WO2016147774A1 - Procédé de mesure et dispositif de mesure - Google Patents

Procédé de mesure et dispositif de mesure Download PDF

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Publication number
WO2016147774A1
WO2016147774A1 PCT/JP2016/054331 JP2016054331W WO2016147774A1 WO 2016147774 A1 WO2016147774 A1 WO 2016147774A1 JP 2016054331 W JP2016054331 W JP 2016054331W WO 2016147774 A1 WO2016147774 A1 WO 2016147774A1
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Prior art keywords
optical blank
blank value
value
prism
light
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PCT/JP2016/054331
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English (en)
Japanese (ja)
Inventor
悠一 京極
野田　哲也
伸浩 山内
史生 長井
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コニカミノルタ株式会社
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Priority to JP2017506150A priority Critical patent/JPWO2016147774A1/ja
Publication of WO2016147774A1 publication Critical patent/WO2016147774A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to a measurement method and a measurement apparatus for measuring a substance to be measured.
  • SPFS Surface Plasmon-field enhanced Fluorescence Spectroscopy
  • a measurement chip having a prism (transparent support), a metal film formed on the prism, and a capturing body (for example, an antibody) fixed on the metal film is used.
  • a specimen containing a substance to be measured is supplied onto the metal film, the substance to be measured is captured by the capturing body (primary reaction).
  • the captured substance to be measured is further labeled with a fluorescent substance (secondary reaction).
  • secondary reaction when the metal film is irradiated with excitation light through the prism at an angle at which surface plasmon resonance occurs, localized field light can be generated on the surface of the metal film.
  • This localized field light selectively excites the fluorescent substance that labels the substance to be measured captured on the metal film, and the fluorescence emitted from the fluorescent substance is observed.
  • fluorescence is detected and the presence or amount of a substance to be measured is measured.
  • an optical blank measurement is performed before performing the secondary reaction.
  • the substance to be measured can be measured with high accuracy by calculating the signal value by subtracting the optical blank value from the fluorescence detection value (hereinafter also simply referred to as “detection value”).
  • the measurement device using SPFS uses a highly sensitive light-receiving sensor such as a photomultiplier tube (PMT) or avalanche photodiode (APD). It is common to do.
  • PMT photomultiplier tube
  • APD avalanche photodiode
  • these high-sensitivity light-receiving sensors are suitable for detecting weak light, but have a problem that high-precision temperature control is required because the light-receiving sensitivity varies greatly with temperature.
  • the present inventors examined using a light receiving sensor (for example, a photodiode (PD)) whose change in light receiving sensitivity is small even when the temperature changes.
  • a light receiving sensor for example, a photodiode (PD)
  • the present inventors use SPFS to measure a small amount of light even when using a light-sensitive sensor such as a PD. It has been found that substances can be measured with high accuracy.
  • the substance to be measured is measured with high accuracy by the attenuation of the amount of autofluorescence emitted from the measurement chip. You may not be able to.
  • An object of the present invention is a measurement method and a measurement apparatus using SPFS, which are affected by attenuation of the amount of autofluorescence emitted from a measurement chip even when high-power excitation light is irradiated. And a measuring method and a measuring apparatus capable of measuring a substance to be measured with high accuracy.
  • a measurement method detects fluorescence emitted from a fluorescent substance that is labeled with a substance to be measured, excited by localized field light based on surface plasmon resonance.
  • a measurement method for measuring a signal value indicating the presence or amount of the substance to be measured comprising a prism made of resin as a dielectric and a metal chip disposed on one surface of the prism And when irradiating the metal film with excitation light through the prism so that surface plasmon resonance occurs in the metal film in a state where the fluorescent material is not present on the metal film, Detecting the emitted light and measuring the first optical blank value; and measuring the first optical blank value, and then the target substance labeled with the fluorescent substance is the metal Fluorescence value is detected by detecting fluorescence emitted from the fluorescent material when the metal film is irradiated with excitation light through the prism so that surface plasmon resonance occurs in the metal film in a state of being present on the surface
  • a measuring device is equipped with a measuring chip having a prism made of resin as a dielectric and a metal film disposed on one surface of the prism, By irradiating the metal film with excitation light through the prism, a fluorescent substance that labels the substance to be measured existing on the metal film is excited by localized field light based on surface plasmon resonance, and emitted from the fluorescent substance.
