the conjugate may be prepared to include a weight substance that promotes gravity sedimentation. That is, in the conjugate containing the weight substance, it is easy to settle on the surface of the liquid sample introduction plate, and the time until the detection operation is started can be shortened. It can be used when the specific gravity of the conjugate is small.
  • the weight substance is not particularly limited as long as it is a substance having such properties, and can be appropriately selected according to the purpose. Examples thereof include known gold nanoparticles.
  • the weight substance is preferably bound to the target substance to form the conjugate. Examples of the binding method include physical adsorption, antigen-antibody reaction, binding with an aptamer, DNA hybridization, biotin-avidin binding. In addition, a known binding method such as a chelate bond or an amino bond can be used.
  • the light irradiation step includes a back side light irradiation step of irradiating the light from the back side of the liquid sample introduction plate when the liquid sample introduction plate is formed of the translucent plate, and the liquid sample introduction plate is A surface-side light irradiating step of irradiating the light from the surface side of the liquid sample introduction plate when formed by either a light-transmitting plate or the reflection plate; and the liquid sample introduction plate is formed by the introduction plate A side surface that irradiates the liquid sample held on the liquid sample introduction plate from the side surface side of the liquid sample introduction plate in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate.
  • the said back surface side light irradiation process it can implement by the said back surface side light irradiation part demonstrated in the said target substance detection apparatus.
  • the surface side light irradiation step can be performed by the surface side light irradiation unit described in the target substance detection device.
  • the side surface side light irradiation step can be performed by the side surface side light irradiation unit described in the target substance detection device.
  • the total reflected light irradiation step can be performed by the total reflected light illumination unit described in the target substance detection device.
  • the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate is moved in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate by applying a magnetic field.
  • the first combined body moving step of moving in either the direction having the vector component or the direction moving away from the liquid sample introduction plate, and the magnetic field from the magnetic field application unit arranged on the back side of the liquid sample introduction plate.
  • the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate by application is drawn onto the surface of the liquid sample introduction plate, and the magnetic field application unit is moved to the liquid while the magnetic field is applied.
  • a second coupled body moving step in which the coupled body is moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the sample introduction plate, and is moved following the movement of the magnetic field application unit; Is any of the steps.
  • the first combined body moving step can be performed by the first magnetic field application unit described in the target substance detection device.
  • the second combined body moving step can be performed by the second magnetic field application unit described in the target substance detection device.
  • the first combined body moving step and the second combined body moving step can be performed repeatedly while sandwiching the optical signal detecting step, respectively, thereby increasing detection accuracy.
  • the relative position of the first magnetic field application unit with respect to the liquid sample introduction plate is changed, and the combined body is moved.
  • the combined body may be moved in a different direction.
  • the movement of the coupled body in different directions can be performed by installing a plurality of the first magnetic field application units and sequentially applying magnetic fields from different directions.
  • the second combined body moving step is repeatedly performed in the second magnetic field applying unit, the magnetic field applying unit is attached to the liquid sample introduction plate in each second combined body moving step.
  • the combined body may be moved by moving in a different direction having a vector component in a direction parallel to the in-plane direction of the surface and changing the direction in which the combined body is moved.
  • the first combined body moving step and the second combined body moving step are interwoven. Can be implemented.
  • the same effect can be obtained by moving the liquid sample introduction plate in a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate when a magnetic field is applied. May be.
  • the optical signal detection step is a step of detecting the movement of the combined body accompanying the combined body moving step by a signal change of an optical signal based on the propagating light or the near-field light.
  • the optical signal detection step is generated from the photoresponsive substance when the combined body receives the propagating light or the near-field light out of the propagating light or the optical signal based on the near-field light.
  • This is a step of detecting only an optical signal based on the conjugate in which the magnetic particles and the photoresponsive substance are bound to one target substance as a signal change target.
  • the optical signal detection step can be performed by the optical signal detection unit described in the target substance detection device.
  • the combined body attracting step is further performed by applying an attracting magnetic field after the liquid sample introduction and holding step and before the combined body moving step. This is a step of drawing all or part of the combined body in the liquid sample once onto the surface of the liquid sample introduction plate.
  • the optical signal is detected in the optical signal detecting step by focusing on the surface of the liquid sample introduction plate or in the vicinity thereof. For example, immediately after the liquid sample is introduced into the liquid sample introduction plate, the conjugate is not drawn to the surface of the liquid sample introduction plate. It is in a floating state in the liquid layer. When the floating coupled body exists outside the imageable range where the optical signal can be detected in the optical signal detection step or outside the near-field light generation region, the coupled body is not detected. Become. Therefore, when the optical signal is detected by the optical signal detection step with focusing on the surface of the liquid sample introduction plate or the vicinity thereof, the liquid sample is introduced into the liquid sample introduction plate, and then the coupling is performed.
  • the combined body moving step is the first combined body moving step
  • the combined body pulling step is further performed to shorten detection preparation time and perform more efficient detection. Is preferred.
  • the combined body drawing step can be performed by the third magnetic field application unit described in the target substance detection device.
  • the conjugate drawing step and in the case of carrying out the first conjugate moving step by moving the conjugate in a direction away from the liquid sample introduction plate, there is no particular limitation, After the liquid sample introduction and holding step, it is preferable to repeat the conjugate drawing step, the conjugate moving step, and the optical signal detection step in this order (multiple magnetic field application).
  • the alternating magnetic field By applying the alternating magnetic field, the optical signals originating from the same combined body are repeatedly detected, so that the detection accuracy can be improved.
  • it is also possible to amplify the optical signal by periodically applying the alternating magnetic field and applying a known lock-in amplifier to the frequency of the optical signal caused by the same combination. Thus, the detection sensitivity can be improved.
  • the target substance detection kit includes a magnetic particle that forms a conjugate with a target substance, a photoresponsive substance that forms the conjugate with the target substance, and a first binding substance that binds the magnetic particle to the target substance. And a second binding substance that binds the photoresponsive substance to the target substance, and the first binding substance and the second binding substance are composed of the same binding substance.
  • the detection liquid is divided into two liquids according to the difference in affinity. There is no need to determine the order of addition. Further, if the detection liquid is prepared and stored as a single liquid, the liquid sample can be prepared simply by adding the detection liquid to the analyte liquid of the target substance, and pretreatment before detection is efficient. Can be done automatically. In particular, since there is no difference in affinity between the first binding substance and the second binding substance, reactions in which these binding substances bind to the target substance occur in parallel, and the target substance and the binding substance are combined. The combined body of the magnetic particles and the photoresponsive substance via a substance can be obtained in a short time, and the time required for the pretreatment before detection can be shortened to further increase the efficiency. it can.
