WO2023107137A1 - Procédé et dispositif de mesure immunologique - Google Patents

Procédé et dispositif de mesure immunologique Download PDF

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Publication number
WO2023107137A1
WO2023107137A1 PCT/US2021/072836 US2021072836W WO2023107137A1 WO 2023107137 A1 WO2023107137 A1 WO 2023107137A1 US 2021072836 W US2021072836 W US 2021072836W WO 2023107137 A1 WO2023107137 A1 WO 2023107137A1
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WIPO (PCT)
Prior art keywords
magnetic
sensor
magnetic field
immunological measurement
measurement target
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PCT/US2021/072836
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English (en)
Inventor
Chihiro Manri
Naoshi Itabashi
Toshiro Saito
Masayoshi Momiyama
Masahiko Ichimura
Naoto Fukatani
Heng Yu
Sebastian J. Osterfeld
Original Assignee
Hitachi High-Tech Corporation
Magarray, Inc.
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Priority to PCT/US2021/072836 priority Critical patent/WO2023107137A1/fr
Publication of WO2023107137A1 publication Critical patent/WO2023107137A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
    • G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • G01R33/093—Magnetoresistive devices using multilayer structures, e.g. giant magnetoresistance sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • G01R33/098—Magnetoresistive devices comprising tunnel junctions, e.g. tunnel magnetoresistance sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/12—Measuring magnetic properties of articles or specimens of solids or fluids
    • G01R33/1269—Measuring magnetic properties of articles or specimens of solids or fluids of molecules labeled with magnetic beads

Definitions

  • the present invention relates to an immunological measurement method, and an immunological measurement apparatus.
  • Biomarkers are indicators that are widely used for knowing the genetic background, physiological state, disorder state or the like of an organism. Biomarkers are measured by using biosensors. In a biosensor, typically, in order to detect a biomarker which is a measurement target, a label that generates a signal to the outside is caused to bind with the biomarker, and the signal is detected by the sensor.
  • Known label types, and detection methods include, for example, optical immunity detection methods in which enzyme, fluorescent molecules, and the like are used as labels.
  • Patent Literature 1 describes a method of evaluating whether or not there is a specimen in samples. It is described that, in this method, a magnetic tunnel junction (MTJ) magnetoresistive element, a first electrode, a second electrode, and a magnetic sensor are used to perform a process in the following manner.
  • the first electrode contacts at least a portion of a surface of the MTJ magnetoresistive element, and extends beyond an edge of the surface of the MTJ magnetoresistive element.
  • the second electrode contacts at least a portion of an opposing surface of the MTJ magnetoresistive element, and extends beyond an edge of the opposing surface of the MTJ magnetoresistive element.
  • Patent Literature 1 in the magnetic sensor, facing surfaces of the extending portions of the first electrode and the second electrodes are non-overlapping.
  • the method described in Patent Literature 1 is characterized in including: generating a signal by causing them to contact a sample; acquiring a signal from the magnetic sensor; and evaluating whether or not there is the specimen in each sample on the basis of the signal.
  • Patent Literature 1 describes that the samples are magnetically labelled before causing them to contact in this method.
  • Patent Literature 2 describes a method for detecting, and/or quantifying target moieties in a sample fluid. This method has a step of providing the sample fluid to a magnetic sensor device, a step of attracting magnetic or magnetizable objects to a sensor chip surface of the magnetic sensor device, the sensor chip surface having binding sites, and a step of applying a moving magnetic field for increasing a binding possibility of magnetic or magnetizable objects onto the binding sites [0006]
  • Fig. 14 is an enlarged view of main parts of a conventional magnetic sensor on which the linear (stripe) groove section 100 is formed.
  • Patent Literature 2 Japanese Unexamined Patent Application Publication (Translation of PCT Application) No. 2016-520802
  • Magnetic sensors have a characteristic that the sensitivity is enhanced as the distances from magnetic particles decrease. Accordingly, for example in Patent Literature 2, the binding possibility of the magnetic particles to the binding sites on the sensor chip top surface is increased by applying the moving magnetic field to stir the magnetic particles. However, as mentioned above, in the case of the magnetic sensor in which the linear groove section 100 is formed, the highest sensitivity is attained at the side wall surfaces 103. Accordingly, there has been a problem that only increasing the binding possibility of the magnetic particles to the binding sites of the top surfaces 101 or the bottom surfaces 102 of the magnetic sensor is not enough to make use of the sensitivity of the magnetic sensor, and it is difficult to detect a biomarker highly sensitively.
  • the present invention has been made in order to solve such problems, and an object of the present invention is to provide an immunological measurement method, and an immunological measurement apparatus for detecting a biomarker highly sensitively .