  • a measurement device for measuring a signal value indicating the presence or amount of the substance to be measured by detecting the detected fluorescence, a holder for holding the measurement chip, and for exciting the fluorescent substance A light irradiator that irradiates the measurement chip held by the holder with excitation light; and when the light irradiator irradiates the measurement chip with excitation light, A light detection unit for detecting the detected light, and a processing unit for processing the detection value obtained by the light detection unit, wherein the fluorescent material is not present on the metal film,
  • the light detection unit detects light emitted from the measurement chip, and the first optical After measuring the blank value and measuring the first optical blank value, surface plasmon resonance occurs in the metal film in a state where the substance to be measured labeled with the fluorescent material exists on the metal film
  • the light irradiation unit irradiates the metal film with excitation light
  • the present invention it is possible to measure a substance to be measured with high sensitivity and high accuracy while suppressing a measurement error caused by autofluorescence attenuation of the measurement chip. For example, according to the present invention, misdiagnosis in clinical examination can be prevented.
  • FIG. 1 is a diagram showing a configuration of a surface plasmon enhanced fluorescence measuring apparatus according to an embodiment of the present invention.
  • FIG. 2 is a flowchart showing an example of an operation procedure of the surface plasmon enhanced fluorescence measuring apparatus.
  • FIG. 3A is a graph showing the relationship between the total irradiation energy of excitation light irradiated to the prism of the measurement chip and the optical blank value, and FIGS. 3B and 3C show the effect of irradiation of excitation light on the prism of the measurement chip. It is a conceptual diagram for demonstrating.
  • SPFS apparatus surface plasmon enhanced fluorescence measurement apparatus
  • SPR surface plasmon resonance
  • FIG. 1 is a diagram showing a configuration of the SPFS apparatus 100 according to the present embodiment.
  • the SPFS device 100 includes a chip holder 110 for detachably holding the measurement chip 10, a light irradiation unit (light irradiation unit) 120 for irradiating the measurement chip 10 with light, A light receiving unit (light detection unit) 130 for detecting light (autofluorescence, plasmon scattered light ⁇ or fluorescence ⁇ ) emitted from the measurement chip 10, a control unit (processing unit) 140 for controlling these, and a measurement chip 10 and a liquid feeding unit (not shown) for feeding liquid.
  • the SPFS device 100 is used with the measurement chip 10 mounted on the chip holder 110. Therefore, the measurement 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 formation surface 22 and an emission surface 23, a metal film 30 formed on the film formation surface 22, and a film formation surface 22.
  • a flow path lid 40 disposed on the metal film 30 is included.
  • the measuring chip 10 is replaced for each measurement (analysis).
  • the prism 20 is transparent to the excitation light ⁇ and is made of a resin that is a dielectric.
  • the prism 20 has an incident surface 21, a film forming surface 22, and an exit surface 23.
  • the incident surface 21 causes the excitation light ⁇ from the light irradiation unit 120 to enter the prism 20.
  • a metal film 30 is disposed on the film formation surface 22.
  • the excitation light ⁇ incident on the inside of the prism 20 is reflected by the metal film 30. More specifically, the light is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30.
  • the emission surface 23 emits the excitation light ⁇ reflected by the metal film 30 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 column having a trapezoidal bottom surface.
  • the surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23.
  • the trapezoid serving as the bottom surface is preferably an isosceles trapezoid.
  • the entrance surface 21 and the exit surface 23 are symmetric, 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 light irradiation unit 120.
  • the angle of the incident surface 21 is set so that the excitation light ⁇ does not enter the incident surface 21 perpendicularly in a scanning range centered on an ideal resonance angle or enhancement angle.
  • the “resonance angle” means an incident angle when the amount of reflected light emitted from the emission surface 23 is minimized when the incident angle of the excitation light ⁇ with respect to the metal film 30 is scanned.
  • the “enhancement angle” refers to 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 referred to as “plasmon scattered light”). This means the angle of incidence when the light quantity of ⁇ is maximized.
  • the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 °.
  • the resin constituting the prism 20 examples include polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin-based polymer, and the like.
  • the resin constituting the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.
  • the prism 20 When the prism 20 is formed using the resin (composition) as described above, the prism 20 usually emits autofluorescence when irradiated with light.
  • the metal film 30 is formed on the film formation surface 22 of the prism 20.