  • FIG. 5 is an explanatory diagram of the target substance detection device 1.
  • the target substance detection apparatus 1 is configured according to a known transmission microscope, and includes a liquid sample introduction plate 2, a light irradiation unit 3, a first magnetic field application unit 4, an imaging device 5a, and It is comprised with the optical signal detection part 5 comprised with the objective lens 5b.
  • the imaging device 5a is configured by, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image.
  • Liquid sample introduction plate 2 the transmitted light T L of the light L liquid sample containing the magnetic particles forming the coupling member and the target substance and the target substance is irradiated from the back side while being introduced onto the surface It is formed of a translucent plate that can propagate as propagating light above the surface.
  • the liquid sample introduction plate 2 itself constitutes a liquid sample holding unit, and after the liquid sample is introduced onto the surface, a cover glass or the like is disposed so as to cover the liquid sample, whereby the liquid sample is disposed. Hold.
  • the light irradiation unit 3 is configured as a back surface side light irradiation unit that can irradiate the light L from the back surface side of the liquid sample introduction plate 2.
  • the first magnetic field application unit 4 is arranged on the surface side of the liquid sample introduction plate 2 and applies a magnetic field to the combination in the liquid sample introduced onto the surface of the liquid sample introduction plate 2. Is configured to move in a direction away from the liquid sample introduction plate 2.
  • the first magnetic field applying unit 4 is formed by an annular electromagnet which through-holes are formed in the center, the optical signal is the through hole based on the transmitted light T L of the light L irradiated from the light irradiation section 3 Through the optical signal detection unit 5, the detection is possible.
  • the optical signal detection unit 5 is arranged on the surface side of the liquid sample introduction plate 2 and can detect a signal change of the optical signal based on the propagation light before and after application of the magnetic field by the first magnetic field application unit 4.
  • the target substance detection method is performed using the target substance detection apparatus 1.
  • the liquid sample is introduced and held on the surface of the liquid sample introduction plate 2 (liquid sample introduction and holding step).
  • the liquid sample includes the magnetic particles and the photoresponsive substance that form a conjugate with the target substance, the first binding substance that binds the magnetic particles to the target substance, and the light. It is important that the same binding substance is used for the first binding substance and the second binding substance, including the second binding substance that binds the responsive substance to the target substance (see FIG. 1 to 3).
  • the imageable range refers to a range in which an optical signal at the focal depth and in the vicinity thereof can be acquired.
  • FIG. 6 schematically shows a state on the surface of the liquid sample introduction plate 2 in the observation field observed by the imaging device 5a at this time.
  • the light transmitted through the liquid sample that propagates above the surface of the liquid sample introduction plate 2 is transmitted.
  • Four optical signals a to d that can be distinguished from the background signal are observed based on a contrast difference with the signal (background signal).
  • the optical signals a and d are observed as light spots, and the optical signals b and c are observed as dark spots.
  • FIG. 7 shows a state in which the substance a ′ for generating the optical signal a and the substance b ′ for generating the optical signal b at this time are viewed from the side surface of the liquid sample introduction plate 2.
  • 7 is a cross-sectional view taken along line AA in FIG.
  • an arrow B in FIG. 7 indicates an imageable range where an optical signal can be acquired.
  • the substance a ′ and the substance b ′ are in a state of being gravity settled on the surface of the liquid sample introduction plate 2.
  • the electromagnet of the first magnetic field application unit 4 is excited to apply the magnetic field to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 2 by applying a magnetic field. And then the combined body is moved in a direction away from the liquid sample introduction plate 2 (a combined body moving step). Next, an optical signal on the surface of the liquid sample introduction plate 2 after the combined body is moved away from the liquid sample introduction plate 2 while maintaining the imageable range and the observation field is acquired by the imaging device 5a. (Optical signal detection step).
  • FIG. 8 schematically shows the state on the surface of the liquid sample introduction plate 2 in the observation field observed by the imaging device 5a after the combined body moving step.
  • the optical signals a and b are before and after the combined body moving step.
  • the optical signal changes, and the optical signals c and d do not change before and after the combined body moving step. From this, it is understood that the substances a ′ and b ′ that generate the optical signals a and b are the combined body including the magnetic particles attracted to the first magnetic field application unit 4 and include the target substance. .
  • the optical signals c and d which are not confirmed before and after the combined body moving step, are scratches on the surface of the liquid sample introduction plate 2, adsorbed on the surface, or impurities present on the surface, light source It can be seen that this is a noise signal such as output fluctuation.
  • FIG. 9 shows a state in which the substance a ′ for generating the optical signal a and the substance b ′ for generating the optical signal b after the combined body moving step are viewed from the side surface of the liquid sample introduction plate 2.
  • 9 is a cross-sectional view taken along line AA in FIG. Further, an arrow B in FIG. 9 indicates an imageable range where an optical signal can be acquired.
  • the substance a ′ and the substance b ′ are moved in a direction away from the liquid sample introduction plate 2 by the application of the magnetic field by the first magnetic field application unit 4.
  • the size of the light spot is observed large before and after the combined body moving step (see FIG. 8). This is based on the depth of focus in a state where the surface of the liquid sample introduction plate 2 is in focus before the combined body moving step, although the substance a ′ is present within the imageable range of the optical signal detection unit 5. Since it is off, the size of the light spot is observed large (see FIG. 9). On the other hand, it is confirmed that the optical signal b disappears after the combined body moving step (see FIG. 8). This is because the substance b ′ has moved out of the imageable range of the optical signal detector 5 (see FIG. 9).
  • the optical signal based on the target substance is adsorbed on the surface of the liquid sample introduction plate 2 or is present on the surface.
  • the target substance can be detected with high accuracy because it can be clearly distinguished and detected from noise signals such as impurities and fluctuations in light source output. Further, even when the contaminants are adsorbed on the surface of the liquid sample introduction plate 2, since the detection can be performed while ignoring its presence, the cleaning process for the liquid sample introduction plate 2 is not necessarily performed for each detection. Efficient detection can be performed without the need to do so.
  • the target substance can be detected efficiently, accurately, and stably.
  • FIG. 10 is an explanatory diagram of the target substance detection apparatus 1A.
  • the target substance detection device 1 ⁇ / b> A is configured by further arranging a third magnetic field application unit 6 with respect to the target substance detection device 1.
