WO2015178099A1 - Procédé de spectroscopie par fluorescence renforcée par plasmon de surface et dispositif pour spectroscopie par fluorescence renforcée par plasmon de surface - Google Patents

Procédé de spectroscopie par fluorescence renforcée par plasmon de surface et dispositif pour spectroscopie par fluorescence renforcée par plasmon de surface Download PDF

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WO2015178099A1
WO2015178099A1 PCT/JP2015/059358 JP2015059358W WO2015178099A1 WO 2015178099 A1 WO2015178099 A1 WO 2015178099A1 JP 2015059358 W JP2015059358 W JP 2015059358W WO 2015178099 A1 WO2015178099 A1 WO 2015178099A1
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substance
metal film
detected
fluorescent
container
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PCT/JP2015/059358
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English (en)
Japanese (ja)
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駿 小島
高敏 彼谷
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コニカミノルタ株式会社
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Priority to JP2016520987A priority Critical patent/JP6414209B2/ja
Publication of WO2015178099A1 publication Critical patent/WO2015178099A1/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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • 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 surface plasmon enhanced fluorescence measurement method and a surface plasmon enhanced fluorescence measurement apparatus.
  • SPFS Surface plasmon-field enhanced fluorescence spectroscopy
  • magnétique particles, a fluorescent substance, and a substance to be detected are reacted at a measurement position on the metal film in the SPFS measurement apparatus where the substance to be detected is to be fixed.
  • a complex containing a fluorescent substance and a substance to be detected is fixed at the measurement position, and the substance to be detected is measured by SPFS.
  • the magnetic particles have a size of, for example, 100 nm to 1 ⁇ m, and unreacted magnetic particles are less likely to be magnetically induced compared to the above complex and easily float in the sample solution prepared for measurement. It is removed by washing together with the reaction components.
  • the fluorescent magnetic particles are aligned at a certain distance from the surface of the metal film by magnetic induction, and SPFS is used. Measure the substance to be detected.
  • SPFS can detect a substance to be detected with high sensitivity, but tends to have a greater influence on measurement results of noise signals due to contaminants in the sample liquid.
  • any of the SPFSs described in Patent Documents 1 and 2 since the sample solution directly touches the measurement position, impurities in the sample solution adsorb to the measurement position non-specifically and are detected as noise signals. There is.
  • the sensitivity of detection of the substance to be detected in the SPFS may be relatively lowered and may be insufficient.
  • This invention aims at suppressing the relative fall of the sensitivity of the to-be-detected substance which the noise signal by a contaminant brings about in SPFS.
  • the present inventors have performed pretreatment for removing contaminants in the sample stock solution in a container independent of the measurement position using fluorescent magnetic particles, and then using fluorescent reaction, the fluorescent magnetic particles was fixed together with the substance to be detected at the above measurement position, and the fluorescence signal was detected to detect high sensitivity and low noise, and the present invention was completed through further studies.
  • the excitation light is irradiated toward the metal film on which the detection target substance labeled with the fluorescent substance is fixed, and the excitation light is applied to the metal film.
  • a surface plasmon enhanced fluorescence measurement method for measuring the substance to be detected by detecting fluorescence of the fluorescent substance excited by an enhanced electric field generated by surface plasmon generated by irradiation of A first step in which a magnetic particle containing a fluorescent substance is loaded with the substance to be detected in a sample stock solution in a container that is independently arranged, and a magnetic field is generated in the container to fix the magnetic particle.
  • a second step of removing the impurities in the sample stock solution in the container to prepare a sample solution for measurement, and a third step of fixing the detected substance in the sample solution to the metal film Process, Including surface plasmon enhanced fluorescence measuring method, it provides.
  • the present invention provides a light source for irradiating excitation light toward a metal film on which a detection target substance labeled with a fluorescent substance is to be fixed, and a fluorescent substance as a means for realizing the above-described object.
  • a photodetector for detecting fluorescence generated by irradiating the metal film with the excitation light when a labeled substance to be detected is fixed; and a substance to be detected to be fixed to the metal film
  • a sample solution preparation unit for preparing a sample solution for measurement in a container arranged independently of the metal film, and for generating a magnetic field in the container according to the work of preparing the sample solution
  • a surface plasmon enhanced fluorescence measuring device comprising:
  • the contaminants are removed from the sample stock solution while the magnetic particles carrying the substance to be detected are fixed by the magnetic field.