  • a sensor having an area having detection sensitivity to a characteristic substance, and an area not having detection sensitivity to the characteristic substance is used, and the immunological measurement method has: a first step of gathering, to the area having detection sensitivity, a measurement target to which the characteristic substance is bound; and a second step of detecting a signal from the characteristic substance bound to the measurement target gathered to the area having detection sensitivity.
  • an immunological measurement method and an immunological measurement apparatus that enable highly-sensitive detection of biomarkers can be provided .
  • Fig. 1 is a schematic diagram depicting the configuration of an immunological measurement apparatus 1 according to a first embodiment .
  • Fig. 2 is a flowchart representing process contents of an immunological measurement method according to the first embodiment .
  • Fig. 3A is an explanatory diagram depicting the overview of the direction of a magnetic field applied at Step S101 depicted in Fig . 2.
  • Fig. 3B is an explanatory diagram depicting the overview of the direction of the magnetic field applied at Step S101 depicted in Fig. 2.
  • Fig. 4A is an explanatory diagram depicting an example of a measurement target 13 bound to a magnetic sensor 21, and a magnetic particle 15 bound to the measurement target 13, that are formed by the immunological measurement method according to the first embodiment.
  • Fig. 4B is an explanatory diagram depicting another example of the measurement target 13 bound to the magnetic sensor 21, and the magnetic particle 15 bound to the measurement target 13, that are formed by the immunological measurement method according to the first embodiment.
  • Fig. 5 is a graph depicting measurement results of Condition 4 in which the configuration of the first embodiment was adopted, and Conditions 1 to 3 in which the configuration of the first embodiment was not adopted.
  • the vertical axis represents magnetic signal strength (a.u. ) .
  • Fig. 6 is a graph depicting results of measurement of the magnetic signal strength that was observed in a case that the strength of a first magnetic field was changed under Condition 4 that was confirmed to produce effects depicted in Fig. 5.
  • the horizontal axis represents the strength (Gauss) of the first magnetic field
  • the vertical axis represents magnetic signal strength (a.u. ) .
  • Fig. 7A is an SEM image obtained by observation of the magnetic particles 15 on the magnetic sensor 21 by a scanning electron microscope (SEM) in a case that the strength of the first magnetic field was 17 Gauss.
  • the lower right scale bar represents 0.5 nm.
  • Fig. 7B is an SEM image obtained by observation of the magnetic particles 15 on the magnetic sensor 21 by the SEM in a case that the strength of the first magnetic field was 55 Gauss.
  • the lower right scale bar represents 0.5 nm.
  • Fig. 7C is an SEM image obtained by observation of the magnetic particles 15 on the magnetic sensor 21 by the SEM in a case that the strength of the first magnetic field was 91 Gauss.
  • the lower right scale bar represents 0.5 nm.
  • Fig. 8 is a graph depicting results obtained by checking whether or not a magnetic signal characteristic of the target measurement target 13 was detected, in a case that the configuration of the first embodiment was adopted.
  • the horizontal axis represents antigen concentration ( ,g/mL)
  • the vertical axis represents magnetic signal strength (a.u. ) .
  • Fig. 9 is a flowchart representing process contents of an immunological measurement method according to a second embodiment .
  • Fig. 10 is a flowchart depicting an example of specific contents of Step Sill in Fig. 9.
  • Fig. 11A is an SEM image obtained by SEM observation of the magnetic sensor 21 before a dissociation reaction solution is added at Step Sill depicted in Fig. 9 (after Step S110) .
  • the lower right scale bar represents 1.0 nm.
  • Fig. 11B is an SEM image obtained by SEM observation of the magnetic sensor 21 after the dissociation reaction solution is added at Step Sill depicted in Fig. 9 (after Step Sill) .
  • the lower right scale bar represents 1.0 nm.
  • Fig. 12 is a graph depicting results obtained by checking whether or not a magnetic signal characteristic of the target measurement target 13 was detected, in a case that the configuration of the second embodiment was adopted.
  • the horizontal axis represents antigen concentration ( ,g/mL)
  • the vertical axis represents magnetic signal strength ( a . u . ) .
  • Fig. 13 is a graph depicting results of measurement of the measurement target 13 included in a crude specimen by the methods explained in the first embodiment, and the second embodiment.
  • the vertical axis represents magnetic signal strength (a.u. ) .
  • Fig. 14 is an enlarged view of main parts of a conventional magnetic sensor on which a linear (stripe) groove section 100 is formed .
  • the present disclosure basically relates to gathering a measurement target to an area having detection sensitivity on a sensor surface in a case that the measurement target is bound (specifically) to a characteristic substance by which labelling or the like can be recognized.