  • description is abbreviate
  • the third magnetic field application unit 6 is arranged on the back side of the liquid sample introduction plate 2 and the combined body in the liquid sample introduced into the liquid sample introduction plate 2 is applied to the liquid sample introduction plate 2 by applying a magnetic field. It can be drawn onto the surface, and here is formed of an annular electromagnet having a through hole, and the light irradiation unit 3 can irradiate light from the back side of the liquid sample introduction plate 2 through the through hole.
  • the conjugate that floats in the liquid layer of the liquid sample after the liquid sample introduction / holding step becomes the Without waiting for gravity sedimentation on the surface, after the liquid sample introduction and holding step and before the combined body moving step, the binding in the liquid sample is performed by applying a pulling magnetic field in the third magnetic field application unit 6. All or a part of the body can be once drawn on the surface of the liquid sample introduction plate 2 (a combined body drawing step). Therefore, according to the first modification of the first embodiment, in addition to the advantages of the first embodiment, the time required for detection is shortened and the target substance is detected more efficiently. be able to.
  • FIG. 11 is explanatory drawing of the target substance detection apparatus 1B.
  • the target substance detection device 1 ⁇ / b> B is configured by arranging a first magnetic field application unit 7 instead of the first magnetic field application unit 4 in the target substance detection device 1.
  • description is abbreviate
  • the first magnetic field application unit 7 is configured by an electromagnet, and is detected on the surface of the liquid sample introduction plate 2 (received by the light irradiation unit 3 on the back surface side, and propagates above the surface).
  • the region in the liquid sample introduction plate 2 is disposed obliquely above the surface of the liquid sample introduction plate 2 and is applied to the surface of the liquid sample introduction plate 2 by applying a magnetic field. Move in a direction having a vector component in a direction parallel to the in-plane direction (first combined body moving step).
  • FIG. 12 schematically shows the state on the surface of the liquid sample introduction plate 2 in the observation field of view observed by the imaging device 5a after the first combined body moving step performed using the first magnetic field application unit 7. Shown in The state before the first combined body moving step is the same as in FIG.
  • the optical signals a and b are The optical signal changes before and after the first combined body moving process, and the optical signals of the optical signals c and d do not change before and after the first combined body moving process. Therefore, according to the second modification of the first embodiment, as in the first embodiment, the substances a ′ and b ′ that generate the optical signals a and b include the target substance, and the optical signal c , D can be determined as noise signals such as scratches on the surface of the liquid sample introduction plate 2, adsorption on the surface or impurities existing on the surface, fluctuations in light source output, and the like.
  • FIG. 13 shows the state when viewed from above.
  • FIG. 13 is a cross-sectional view taken along line AA in FIG. Further, an arrow B in FIG. 13 indicates an imageable range where an optical signal can be acquired.
  • the substance a ′ and the substance b ′ are each parallel to the in-plane direction of the surface of the liquid sample introduction plate 2 by the magnetic field attracted from the oblique upper side by the first magnetic field application unit 7.
  • the first embodiment in which the substance a ′ and the substance b ′ are moved only in the direction away from the liquid sample introduction plate 2 and the first combined body moving step. The situation after is different.
  • the optical signal b is the result of the disappearance of the optical signal in both figures.
  • the optical signal a in the case shown in FIG. 8, only the target substance is detected based on the size change, and the target substance cannot be detected based on the movement, whereas in the case shown in FIG.
  • the case shown in FIG. 12 is easier to detect the target substance in that the target substance can be detected based on movement in addition to the detection of the target substance based on the change. Therefore, in the second modification of the first embodiment, the target substance can be detected with higher accuracy.
  • FIG. 14 is an explanatory diagram of the target substance detection device 10.
  • the target substance detection apparatus 10 is configured according to a known transmission microscope, and includes a liquid sample introduction plate 12, a light irradiation unit 13, a second magnetic field application unit 18, and an imaging device 15a. And the optical signal detection unit 15 including the objective lens 15b.
  • the liquid sample introduction plate 12, the light irradiation unit 13, and the optical signal detection unit 15 are configured in the same manner as the liquid sample introduction plate 2, the light irradiation unit 3, and the optical signal detection unit 5 in the target substance detection device 1 of the first embodiment.
  • the target substance detection apparatus 10 is different from the target substance detection apparatus 1 in that a second magnetic field application unit 18 is provided instead of the first magnetic field application unit 4.
  • a second magnetic field application unit 18 is provided instead of the first magnetic field application unit 4.
  • the second magnetic field application unit 18 is disposed on the back surface side of the liquid sample introduction plate 12, and the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 12 is liquidated by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 12 and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12 with the magnetic field applied.
  • the second magnetic field applying unit 18 is formed out with sliding member for sliding said permanent magnet and the permanent magnet of annular through holes are formed in the direction of the X 1 or X 2 (not shown),
  • the light irradiation unit 13 can irradiate light from the back side of the liquid sample introduction plate 12 through the through hole.
  • the combined sample is moved by using the second magnetic field application unit 18 as a magnetic field application unit, and the liquid sample introduced onto the surface of the liquid sample introduction plate 12 by application of the magnetic field from the second magnetic field application unit 18.
  • the combined body in the liquid sample introduction plate 12 is attracted to the surface of the liquid sample introduction plate 12 and the second magnetic field application unit 18 is applied in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12 with the magnetic field applied.
  • the movement is performed in a direction having a vector component, and the combined body is moved following the movement of the second magnetic field applying unit 18 (second combined body moving step).
  • the 2nd magnetic field application part 18 is comprised by the some member arrange
  • the second magnetic field application unit 18 in the second combined body moving step, all or a part of the combined body in the liquid sample is applied on the surface of the liquid sample introduction plate 12 by applying the magnetic field. Therefore, after the liquid sample introduction and holding step, there is no need to wait for the conjugate floating in the liquid layer of the liquid sample to gravity settle on the surface of the liquid sample introduction plate 12.
  • FIG. 15 schematically shows a state on the surface of the liquid sample introduction plate 12 in the observation field of view observed by the imaging device 15a after the second combined body moving step.
  • the state before the second combined body moving step is the same as in FIG.
  • the optical signals a and b are obtained before and after the second combined body moving step.
  • the optical signals c and d do not change before and after the second combined body moving step.
  • the substances a ′ and b ′ that generate the optical signals a and b include the target substance, and the optical signals c and d are scratches on the surface of the liquid sample introduction plate 12, It can be determined that the signal is a noise signal such as a foreign matter adsorbed on the surface or a contaminant present on the surface, or a fluctuation in light source output.