  • the adsorption of impurities to the measurement position is prevented. Therefore, in SPFS, a relative decrease in the sensitivity of the substance to be detected caused by a noise signal due to impurities is suppressed.
  • a surface plasmon enhanced fluorescence measurement method (hereinafter also referred to as “SPFS”) according to an embodiment of the present invention irradiates excitation light toward a metal film on which a target substance labeled with a fluorescent substance is fixed, The presence or amount of the detected substance is detected by detecting the fluorescence of the fluorescent substance that is excited by the enhanced electric field generated by the surface plasmon generated by irradiation of the metal film with excitation light. Measure etc.
  • the excitation light is light that directly or indirectly excites a fluorescent substance that labels the substance to be detected. If the excitation light is light that directly excites the fluorescent substance, the target substance is directly irradiated, and if the excitation light is light that indirectly excites the fluorescent substance, a place where the excitation of the fluorescent substance is generated (for example, a metal film) The back surface of the diffraction grating or metal film in FIG.
  • the SPFS includes a prism coupling SPFS (PC-SPFS) that uses a prism for coupling excitation light and surface plasmon, and a grating cup that uses a diffraction grating for coupling excitation light and surface plasmon. It can also be applied to ring SPFS (GC-SPFS).
  • PC-SPFS prism coupling SPFS
  • GC-SPFS ring SPFS
  • the SPFS according to the present embodiment includes at least first to third steps.
  • magnetic particles containing a fluorescent substance hereinafter also referred to as “fluorescent magnetic particles” are detected in the sample stock solution in a container that is arranged independently of the metal film. Load the substance.
  • “Independently arranged” means that the container is arranged so that a liquid flow path from the container directly to the metal film is not formed.
  • the container is disposed away from the metal film in the plane direction of the metal film.
  • sample solution refers to a solution containing a substance to be detected (to be used for SPFS) immediately before being fixed to a metal film described later
  • sample stock solution refers to a substance to be detected. This refers to the liquid that is contained before or during the treatment in the present invention.
  • the magnetic particles are particles that are fixed in the container by the magnetic field of the magnetic field generator when the sample solution is prepared or impurities are removed.
  • Examples of the magnetic particles include magnetic particles, magnetic particles coated with a resin component, and resin particles in which magnetic particles are dispersed.
  • the particle diameter of the magnetic particles can be appropriately determined within the range fixed by the magnetic field, and the number average particle diameter is preferably 10 to 300 nm, and more preferably 10 to 150 nm.
  • the above fluorescent substance is a substance that generates fluorescence when excited by an electric field enhanced by surface plasmons.
  • the fluorescent substance may be a single molecule or a polymer containing a molecular structure (such as a substituent) that emits fluorescence.
  • the fluorescent substance may be carried on the surface of the magnetic particle, may be contained in the magnetic particle, or may cover the surface of the magnetic particle.
  • fluorescent substance examples include fluorescein family fluorescent dyes (for example, Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (for example, manufactured by Life Technologies Japan Co., Ltd.), hexachlorofluorescein family fluorescent dyes ( For example, Life Technologies Japan Co., Ltd.), Coumarin Family Fluorescent Dye (for example, Life Technologies Japan KK), Rhodamine Family Fluorescent Dye (for example, GE Healthcare Japan Ltd.), Cyanine Family Fluorescence Dyes, indocarbocyanine family fluorescent dyes, oxazine family fluorescent dyes, thiazine family fluorescent dyes, squaraine family fluorescent dyes, chelating runs Nido family fluorescent dye, BODIPY family fluorescent dye (manufactured by Life Technologies Japan Ltd.
  • fluorescein family fluorescent dyes for example, Integrated DNA Technologies
  • polyhalofluorescein family fluorescent dyes for example, manufactured by Life Technologies Japan Co., Ltd.
  • BODIPY is a registered trademark of the company
  • naphthalenesulfonic acid family fluorescent dye pyrene family fluorescent dye
  • Organic fluorescence such as phenylmethane family fluorescent dye
  • Alexa Fluor dye series for example, Life Technologies Japan Co., Ltd.