  • an interaction surface plasmon resonance; SPR
  • SPR surface plasmon resonance
  • the material of the metal film 30 is not particularly limited as long as it is a metal that causes 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 the method 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 capturing body for capturing the substance to be measured is fixed to the surface of the metal film 30 that does not face the prism 20. By fixing the capturing body, it becomes possible to selectively measure the substance to be measured.
  • at least a part of the surface of the metal film 30 is set as a reaction field.
  • the central portion of the surface of the metal film 30 is set as the reaction field.
  • a trap is uniformly fixed in the reaction field.
  • the type of the capturing body is not particularly limited as long as the substance to be measured can be captured.
  • the capturing body is an antibody or a fragment thereof that can specifically bind to the substance to be measured.
  • the channel lid 40 is disposed on the surface of the metal film 30 that does not face the prism 20 with the channel 41 interposed therebetween.
  • the channel lid 40 may be disposed on the film formation surface 22 with the channel 41 interposed therebetween.
  • the flow path lid 40 and the metal film 30 (and the prism 20) form a flow path 41 through which a liquid such as a specimen, a fluorescent labeling liquid, and a cleaning liquid flows.
  • the reaction field is exposed in the flow path 41.
  • Both ends of the channel 41 are connected to an inlet and an outlet (both not shown) formed on the upper surface of the channel lid 40, respectively. When liquids are injected into the channel 41, these liquids contact the reaction field capturing body in the channel 41.
  • the channel lid 40 is a resin member made of a material that is transparent to light (plasmon scattered light ⁇ and fluorescence ⁇ ) emitted from the reaction field of the metal film 30.
  • the material of the channel lid 40 is not particularly limited as long as it is transparent to these lights. As long as these lights can be guided to the light receiving unit 130, a part of the flow path lid 40 may be formed of an opaque material.
  • the channel lid 40 is bonded to the metal film 30 or the prism 20 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.
  • the resonance angle (and the enhancement angle near the pole) is generally determined by the design of the measurement chip 10.
  • the design factors are 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 resonance angle and the enhancement angle are shifted by the substance to be measured trapped on the metal film 30, but the amount is less than several degrees.
  • the SPFS device 100 includes the chip holder 110, the light irradiation unit (light irradiation unit) 120, the light receiving unit (light detection unit) 130, and the control unit (processing unit) 140.
  • the chip holder 110 holds the measurement chip 10 at a predetermined position.
  • the measurement chip 10 is irradiated with the excitation light ⁇ from the light irradiation unit 120 while being held by the chip holder 110.
  • the light irradiation unit 120 irradiates excitation light ⁇ (single mode laser light) toward the incident surface 21 of the prism 20 of the measurement chip 10 held by the chip holder 110. More specifically, the light source unit 121 emits the excitation light ⁇ to the back surface of the metal film 30 corresponding to the region where the capturing body is fixed from the prism 20 side of the measurement chip 10 so as to have a total reflection angle. .
  • the light irradiation unit 120 includes a light source unit 121 that emits excitation light ⁇ , an angle adjustment unit 122 that adjusts the incident angle of the excitation light ⁇ with respect to the interface (deposition surface 22) between the prism 20 and the metal film 30, and a light source unit. And a light source control unit 123 that controls various devices included in the device 121.
  • the light source unit 121 emits excitation light ⁇ .
  • the light source unit 121 includes a light source of excitation light ⁇ , a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).
  • the type of the light source is not particularly limited, but it is preferably a high-power light source from the viewpoint of using a non-sensitive photodetector such as a photodiode (PD) as the light receiving sensor 135.
  • the light source is, for example, a laser diode (LD).
  • Other examples of light source types include light emitting diodes, mercury lamps, and other laser light sources.
  • the light emission power of the light source is 1 mW / mm 2 or more.
  • the excitation light ⁇ emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like.
  • the excitation light ⁇ emitted from the light source is converted into monochromatic light by a diffraction grating or the like.
  • the excitation light ⁇ emitted from the light source is not linearly polarized light, the excitation 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 band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom means.
  • the beam shaping optical system may include all of these or a part thereof.
  • the bandpass filter turns the excitation light ⁇ emitted from the light source into a narrow band light having only the center 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 completely linearly polarized light.
  • the half-wave plate adjusts the polarization direction of the excitation light ⁇ so that the P-wave component light is incident on the metal film 30.
  • the slit and zoom means adjust the beam diameter, contour shape, and the like of the excitation light ⁇ so that the shape of the irradiation spot on the back surface of the metal film 30 is 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. The APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source to be constant.