  • FIG. 16 shows a state when the substance a ′ that generates the optical signal a and the substance b ′ that generates the optical signal b are viewed from the side surface of the liquid sample introduction plate 12 after the second combined body moving step.
  • FIG. 16 is a cross-sectional view taken along line AA in FIG. Note that an arrow B in FIG. 16 indicates an imageable range where an optical signal can be acquired.
  • the substance a ′ and the substance b ′ are attracted onto the surface of the liquid sample introduction plate 12 by application of the magnetic field from the second magnetic field application unit 18, and then applied with the second magnetic field. Based on the movement (direction X 1 or X 2 in FIG.
  • the second magnetic field application section 18 Is moved in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 12.
  • 15 and 16 show an example in which the substance a ′ and the substance b ′ move within the observation field of view, but the second magnetic field application unit 18 is arranged in the in-plane direction of the surface of the liquid sample introduction plate 12.
  • the optical system is irradiated with light from the back side of the liquid sample introduction plates 2 and 12 according to the configuration of a known upright microscope, and the front side
  • the optical signal based on the propagating light transmitted to the optical signal is detected by the optical signal detectors 5 and 15, and light is irradiated from the surface side of the liquid sample introduction plate according to the configuration of a known inverted microscope.
  • the optical signal based on the propagating light transmitted to the back side may be detected by the optical signal detection unit arranged on the back side.
  • FIG. 17 is an explanatory diagram of the target substance detection device 20.
  • the target substance detection device 20 is configured according to a known epi-illumination microscope, and includes a liquid sample introduction plate 22, a light irradiation unit 23, a first magnetic field application unit 24, an imaging device 25a,
  • the optical signal detector 25 includes an objective lens 25b and a half mirror (such as a dichroic mirror) 25c.
  • the imaging device 25a is configured with, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image.
  • the half mirror 25c is also used as an optical element of the light irradiation unit 23 for introducing irradiation light onto the surface of the liquid sample introduction plate 22 by reflection.
  • the liquid sample introduction plate 22 is formed by the propagation possible reflector to said surface upward reflected light R L as the propagation of the light L emitted from the front surface side while being introduced onto the surface
  • the liquid sample introduction plate 22 itself constitutes the liquid sample holding unit, and after the liquid sample is introduced onto the surface, a cover glass or the like is disposed so as to cover the liquid sample, whereby the liquid is introduced. Hold the sample.
  • the light irradiation unit 23 is configured as a surface-side irradiation unit that can irradiate the light L from the surface side of the liquid sample introduction plate 22 by the reflected light from the half mirror 25c.
  • the first magnetic field application unit 24 is arranged on the surface side of the liquid sample introduction plate 22 and applies a magnetic field to the conjugate in the liquid sample introduced onto the surface of the liquid sample introduction plate 22. Is configured to move away from the liquid sample introduction plate 22.
  • the first magnetic field application unit 24 is formed of an annular electromagnet having a through hole formed in the center, and the light L emitted from the light irradiation unit 23 is applied to the liquid sample introduction plate 22 through the through hole. optical signal based on the reflected light R L of the light L while being capable radiation is detectable by the optical signal detection unit 25 through the through hole.
  • the optical signal detection unit 25 is arranged on the surface side of the liquid sample introduction plate 22 and can detect a signal change of the optical signal based on the propagation light before and after application of the magnetic field by the first magnetic field application unit 24.
  • the Note that the liquid sample introduction plate 22, the light irradiation unit 23, and the optical signal detection unit 25 (the imaging device 25a, the objective lens 25b, and the half mirror 25c) can be configured according to a known episcopic microscope.
  • the target substance detection method according to the third embodiment is performed using the target substance detection device 20.
  • the liquid sample is introduced and held on the surface of the liquid sample introduction plate 22 (liquid sample introduction and holding step).
  • the liquid sample includes the magnetic particles and the photoresponsive substance that form a conjugate with the target substance, the first binding substance that binds the magnetic particles to the target substance, and the light. It is important that the same binding substance is used for the first binding substance and the second binding substance, including the second binding substance that binds the responsive substance to the target substance (see FIG. 1 to 3).
  • the light L irradiated from the light irradiation unit 23 is liquidated via the half mirror 25c.
  • the surface of the sample introduction plate 22 is irradiated (light irradiation step)
  • the irradiation objective lens 25b is adjusted to place the surface or the vicinity thereof within the imageable range
  • the imaging device 25a emits the light L on the surface.
  • An optical signal based on the reflected light RL is acquired (optical signal detection step).
  • the electromagnet of the first magnetic field application unit 24 is excited to apply the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 22 by applying a magnetic field. And the combined body is moved in a direction away from the liquid sample introduction plate 22 (first combined body moving step).
  • an optical signal on the surface of the liquid sample introduction plate 22 after the combined body is moved in a direction away from the liquid sample introduction plate 22 while maintaining the imageable range and the observation field of view is acquired by the imaging device 25a. (Optical signal detection step).
  • optical signals before and after the first combined body moving step in the optical signal detecting step are obtained as shown in FIGS. 6 and 8, and light based on the target substance is obtained.
  • the signal can be detected by clearly distinguishing it from noise signals such as scratches on the surface of the liquid sample introduction plate 22, adsorption on the surface or contaminants existing on the surface, fluctuations in the light source output, and the like. Therefore, according to the third embodiment, the target substance can be detected with high accuracy. Further, even when the contaminants are adsorbed on the surface of the liquid sample introduction plate 22, since the detection can be performed while ignoring its presence, the cleaning process for the liquid sample introduction plate 22 is not necessarily performed for each detection. Efficient detection can be performed without the need to do so.
  • an optical signal generated based on various phenomena such as the scattered light, the reflected light, the fluorescence, and the light absorption can be handled as an identification signal, and can be expected to be used in a wide range of fields.
  • a phenomenon of disappearing in addition to the defocus can be used, so that the change of the optical signal can be clearly captured.
  • the target substance since the same binding substance is used for the first binding substance and the second binding substance, the target substance can be detected efficiently, accurately, and stably.
  • FIG. 18 is an explanatory diagram of the target substance detection device 20A.
  • a third magnetic field application unit 26 is further arranged with respect to the target substance detection device 20, and the first magnetic field application unit 24 is replaced with the first magnetic field.
  • the application unit 27 is arranged.
  • description is abbreviate
  • the third magnetic field application unit 26 is formed of an electromagnet, and is disposed on the back side of the liquid sample introduction plate 22, and the combination in the liquid sample introduced into the liquid sample introduction plate 22 is liquidated by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 22.