  • the characteristic substance bound with the measurement target is not limited to magnetic particles, but may be a charged substance (e.g. ionizing particles, electron attached particles, etc. ) , a luminescent substance (e.g. fluorescent labelling, etc. ) , or the like.
  • a sensor having an area having detection sensitivity to the charged substance e.g. an ion sensitive area in an ion sensitive sensor
  • a sensor having an area having detection sensitivity to the luminescent substance e.g. an area having optical sensitivity in an optical sensor
  • the technology of the present disclosure can be applied by gathering a measurement target by Coulomb force, magnetic force or the like to the area having detection sensitivity to the charged substance described above.
  • a strong signal can be obtained by detecting the measurement target by using a sensor having detection sensitivity to the charged substance.
  • application examples in which what are described above are combined include an example in which a measurement target bound with (e.g. specifically fluorescent- labelled by) a luminescent substance is given a charged substance or magnetic particles.
  • a measurement target bound with (e.g. specifically fluorescent- labelled by) a luminescent substance is given a charged substance or magnetic particles.
  • Coulomb force or magnetic force to an effective sensitive area of a sensor having detection sensitivity to a luminescent substance, a strong signal of light emission therefrom can be obtained by using an optical sensor.
  • a sensor has an area having detection sensitivity, that is, an area where measurement is possible highly sensitively (at a high S/N ratio) without being influenced by a high voltage or magnetic force (disturbance such as noise) , and an area not having detection sensitivity.
  • the present disclosure relates to a method including a first step of gathering a measurement target bound with a characteristic substance to an area having detection sensitivity on a sensor surface, and a second step of detecting a signal from the characteristic substance bound to the measurement target gathered to the area having detection sensitivity .
  • the present disclosure merely depicts an example in order to explain the idea of a method in which a characteristic substance bound to a measurement target is effectively gathered to an area having detection sensitivity to the characteristic substance on a sensor surface, and thereafter a signal is obtained, and does not limit the combination of a characteristic substance and a sensor to the combination of magnetic particles, and a magnetic sensor that are explained below.
  • the present disclosure relates to a technology of performing, before a measurement step (second step) , a step (first step) of effectively gathering some characteristic substance bound to a measurement target to an area having detection sensitivity on a sensor surface in order to recognize the characteristic substance, and it does not matter what a measurement target, a characteristic substance, a method by which they are bound, a sensor, and the like are.
  • Fig. 1 is a schematic diagram depicting the configuration of an immunological measurement apparatus 1 according to a first embodiment.
  • Fig. 2 is a flowchart representing process contents of an immunological measurement method according to the first embodiment.
  • Fig. 3A, and Fig. 3B are explanatory diagrams depicting the overview of the direction of a magnetic field applied at Step S101 depicted in Fig. 2.
  • Fig. 4A is an explanatory diagram depicting an example of a measurement target 13 bound to a magnetic sensor 21, and a magnetic particle 15 bound to the measurement target 13, that are formed by the immunological measurement method according to the first embodiment.
  • Fig. 1 is a schematic diagram depicting the configuration of an immunological measurement apparatus 1 according to a first embodiment.
  • Fig. 2 is a flowchart representing process contents of an immunological measurement method according to the first embodiment.
  • Fig. 3A, and Fig. 3B are explanatory diagrams depicting the overview of the direction of a magnetic field applied at Step S101 depicted in
  • FIG. 4B is an explanatory diagram depicting another example of the measurement target 13 bound to the magnetic sensor 21, and the magnetic particle 15 bound to the measurement target 13, that are formed by the immunological measurement method according to the first embodiment.
  • the immunological measurement apparatus 1 executes immunological measurement by executing the immunological measurement method according to the first embodiment.
  • the immunological measurement apparatus 1 includes a magnetic measurement apparatus 2, a control section 3, and a computer 4 (e.g. a PC, etc. ) .
  • a computer 4 e.g. a PC, etc.
  • the magnetic measurement apparatus 2 has a magnetic field applying apparatus 22 that applies a magnetic field toward the magnetic sensor 21.
  • a measurement target 13, and magnetic particles 15 to be bound to the measurement target 13 are placed in the magnetic sensor 21.
  • a capture antibody 12 that binds with the measurement target 13 is fixed to the substrate surface of the magnetic sensor 21 in advance.
  • the fixation of the capture antibody 12 to the substrate surface of the magnetic sensor 21 can be performed by a typical technique used in immunological measurement.
  • the fixation can be performed by applying a solution containing the capture antibody 12 onto the substrate surface of the magnetic sensor 21, and causing a solvent thereof to evaporate.