  • the temperature adjustment mechanism is, for example, a heater or a Peltier element.
  • the wavelength and energy of the light emitted from the light source may vary depending on the temperature. For this reason, the wavelength and energy of the light emitted from the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting mechanism.
  • the angle adjusting unit 122 adjusts the incident angle of the excitation light ⁇ to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)).
  • the angle adjusting unit 122 irradiates the excitation light ⁇ to a predetermined position (the back side of the reaction field) of the metal film 30 (deposition surface 22) at a predetermined incident angle and the optical axis of the excitation light ⁇ and the chip holder 110. And rotate relative to each other.
  • the angle adjustment unit 122 rotates the light source unit 121 around an axis orthogonal to the optical axis of the excitation light ⁇ .
  • the position of the rotation axis is set so that the irradiation position on the metal film 30 (deposition surface 22) hardly moves even when the incident angle is scanned.
  • the position of the rotation center is set near the intersection of the optical axes of the two excitation lights ⁇ at both ends of the scanning range of the incident angle (between the irradiation position on the film forming surface 22 and the incident surface 21 of the prism 20).
  • the deviation of the irradiation position can be minimized.
  • the light source control unit 123 controls various devices included in the light source unit 121 to adjust the power of the excitation light ⁇ from the light source unit 121, the irradiation time, and the like.
  • the light source control unit 123 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • the light receiving unit 130 is disposed so as to face the surface of the metal film 30 of the measurement chip 10 held by the chip holder 110 that does not face the prism 20.
  • the light receiving unit 130 detects autofluorescence emitted from the measurement chip 10 and light (plasmon scattered light ⁇ or fluorescence ⁇ ) emitted from the metal film 30.
  • the light receiving unit 130 includes a first lens 132, an optical filter 133, a second lens 134, and a light receiving sensor 135 disposed in the light receiving optical system unit 131, a position switching mechanism 136, and an optical sensor control unit 137.
  • the first lens 132 is, for example, a condensing lens, and condenses light emitted from the metal film 30.
  • the second lens 134 is, for example, an imaging lens, and forms an image of the light collected by the first lens 132 on the light receiving surface of the light receiving sensor 135. The optical path between both lenses is substantially parallel.
  • the light receiving sensor 135 detects autofluorescence, plasmon scattered light ⁇ and fluorescence ⁇ emitted from the measurement chip 10.
  • the type of the light receiving sensor 135 is not particularly limited as long as the above object can be achieved, but it is preferable that the variation in the measured value is small even if the amount of received light increases.
  • the light receiving sensor 135 is, for example, a photodiode (PD).
  • PD photodiode
  • the present inventors increase the power of the light source of the excitation light ⁇ (for example, 1 mW / mm 2 or more), so that the SPFS can be used even when the light receiving sensor 135 that is not highly sensitive like PD is used. It has been confirmed that it is possible to measure a very small amount of a substance to be measured with high accuracy.
  • the position switching mechanism 136 switches the position of the optical filter 133 on or off the optical path in the light receiving optical system unit 131. Specifically, when the light receiving sensor 135 measures the optical blank value or the fluorescence value, the optical filter 133 is disposed on the optical path in the light receiving optical system unit 131, and when the light receiving sensor 135 detects the plasmon scattered light ⁇ , the optical filter 133 is optical. A filter 133 is disposed outside the optical path.
  • the light sensor control unit 137 detects the output value of the light receiving sensor 135, manages the sensitivity of the light receiving sensor 135 based on the detected output value, and controls the sensitivity of the light receiving sensor 135 to obtain an appropriate output value.
  • the optical sensor control unit 137 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • the control unit 140 controls the angle adjustment unit 122, the light source control unit 123, the position switching mechanism 136, and the optical sensor control unit 137.
  • the control unit 140 also functions as a processing unit for estimating an optical blank value included in the fluorescence value based on the detection result of the light receiving sensor 135 and calculating a signal value indicating the presence or amount of the substance to be measured. To do.
  • the control unit 140 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • FIG. 2 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus.
  • the SPFS apparatus 100 according to the present embodiment measures a substance to be measured according to one of two operation procedures according to the nature of autofluorescence emitted from the prism 20.
  • step S10 preparation for measurement is performed (step S10).
