WO2021014864A1 - Procédé de mesure et dispositif de mesure - Google Patents

Procédé de mesure et dispositif de mesure Download PDF

Info

Publication number
WO2021014864A1
WO2021014864A1 PCT/JP2020/024579 JP2020024579W WO2021014864A1 WO 2021014864 A1 WO2021014864 A1 WO 2021014864A1 JP 2020024579 W JP2020024579 W JP 2020024579W WO 2021014864 A1 WO2021014864 A1 WO 2021014864A1
Authority
WO
WIPO (PCT)
Prior art keywords
signal value
measured
blood
substance
value
Prior art date
Application number
PCT/JP2020/024579
Other languages
English (en)
Japanese (ja)
Inventor
貴紀 村山
Original Assignee
コニカミノルタ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by コニカミノルタ株式会社 filed Critical コニカミノルタ株式会社
Priority to JP2021533879A priority Critical patent/JP7469309B2/ja
Publication of WO2021014864A1 publication Critical patent/WO2021014864A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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 measuring method and a measuring device for measuring the amount of a substance to be measured in a liquid component of blood.
  • SPR Surface plasmon resonance
  • SPFS surface Plasmon-field enhanced Fluorescence Spectroscopy
  • a trap for example, a primary antibody capable of specifically binding to a substance to be measured is immobilized on a metal membrane to form a reaction field for specifically capturing the substance to be measured.
  • a sample for example, blood
  • a capture substance labeled with a fluorescent substance for example, a secondary antibody
  • the substance to be measured bound to the reaction field is labeled with the fluorescent substance.
  • the fluorescent substance labeling the substance to be measured is excited by the electric field enhanced by SPR and emits fluorescence. Therefore, by detecting fluorescence, the presence or amount of the substance to be measured can be detected.
  • the fluorescent substance is excited by the electric field enhanced by SPR, the substance to be measured can be measured with high sensitivity.
  • the measured value is the mass of the substance to be measured per unit volume of the liquid component (plasma or serum) in blood. It is indicated by the amount of signal corresponding to it. Since the proportion of liquid component in blood varies from person to person, it is not possible to uniformly convert the measured value of blood (whole blood) into the measured value of liquid component. Therefore, when blood is used as a sample, the hematocrit value (ratio of the volume of blood cells in the blood) of the blood is measured, and the hematocrit value is used to measure the blood component (plasma or serum). Is being converted to.
  • Patent Document 1 when measuring the amount of a substance to be measured in a liquid component of blood using surface plasma resonance, the conversion coefficient is calculated using the hematocrit value measured using surface plasma resonance.
  • a measuring method for calculating and converting a measured value of blood into a measured value of a liquid component (plasma or serum) is described.
  • a device for measuring the hematocrit value such as a centrifuge or a device for measuring electric conductivity or hemoglobinometry. , It is said that the amount of the substance to be measured in the liquid component of blood can be measured.
  • Patent Document 1 The inventor of the present application has found that the measurement method described in Patent Document 1 has room for improvement in the accuracy of the measured value of the liquid component of blood calculated from the measured value of blood using the conversion coefficient.
  • An object of the present invention is to provide a measuring method and a measuring device capable of measuring the amount of a substance to be measured in a liquid component of blood with higher accuracy.
  • the measuring method is a method for measuring the amount of the substance to be measured in the liquid component of blood, and has a reaction field in which the trap is immobilized.
  • the step of providing a diluted solution of blood on the reaction field of the measuring chip and binding the substance to be measured and the trapped body contained in the diluted solution and the step of binding the substance to be measured and the trapped body are combined.
  • the first signal By multiplying the value by the conversion coefficient c represented by the following equation (1), the first signal value is converted into the second signal value.
  • df is the dilution ratio of the diluent calculated by the following formula (2)
  • Ht is the hematocrit value of the blood
  • k is the type of the substance to be measured and the dilution ratio df. It is a predetermined coefficient according to.
  • a is the amount of blood in the diluent and e is the amount of liquid other than blood in the diluent.
  • the measuring device is a device for measuring the amount of the substance to be measured in the liquid component of blood, and is a reaction field in which the trap is immobilized.
  • a first signal value indicating the amount of the substance to be measured in the diluted solution of blood is calculated from the detection unit that detects the optical change caused by the coupling with the detection unit and the detection result of the detection unit, and the first signal value is calculated.
  • It has a processing unit that converts a signal value of 1 into a second signal value indicating the amount of the substance to be measured in the liquid component of the blood, and the processing unit converts the first signal value into the first signal value.
  • the first signal value is multiplied by the conversion coefficient c represented by the following formula (1) to convert the first signal value into the second signal value.
  • Convert. In the formula (1), df is the dilution ratio of the diluent calculated by the following formula (2), Ht is the hematocrit value of the blood, and k is the type of the substance to be measured and the dilution ratio df. It is a predetermined coefficient according to.)
  • a is the amount of blood in the diluent and e is the amount of liquid other than blood in the diluent.
  • the substance to be measured in the liquid component of blood can be measured with higher accuracy.
  • FIG. 1 is a schematic view showing the configuration of a surface plasmon excitation-enhanced fluorescence analyzer according to an embodiment.
  • FIG. 2 is a flowchart showing an example of the operation procedure of the apparatus shown in FIG.
  • FIG. 3 is a graph showing the predicted value and the measured value of the signal value of the substance to be measured at each hematocrit value.
  • FIG. 4A is a graph showing the relationship between the hematocrit value of blood and the signal value corrected by using the conventional conversion coefficient c'.