  • Alexa Fluor is a registered trademark of Molecular Probes
  • CF dye series for example, manufactured by Biotium
  • the fluorescent material examples include rare earth complex fluorescent dyes such as Eu and Tb (for example, ATBTA-Eu 3+ ), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), Fluorescent proteins such as yellow fluorescent protein (YFP), red fluorescent protein (DsRed), or allophycocyanin (APC), and fluorescent fine particles such as latex and silica are included.
  • rare earth complex fluorescent dyes such as Eu and Tb (for example, ATBTA-Eu 3+ ), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), Fluorescent proteins such as yellow fluorescent protein (YFP), red fluorescent protein (DsRed), or allophycocyanin (APC), and fluorescent fine particles such as latex and silica are included.
  • the above-mentioned fluorescent substance can be appropriately determined according to other requirements in SPFS.
  • the maximum fluorescence wavelength is set in the near infrared region such as Cy5 or Alexa Fluor 647 in order to minimize the influence of light absorption by iron derived from blood cell components in the blood. It is desirable to use a fluorescent dye having the same.
  • examples of the fluorescent magnetic particles include “polymer-coated magnetic fine particles holding a fluorescent substance” described in International Publication No. 09/072457.
  • sample stock solution examples include biological samples such as blood, serum, plasma, lymph, saliva, cerebrospinal fluid, urine, biosynthetic components such as cell culture fluid, and diluted solutions thereof.
  • the substance to be detected is one or more components in the sample stock solution.
  • Examples of the substance to be detected include biological substances such as proteins, hormones, nucleic acids, exosomes, and cells.
  • the substance to be detected may be supported on the fluorescent magnetic particles by a chemical method such as hydrogen bonding or binding by intermolecular force.
  • the fluorescent substance may be supported by biochemical specific binding such as antigen-antibody reaction. It can be suitably supported on magnetic particles.
  • the fluorescent magnetic particles have a first capturing body that specifically binds to the substance to be detected on the surface thereof.
  • the substance to be detected can be specifically bound to the first capturing body, and the substance to be detected can be supported on the fluorescent magnetic particles.
  • the first capture body can be appropriately determined according to the substance to be detected, and examples of the first capture body include antibodies, nucleic acids, enzymes, streptavidin, biotin, and lectins. Specific binding between the first capturing body and the substance to be detected can be performed by a conventional method such as mixing at room temperature.
  • a magnetic field is generated in the container to fix the fluorescent magnetic particles, thereby removing impurities in the sample stock solution in the container to prepare the sample liquid.
  • the magnetic field is generated so as to keep the magnetic particles in a part of the container.
  • the device that generates the magnetic field preferably generates the magnetic field at an arbitrary timing, and examples of the device include an electromagnet, a permanent magnet, and a transport device.
  • the transport device may approach and separate the permanent magnet from the container in a timely manner, or may approach and separate the container from the permanent magnet.
  • the contaminant is a component that can be adsorbed to the metal film or the third capturing body other than the target substance in the sample stock solution, and is appropriately selected according to the target substance, the metal film, or the third capturing body. Decided.
  • contaminants include heterophilic antibodies, immunoglobulins, chyle, bilirubin, albumin, rheumatoid factor and hemoglobin.
  • the removal of the contaminants from the sample stock solution includes, for example, discharging the liquid from the container when the fluorescent magnetic particles are fixed by the magnetic field, supplying the cleaning liquid, and cleaning liquid from the container when the fluorescent magnetic particles are fixed by the magnetic field. It is possible to do so by discharging.
  • the cleaning liquid supplied into the container is preferably stirred from the viewpoint of enhancing the cleaning effect.
  • the cleaning liquid is appropriately determined from the range in which the impurities can be discharged.
  • the cleaning solution preferably has the same or similar properties as the sample stock solution or the liquid component of the sample solution from the viewpoint of removing impurities without adversely affecting the binding of the substance to be detected with the fluorescent magnetic particles.
  • the cleaning solution include a buffer solution such as PBS or TBS, and a solution obtained by adding a surfactant to the buffer solution.
  • the substance to be detected in the sample stock solution (the sample solution) from which the impurities have been removed is fixed to the metal film.
  • the metal film has a thickness of 30 to 70 nm, for example.
  • Examples of the material of the metal film include gold, silver, aluminum, copper, and platinum.
  • the metal film can be produced by a known method such as sputtering, plating or vapor deposition.
  • the metal film may have a diffraction grating on its surface.