  • the third magnetic field application unit 26 as in the case of using the target substance detection device 20, after the liquid sample introduction and holding step, the combined body floating in the liquid layer of the liquid sample is a liquid sample introduction plate. Without waiting for gravity sedimentation on the surface of 22, after the liquid sample introduction and holding step, and before the combined body moving step, by applying a drawing magnetic field in the third magnetic field application unit 26, All or a part of the combined body can be once drawn on the surface of the liquid sample introduction plate 22 (a combined body drawing step). Therefore, according to the modification of the third embodiment, in addition to the advantages of the third embodiment, the time required for detection can be shortened and the target substance can be detected more efficiently. .
  • the first magnetic field application unit 27 is composed of an electromagnet, and is arranged on the side of the liquid sample introduction plate 22, and the coupling in the liquid sample introduced onto the surface of the liquid sample introduction plate 22.
  • the body is moved in a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate 2 by applying a magnetic field (first combined body moving step).
  • first magnetic field application unit 27 is used instead of the first magnetic field application unit 24
  • optical signals before and after the first combined body movement step in the optical signal detection step are obtained as shown in FIGS.
  • the optical signal based on the target substance is clearly distinguished from noise signals such as scratches on the surface of the liquid sample introduction plate 22, adsorption on the surface or impurities existing on the surface, fluctuations in light source output, etc. Can be detected.
  • FIG. 19 is an explanatory diagram of the target substance detection device 30.
  • the target substance detection device 30 is configured according to a known epi-illumination microscope, and includes a liquid sample introduction plate 32, a light irradiation unit 33, a second magnetic field application unit 38, and an imaging device 35a. And an optical signal detection unit 35 including an objective lens 35b and a half mirror 35c.
  • the liquid sample introduction plate 32, the light irradiation unit 33, and the optical signal detection unit 35 are configured in the same manner as the liquid sample introduction plate 22, the light irradiation unit 23, and the optical signal detection unit 25 in the target substance detection device 20 of the third embodiment.
  • the target substance detection device 30 is different from the target substance detection device 20 in that a second magnetic field application unit 38 is provided instead of the first magnetic field application unit 24.
  • a second magnetic field application unit 38 is provided instead of the first magnetic field application unit 24.
  • the second magnetic field application unit 38 is arranged on the back surface side of the liquid sample introduction plate 32 and applies the liquid to the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 32 by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 32 and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 32 with the magnetic field applied.
  • the second magnetic field applying unit 38 is formed out with sliding member for sliding said permanent magnet and the permanent magnet in the direction of the X 1 or X 2 (not shown).
  • the combined body in the liquid sample introduction plate 32 is attracted to the surface of the liquid sample introduction plate 32 and the second magnetic field application unit 38 is applied in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 32 with the magnetic field applied.
  • the movement is performed in a direction having a vector component, and the combined body is moved following the movement of the second magnetic field applying unit 38 (second combined body moving step).
  • the 2nd magnetic field application part 38 is comprised by the some member arrange
  • the second magnetic field application unit 38 in the second combined body moving step, all or part of the combined body in the liquid sample is applied on the surface of the liquid sample introduction plate 32 by applying the magnetic field. Therefore, it is not necessary to wait for the combined substance floating in the liquid layer of the liquid sample to gravity settle on the surface of the liquid sample introduction plate 2 after the liquid sample introduction and holding step.
  • optical signals before and after the second combined body moving step in the optical signal detecting step are obtained as shown in FIGS. 6 and 15, and light based on the target substance is obtained.
  • the signal can be detected by clearly distinguishing it from noise signals such as scratches on the surface of the liquid sample introduction plate 32, adsorption on the surface or impurities existing on the surface, and fluctuations in the light source output.
  • FIG. 15 shows an example in which the substance a ′ and the substance b ′ move within the observation field.
  • the second magnetic field application unit 38 is parallel to the in-plane direction of the surface of the liquid sample introduction plate 32.
  • the distance a is longer than the length of the one side.
  • the substance b ′ can be moved out of the observation field, and highly accurate detection based on the disappearance of the optical signals a and b can be performed.
  • optical signal based on the conjugate examples are shown in FIGS. 6, 8, 12, and 15, and it has been described that the optical signal is caused by the scattered light, the reflected light, the fluorescence, This is for convenience of drawing display, and the optical signal may be an optical signal resulting from the transmitted light due to the phase difference, the differential interference, or the like.
  • the optical signal may be an optical signal resulting from the transmitted light due to the phase difference, the differential interference, or the like.
  • the position movement, the defocus, and the disappearance examples are shown in FIGS. 8, 12, and 15, and the position movement, the defocus, and the disappearance have been described.
  • FIG. 20 is an explanatory diagram of the target substance detection device 40.
  • the target substance detection device 40 is configured according to a known waveguide mode sensor, and includes a liquid sample introduction plate 42, a light irradiation unit including a light source 43a and an optical prism 43b, and a third A magnetic field application unit 46, a first magnetic field application unit 47, and an optical signal detection unit 45 (imaging device) are included.
  • the imaging device is constituted by, for example, a known CCD image sensor or the like, and can acquire a two-dimensional image.
  • the third magnetic field applying unit 46 controls the on / off of the magnetic field application by moving the permanent magnet and the permanent magnet away from or close to the optical prism 43b (see directions Y 1 and Y 2 in FIG. 20).
  • the liquid sample introduction plate 42 is formed by a moving member (not shown), arranged on the back side of the liquid sample introduction plate 42 and the combined body in the liquid sample introduced into the liquid sample introduction plate 42 by applying a magnetic field. It is possible to draw on the surface.
  • the liquid sample introduction plate 42 is capable of generating near-field light above the surface when the liquid sample E is introduced onto the surface and irradiated with the light L irradiated on the surface under total reflection conditions. It is formed with a detection plate. Further, the liquid sample introduction plate 42 itself constitutes the liquid sample holding unit, and after the liquid sample E is introduced onto the surface, the cover glass G is disposed so as to cover the liquid sample. A liquid sample E is held.
  • the light irradiation unit is configured as a total reflection light irradiation unit that can irradiate the surface of the liquid sample introduction plate 42 with the light L from the light source 43a through the optical prism 43b under total reflection conditions. .
  • the total reflection light irradiation unit introduces the light L emitted from the light source 43a under the total reflection condition to the surface of the liquid sample introduction plate 42 via a grating, for example, instead of the optical prism 43b. It can also be configured.