  • FIG. 4B is a graph showing the relationship between the concentration of the substance to be measured and the signal value corrected by using the conventional conversion coefficient c'.
  • FIG. 5 is a graph showing the relationship between the theoretical value and the measured value of the signal value at each hematocrit value.
  • the amount of the substance to be measured contained in blood (whole blood: blood not separated and diluted) is measured by surface plasmon excitation enhanced fluorescence spectroscopy (SPFS).
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • FIG. 1 is a schematic view showing the configuration of a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) 100 according to an embodiment of the present invention.
  • the SPFS device 100 includes an excitation light irradiation unit 110, a reflected light detection unit 120, a fluorescence detection unit 130, a liquid feed unit 140, a transfer unit 150, and a control unit 160.
  • the SPFS device 100 is used with the measuring chip 10 mounted on the chip holder 152 of the transport unit 150. Therefore, the measuring chip 10 will be described first, and then each component of the SPFS device 100 will be described.
  • the measuring chip 10 includes a prism 20 having an incident surface 21, a film forming surface 22, and an emitting surface 23, a metal film 30 formed on the film forming surface 22, and a flow arranged on the film forming surface 22 or the metal film 30. It has a road cover 40 and. Normally, the measuring chip 10 is replaced at each analysis.
  • the measuring chip 10 is preferably a structure in which each piece has a length of several mm to several cm, but even a smaller structure or a larger structure that is not included in the category of “chip” may be used. Good.
  • the prism 20 is made of a dielectric material that is transparent to the excitation light ⁇ .
  • the prism 20 has an incident surface 21, a film forming surface 22, and an emitting surface 23.
  • the incident surface 21 causes the excitation light ⁇ from the excitation light irradiation unit 110 to enter the inside of the prism 20.
  • a metal film 30 is arranged on the film-forming surface 22.
  • the excitation light ⁇ incident on the inside of the prism 20 is reflected by the back surface of the metal film 30 to become reflected light ⁇ . More specifically, the excitation light ⁇ is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30 to become reflected light ⁇ .
  • the exit surface 23 emits the reflected light ⁇ to the outside of the prism 20.
  • the shape of the prism 20 is not particularly limited.
  • the shape of the prism 20 is a pillar body having a trapezoidal bottom surface.
  • the surface corresponding to one base of the trapezoid is the film forming surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the exit surface 23.
  • the trapezoid to be the bottom surface is preferably an isosceles trapezoid. As a result, the incident surface 21 and the exit surface 23 become symmetrical, and the S wave component of the excitation light ⁇ is less likely to stay in the prism 20.
  • the incident surface 21 is formed so that the excitation light ⁇ does not return to the excitation light irradiation unit 110.
  • the light source of the excitation light ⁇ is a laser diode (hereinafter, also referred to as “LD”)
  • LD laser diode
  • the angle of the incident surface 21 is set so that the excitation light ⁇ is not incident perpendicularly to the incident surface 21 in the scanning range centered on the ideal resonance angle or enhancement angle.
  • the "resonance angle” means the incident angle when the amount of reflected light ⁇ emitted from the exit surface 23 is minimized when the incident angle of the excitation light ⁇ with respect to the metal film 30 is scanned.
  • the "enhanced angle” is scattered light having the same wavelength as the excitation light ⁇ emitted above the measurement chip 10 when the incident angle of the excitation light ⁇ with respect to the metal film 30 is scanned (hereinafter, “plasmon scattered light””. It means the incident angle when the amount of light of ⁇ is maximized.
  • the angle between the incident surface 21 and the film forming surface 22 and the angle between the film forming surface 22 and the emitting surface 23 are both about 80 °.
  • the resonance angle (and the augmentation angle in the immediate vicinity thereof) is roughly determined by the design of the measurement chip 10.
  • the design elements include the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light ⁇ , and the like.
  • the substance to be measured captured on the metal film 30 shifts the resonance angle and the enhancement angle, but the amount is less than a few degrees.
  • the prism 20 has not a little birefringence characteristic.
  • materials for the prism 20 include resin and glass.
  • the material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.
  • the metal film 30 is arranged on the film forming surface 22 of the prism 20.
  • an interaction occurs between the photons of the excitation light ⁇ incident on the film forming surface 22 under total reflection conditions and the free electrons in the metal film 30, and a localized field is generated on the surface of the metal film 30.
  • Light commonly referred to as “evanescent light” or “proximity field light” can be produced.
  • the material of the metal film 30 is not particularly limited as long as it is a metal capable of causing surface plasmon resonance.
  • Examples of the material of the metal film 30 include gold, silver, copper, aluminum, and alloys thereof.
  • the metal film 30 is a gold thin film.
  • the method for forming the metal film 30 is not particularly limited. Examples of methods for forming the metal film 30 include sputtering, vapor deposition, and plating.
  • the thickness of the metal film 30 is not particularly limited, but is preferably in the range of 30 to 70 nm.
  • a trapping body for capturing the substance to be measured is immobilized on the surface of the metal film 30 that does not face the prism 20 (the surface of the metal film 30). By immobilizing the trap, it becomes possible to selectively measure the substance to be measured.
  • the trap is uniformly immobilized in a predetermined region (reaction field) on the metal film 30.
  • the type of capture body is not particularly limited as long as it can capture the substance to be measured.
  • the trap is an antibody or fragment thereof specific for the substance to be measured.
  • the flow path lid 40 is arranged on the metal film 30.
  • the flow path lid 40 may be arranged on the film forming surface 22.
  • a flow path groove is formed on the back surface of the flow path lid 40, and the flow path lid 40 forms a flow path 41 through which the liquid flows together with the metal film 30 (and the prism 20).