  • the SPFS according to the present embodiment can be applied to GC-SPFS.
  • the diffraction grating generates an evanescent wave when irradiated with excitation light.
  • the diffraction grating may be, for example, a one-dimensional diffraction grating or a two-dimensional diffraction grating.
  • the substance to be detected may be fixed to the measurement position of the metal film by binding by hydrogen bond or intermolecular force, but can be suitably fixed to the metal film by specific binding such as antigen-antibody reaction. It is.
  • the metal film has, on the surface thereof, a third capturing body that captures the second capturing body that specifically binds to the substance to be detected, and the second capturing body. From the viewpoint of removing unreacted magnetic particles, it is preferable to fix the substance to be detected to the metal film by capturing with the third capturing body.
  • the SPFS according to the present embodiment further includes a fourth step. In the fourth step, the second capturing body is bound to the substance to be detected carried on the fluorescent magnetic particles.
  • the second capturing body can be appropriately determined according to the substance to be detected and the third capturing body.
  • the second capturing body includes a site that specifically binds to the substance to be detected and a site that specifically binds to the third capturing body.
  • the second capturing body include a biotin-labeled antibody and a biotin-labeled nucleic acid.
  • the third capturing body can be appropriately determined according to the second capturing body.
  • the third capturing body include an antibody, avidin, modified avidin and a nucleic acid specific to the second capturing body.
  • the modified avidin is obtained by modifying a part of the amino acid sequence of avidin, and examples of the modified avidin include streptavidin and neutral avidin.
  • the third capturing body may be directly fixed to the metal film, or may be fixed to the metal film via another substance.
  • the other substance include a polymer compound obtained by polymerization of one or more monomers.
  • monomers include glucose, carboxymethylated glucose, and vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic diesters, maleic diesters , Fumaric acid diesters, allyl compounds, vinyl ethers, and vinyl ketones.
  • the above polymer compounds include hydrophilic polymer compounds such as dextran and dextran derivatives, vinyl esters, acrylic acid esters, methacrylic acid esters, olefins, styrenes, crotonic acid esters, itaconic acid diesters, maleic compounds. It is more preferable to include a hydrophobic polymer compound obtained by polymerizing at least one selected from the group of hydrophobic monomers consisting of acid diesters, fumaric acid diesters, allyl compounds, vinyl ethers and vinyl ketones.
  • dextran such as carboxymethyldextran (CMD) is particularly preferable from the viewpoint of biocompatibility, suppression of nonspecific adsorption reaction, and high hydrophilicity.
  • CMD carboxymethyldextran
  • the second step and the fourth step can be performed at an arbitrary time before the third step.
  • the first step and the second step may be performed simultaneously, or the first step, the second step, and the fourth step may be performed simultaneously, or The second step may be performed after the first step and the fourth step.
  • the SPFS according to the present embodiment may further include other steps than the first to fourth steps described above as long as the effect of the present invention is obtained.
  • the SPFS according to the present embodiment can be performed using a normal SPFS apparatus except that it has a magnetic field generator for preparing the sample solution (removal of impurities from the sample stock solution).
  • the SPFS according to the present embodiment can be performed using an SPFS apparatus as shown in FIG.
  • FIG. 1 is a diagram schematically showing a configuration of an example of an SPFS apparatus capable of performing SPFS according to the present embodiment.
  • the SPFS device 100 is a device for PC-SPFS. As shown in FIG. 1, the SPFS apparatus 100 includes an excitation optical system unit 110, a light receiving optical system unit 120, a sample solution preparation unit 130, and a control unit (CU) 160.
  • the SPFS apparatus 100 includes an excitation optical system unit 110, a light receiving optical system unit 120, a sample solution preparation unit 130, and a control unit (CU) 160.
  • CU control unit
  • the excitation optical system unit 110 includes a light source unit 111, an angle adjustment mechanism (ARS) 112, and a light source control unit (ICU) 113.
  • ARS angle adjustment mechanism
  • ICU light source control unit
  • the light source unit 111 includes, for example, a laser diode (hereinafter also referred to as “LD”), a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).
  • 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 unit.
  • the APC mechanism includes means for detecting the amount of excitation light ⁇ after collimation (for example, a device for branching excitation light ⁇ and a photodiode for detecting the amount of branched light).
  • the temperature adjustment mechanism is, for example, a heater or a Peltier element.