  • the magnetic sensor 21 is a device that generates an electric signal (a signal, a magnetic signal) in accordance with the magnetic particles 15 associated with the sensor surface.
  • the magnetic sensor 21 is preferably able to detect a magnetic signal according to an applied magnetic field.
  • the magnetic sensor 21 is preferably able to detect a magnetic signal according to a first magnetic field, and a magnetic signal according to a second magnetic field that are described later. By doing so, it is possible to check whether or not magnetic fields are applied appropriately in the first magnetic field of a first step, and the second magnetic field at a second step that are described later.
  • Examples of the magnetic sensor 21 include magnetoresistive sensor devices including giant magnetoresistive (GMR) devices, for example, but these are not the sole examples. It is also possible that the GMR devices include spin valve detectors, and magnetic tunnel junction (MTJ) detectors, but these are not the sole examples .
  • GMR giant magnetoresistive
  • MTJ magnetic tunnel junction
  • a Helmholtz coil in which two identical coils are arranged coaxially can be used, but this is not the sole example.
  • the control section 3 controls the magnetic measurement apparatus 2 in accordance with control contents input from the computer 4, and performs application of magnetic fields by the magnetic field applying apparatus 22, and detect ion/measurement of magnetic signals from the magnetic particles 15.
  • the computer 4 has: a central processing unit (not depicted in the figure) that executes programs for controlling the control section 3, processing obtained magnetic signals, and so on; a storage section (not depicted in the figure) that stores various types of program, data, and the like; an input section (not depicted in the figure) such as a mouse or a keyboard; and a display section (not depicted in the figure) such as a screen or a printer, but these are not the sole examples. [0027]
  • the immunological measurement apparatus 1 having such a configuration implements the immunological measurement method by a procedure like the one mentioned next, for example.
  • the magnetic sensor 21 having the capture antibody 12 fixed to a substrate surface thereof is set at a predetermined position on the magnetic measurement apparatus 2 included in the immunological measurement apparatus 1.
  • the magnetic sensor 21 (sensor) has an area having detection sensitivity to a characteristic substance such as the magnetic particles 15, and an area not having detection sensitivity to the characteristic substance .
  • Step S101 the characteristic substance (e.g. the magnetic particles 15) is gathered to the area having detection sensitivity.
  • Step S101 can be performed, for example, by the magnetic field applying apparatus 22 applying a magnetic field (first magnetic field) to the magnetic sensor 21 (first step) .
  • the magnetic sensor 21 has a linear (stripe) groove section 21a, for example.
  • the groove section 21a has top surfaces 21c, bottom surfaces 21d, and side wall surfaces 21b that are present between the top surfaces 21c, and the bottom surfaces 21d, rise from the bottom surfaces 21d, and continue to the top surfaces 21c.
  • the groove section 21a can be formed such that the dimension of the top surfaces 21c is 50 to 5000 nm, the dimension of the bottom surfaces 21d is 50 to 5000 nm, the dimension (height) of the side wall surface 21b is 10 to 500 nm, and so on, for example, but these are not the sole examples, and the dimensions can be set as desired. [0029]
  • the direction of the magnetic field applied at Step S101 is preferably a direction including a component 501 perpendicular to a direction 500 parallel to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21.
  • magnetic poles are generated upright relative to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21, and so the density of the magnetic particles 15 on the side wall surfaces 21b can be increased as desired. That is, the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21 correspond to the area having detection sensitivity, and a lot of the magnetic particles 15, which are the characteristic substance, can be gathered to the area (side wall surfaces 21b) . Accordingly, the detection sensitivity becomes high if detection is performed in this area. Note that, in this case, the top surfaces 21c, and the bottom surfaces 21d of the groove section 21a of the magnetic sensor 21 correspond to the area not having detection sensitivity. [0030]
  • the strength of the first magnetic field is preferably set such that the first magnetic field is capable of both attracting the magnetic particles 15 to the side wall surfaces 21b of the groove section 21a on the surface of the magnetic sensor 21, and stirring a sample solution by the magnetic particles 15 in an immunological reaction ( ant igen/ant ibody reaction) depicted in Fig. 4A, and Fig. 4B.
  • the strength of the first magnetic field is preferably set to a strength that attracts the magnetic particles 15 to the side wall surfaces 21b on the surface of the magnetic sensor 21, and further to a strength that is optimum for stirring of the immunological reaction.
  • the strength of the first magnetic field is preferably set by conducting tests in advance in accordance with the configuration size of the magnetic sensor 21 to be used.
  • the application of the first magnetic field is preferably performed intermittently or continuously until the second step described later is started .