  • liquids include blood, plasma, serum containing a substance to be measured or a diluted solution thereof, a labeling solution containing a capture substance labeled with a fluorescent substance, a reference solution, a washing solution and the like.
  • the trapped body immobilized on the metal film 30 is exposed in the flow path 41. Both ends of the flow path 41 are connected to an injection port and an discharge port (not shown) formed on the upper surface of the flow path lid 40, respectively.
  • the flow path lid 40 is preferably made of a material that is transparent to the fluorescent ⁇ emitted from the metal film 30 and the plasmon scattered light ⁇ .
  • the material of the flow path lid 40 include a resin. If the portion that extracts the fluorescent ⁇ and the plasmon scattered light ⁇ to the outside is transparent to the fluorescent ⁇ and the plasmon scattered light ⁇ , the other portion of the flow path lid 40 may be formed of an opaque material.
  • the flow path lid 40 is joined to the metal film 30 or the prism 20 by, for example, bonding with double-sided tape or an adhesive, laser welding, ultrasonic welding, crimping using a clamp member, or the like.
  • the excitation light ⁇ is incident on the prism 20 from the incident surface 21.
  • the excitation light ⁇ incident on the prism 20 is incident on the metal film 30 at a total reflection angle (angle at which SPR occurs).
  • the localized field light excites the fluorescent substance that labels the substance to be measured existing on the metal film 30, and emits fluorescent ⁇ .
  • the SPFS device 100 measures the amount of the substance to be measured by measuring the amount of light of the fluorescent ⁇ emitted from the fluorescent substance.
  • the SPFS device 100 includes an excitation light irradiation unit 110, a reflected light detection unit 120, a fluorescence detection unit 130, a liquid feed unit 140, a transfer unit 150, and a control unit 160.
  • the excitation light irradiation unit 110 and the fluorescence detection unit 130 (when measuring the substance to be measured by SPFS) or the reflected light detection unit 120 (when measuring the substance to be measured by the SPR method as described later) are the chip holder 152. It functions as a detection unit for detecting an optical change caused by a bond between a substance to be measured contained in blood and a trap in the measuring chip 10 held in the blood.
  • the excitation light irradiation unit 110 irradiates the metal film 30 of the measurement chip 10 held in the chip holder 152 with the excitation light ⁇ from the prism 20 side.
  • the excitation light irradiation unit 110 emits only the P wave with respect to the metal film 30 toward the incident surface 21 so that the incident angle with respect to the metal film 30 is an angle that causes SPR.
  • the "excitation light” is light that directly or indirectly excites a fluorescent substance.
  • the excitation light ⁇ is light that generates localized field light that excites a fluorescent substance on the surface of the metal film 30 when the metal film 30 is irradiated through the prism 20 at an angle at which SPR is generated.
  • the excitation light irradiation unit 110 includes a light source unit 111, an angle adjusting mechanism 112, and a light source control unit 113.
  • the light source unit 111 emits excitation light ⁇ that is collimated and has a constant wavelength and light amount so that the shape of the irradiation spot on the back surface of the metal film 30 is substantially circular.
  • the light source unit 111 includes, for example, a light source for excitation light ⁇ , a beam shaping optical system, an APC mechanism, and a temperature adjusting mechanism (all not shown).
  • the type of light source is not particularly limited, and is, for example, a laser diode (LD).
  • Other examples of light sources include light emitting diodes, mercury lamps, and other laser light sources.
  • the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like.
  • the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like.
  • the light emitted from the light source is not linearly polarized light, the light emitted from the light source is converted into linearly polarized light by a polarizer or the like.
  • the beam shaping optical system includes, for example, a collimator, a bandpass filter, a linear polarizing filter, a half-wave plate, a slit, a zoom means, and the like.
  • the beam shaping optical system may include all of these, or may include some of them.
  • the collimator collimates the excitation light ⁇ emitted from the light source.
  • the bandpass filter converts the excitation light ⁇ emitted from the light source into narrow-band light having only a central wavelength. This is because the excitation light ⁇ from the light source has a slight wavelength distribution width.
  • the linear polarization filter turns the excitation light ⁇ emitted from the light source into fully linearly polarized light.
  • the half-wave plate adjusts the polarization direction of the excitation light ⁇ so that the P wave component is incident on the metal film 30.
  • the slit and the zoom means adjust the beam diameter and contour shape of the excitation light ⁇ so that the shape of the irradiation spot on the back surface of the metal film 30 becomes a circle of a predetermined size.
  • the APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light ⁇ with a photodiode (not shown) or the like. Then, the APC mechanism controls the output of the light source to be constant by controlling the input energy with the regression circuit.
  • the temperature control mechanism is, for example, a heater or a Peltier element.
  • the wavelength and energy of the emitted light from the light source may vary with temperature. Therefore, by keeping the temperature of the light source constant by the temperature adjusting mechanism, the wavelength and energy of the emitted light of the light source are controlled to be constant.
  • the angle adjusting mechanism 112 adjusts the incident angle of the excitation light ⁇ with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (deposition surface 22)).
  • the angle adjusting mechanism 112 makes the optical axis of the excitation light ⁇ and the chip holder 152 relative to each other in order to irradiate the excitation light ⁇ toward a predetermined position of the metal film 30 at a predetermined incident angle via the prism 20. Rotate.
  • the angle adjusting mechanism 112 rotates the light source unit 111 around an axis orthogonal to the optical axis of the excitation light ⁇ (the axis perpendicular to the paper surface in FIG. 1).
  • the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even if the incident angle is scanned.
  • the angle at which the amount of plasmon scattered light ⁇ is maximized is the enhancement angle.