  • the angle adjustment mechanism 112 is a device for changing the irradiation angle of the excitation light ⁇ , and includes, for example, a rotation support member that rotates the light source unit 111, a robot arm, or the like.
  • the light source control unit 113 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 source control unit 113 operates and controls the above-described various devices that constitute the light source unit 111, and controls emission of emitted light from the light source unit 111.
  • the light receiving optical system unit 120 includes, for example, a light receiving unit 121, a position switching mechanism (PSS) 122, and a sensor control unit (SCU) 123.
  • PSS position switching mechanism
  • SCU sensor control unit
  • the light receiving unit 121 includes, for example, a first lens 124, an optical filter 125, a second lens 126, and a light receiving sensor 127 that are sequentially arranged in the normal direction of the metal film (deposition surface 22) of the analysis chip 10.
  • the first lens 124 is, for example, a condensing lens for condensing light emitted from the metal film 30, and the second lens 126 receives, for example, the light condensed by the first lens 124.
  • This is an imaging lens for re-imaging on the light receiving surface of the sensor 127.
  • An optical filter 125 is disposed between the two lenses.
  • the optical filter 125 is, for example, a filter made of a multilayer film that removes a predetermined light component by reflecting it.
  • the light receiving sensor 127 is a light receiving sensor for detecting the amount of the fluorescent light, and has a high sensitivity capable of detecting weak fluorescence from a small amount of a substance to be detected.
  • the light receiving sensor 127 is, for example, a head-on type photomultiplier tube (PMT).
  • the position switching mechanism 122 is, for example, a known mechanism that causes the optical filter 125 to advance and retreat from the optical path by a rotational motion, such as a rotation drive unit and a turntable or rack and pinion having the optical filter 125. It is comprised by.
  • the sensor 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 sensor control unit 123 controls the detection of the light amount by the light receiving sensor 127 and the management and change of the detection range of the light receiving sensor 127.
  • the sample solution preparation unit 130 supplies the sample stock solution, chemical solution, or cleaning solution to the container 131 and supplies the sample solution or cleaning solution to the analysis chip 10.
  • the sample solution preparation unit 130 includes, for example, a container 131, a syringe pump 132, a magnet 137, and a sample solution preparation mechanism (SPS) 133.
  • SPS sample solution preparation mechanism
  • the container 131 includes, for example, a container for preparing a sample solution, and a container for storing a sample stock solution, various chemical solutions, and a cleaning solution.
  • the syringe pump 132 includes a syringe 134 and a plunger 135 that can reciprocate inside the syringe 134. By the reciprocating motion of the plunger 135, the sample stock solution, the chemical solution or the cleaning solution is sucked and discharged quantitatively.
  • the magnet 137 is a permanent magnet, for example.
  • the magnet 137 is disposed so as to be able to approach and separate from the container 131.
  • the sample solution preparation mechanism 133 includes, for example, a driving device for the plunger 135, a moving device for the syringe pump 132, and a moving device for the magnet 137.
  • the drive device of the plunger 135 is a device for reciprocating the plunger 135 and includes, for example, a stepping motor.
  • the driving device including the stepping motor is preferable from the viewpoint of managing the liquid feeding amount and the liquid feeding speed of the syringe pump 132 uniformly and managing the residual liquid amount of the analysis chip 10 constant.
  • the moving device of the syringe pump 132 is a device that freely moves the syringe pump 132 in two directions, for example, an axial direction (for example, a vertical direction) of the syringe 134 and a direction crossing the axial direction (for example, a horizontal direction). .
  • the moving device of the syringe pump 132 is configured by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.
  • the moving device of the magnet 137 is configured similarly to the moving device of the syringe pump 132.
  • the control unit 160 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 160 controls the angle adjustment mechanism 112, the light source control unit 113, the position switching mechanism 122, the sensor control unit 123, and the sample solution preparation mechanism 133, and causes the above-described mechanism to perform various operations.
  • the control unit 160 appropriately sets numerical values necessary for the above operation, such as the detection range of the light receiving sensor 127.
  • an analysis chip 10 is mounted on a chip holder (not shown) in measuring a substance to be detected.
  • the chip holder is configured to transport the analysis chip 10 to either the sample solution preparation unit 130 or the measurable position of the SPFS device 100.