  • Step S102 in Fig. 2 a sample solution containing the measurement target 13 is added to the magnetic sensor 21.
  • the measurement target 13 is bound to the capture antibody 12 fixed to the surface of the magnetic sensor 21.
  • the capture antibody 12 used can be of any type as long as it is an antibody of an antigen.
  • a detection antibody 14 directly or indirectly bound with the magnetic particles 15 binds specifically to the measurement target 13. Because the first magnetic field is applied intermittently or continuously, it is possible to attract the measurement target 13 bound with the magnetic particles 15 (characteristic substance) , and gather the measurement target 13 to the side wall surfaces 21b (i.e. the area having detection sensitivity) of the groove section 21a of the magnetic sensor 21 (first step) .
  • the side wall surfaces 21b i.e. the area having detection sensitivity
  • the addition of the sample solution may be performed by mixing the measurement target 13, the detection antibody 14, the magnetic particles 15, and the like in this order, or may be performed by adding a sample solution obtained by mixing all of them in advance.
  • the measurement target 13 may be added, and then the detection antibody 14, and the magnetic particles 15 may be added simultaneously.
  • the magnetic particles 15 may be ferromagnetic particles or paramagnetic particles. Particles including various magnetic materials such as ferrite or alnico can be applied.
  • the magnetic particles 15, and the detection antibody 14 can bind to each other indirectly by using a captured material 16, a capturing material 17, and the like as depicted in Fig. 4A.
  • the captured material 16 include biotin, and the like, for example.
  • examples of the capturing material 17 include avidin, streptavidin, an anti-biotin antibody, and the like, for example. That is, in the present embodiment, a labelling process of causing the magnetic particles 15 to bind to the detection antibody 14 in advance may be performed by using the captured material 16, and the capturing material 17.
  • the detection antibody 14 can be caused to bind directly to the magnetic particles 15 in advance, as depicted in Fig. 4B.
  • the solution after the sample addition may be stirred in order to enhance the binding reaction efficiency.
  • stirring method an existing method such as horizontal stirring, vertical stirring, rotational stirring, air blow stirring, ultrasonic stirring or the like can be used.
  • Step S103 in Fig. 2 a magnetic signal (signal) from the magnetic particles 15 (characteristic substance) bound to the measurement target 13 that is gathered to the side wall surfaces 21b (i.e. the area having detection sensitivity) of the groove section 21a of the magnetic sensor 21 is detected (second step) .
  • the second step can be performed by applying a magnetic field (second magnetic field) to the magnetic sensor 21 from the magnetic field applying apparatus 22, similarly to the first step. That is, the magnetic field applying apparatus 22 applies the second magnetic field for measuring, by the magnetic sensor 21, a magnetic signal of the magnetic particles 15 bound to the measurement target 13.
  • the strength of the second magnetic field is preferably set to a magnetic field strength optimum for detecting the magnetic signal.
  • the magnetic field strength of the magnetic sensor 21 suited for detection differs depending on the configuration size of the sensor. Accordingly, the strength of the second magnetic field is preferably set by conducting tests in advance in accordance with the configuration size of the magnetic sensor 21 to be used. Note that, in order to allow the reaction of binding of the magnetic particles 15 to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21 to proceed, Step S103 is started after the passage of a certain length of time after the immunological reaction is started at Step S102. The length of time required for this may be set in accordance with the magnetic sensor 21, the measurement target 13, or the like to be used.
  • Fig. 5 is a graph depicting measurement results of Condition 4 in which the configuration of the first embodiment was adopted, and Conditions 1 to 3 in which the configuration of the first embodiment was not adopted.
  • Table 1 depicts those measurement conditions. As depicted in Table 1, under Conditions 1, and 2, the strengths of the first magnetic field, and the second magnetic field were the same. That is, under Conditions 1, and 2, measurement was performed without separating the step (first step) of gathering the measurement target 13 bound with the magnetic particles 15 to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21, and the step (second step) of detecting, by the magnetic sensor 21, a magnetic signal from the magnetic particles 15 bound to the measurement target 13. Under Conditions 3, and 4, measurement was performed by separating these steps.
  • the magnetic sensor 21 used in this example is one that produces a magnetic field strength of 17 Gauss as a magnetic field strength suited for detection of magnetic signals.
  • CEA which is a tumor marker
  • a product available from Miltenyi Biotec Inc. was used as the magnetic particles 15.
  • a measurement apparatus available from MagArray Inc. was used as the immunological measurement apparatus 1.
  • An anti-CEA antibody was used as the capture antibody 12.
  • Biotin was used as the captured material 16.
  • An anti-biotin antibody was used as the capturing material 17.