  • the basic incident conditions of the excitation light ⁇ are determined by the material and shape of the prism 20 of the measuring chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the flow path 41, and the like, but the fluorescent substance in the flow path 41.
  • the optimum incident conditions vary slightly depending on the type and amount of light, the shape error of the prism 20, and the like. Therefore, it is preferable to obtain the optimum enhancement angle for each measurement.
  • the light source control unit 113 controls various devices included in the light source unit 111 to control the emission of the excitation light ⁇ from the light source unit 111.
  • the light source control unit 113 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.
  • the reflected light detection unit 120 when the excitation light irradiation unit 110 irradiates the metal film 30 of the measurement chip 10 with the excitation light ⁇ in order to measure the resonance angle or the like, the metal film 30 (the film-forming surface of the prism 20) The amount of the excitation light ⁇ (reflected light ⁇ ) reflected in 22) is detected.
  • the reflected light detection unit 120 includes a light receiving sensor 121, an angle adjusting mechanism 122, and a sensor control unit 123.
  • the light receiving sensor 121 is arranged at a position where the reflected light ⁇ is incident, and measures the amount of the reflected light ⁇ .
  • the type of the light receiving sensor 121 is not particularly limited.
  • the light receiving sensor 121 is a photodiode (PD).
  • the angle adjusting mechanism 122 adjusts the position (angle) of the light receiving sensor 121 according to the incident angle of the excitation light ⁇ with respect to the metal film 30.
  • the angle adjusting mechanism 122 relatively rotates the light receiving sensor 121 and the chip holder 152 so that the reflected light ⁇ is incident on the light receiving sensor 121.
  • the sensor control unit 123 controls detection of the output value of the light receiving sensor 121, management of the sensitivity of the light receiving sensor 121 based on the detected output value, change of the sensitivity of the light receiving sensor 121 in order to obtain an appropriate output value, and the like.
  • the sensor control unit 123 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.
  • the fluorescence detection unit 130 detects the amount of light emitted from the vicinity of the surface of the metal film 30 that does not face the prism 20 when the excitation light irradiation unit 110 irradiates the metal film 30 of the measurement chip 10 with the excitation light ⁇ . To do. Specifically, the fluorescence detection unit 130 detects the fluorescence ⁇ generated by irradiating the metal film 30 with the excitation light ⁇ . If necessary, the fluorescence detection unit 130 also detects the plasmon scattered light ⁇ generated by irradiating the metal film 30 with the excitation light ⁇ .
  • the fluorescence detection unit 130 includes a light receiving unit 131, a position switching mechanism 132, and a sensor control unit 133.
  • the light receiving unit 131 is arranged in the normal direction of the metal film 30 of the measuring chip 10.
  • the light receiving unit 131 includes a first lens 134, an optical filter 135, a second lens 136, and a light receiving sensor 137.
  • the first lens 134 is, for example, a condensing lens, which condenses the light emitted from the metal film 30.
  • the second lens 136 is, for example, an imaging lens, and the light collected by the first lens 134 is imaged on the light receiving surface of the light receiving sensor 137.
  • the optical path between the two lenses is a substantially parallel optical path.
  • the optical filter 135 is arranged between the two lenses.
  • the optical filter 135 guides only the fluorescence component to the light receiving sensor 137 and removes the excitation light component (plasmon scattered light ⁇ ) in order to detect the fluorescence ⁇ at a high S / N ratio.
  • the optical filter 135 include an excitation light reflection filter, a short wavelength cut filter and a bandpass filter.
  • the optical filter 135 is, for example, a filter including a multilayer film that reflects a predetermined light component, or a colored glass filter that absorbs a predetermined light component.
  • the light receiving sensor 137 detects fluorescence ⁇ and plasmon scattered light ⁇ .
  • the light receiving sensor 137 has high sensitivity capable of detecting weak fluorescence ⁇ from a minute amount of the substance to be measured.
  • the light receiving sensor 137 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).
  • the position switching mechanism 132 switches the position of the optical filter 135 on or out of the optical path of the light receiving unit 131. Specifically, when the light receiving sensor 137 detects the fluorescence ⁇ , the optical filter 135 is arranged on the optical path of the light receiving unit 131, and when the light receiving sensor 137 detects the plasmon scattered light ⁇ , the optical filter 135 is placed on the light receiving unit 131. Place it outside the optical path of.
  • the sensor control unit 133 controls detection of the output value of the light receiving sensor 137, management of the sensitivity of the light receiving sensor 137 based on the detected output value, change of the sensitivity of the light receiving sensor 137 in order to obtain an appropriate output value, and the like.
  • the sensor control unit 133 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.
  • the liquid feeding unit 140 supplies a sample (diluted blood), a labeling liquid, a reference liquid, a washing liquid, and the like into the flow path 41 of the measuring chip 10 held in the chip holder 152.
  • the liquid feed unit 140 includes a liquid chip 141, a syringe pump 142 and a liquid feed pump drive mechanism 143.
  • the liquid chip 141 is a container for containing liquids such as a sample (blood or a diluted solution of blood), a labeling solution, a reference solution, and a washing solution.
  • a plurality of containers are usually arranged according to the type of liquid, or a chip in which a plurality of containers are integrated is arranged.
  • the syringe pump 142 is composed of a syringe 144 and a plunger 145 capable of reciprocating in the syringe 144.
  • the reciprocating motion of the plunger 145 quantitatively sucks and drains the liquid. If the syringe 144 is replaceable, cleaning of the syringe 144 becomes unnecessary. Therefore, it is preferable from the viewpoint of preventing the mixing of impurities. If the syringe 144 is not configured to be replaceable, the syringe 144 can be used without replacement by further adding a configuration for cleaning the inside of the syringe 144.
  • the liquid feed pump drive mechanism 143 includes a drive device for the plunger 145 and a moving device for the syringe pump 142.