  • the first magnetic field application unit 47 controls on / off of the magnetic field application by moving the permanent magnet and the permanent magnet away from or close to the optical prism 43b (see directions X 1 and X 2 in FIG. 20). Formed by a slide moving member (not shown), disposed on the side of the liquid sample introduction plate 42, and the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 42 by applying a magnetic field.
  • the liquid sample introduction plate 42 is configured to move in a direction having a vector component in a direction parallel to the in-plane direction of the surface.
  • the target substance detection method according to the fifth embodiment is performed.
  • the liquid sample is introduced and held on the surface of the liquid sample introduction plate 42 (liquid sample introduction and holding step).
  • the liquid sample includes the magnetic particles and the photoresponsive substance that form a conjugate with the target substance, the first binding substance that binds the magnetic particles to the target substance, and the light. It is important that the same binding substance is used for the first binding substance and the second binding substance, including the second binding substance that binds the responsive substance to the target substance (see FIG. 1 to 3).
  • the combined body floating in the liquid layer of the liquid sample is attracted onto the surface of the liquid sample introduction plate 42.
  • the light L emitted from the light source 43a is applied to the surface of the liquid sample introduction plate 42 through the optical prism 43b under a total reflection condition (light irradiation process), and the optical signal detector 45 applies the light on the surface.
  • An optical signal S based on near-field light is acquired (optical signal detection step).
  • the slide moving member in the third magnetic field applying unit 46 is operated to turn off the magnetic field from the third magnetic field applying unit 46
  • the slide moving member in the first magnetic field applying unit 47 is operated to operate the first magnetic field applying unit 47.
  • the magnetic field from the magnetic field application unit 47 is turned on, and the combined body in the liquid sample introduced onto the surface of the liquid sample introduction plate 42 is drawn toward the first magnetic field application unit 47 by applying a magnetic field, and The coupled body is moved in a direction having a vector component parallel to the in-plane direction of the surface of the liquid sample introduction plate 42 by applying a magnetic field (first coupled body moving step).
  • the optical signal on the surface of the liquid sample introduction plate 42 after the combined body is moved while maintaining the observation visual field is acquired by the optical signal detection unit 45 (optical signal detection step).
  • the optical signals before and after the first combined body moving step in the optical signal detecting step are obtained as shown in FIGS. 21 and 22, and the optical signal based on the target substance is obtained.
  • e and g can be detected by clearly distinguishing them from noise signals f such as scratches on the surface of the liquid sample introduction plate 42, adsorption on the surface or impurities existing on the surface, fluctuations in the light source output, and the like.
  • FIG. 21 is a diagram showing a state on the surface of the liquid sample introduction plate 42 before the conjugate moving step
  • FIG. 22 is a diagram on the surface of the liquid sample introduction plate 42 after the conjugate moving step.
  • the optical signal obtained by using the near-field light has a dark field as a background due to the attenuation of the near-field light. Based on this, the target substance is detected.
  • the appearance of an optical signal based on movement from outside the observation field of view can also be a detection target.
  • the detection can be performed while ignoring the presence of the impurities, and therefore the cleaning process for the liquid sample introduction plate 42 is not necessarily performed for each detection. Efficient detection can be performed without the need to do so.
  • an optical signal generated based on the phenomenon such as the scattered light and the fluorescence can be handled as an identification signal.
  • the appearance / disappearance phenomenon can also be used as the mode of change of the optical signal, so that the change of the optical signal can be clearly captured.
  • the target substance can be detected efficiently, accurately, and stably.
  • FIG. 23 is an explanatory diagram of the target substance detection device 50.
  • the target substance detection device 50 is configured according to a known waveguide mode sensor, and includes a liquid sample introduction plate 52, a light irradiation unit including a light source 53a and an optical prism 53b, a second The magnetic field applying unit 58 and the optical signal detecting unit 55 are configured.
  • the liquid sample introduction plate 52, the light irradiation unit, and the optical signal detection unit 55 are configured in the same manner as the liquid sample introduction plate 42, the light irradiation unit, and the optical signal detection unit 45 in the target substance detection device 40 of the fifth embodiment.
  • the target substance detection device 50 is different from the target substance detection device 40 in that a second magnetic field application unit 58 is provided in place of the first magnetic field application unit 47 and the third magnetic field application unit 46. .
  • a second magnetic field application unit 58 is provided in place of the first magnetic field application unit 47 and the third magnetic field application unit 46.
  • the second magnetic field application unit 58 is arranged on the back surface side of the liquid sample introduction plate 52 and applies the liquid to the combination in the liquid sample introduced onto the surface of the liquid sample introduction plate 52 by applying a magnetic field. It can be drawn onto the surface of the sample introduction plate 52 and can be moved in a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 52 with the magnetic field applied.
  • the second magnetic field applying unit 58 is formed out with sliding member for sliding said permanent magnet and the permanent magnet in the direction of the X 1 or X 2 (not shown).
  • the combined sample is moved by using the second magnetic field application unit 58 as a magnetic field application unit, and the liquid sample introduced onto the surface of the liquid sample introduction plate 52 by application of the magnetic field from the second magnetic field application unit 58.
  • the combined body in the liquid sample introduction plate 52 is attracted to the surface of the liquid sample introduction plate 52 and the second magnetic field application unit 58 is applied in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate 52 with the magnetic field applied.
  • the movement is performed in the direction having the vector component, and the combined body is moved following the movement of the second magnetic field applying unit 58 (second combined body moving step).
  • the 2nd magnetic field application part 58 is comprised by the some member arrange
  • the second magnetic field application unit 58 in the second combined body moving step, all or a part of the combined body in the liquid sample is placed on the surface of the liquid sample introduction plate 52 by applying the magnetic field. Therefore, it is not necessary to wait for the combined substance floating in the liquid layer of the liquid sample to gravity settle on the surface of the liquid sample introduction plate 52 after the liquid sample introduction and holding step.
  • FIGS. 24 and 25 optical signals before and after the second conjugate moving step in the optical signal detecting step are obtained as shown in FIGS. 24 and 25, and the optical signal based on the target substance is obtained. h can be detected by clearly distinguishing it from a noise signal i such as a scratch on the surface of the liquid sample introduction plate 52, adsorption on the surface or impurities existing on the surface, and fluctuations in the light source output.
  • FIG. 24 is a diagram showing a state on the surface of the liquid sample introduction plate 52 before the conjugate moving step
  • FIG. 25 is a diagram on the surface of the liquid sample introducing plate 52 after the conjugate moving step.
  • the target substance detection devices of the fifth and sixth embodiments are configured according to the configuration of the waveguide mode sensor, as a modification of these embodiments, liquid sample introduction plates 42 and 52 are provided.