  • Fig. 6 is a graph depicting results of measurement of the magnetic signal strength that was observed in a case that the strength of the first magnetic field was changed under Condition 4 that was confirmed to produce effects depicted in Fig. 5.
  • the strength of the second magnetic field was kept at 17 Gauss suited for detecting a magnetic signal in any of the cases.
  • Other measurement conditions the antibody, apparatus, and the like that were used
  • the strength of the first magnetic field is preferably made stronger than the strength of the second magnetic field which is set to a magnetic field strength suited for detection of a magnetic signal.
  • Fig. 7A is an SEM image obtained by observation of the magnetic particles 15 on the magnetic sensor 21 by the SEM in a case that the strength of the first magnetic field was 17 Gauss.
  • Fig. 7B is an SEM image obtained by observation of the magnetic particles 15 on the magnetic sensor 21 by the SEM in a case that the strength of the first magnetic field was 55 Gauss.
  • Fig. 7C is an SEM image obtained by observation of the magnetic particles 15 on the magnetic sensor 21 by the SEM in a case that the strength of the first magnetic field was 91 Gauss.
  • Table 2 depicts results, summarized for each site of the magnetic sensor 21, of the density (the number of magnetic particles per area size 1 m 2 of the magnetic sensor 21) of the magnetic particles 15 at each magnetic field strength that was observed in the SEM images depicted in Fig. 7A to Fig. 7C. [0043] [Table 2]
  • Fig. 8 is a graph depicting results obtained by checking whether or not a magnetic signal characteristic of the target measurement target 13 was detected, in a case that the configuration of the first embodiment was adopted.
  • the magnetic field strength depicted in Fig. 8 represents the strength of the first magnetic field applied at Step S101 in Fig. 2.
  • the strength of the second magnetic field was kept at 17 Gauss in all the cases.
  • the magnetic signal strength is correlated with the concentration (antigen concentration) of the measurement target 13. Accordingly, it was confirmed that specificity could be ensured in a case that the configuration of the first embodiment was adopted.
  • a magnetic field strength suited for the first step of gathering the measurement target 13 bound with the magnetic particles 15 which are a characteristic substance to the side wall surfaces 21b of the groove section 21a which are the area having detection sensitivity in the magnetic sensor 21, and a magnetic field strength suited for the second step of detecting a signal (a magnetic signal from the magnetic particles 15) from the characteristic substance bound to the measurement target 13 gathered to the area having detection sensitivity are applied.
  • the immunological measurement method, and the immunological measurement apparatus 1 according to the first embodiment make it possible to attract, and gather the magnetic particles 15 to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21, and furthermore make it possible to detect a magnetic signal at a magnetic field strength suited for detection from the magnetic particles 15 bound, via at least the measurement target 13, to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21. Accordingly, the immunological measurement method, and the immunological measurement apparatus 1 according to the first embodiment make it possible to detect the measurement target 13 (biomarker) highly sensitively.
  • Fig. 9 is a flowchart representing process contents of an immunological measurement method according to the second embodiment.
  • cleaning of the magnetic sensor 21 is performed, and a magnetic signal of a dissociation reaction of the magnetic particles 15 is detected.
  • the immunological measurement method according to the second embodiment is different from the immunological measurement method according to the first embodiment in that these are not performed in the immunological measurement method according to the first embodiment.
  • basic process contents in the immunological measurement method according to the second embodiment are the same as those of the immunological measurement method according to the first embodiment depicted in Fig. 2, and so overlapping contents of these methods are omitted or simplified in the following explanation in some cases.
  • Step S100 in Fig. 9 the magnetic sensor 21 having the capture antibody 12 fixed to a substrate surface thereof is set at a predetermined position on the magnetic measurement apparatus 2.
  • Step S101 the magnetic field applying apparatus 22 applies a magnetic field (first magnetic field) to the magnetic sensor 21 (first step) .
  • Step S102 a sample solution containing the measurement target 13 is added to the magnetic sensor 21. Thereby, the measurement target 13 is bound to the capture antibody 12 fixed to the surface of the magnetic sensor 21.
  • Step S110 the measurement target 13, and the magnetic particles 15 that are not bound onto the magnetic sensor 21 are cleaned.
  • the sample solution is eliminated, and a cleaning solution is added.
  • a cleaning solution a cleaning solution that is used in a typical immunological reaction such as PBS or PBS containing 0.5% Tween can be used.
  • Step S110 is preferably started after the passage of a certain length of time after the immunological reaction is started by the addition of the sample solution at Step S102. The length of time required for this can be set as appropriate in accordance with the magnetic sensor 21 or measurement target 13 to be used.