  • the analysis chip 10 includes a prism 20, a metal film 30 formed on the film formation surface 22 of the prism 20, and a flow path lid 40 disposed on the film formation surface 22 or the metal film 30.
  • the prism 20 has an entrance surface 21, a film formation surface 22, and an exit surface 23, and is made of a dielectric that is transparent to the excitation light ⁇ .
  • the prism 20 has a number of birefringence characteristics. Examples of the material of 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 shape of the prism 20 is, for example, 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. Thereby, 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 resonance angle (and the enhancement angle in the vicinity thereof) is roughly determined by the design of the analysis chip 10. Design factors include prism refractive index, metal refractive index, metal film thickness, metal extinction coefficient, excitation wavelength, and the like.
  • the resonance angle and the enhancement angle are shifted according to the amount of the substance to be detected fixed to the metal film, but the shift amount is less than several degrees.
  • the incident surface 21 is formed so that the excitation light ⁇ does not return to the excitation optical system unit 110 from the viewpoint of preventing fluctuations in the wavelength or output of the excitation light ⁇ .
  • 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 metal film 30 is, for example, a gold deposition film, and the thickness of the metal film 30 is, for example, 50 nm.
  • a third capturing body which will be described later, is fixed to the surface of the metal film 30, and the third capturing body is covered with, for example, a moisturizing agent.
  • the channel lid 40 is disposed on the surface of the metal film 30 that does not face the prism 20 with the channel interposed therebetween.
  • the flow path lid 40 may be disposed on the film formation surface 22 with the flow path interposed therebetween.
  • the channel lid 40 forms a channel through which liquid flows together with the metal film 30 (and the prism 20).
  • the flow path cover 40 is made of a material that is transparent to light (plasmon scattered light and fluorescence) emitted from the metal film 30.
  • An example of the material of the flow path lid 40 includes a resin.
  • the flow path cover 40 only needs to be able to guide these lights to the light receiving optical system unit 120.
  • a part of the channel lid 40 may be made of an opaque material as long as at least the surface from which the fluorescence from the fluorescent substance labeled with the substance to be detected is extracted is optically transparent.
  • the flow path 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.
  • FIG. 2 is a diagram schematically showing an intermediate state of an example of SPFS according to the present embodiment.
  • FIG. 3 is a diagram schematically showing a cross section of the fluorescent magnetic particles in the present embodiment.
  • FIG. 4 is a flowchart illustrating an example of a main operation procedure of the SPFS apparatus 100.
  • the control unit 160 causes the fluorescent magnetic particles 400 to be supplied from the container 131 containing the fluorescent magnetic particles 400 to the preparation container 131 (FIG. 2A), and then from the container 131 containing the sample stock solution.
  • the sample stock solution is supplied to the preparation container 131 and allowed to react for a certain period of time ((B) in FIG. 2, step 401 in FIG. 4).
  • the sample stock solution includes a substance to be detected 500 and a contaminant 600.
  • the fluorescent magnetic particle 400 includes a magnetic particle 410, a fluorescent layer 420 that covers the surface of the magnetic particle 410, and a first capturing body 430 that is disposed on the surface of the fluorescent layer 420.
  • the volume average particle diameter of the fluorescent magnetic particles 400 is, for example, 0.2 ⁇ m.
  • the magnetic particles 410 are, for example, resin particles in which magnetic fine particles are dispersed
  • the fluorescent layer 420 is a resin layer containing, for example, a fluorescent dye such as Cy5
  • the first capturing body 430 is a sample stock solution.
  • An antibody for example, an anti-PSA antibody that specifically binds to the substance to be detected 500 therein.
  • the substance to be detected 500 specifically binds to the first capturing body 430 of the fluorescent magnetic particle 400 by mixing (reaction) of the fluorescent magnetic particle 400 and the sample stock solution.
  • reaction reaction
  • the detected substance in the sample stock solution is supported on the magnetic particles having the fluorescent substance, and the first complex 450 of the detected substance 500 and the fluorescent magnetic particle 400 is formed.
  • control unit 160 places the magnet 137 in the vicinity of the peripheral wall of the preparation container 131 (FIG. 2C, step 402 in FIG. 4), and the first complex 450 in the preparation container 131. Is drawn to the wall surface of the container 131 and the liquid in the container 131 is sucked and removed ((D) in FIG. 2, step 403 in FIG. 4). Along with the removal of the liquid, the foreign matter 600 in the container 131 is also removed from the container 131.