  • the driving device of the syringe pump 142 is a device for reciprocating the plunger 145, and includes, for example, a stepping motor.
  • a drive device including a stepping motor is preferable from the viewpoint of controlling the residual liquid amount of the measuring tip 10 because the liquid feeding amount and the liquid feeding speed of the syringe pump 142 can be controlled.
  • the moving device of the syringe pump 142 for example, freely moves the syringe pump 142 in two directions, an axial direction (for example, a vertical direction) of the syringe 144 and a direction crossing the axial direction (for example, a horizontal direction).
  • the moving device of the syringe pump 142 is composed of, for example, a robot arm, a two-axis stage, or a vertically movable turntable.
  • the liquid feeding unit 140 sucks various liquids from the liquid tip 141 and supplies them into the flow path 41 of the measuring tip 10. At this time, by moving the plunger 145, the liquid reciprocates in the flow path 41 in the measurement chip 10, and the liquid in the flow path 41 is agitated. As a result, it is possible to make the concentration of the liquid uniform and promote the reaction (for example, the antigen-antibody reaction) in the flow path 41. From the viewpoint of performing such an operation, the injection port of the measuring tip 10 is protected by a multilayer film, and the measuring tip 10 and the syringe 144 can seal the injection port when the syringe 144 penetrates the multilayer film. It is preferably configured.
  • the liquid in the flow path 41 is sucked again by the syringe pump 142 and discharged to the liquid tip 141 or the like.
  • reactions with various liquids, washing, and the like can be carried out, and the substance to be measured labeled with the fluorescent substance can be arranged in the reaction field in the flow path 41.
  • the transport unit 150 transports the measuring tip 10 to the measuring position or the liquid feeding position and fixes it.
  • the “measurement position” means that the excitation light irradiation unit 110 irradiates the measurement chip 10 with the excitation light ⁇ , and the reflected light ⁇ , fluorescence ⁇ , or plasmon scattered light ⁇ generated accordingly is detected by the reflected light detection unit 120 or fluorescence detection. This is the position detected by the unit 130.
  • the "liquid feeding position” is a position where the liquid feeding unit 140 supplies the liquid into the flow path 41 of the measuring chip 10 or removes the liquid in the flow path 41 of the measuring chip 10.
  • the transport unit 150 includes a transport stage 151 and a tip holder 152.
  • the tip holder 152 is fixed to the transport stage 151 and holds the measuring tip 10 detachably.
  • the shape of the chip holder 152 is such that it can hold the measurement chip 10 and does not obstruct the optical path of the excitation light ⁇ , the reflected light ⁇ , the fluorescence ⁇ , and the plasmon scattered light ⁇ .
  • the chip holder 152 is provided with an opening for passing the excitation light ⁇ , the reflected light ⁇ , the fluorescence ⁇ , and the plasmon scattered light ⁇ .
  • the transport stage 151 moves the tip holder 152 in one direction and vice versa.
  • the transport stage 151 also has a shape that does not obstruct the optical paths of the excitation light ⁇ , the reflected light ⁇ , the fluorescence ⁇ , and the plasmon scattered light ⁇ .
  • the transport stage 151 is driven by, for example, a stepping motor or the like.
  • the control unit 160 controls the angle adjustment mechanism 112, the light source control unit 113, the angle adjustment mechanism 122, the sensor control unit 123, the position switching mechanism 132, the sensor control unit 133, the liquid feed pump drive mechanism 143, and the transfer stage 151. Further, the control unit 160 calculates a first signal value indicating the amount of the substance to be measured in the blood diluent from the detection result of the fluorescence detection unit 130 (detection unit), and calculates the calculated first signal value. It also functions as a processing unit that converts the amount of the substance to be measured in the liquid component of blood into a second signal value.
  • the control unit 160 uses the first signal value as a conversion coefficient c obtained by correcting the hematocrit value of blood according to the type of the substance to be measured and the dilution ratio of the blood diluent. By multiplying, the first signal value is converted into the second signal value.
  • the control unit 160 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.
  • the conversion coefficient c includes a coefficient k for correcting the hematocrit value of blood according to the type of the substance to be measured and the dilution rate of the blood diluent (see formula (1)).
  • the coefficient k is predetermined before the step of converting the first signal value into the second signal value, and changes depending on the type of the substance to be measured and the dilution rate of the blood diluent.
  • FIG. 2 is a flowchart showing an example of the operation procedure of the SPFS device 100.
  • the measuring chip 10 is installed in the chip holder 152 of the SPFS device 100.
  • the inside of the flow path 41 is washed so that the trapping body can appropriately capture the substance to be measured contained in the sample (diluted blood). Remove the moisturizer.
  • a blood diluent (sample) is provided on the reaction field of the metal film 30 of the measurement chip, and the substance to be measured contained in the blood diluent is bound to the trap (primary reaction; step S20).
  • the "diluted solution” means a liquid obtained by diluting blood with a liquid other than blood.
  • the control unit 160 operates the transfer stage 151 to move the measuring chip 10 to the liquid feeding position. After that, the control unit 160 operates the liquid feeding unit 140 to remove the liquid in the flow path 41 of the measuring chip 10 and introduce the diluted liquid of blood in the liquid chip 141 into the flow path 41.
  • the substance to be measured is captured on the reaction field of the metal film 30 by the antigen-antibody reaction. After that, the diluted solution of blood in the flow path 41 is removed, and the inside of the flow path is washed with a washing solution.