  • the cleaning at this Step S110 can be performed by giving a cleaning mechanism (not depicted in the figure) to the immunological measurement apparatus 1.
  • the cleaning mechanism include, for example, a mechanism including: a cleaning solution tank in which the cleaning solution is put; a feeding apparatus that feeds the cleaning solution from within the cleaning solution tank by application of a pressure, suctioning, or the like; and a chip that supplies, to the magnetic sensor 21, the cleaning solution fed from within the cleaning solution tank.
  • Examples of such a cleaning mechanism include, specifically, a cleaning solution tank in which the cleaning solution is put, and an electrically driven pipet automated system connected to the cleaning solution tank.
  • Step S103 the second magnetic field is applied, and a magnetic signal from the magnetic particles 15 bound to the measurement target 13 gathered to the side wall surfaces 21b of the groove section 21a of the magnetic sensor 21 is detected (second step) .
  • the strength of the second magnetic field is preferably set to a magnetic field strength optimum for the magnetic sensor 21 to detect the magnetic signal.
  • the strength of the second magnetic field is preferably set by conducting tests in advance in accordance with the configuration of the magnetic sensor to be used.
  • Step Sill a dissociation reaction solution for dissociating the magnetic particles 15 from the magnetic sensor 21 is added, and simultaneously a magnetic signal is detected.
  • Fig. 10 is a flowchart depicting an example of specific contents of Step Sill in Fig. 9.
  • Step S103 in Fig. 9 After the second magnetic field is applied at Step S103 in Fig. 9, detection of a magnetic signal by the magnetic measurement apparatus 2 is started as depicted as Step Sllla in Fig. 10.
  • the magnetic signal measured here is a magnetic signal observed in a state that the magnetic particles 15 are bound to the magnetic sensor 21.
  • Step Slllb storage of the magnetic signal, and display of the magnetic signal on a screen of the computer 4 are executed, and thereafter the processing process proceeds to Step Slllc.
  • Step Slllc by adding the dissociation reaction solution, the magnetic particles 15 bound to the magnetic sensor 21 are dissociated.
  • the dissociation reaction solution an existing acid solution such as a glycine hydrochloride buffer solution can be used. After the addition of the dissociation reaction solution, the solution may be stirred in order to enhance the dissociation reaction efficiency.
  • an existing method such as horizontal stirring, vertical stirring, rotational stirring, air blow stirring, ultrasonic stirring or the like can be used. By doing so, explaining in terms of the example depicted in Fig.
  • dissociation occurs at least between the capture antibody 12, and the measurement target 13, between the measurement target 13, and the detection antibody 14, or between the captured material 16, and the capturing material 17, and the magnetic particles 15 are dissociated from the magnetic sensor 21.
  • dissociation occurs at least between the capture antibody 12, and the measurement target 13, or between the measurement target 13, and the detection antibody 14, and the magnetic particles 15 are dissociated from the magnetic sensor 21.
  • Step Sllld the magnetic sensor 21 detects a magnetic signal simultaneously with the start of the addition of the dissociation reaction solution.
  • the processing process proceeds to Step Sllle, and storage of the magnetic signal, and display of the magnetic signal on the screen of the computer 4 are executed.
  • the display at Step Sllle may be display of the negative values, which are the raw data, or may be positive values obtained by converting the negative values into their absolute values.
  • the values obtained here represent that the magnetic particles 15 that had been bound specifically to the magnetic sensor 21 via an antibody are dissociated from the magnetic sensor 21 along with dissociation of the antibody from a binding target due to the addition of the dissociation reaction solution.
  • the magnetic particles 15 that are bound non-specif ically to the magnetic sensor 21 are different from those that are bound specifically in terms of the manner, type, and the like of binding, and so it is considered that they are not dissociated (are hardly dissociated) depending on the dissociation reaction solution described before.
  • the values obtained here accurately represent the amount of the magnetic particles 15 that had been bound specifically to the magnetic sensor 21.
  • timing of the magnetic signal detection at Step Sllld may be set by conducting tests in advance to find a length of time that is required for the magnetic particles 15 to dissociate.
  • magnetic signals detected from Step Sllla to Step Sllle may be entirely or partially stored as temporal changes, and the temporal changes may be displayed on a PC screen, and so on.
  • Fig. 11A is an SEM image obtained by SEM observation of the magnetic sensor 21 before the dissociation reaction solution is added at Step Sill depicted in Fig. 9 (after Step S110) .
  • Fig. 11B is an SEM image obtained by SEM observation of the magnetic sensor 21 after the dissociation reaction solution is added at Step Sill depicted in Fig. 9 (after Step Sill) .