  • the measurement chip 10 is installed in the chip holder 110 of the SPFS device 100. Further, when a humectant is present in the flow channel 41 of the measurement chip 10, the humectant is removed by washing the flow channel 41 so that the capturing body can appropriately capture the substance to be measured.
  • control unit 140 controls the position switching mechanism 136 to move the optical filter 133 out of the optical path of the light receiving optical system unit 131.
  • control unit 140 controls the optical sensor control unit 137 so that the light receiving sensor 135 detects the plasmon scattered light ⁇ .
  • the control unit 140 obtains data including the relationship between the incident angle of the excitation light ⁇ and the intensity of the plasmon scattered light ⁇ .
  • the irradiation energy of the excitation light ⁇ irradiated on the prism 20 is also stored in the control unit 140.
  • the control unit 140 analyzes the data and determines an incident angle (enhancement angle) that maximizes the intensity of the plasmon scattered light ⁇ .
  • the enhancement angle is determined by the material and shape of the prism 20, the thickness of the metal film 30, the refractive index of the liquid in the flow channel 41, etc. It varies slightly due to various factors such as. For this reason, it is preferable to determine the enhancement angle every time measurement is performed.
  • the enhancement angle is determined on the order of about 0.1 °.
  • the incident angle of the excitation light ⁇ with respect to the metal film 30 (deposition surface 22) is set to the enhancement angle determined in step S20 (step S30). Specifically, the control unit 140 controls the angle adjustment unit 122 to set the incident angle of the excitation light ⁇ with respect to the metal film 30 (deposition surface 22) as an enhancement angle. In the subsequent steps, the incident angle of the excitation light ⁇ with respect to the metal film 30 (deposition surface 22) remains the enhancement angle.
  • the “optical blank value” means the amount of background light emitted above the measurement chip 10. This background light is mainly caused by autofluorescence emitted from the measurement chip 10 (prism 20) when the excitation light ⁇ is irradiated.
  • the first optical blank value is an optical blank value measured to estimate an optical blank value (second optical blank value) included in the fluorescence value in the step of measuring the fluorescence value described later (step S70).
  • the substance to be measured in the sample is reacted with the capturing body (primary reaction; step S50). Specifically, the sample is injected into the channel 41 on the liquid feeding unit side, and the sample and the capturing body are brought into contact with each other. When the substance to be measured exists in the specimen, at least a part of the substance to be measured is captured by the capturing body. Thereafter, the inside of the flow path 41 is washed with a buffer solution or the like to remove substances not captured by the capturing body.
  • the type of specimen is not particularly limited. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof.
  • a fluorescent labeling solution is provided in the channel 41.
  • the fluorescent labeling solution is, for example, a buffer solution containing an antibody (secondary antibody) labeled with a fluorescent substance.
  • the fluorescent labeling liquid comes into contact with the substance to be measured, and the substance to be measured is labeled with the fluorescent substance. Thereafter, the inside of the flow path 41 is washed with a buffer solution or the like to remove free fluorescent substances.
  • control unit 140 controls the optical sensor control unit 137 to detect the fluorescence ⁇ emitted from the fluorescent substance that labels the substance to be measured by the light receiving sensor 135.
  • the measured fluorescence value is transmitted to the control unit (processing unit) 140 and stored together with the irradiation energy of the excitation light ⁇ .
  • the processing unit 140 estimates an optical blank value (second optical blank value) included in the fluorescence value obtained in the step of measuring the fluorescence value (step S70) (step S80).
  • the control unit (processing unit) 140 is based on the following formula (1), which is a logarithmic approximation formula including an attenuation coefficient measured in advance, and the first optical blank value obtained in step S40.
  • a second optical blank value is calculated.
  • y is an optical blank value (first optical blank value or second optical blank value) when the prism 20 is irradiated with the excitation light ⁇ with the total irradiation energy x.
  • a is an attenuation coefficient of the amount of autofluorescence emitted from the prism 20.
  • b is a constant.
  • the prism 20 is irradiated before the step (step S40) of substituting the first optical blank value measured in step S40 for y and measuring the first optical blank value.
  • a constant b in this embodiment, b corresponds to the first optical blank value when x is 1 mW ⁇ second / mm 2 ) is obtained. be able to.
  • x in the step (step S40) of measuring the irradiation energy of the excitation light ⁇ and the first optical blank value when the incident angle is the enhancement angle in the step of measuring the enhancement angle (step S20).