  • the enhancement angle is determined (step S30). Specifically, the control unit 160 operates the liquid feeding unit 140 to replace the cleaning liquid in the flow path 41 of the measuring chip 10 with a reference liquid for measurement (for example, a buffer liquid). Next, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the measurement position. After that, the control unit 160 operates the fluorescence detection unit 130 to detect the plasmon scattered light while operating the excitation light irradiation unit 110 to scan the incident angle of the excitation light ⁇ with respect to the metal film 30. At this time, the control unit 160 operates the position switching mechanism 132 to arrange the optical filter 135 outside the optical path of the light receiving unit 131. Then, the control unit 160 determines the incident angle of the excitation light ⁇ when the amount of the plasmon scattered light is maximum as the enhancement angle. The determined augmentation angle is recorded in the control unit 160.
  • a reference liquid for measurement for example, a buffer liquid.
  • the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the measurement position
  • the optical blank value is measured (step S40).
  • the "optical blank value” means the amount of background light emitted above the measuring chip 10 in the measurement of fluorescence ⁇ (step S60).
  • the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 to irradiate the metal film 30 with the excitation light ⁇ and record the output value (optical blank value) of the light receiving sensor 137. To do.
  • the control unit 160 operates the angle adjusting mechanism 112 to set the incident angle of the excitation light ⁇ to the enhancement angle.
  • the control unit 160 controls the position switching mechanism 132 to arrange the optical filter 135 in the optical path of the light receiving unit 131.
  • the measured optical blank value is recorded in the control unit 160.
  • the substance to be measured captured on the reaction field of the metal film 30 is labeled with a fluorescent substance (secondary reaction; step S50).
  • the control unit 160 operates the transfer stage 151 to move the measuring chip 10 to the liquid feeding position.
  • the control unit 160 operates the liquid feeding unit 130 to remove the reference liquid in the flow path 41 of the measuring chip 10, and introduces a liquid (labeling liquid) containing a trap labeled with a fluorescent substance. ..
  • the substance to be measured captured on the metal film 30 is labeled with a fluorescent substance by the antigen-antibody reaction. After that, the labeling liquid in the flow path 41 is removed, and the inside of the flow path is washed with a cleaning liquid.
  • the amount of fluorescent ⁇ light is measured to obtain a first signal value indicating the amount of the substance to be measured in the diluted blood solution.
  • the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the measurement position. After that, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 to irradiate the metal film 30 with the excitation light ⁇ and record the output value of the light receiving sensor 137.
  • control unit 160 operates the angle adjusting mechanism 112 to set the incident angle of the excitation light ⁇ to the enhancement angle. Further, the control unit 160 controls the position switching mechanism 132 to arrange the optical filter 135 in the optical path of the light receiving unit 131. The control unit 160 subtracts the optical blank value from the detected value and calculates a first signal value indicating the amount of the substance to be measured in the blood diluent. The first signal value is converted into the amount and concentration of the substance to be measured, if necessary. The first signal value is recorded in the control unit 160.
  • the first signal value measured in step S60 is converted into a second signal value indicating the amount of the substance to be measured in the liquid component of blood (step).
  • the control unit 160 converts the first signal value into the second signal value by multiplying the first signal value by the conversion coefficient c represented by the following equation (1).
  • the first signal value and the second signal value may be the amount of light of the fluorescent ⁇ , or may be converted values such as the amount and concentration of the substance to be measured.
  • df is the dilution ratio of the diluent calculated by the following formula (2)
  • Ht is the hematocrit value of blood
  • k is the type of the substance to be measured and the dilution ratio df. It is a predetermined coefficient.
  • a is the amount of blood in the diluent and e is the amount of liquid other than blood in the diluent.
  • the conversion coefficient c includes a coefficient k for correcting the hematocrit value of blood according to the type of the substance to be measured and the dilution rate of the blood diluent.
  • the coefficient k is predetermined before the step of converting the first signal value into the second signal value, and changes depending on the type of the substance to be measured and the dilution rate of the blood diluent.
  • the coefficient k is a predicted value obtained by calculating a signal value indicating the amount of the substance to be measured in the blood when the hematocrit value of the blood containing the substance to be measured is changed at a predetermined concentration in the liquid component, and a value in the blood.
  • the coefficient k is determined as the slope of an approximate straight line in a graph plotting expected and measured values (see FIG. 5).
  • the coefficient k is about 0.79.
  • the coefficient k is about 0.80.
  • the coefficient k is about 0.95.
  • the amount of the substance to be measured in the blood before dilution can be calculated more accurately from the first signal value indicating the amount of the substance to be measured in the diluted solution of blood. it can.
  • a signal value indicating the amount of myocardial troponin I (measured substance) in a diluted solution obtained by diluting blood (plasma) having a hematocrit value of 0% twice is measured.
  • This signal value is used as a reference signal value.
  • the hematocrit value of blood containing myocardial troponin I at the same concentration in the liquid component (plasma) is increased to 20%, 40% and 60%, respectively, when the reference signal value is 100%, the diluted solution of blood
  • the calculated signal values (theoretical values) indicating the amount of myocardial troponin I in the blood plasma are reduced to 89%, 75%, and 57%, respectively (see FIG. 3). This calculation can be easily performed considering that increasing the hematocrit value reduces the proportion of the liquid component (plasma) in the blood.
  • the liquid component of blood is used by using the conversion coefficient c'of the formula (the formula (3) below) described in Patent Document 1.
  • the concentration of the substance to be measured (myocardial troponin I) after the correction tends to be higher than the actual concentration, as shown in FIG. 4A. This tendency increases as the hematocrit value increases.
  • the results of blood having a concentration of myocardial troponin I (cTnI) in the liquid component (plasma) of 11.9 ng / L are shown in circles ( ⁇ ), and myocardial troponin I in the liquid component (plasma).