  • These SEM images represent results of measurement of magnetic signals observed when the strength of the first magnetic field was 91 Gauss, and the strength of the second magnetic field was 17 Gauss. It was confirmed from Fig. 11A and Fig. 11B that the magnetic particles 15 are dissociated from the magnetic sensor 21 due to the dissociation reaction.
  • Fig. 12 is a graph depicting results obtained by checking whether or not a magnetic signal characteristic of the target measurement target 13 was detected, in a case that the configuration of the second embodiment was adopted.
  • This example represents results of measurement of magnetic signals observed when the strength of the first magnetic field was 91 Gauss, and the strength of the second magnetic field was 17 Gauss. It was confirmed, as depicted in Fig. 12, that the magnetic signal strength is correlated with the concentration (antigen concentration) of the measurement target 13. Accordingly, it was confirmed that specificity could be ensured in a case that the configuration of the second embodiment was adopted.
  • Fig. 13 is a graph depicting results of measurement of the measurement target 13 included in a crude specimen by the methods explained in the first embodiment, and the second embodiment.
  • Crude specimens are body fluids such as blood or cerebrospinal fluid, specimens that are obtained by grinding tissues, and samples containing impurities such as culture solutions for cells, bacteria or the like, and blood was used here. Note that because crude specimens contain a lot of impurities, non-specific binding of the magnetic particles 15 occurs in some cases depending on the specimens.
  • the graph according to the first embodiment in Fig. 13 represents results of measurement of magnetic signals observed when the strength of the first magnetic field was 91 Gauss, and the strength of the second magnetic field was 17 Gauss.
  • the graph according to the second embodiment in Fig. 13 is based on a magnetic signal strength whose raw data represents negative values that were obtained by adding the dissociation reaction solution after measurement was performed with the strength of the first magnetic field at 91 Gauss, and the strength of the second magnetic field at 17 Gauss (Step S103/Step Sllla) .
  • a glycine hydrochloride buffer solution pH1.5
  • the magnetic signal strength became lower in the method of the second embodiment, than in the method of the first embodiment.
  • the examination of this confirms that while a magnetic signal detected in the method of the first embodiment includes a magnetic signal from the magnetic particles 15 bound non-specif ically to the magnetic sensor 21, a magnetic signal detected in the method of the second embodiment includes only a magnetic signal attributable to the magnetic particles 15 bound specifically to the magnetic sensor 21.
  • the results of the method of the second embodiment were obtained due to the fact that while the binding state of the capture antibody 12, the measurement target 13, and the detection antibody 14 bound to the magnetic sensor 21 are changed (proteins are modified in some cases) by using an highly acidic solution as the dissociation reaction solution for the dissociation reaction at Step Sill (Step Slllc) , and only the magnetic particles 15 bound specifically to the measurement target 13 are dissociated, it is difficult for the magnetic particle 15 bound non-specif ically to the magnetic sensor 21 to be dissociated by the dissociation reaction solution.
  • the immunological measurement method, and the immunological measurement apparatus 1 according to the second embodiment make it possible to detect the measurement target 13 (biomarker) highly sensitively for reasons similar to those for the immunological measurement method, and the immunological measurement apparatus 1 according to the first embodiment.
  • the immunological measurement method, and the immunological measurement apparatus 1 according to the second embodiment make it possible to detect the measurement target 13 (biomarker) more precisely because it is possible to detect only the magnetic particles 15 bound specifically to the measurement target 13.
  • Control section 4 Computer

Abstract

L'invention concerne un procédé et un dispositif de mesure immunologique qui permettent une détection hautement sensible de biomarqueurs. Dans un procédé de mesure immunologique selon la présente invention, on utilise un capteur comportant une zone présentant une sensibilité de détection à une substance caractéristique, ainsi qu'une zone dépourvue de la sensibilité de détection à la substance caractéristique, et le procédé de mesure immunologique comprend : une première étape consistant à recueillir, dans la zone présentant la sensibilité de détection, une cible de mesure à laquelle la substance caractéristique est liée ; et une seconde étape consistant à détecter un signal en provenance de la substance caractéristique liée à la cible de mesure recueillie dans la zone présentant la sensibilité de détection. De plus, un appareil de mesure immunologique 1 selon la présente invention comprend un capteur qui comporte une zone présentant une sensibilité de détection à une substance caractéristique, ainsi qu'une zone dépourvue de la sensibilité de détection à la substance caractéristique, et qui détecte un signal en provenance de la substance caractéristique liée à une cible de mesure recueillie dans la zone présentant la sensibilité de détection.
PCT/US2021/072836 2021-12-09 2021-12-09 Procédé et dispositif de mesure immunologique WO2023107137A1 (fr)

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