  • the detection plate used in the surface plasmon resonance sensor and the optical system as an optical system used in the surface plasmon resonance sensor the detection of the target substance based on the movement of the conjugate is performed similarly to these embodiments. It can be performed.
  • an optical system that uses near-field light generated by total reflection for illumination such as an optical system of a known total reflection microscope, can also be used.
  • the liquid sample introduction plate is configured using the translucent plate, the reflection plate, and the detection plate, but may be configured using the introduction plate. Good.
  • the side light irradiation unit is employed as the light irradiation unit, and in the optical signal detection unit disposed on the front surface side or the back surface side of the liquid sample introduction plate, scattered light from the combined body, It can be set as the structure which detects reflected light etc.
  • the optical signal detection unit arranged on the side of the liquid sample introduction plate (the side opposite to the side on which the side light irradiation unit of the liquid sample introduction plate is arranged), It can be set as the structure which detects light absorption, transmitted light, etc.
  • Example 1 As Example 1, the target substance detection test using the target substance detection apparatus (external force support type sensor) was performed as follows.
  • Example 1 a target substance detection apparatus manufactured according to the configuration of the target substance detection apparatus 40 of the fifth embodiment shown in FIG. 20 is used.
  • the liquid sample introduction plate 42 a planar waveguide chip in which a 25 nm thick Si layer and a 343 nm thick SiO 2 layer are laminated in this order on a 0.725 mm thick SiO 2 substrate is used. It was.
  • a light source 43a is connected to a green LED light source (Thorlabs, model number M530F2) by connecting an optical fiber having a core diameter of 600 ⁇ m with a collimating lens attached to the exit end, and an air slit having a width of 500 ⁇ m disposed at the end of the exit end.
  • a prism 43b made of SiO 2 glass having a base angle of 35 degrees is disposed on the back surface of the liquid sample introduction plate 42 in an optically close contact with the incident surface of the prism 43b at an angle parallel to the surface of the liquid sample introduction plate 42. In this case, the light from the light source 43a is made incident.
  • virus-like particles of Norovirus were used.
  • magnetic particles magnetic beads having a diameter of 25 nm (NANOCS, Magnetic Nanoparticles, Protein G Labeled, model number MP25-PG) were used.
  • photoresponsive substance 60 nm diameter gold nanoparticles (Cytodiagnostics, 60 nm NHS-Activated Gold Nanoparticle Conjugation Kit, model number CGN10K-60) were used.
  • binding substance an anti-Norovirus monoclonal antibody was used.
  • the detection liquid one liquid
  • the detection liquid one liquid
  • this detection liquid is used as an analyte liquid of the target substance. Prepared by mixing.
  • the usage-amount of the said detection liquid is 100 microliters, and the density
  • the amount of the analyte liquid used is 100 ⁇ L, and the concentration of the target substance in the analyte liquid is 1 fg / L.
  • a specific method for detecting the target substance is as follows. First, 30 ⁇ L of the liquid sample was introduced into the liquid sample holder formed by installing a 1 mm thick silicon rubber sheet having a 6 mm diameter through hole on the liquid sample introduction plate 42. After introducing the liquid sample, a cover glass G is disposed to cover the liquid sample holding unit, and the third magnetic field applying unit 46 disposed on the bottom surface side of the prism 43b is turned on, whereby the magnetic particles are liquidated. The sample was drawn on the surface of the sample introduction plate 42. The third magnetic field application unit 46 was a neodymium magnet fixed to a slide member.
  • a near field is formed on the surface of the liquid sample introduction plate 42 by irradiation of the incident light, and a neodymium magnet fixed to the slide member disposed on the side surface of the prism 43b is used.
  • the first magnetic field application unit 47 was turned on, and the state on the surface of the liquid sample introduction plate 42 was observed with a moving image by the optical signal detection unit 45 to detect the optical signal.
  • an optical microscope equipped with a 4 ⁇ objective lens and an uncooled CMOS camera manufactured by Basler, model number acA2440-35uc
  • the optical signal detection unit 45 imaging device
  • a CMOS camera provided with a 4 ⁇ objective lens is used. Since the gold nanoparticles used as the photoresponsive substance cause localized plasmon resonance due to light irradiation by the light source 43a, the imaging device uses the photoresponsive substance enhanced by the localized plasmon resonance. Scattered light is observed.
  • the detection of the target substance by the target substance detection method according to the first embodiment includes the first detection performed within 5 minutes after mixing the detection liquid with the analyte liquid of the target substance, and the prepared liquid sample. Was stored twice in the refrigerator for the second detection.
  • an anti-norovirus polyclonal antibody is used, except that the anti-norovirus polyclonal antibody is bound to the magnetic particles, and the anti-norovirus monoclonal antibody is bound to the photoresponsive substance.
  • the optical signal is detected by the same method as the target substance detection method according to Example 1, and the magnetic material obtained by binding the anti-norovirus polyclonal antibody to the analyte liquid of the target substance first.
  • the liquid sample is prepared by adding the photoresponsive substance bound with the anti-norovirus monoclonal antibody, and the first detection and preparation performed after 15 minutes as the reaction time, The liquid sample was stored twice in the refrigerator for the second detection.
  • the amount of the first liquid containing the anti-norovirus polyclonal antibody bound to the magnetic particles is 50 ⁇ L, and the concentration of the anti-norovirus polyclonal antibody in the first liquid is 0.1 pg / L. .
  • the amount of the second solution containing the anti-norovirus monoclonal antibody bound to the photoresponsive substance is 50 ⁇ L, and the concentration of the anti-norovirus monoclonal antibody in the second solution is 2.5 pg / L. It is.
  • the amount of the analyte liquid used and the concentration of the target substance in the analyte liquid are the same as in Example 1.
  • FIG. 26A shows an image at one time point in the moving image of the first detection by the target substance detection method according to the first embodiment
  • FIG. 26B shows the first time by the target substance detection method according to the first embodiment.
  • An image after 7 seconds from the one time point in the moving image of detection is shown.
  • FIG. 27A shows an image at one time point in the moving image of the second detection by the target substance detection method according to the first embodiment
  • FIG. 27B shows the target substance detection method according to the first embodiment.
  • FIG. 28A shows an image at one time point in the moving image of the first detection by the target substance detection method according to Comparative Example 1
  • FIG. 28B shows the target substance detection method according to Comparative Example 1.
  • An image after 6.3 seconds from the one time point in the moving image detected for the first time is shown.