  • the result of blood having a concentration of (cTnI) of 69.5 ng / L is a triangle ( ⁇ ), and the result of blood having a concentration of myocardial troponin I (cTnI) in the liquid component (plasma) is a square ( ⁇ ).
  • the result of blood having a concentration of myocardial troponin I (cTnI) in the liquid component (plasma) of 81333.3 ng / L is shown by a diamond ( ⁇ ).
  • df is the dilution ratio of the diluent calculated by the formula (2)
  • Ht is the hematocrit value of blood.
  • FIG. 4B is a graph in which the data shown in FIG. 4A is re-plotted with the horizontal axis representing the concentration of myocardial troponin I (cTnI) and the vertical axis representing the rate of change of the signal value.
  • concentration of the substance to be measured when the concentration of the substance to be measured is low, the concentration of the substance to be measured (myocardial troponin I) after correction tends to be higher than the actual concentration.
  • the result with a hematocrit value of 0% is a circle ( ⁇ )
  • the result with a hematocrit value of 20% is a triangle ( ⁇ )
  • the result with a hematocrit value of 40% is a quadrangle ( ⁇ ).
  • the result of 60% is shown by a diamond ( ⁇ ).
  • the conversion coefficient c is taken into consideration in consideration of the relationship between the theoretical value and the actually measured value at each hematocrit value shown in FIG.
  • the hematocrit value is corrected according to the type of the substance to be measured and the dilution rate df.
  • FIG. 5 is a graph showing the relationship between the theoretical value and the measured value of the signal value at each hematocrit value. In this graph, the theoretical value is on the horizontal axis and the measured value is on the vertical axis.
  • the slope of the approximate straight line passing through each point should be 1, but as mentioned above, the slope of the approximate straight line is smaller than the theoretical value. It is 0.79.
  • this slope value (0.79 in this example) is used as the coefficient k for correcting the hematocrit value in the formula for calculating the conversion coefficient c.
  • the concentration of the substance to be measured when the concentration of the substance to be measured is low, the concentration of the substance to be measured (myocardial troponin I) after correction tends to be higher than the actual concentration (see FIG. 4B). Therefore, depending on the type of the substance to be measured, the hematocrit value Ht should be corrected using the coefficient k only when the amount of the substance to be measured is small, and the hematocrit value Ht should not be corrected when the amount of the substance to be measured is not small. It may be. That is, when the first signal value is less than a predetermined threshold value, the first signal value is obtained by multiplying the first signal value by the conversion coefficient c represented by the above equation (1).
  • the first signal value Converted to a second signal value, and when the first signal value is equal to or higher than a predetermined threshold value, the first signal value is multiplied by the conversion coefficient c'represented by the above equation (3). Then, the first signal value may be converted into the second signal value.
  • the threshold concentration of myocardial troponin I is 13 ng / L.
  • the method for measuring the hematocrit value of blood is not particularly limited, and can be appropriately selected from known methods such as the microhematocrit method, the wintrobe method, the electrical resistance method, and the method described in Patent Document 1.
  • the amount of the substance to be measured in the liquid component of blood can be measured.
  • the measuring method and measuring device using SPFS have been described in the above embodiment, the measuring method and measuring device according to the present invention are not limited to the measuring method and measuring device using SPFS.
  • the measuring method and measuring device according to the present invention may be a measuring method and measuring device using the SPR method.
  • the measuring device 100 measures the substance to be measured by measuring the amount of reflected light ⁇ as the first signal value without measuring the amount of light of fluorescence ⁇ . Therefore, the measurement of the optical blank value (step S40) and the secondary reaction (step S50) are unnecessary.
  • the measuring method and measuring device according to the present invention may measure the amount of the substance to be measured contained in blood without utilizing surface plasmon resonance (SPR).
  • Plasma contained myocardial troponin I (substance to be measured) at the same concentration and the hematocrit value was adjusted to 0%, 20%, 40% and 60%.
  • the same amount of physiological saline was added to each of these bloods to prepare a 2-fold diluted solution.
  • the first signal value indicating the concentration of myocardial troponin I in the diluted blood solution was measured.
  • the first signal value was converted into a second signal value by multiplying the obtained first signal value by the conversion coefficient c represented by the above equation (1).
  • the coefficient k was 0.79.
  • the first signal value was converted into the second signal value by multiplying the obtained first signal value by the conversion coefficient c'expressed by the above formula (3).
  • each blood has a different hematocrit value, but the concentration of myocardial troponin I in the liquid component (plasma) is the same. Therefore, originally, all the second signal values should be the same value.
  • Table 2 shows the hematocrit value, the conversion coefficient c'of the comparative example, the second signal value of the comparative example (correction value by the conversion coefficient c'), the conversion coefficient c of the example, and the second of the examples.
  • the signal value (correction value by the conversion coefficient c) is shown.
  • the second signal value of the comparative example and the second signal value of the example both show relative values when the value of blood (plasma) having a hematocrit value of 0% is taken as 100%.
  • the conversion coefficient c including the coefficient k represented by the equation (1) the conversion coefficient c'excluding the coefficient k represented by the equation (3) is used more correctly than in the case of using the conversion coefficient c'.
  • the signal value of 2 could be calculated.
  • the measuring method and measuring device according to the present invention are useful for, for example, clinical examinations because they can measure a substance to be measured in blood with high reliability.