  • y i is an optical blank value when the excitation light ⁇ is irradiated to the prism 20 with the total irradiation energy x i .
  • n is the number of measurements of the optical blank value.
  • the fluorescence value mainly includes a fluorescence component (signal value) derived from a fluorescent material that labels the substance to be measured and a fluorescence component (second optical blank value) derived from autofluorescence of the measurement chip 10. Therefore, the control unit 140 can calculate a signal value that correlates with the amount of the substance to be measured by subtracting the second optical blank value estimated in step S80 from the fluorescence value obtained in step S70.
  • the signal value can be converted into the amount or concentration of the substance to be measured by a calibration curve prepared in advance.
  • the second operation procedure may be employed when the attenuation coefficient of the amount of autofluorescent light emitted from the prism 20 is measured in advance. For example, the attenuation coefficient is not measured in advance, or the prism 20 This is adopted when there is a large variation in the amount of autofluorescence emitted from the prism 20.
  • the second operation procedure differs from the first operation procedure only in the step of measuring the first optical blank value (step S40 ') and the step of estimating the second optical blank value (step S80'). Therefore, description of steps other than step S40 and step S80 is omitted.
  • the first optical blank value is measured a plurality of times (step S40 ').
  • the first optical blank value is measured in the same manner as in Step S40, and the plurality of measured first optical blank values are transmitted to the control unit (processing unit) 140 and stored together with the total irradiation energy of the excitation light ⁇ .
  • the process part 140 uses the some 1st optical blank value obtained by process S40 ', and uses the 2nd optical blank value contained in the fluorescence value obtained at the process (process S70) of measuring a fluorescence value.
  • Estimate (step S80 ′) Specifically, the control unit (processing unit) 140 performs the first calculation based on the following equation (4), which is a logarithmic approximation equation including an attenuation coefficient, and a plurality of first optical blank values obtained in step S40 ′. Two optical blank values are calculated. In this case, the attenuation coefficient a and the constant b are calculated by the least square method using a plurality of first optical blank values.
  • y is an optical blank value (first optical blank value or second optical blank value) when the prism 20 is irradiated with the excitation light ⁇ with the total irradiation energy x.
  • a is an attenuation coefficient of the amount of autofluorescence emitted from the prism 20, and is a value expressed by the following equations (5) and (7).
  • b is a value represented by the following formulas (6) and (7).
  • [In formulas (5) to (7), y i is the first optical blank value when the prism 20 is irradiated with the excitation light ⁇ with the total irradiation energy x i .
  • n is the number of measurements of the first optical blank value.
  • the attenuation coefficient a and the constant b can be obtained. Accordingly, the step of measuring the fluorescence value (step S80 ′) by substituting the total irradiation energy of the excitation light ⁇ irradiated to the prism 20 before x in the equation (4) before the step of measuring the fluorescence value (step S70). ) Can be estimated.
  • the number n of times the first optical blank value is measured is preferably as large as possible, and can be appropriately set according to the required measurement accuracy.
  • the SPFS apparatus 100 can measure a substance to be measured with high accuracy while suppressing a measurement error due to autofluorescence decay.
  • the excitation light ⁇ it is preferable that the irradiation conditions are the same. For example, in both steps, it is preferable to irradiate the measurement chip 10 with excitation light having the same power, wavelength, and polarization direction through the overlapping optical paths using the same light source.
  • the primary reaction (step S50) and the secondary reaction (step S60) are continuously performed, and the measurement chip 10 is received between the two steps from the liquid delivery unit side to the light irradiation unit 120 and the light receiving unit. It is not moved to the unit 130 side. For this reason, the total time concerning detection can be shortened by the moving time of the measuring chip 10. In addition, the measurement accuracy can be improved by keeping the primary reaction time, the secondary reaction time, and the interval time between the primary reaction and the secondary reaction constant.
  • step S20 to step S80 is not limited to the above order.
  • the measurement of the enhancement angle (step S20), the setting of the incident angle to the enhancement angle (step S30), and the measurement of the first optical blank value (steps S40 and S40 ′) are performed. May be.
  • the enhancement angle and the optical blank in a state where the substance to be measured is captured by the capturing body. The value can be measured.
  • the order of performing the process of estimating the second blank value is not particularly limited as long as it is before the process of calculating the signal value (process S90).
  • the step of estimating the second blank value may be performed before the step of measuring the fluorescence value (step S70). In this case, an estimated value is used as the total irradiation energy of the excitation light ⁇ before the step of measuring the fluorescence value.