  • FIG. 29A shows an image at one point in the moving image of the second detection by the target substance detection method according to Comparative Example 1
  • FIG. 29B shows the target substance detection method according to Comparative Example 1.
  • An image after 2.7 seconds from the one time point in the moving image detected for the second time is shown.
  • 26 (a) to 29 (b) the background is white and the optical signal detection position is black.
  • the field of view in each of FIGS. 26A to 29B is approximately 1.9 mm ⁇ 1.6 mm.
  • the circled portions indicate the light spots that have moved before and after the movement of the magnet. It is confirmed that the light spot is moving in the direction (direction in which the magnetic particles are attracted by the first magnetic field application unit 47).
  • the timing for acquiring the image shown in each figure (b) is random, but this moves among a plurality of images acquired for each elapsed time. This is because a light spot that can be clearly confirmed is selected.
  • the target substance detection methods according to Example 1 and Comparative Example 1 were performed twice each for a total of four times.
  • measurement results obtained by a three-point measurement method for observing the state before and after the movement of the magnet at arbitrary three locations having different fields of view will be described.
  • the three-point measurement method the number of light spots moved during 40 seconds of camera observation is measured, and the measurement result is evaluated by the average value of the number of light spots moved at three locations.
  • a measurement result with the average value being 2.7 was obtained in the first detection by the target substance detection method according to Example 1.
  • a measurement result having the average value of 2.7 was obtained in the second detection by the target substance detection method according to Example 1, as in the first detection.
  • a measurement result with the average value being 3.0 was obtained in the first detection by the target substance detection method according to Comparative Example 1.
  • a measurement result was obtained in which the average value was significantly lower than the average value of the first detection and the average value was 0.3.
  • a control test was performed using a control liquid sample in which a phosphate buffer solution containing no target substance was used instead of the sample liquid of the target substance. Since no moving light spot is confirmed, it is concluded that the moved light spot is a light spot based on a conjugate in which the magnetic substance and the photoresponsive substance are bound to the target substance via the binding substance. Can do.
  • the average value is used in the target substance detection method according to Example 1 using the liquid sample in which the same binding substance (anti-norovirus monoclonal antibody) is bound to the magnetic particle and the photoresponsive substance. Is the same in the first detection and the second detection (both are 2.7), there is no fluctuation with the passage of time, and a stable detection result can be obtained.
  • the target substance detection method according to Comparative Example 1 using the liquid sample in which the binding substances (anti-Noro monoclonal antibody and anti-Norovirus polyclonal antibody) that are different between the magnetic particles and the photoresponsive substance are bound Since the average value (0.3) of the first detection is substantially lower than the average value (3.0) of the first detection and becomes almost zero, the detection result is unstable due to fluctuations with time. It turns out that it is. Further, the average value (2.7) of the first detection by the target substance detection method according to Example 1 is compared with the average value (3.0) of the first detection by the target substance detection method according to Comparative Example 1.
  • the target substance detection method according to Example 1 Since the average value (2.7) of the second detection by the target substance detection method according to Example 1 is not inferior at the same time, the target substance detection method according to Example 1 has a high-precision detection result. It can be seen that can be obtained stably. In addition, in the target substance detection method according to Example 1, since the liquid sample in which the same binding substance (anti-Norovirus monoclonal antibody) is bound to the magnetic particles and the photoresponsive substance is used, the binding substance is used.

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Abstract

Le problème décrit par la présente invention est de fournir un procédé de détection de substance cible et un kit de détection de substance cible qui peuvent détecter de manière efficace, très précise et stable une substance cible à l'aide d'un capteur assisté par force externe. À cet effet, selon la présente invention, un échantillon liquide utilisé pour un capteur assisté par force externe comprend : des particules magnétiques (M) et une substance sensible à la lumière (O) qui forment respectivement des corps de liaison avec une substance cible (T) ; une première substance de liaison B1 qui lie les particules magnétiques (M) à la substance cible T ; et une seconde substance de liaison B2 qui lie la substance sensible à la lumière (O) à la substance cible (T). La même substance de liaison est utilisée pour la première substance de liaison B1 et la seconde substance de liaison B2.
PCT/JP2019/020765 2018-06-08 2019-05-24 Procédé de détection d'une substance cible et kit de détection d'une substance cible Ceased WO2019235270A1 (fr)

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Citations (7)

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Publication number Priority date Publication date Assignee Title
JP2002504993A (ja) * 1997-05-16 2002-02-12 アボツト・ラボラトリーズ 磁気応答性試薬を使用する磁気補助結合アッセイ
CN102841198A (zh) * 2012-09-18 2012-12-26 武汉大学 一种灵敏简便检测细菌的方法
WO2014007248A1 (fr) * 2012-07-06 2014-01-09 凸版印刷株式会社 Système de détection pour substance de test
WO2017187744A1 (fr) * 2016-04-28 2017-11-02 国立研究開発法人産業技術総合研究所 Procédé de détection optique et dispositif de détection optique
JP2017219512A (ja) * 2016-06-10 2017-12-14 国立研究開発法人産業技術総合研究所 光学的測定方法及び測定装置
WO2018100779A1 (fr) * 2016-11-30 2018-06-07 国立研究開発法人産業技術総合研究所 Dispositif de détection d'une substance cible et procédé de détection d'une substance cible
WO2018100780A1 (fr) * 2016-11-30 2018-06-07 国立研究開発法人産業技術総合研究所 Dispositif de détection d'une substance cible et procédé de détection d'une substance cible

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002504993A (ja) * 1997-05-16 2002-02-12 アボツト・ラボラトリーズ 磁気応答性試薬を使用する磁気補助結合アッセイ
WO2014007248A1 (fr) * 2012-07-06 2014-01-09 凸版印刷株式会社 Système de détection pour substance de test
CN102841198A (zh) * 2012-09-18 2012-12-26 武汉大学 一种灵敏简便检测细菌的方法
WO2017187744A1 (fr) * 2016-04-28 2017-11-02 国立研究開発法人産業技術総合研究所 Procédé de détection optique et dispositif de détection optique
JP2017219512A (ja) * 2016-06-10 2017-12-14 国立研究開発法人産業技術総合研究所 光学的測定方法及び測定装置
WO2018100779A1 (fr) * 2016-11-30 2018-06-07 国立研究開発法人産業技術総合研究所 Dispositif de détection d'une substance cible et procédé de détection d'une substance cible
WO2018100780A1 (fr) * 2016-11-30 2018-06-07 国立研究開発法人産業技術総合研究所 Dispositif de détection d'une substance cible et procédé de détection d'une substance cible

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