Abstract

La présente invention concerne un procédé de mesure comprenant : une étape pour fournir un fluide dilué du sang sur un champ de réaction qui est inclus dans une puce de mesure et dans lequel est fixé un corps de capture, et lier ensemble le corps de capture et une substance à mesurer contenue dans le fluide dilué ; une étape d'acquisition d'une première valeur de signal indiquant la quantité de substance mesurée dans le fluide dilué dans un état dans lequel la substance mesurée et le corps de capture sont liés ensemble et le fluide dilué n'est pas présent sur le champ de réaction ; et une étape de conversion de la première valeur de signal en une deuxième valeur de signal indiquant la quantité de la substance qui est mesurée dans un composant fluide du sang. Dans l'étape de conversion de la première valeur de signal en la deuxième valeur de signal, la première valeur de signal est convertie en la deuxième valeur de signal par multiplication de la première valeur de signal par un coefficient de conversion c obtenu en corrigeant la valeur de l'hématocrite du sang en fonction, par exemple, du type de la substance mesurée.
PCT/JP2020/024579 2019-07-24 2020-06-23 Procédé de mesure et dispositif de mesure WO2021014864A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2021533879A JP7469309B2 (ja) 2019-07-24 2020-06-23 測定方法および測定装置

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019136305 2019-07-24
JP2019-136305 2019-07-24

Publications (1)

Publication Number Publication Date
WO2021014864A1 true WO2021014864A1 (fr) 2021-01-28

Family

ID=74193416

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/024579 WO2021014864A1 (fr) 2019-07-24 2020-06-23 Procédé de mesure et dispositif de mesure

Country Status (2)

Country Link
JP (1) JP7469309B2 (fr)
WO (1) WO2021014864A1 (fr)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002071685A (ja) * 2000-09-01 2002-03-12 Horiba Ltd テオフィリン測定装置
JP2004503779A (ja) * 2000-06-12 2004-02-05 シスメックス株式会社 免疫測定方法及び免疫測定装置
WO2007007849A1 (fr) * 2005-07-14 2007-01-18 Matsushita Electric Industrial Co., Ltd. Analyseur et procédé d’analyse
JP2011137816A (ja) * 2009-12-30 2011-07-14 Lifescan Inc 初期充填速度に基づく全血ヘマトクリット値を測定するためのシステム、デバイス、及び方法
US20110201312A1 (en) * 2010-02-12 2011-08-18 Neustar, Inc. Method for distributing contact information between applications
WO2013147200A1 (fr) * 2012-03-29 2013-10-03 積水メディカル株式会社 Procédé de mesure de valeur hématocrite
WO2015129615A1 (fr) * 2014-02-25 2015-09-03 コニカミノルタ株式会社 Procédé et dispositif de mesure
JP2016519316A (ja) * 2013-05-13 2016-06-30 ビオメリューＢｉｏｍｅｒｉｅｕｘ 分析物の血漿中濃度を全血試料で直接測定するための方法
WO2018034208A1 (fr) * 2016-08-18 2018-02-22 コニカミノルタ株式会社 Procédé de mesure
JP2018031730A (ja) * 2016-08-26 2018-03-01 コニカミノルタ株式会社 ヘマトクリット値の測定方法、ヘマトクリット値の測定装置、被測定物質の量の測定方法、および被測定物質の量の測定装置

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004503779A (ja) * 2000-06-12 2004-02-05 シスメックス株式会社 免疫測定方法及び免疫測定装置
JP2002071685A (ja) * 2000-09-01 2002-03-12 Horiba Ltd テオフィリン測定装置
WO2007007849A1 (fr) * 2005-07-14 2007-01-18 Matsushita Electric Industrial Co., Ltd. Analyseur et procédé d’analyse
JP2011137816A (ja) * 2009-12-30 2011-07-14 Lifescan Inc 初期充填速度に基づく全血ヘマトクリット値を測定するためのシステム、デバイス、及び方法
US20110201312A1 (en) * 2010-02-12 2011-08-18 Neustar, Inc. Method for distributing contact information between applications
WO2013147200A1 (fr) * 2012-03-29 2013-10-03 積水メディカル株式会社 Procédé de mesure de valeur hématocrite
JP2016519316A (ja) * 2013-05-13 2016-06-30 ビオメリューＢｉｏｍｅｒｉｅｕｘ 分析物の血漿中濃度を全血試料で直接測定するための方法
WO2015129615A1 (fr) * 2014-02-25 2015-09-03 コニカミノルタ株式会社 Procédé et dispositif de mesure
WO2018034208A1 (fr) * 2016-08-18 2018-02-22 コニカミノルタ株式会社 Procédé de mesure
JP2018031730A (ja) * 2016-08-26 2018-03-01 コニカミノルタ株式会社 ヘマトクリット値の測定方法、ヘマトクリット値の測定装置、被測定物質の量の測定方法、および被測定物質の量の測定装置

Also Published As

Publication number Publication date
JPWO2021014864A1 (fr) 2021-01-28
JP7469309B2 (ja) 2024-04-16

Similar Documents

Publication Publication Date Title
JP6369533B2 (ja) 測定方法および測定装置
JP6587024B2 (ja) 検出方法および検出装置
JP6777090B2 (ja) 表面プラズモン共鳴蛍光分析方法および表面プラズモン共鳴蛍光分析装置
JP6856074B2 (ja) 測定方法、測定装置および測定システム
US11366130B2 (en) Detection device and detection method
JP6848975B2 (ja) 測定方法
WO2017057136A1 (fr) Spectroscopie de fluorescence améliorée par champ de plasmons de surface et kit de mesure
JP6760384B2 (ja) 測定方法
JP6372186B2 (ja) 検出装置、検出方法、検出チップおよびキット
JP6460104B2 (ja) 測定チップ、測定方法および測定装置
JP6520950B2 (ja) 反応方法、検出方法および検出装置
JP6805637B2 (ja) ヘマトクリット値の測定方法、ヘマトクリット値の測定装置、被測定物質の量の測定方法、および被測定物質の量の測定装置
JP6888548B2 (ja) 測定方法
WO2021014864A1 (fr) Procédé de mesure et dispositif de mesure
JPWO2018016383A1 (ja) 検出方法および検出装置
WO2020241341A1 (fr) Procédé de mesure et dispositif de mesure
JP6634784B2 (ja) 測定方法および測定装置
JP6493412B2 (ja) 検出装置および検出方法

Legal Events

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

Ref document number: 20843644

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021533879

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20843644

Country of ref document: EP

Kind code of ref document: A1