  • OB1 indicates the first optical blank value
  • OB2 indicates the second optical blank value
  • F indicates the fluorescence value
  • S indicates the signal value.
  • OB (OB1 and OB2), F, and S satisfy the following formula (8).
  • the optical blank value decreased logarithmically (or exponentially) as the excitation light ⁇ was irradiated. From this result, it can be seen that the amount of autofluorescence emitted from the measurement chip 10 decreases as the resin member of the measurement chip 10 is irradiated with light.
  • the first optical blank value OB1 and the fluorescence value F are measured with a light amount as shown in FIG. 3B (for convenience of explanation, the light amount of the optical blank value is increased).
  • the fluorescence value F is compared with the measurement of the first optical blank value OB1.
  • the second optical blank value OB2 at the time of measurement is also attenuated. Therefore, as shown in FIG.
  • the second optical blank value OB2 at the time of measuring the fluorescence value F can be estimated based on the measured value of the first optical blank value OB1. . Even if the amount of autofluorescence emitted from the measurement chip 10 is attenuated by the irradiation of the excitation light ⁇ , a signal value indicating the presence or amount of the substance to be measured can be calculated with high accuracy.
  • the measuring method and measuring apparatus which concern on this invention are not limited to this aspect.
  • the second optical blank value may be estimated using an exponential approximation expression represented by the following expression (9) instead of the above expression (1) or (4).
  • the following formulas (10) and (12) are used instead of the above formulas (2) and (3), and the above formula (5 ) To (7), the following formulas (10) to (12) are used.
  • y is an optical blank value (first optical blank value or second optical blank value) when the prism 20 is irradiated with the excitation light ⁇ with the total irradiation energy x.
  • a is an attenuation coefficient of the amount of autofluorescence emitted from the prism 20, and is a value expressed by the following equations (10) and (12).
  • b is a value represented by the following formulas (11) and (12).
  • [In formulas (10) to (12), y i is the (first) optical blank value when the prism 20 is irradiated with the excitation light ⁇ with the total irradiation energy x i .
  • n is the number of measurements of the (first) optical blank value.
  • the measuring method and measuring apparatus can measure a substance to be measured with high sensitivity and high accuracy, and are useful for clinical examinations, for example.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

Selon la présente invention, une puce de mesure ayant un prisme formé d'une résine qui est un corps diélectrique, et ayant un film métallique disposé sur une surface du prisme, est préparée. Ensuite, une première valeur d'ébauche optique est mesurée. Ensuite, la fluorescence émise à partir d'une matière fluorescente est détectée pour mesurer une valeur de fluorescence. Une seconde valeur d'ébauche optique contenue dans la valeur de fluorescence est calculée sur la base de la première valeur d'ébauche optique. Enfin, la seconde valeur d'ébauche optique est soustraite de la valeur de fluorescence pour calculer une valeur de signal.
PCT/JP2016/054331 2015-03-18 2016-02-15 Procédé de mesure et dispositif de mesure WO2016147774A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100271830A1 (en) * 2007-10-05 2010-10-28 Naoyuki Morisada Optical glass and optical apparatus using the same
JP2012507721A (ja) * 2008-10-30 2012-03-29 ユニヴァーシティ オブ ワシントン 使い捨てマイクロ流体デバイス用基板
WO2012141043A1 (fr) * 2011-04-14 2012-10-18 コニカミノルタホールディングス株式会社 Structure de support et dispositif de mesure
WO2014171139A1 (fr) * 2013-04-16 2014-10-23 コニカミノルタ株式会社 Procédé de détection d'anomalie de mesure et dispositif de mesure de fluorescence à champ de plasmon de surface exalté

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100271830A1 (en) * 2007-10-05 2010-10-28 Naoyuki Morisada Optical glass and optical apparatus using the same
JP2012507721A (ja) * 2008-10-30 2012-03-29 ユニヴァーシティ オブ ワシントン 使い捨てマイクロ流体デバイス用基板
WO2012141043A1 (fr) * 2011-04-14 2012-10-18 コニカミノルタホールディングス株式会社 Structure de support et dispositif de mesure
WO2014171139A1 (fr) * 2013-04-16 2014-10-23 コニカミノルタ株式会社 Procédé de détection d'anomalie de mesure et dispositif de mesure de fluorescence à champ de plasmon de surface exalté

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