  • the controller 160 injects the cleaning liquid into the container 131, disposes the magnet 137 away from the peripheral surface of the container 131, disperses the first composite 450 in the cleaning liquid in the container 131 for a certain period of time, and again
  • the magnet 137 is disposed in the vicinity of the peripheral surface of the container 131, the first composite 450 is attracted to the wall surface of the container 131, and the cleaning liquid in the container 131 is sucked and removed.
  • the control unit 160 for example, repeats the cleaning liquid suction operation (cleaning operation) from the injection of the cleaning liquid three times (step 404 in FIG. 4). By this operation, the impurities 600 in the sample stock solution are substantially removed from the container 131.
  • control unit 160 supplies the solution of the biotinylated antibody 700 that is the second capturing body to the preparation container 131 from the container 131 that stores the drug solution ((E) in FIG. 2, step 405 in FIG. 4). ).
  • the substance to be detected of the first complex 450 and the biotinylated antibody 700 are specifically bound, and the second complex 650 of the biotinylated antibody 700, the substance to be detected 500, and the fluorescent magnetic particle 400 is formed. Composed.
  • the controller 160 places the magnet 137 in the vicinity of the peripheral wall of the preparation container 131 (step 406 in FIG. 4), and pulls the second composite 650 in the preparation container 131 to the wall surface of the container 131. Then, the liquid in the container 131 is removed by suction (step 407 in FIG. 4). Along with the removal of this liquid, excess biotinylated antibody 700 in the container 131 is removed from the container 131 ((F) in FIG. 2).
  • the controller 160 injects the cleaning liquid into the container 131, disposes the magnet 137 away from the peripheral surface of the container 131, disperses the second composite 650 in the cleaning liquid in the container 131, and again The magnet 137 is disposed in the vicinity of the peripheral surface of the container 131, the second composite 650 is drawn to the wall surface of the container 131, and the cleaning liquid in the container 131 is sucked and removed.
  • the control unit 160 repeats the cleaning operation three times (step 408 in FIG. 4). By this operation, the excess biotinylated antibody 700 in the sample stock solution is substantially removed from the container 131. Then, the control unit 160 supplies an appropriate amount of the cleaning liquid to the preparation container 131. In this way, a sample solution for measurement is prepared.
  • the control unit 160 transports the analysis chip 10 to the operation position of the sample solution preparation unit 130, and removes the moisturizing agent applied to the surface of the metal film 30 of the analysis chip 10 ((G) in FIG. 2). Step 409 in FIG. 4).
  • a moisturizing agent may be further applied to the metal film 30. By removing the moisturizing agent, the third capturing body 800 appears on the surface of the metal film 30 in a state having high activity.
  • the third capturer 800 is, for example, carboxymethyldextran 820 containing streptavidin 810.
  • the control unit 160 causes the analysis chip 10 to supply the sample solution and react for a certain time (step 410 in FIG. 4).
  • the biotinylated antibody 700 of the second complex 650 in the sample solution specifically binds to the streptavidin 810 of the third capture body 800, and is attached to the third capture body 800 in the analysis chip 10 in the sample solution.
  • the control unit 160 causes the analysis chip 10 to repeat, for example, the above cleaning operation five times (step 411 in FIG. 4). By this operation, surplus fluorescent magnetic particles 400 are substantially removed from the analysis chip 10.
  • the control unit 160 injects a buffer solution (cleaning solution) into the analysis chip 10 to fill the flow path between the metal film 30 and the flow path cover 40 with the buffer solution, and the prism 20 and the metal film 30 in the analysis chip 10.
  • the fluorescent material in the second complex is excited by the surface plasmon on the metal film 30 by causing the excitation light ⁇ to be incident on the interface with the substrate at an incident angle greater than the total reflection angle and excited by the enhanced electric field. Is detected by the light receiving sensor 127 ((H) in FIG. 2, step 412 in FIG. 4).
  • the fluorescence of the fluorescent material of the fluorescent magnetic particle 400 by the SPFS is detected with high sensitivity.
  • a substance that non-specifically binds to the third capturing body 800 is substantially present on the surface of the metal film 30. Therefore, other fluorescence that becomes a noise signal is not substantially detected.
  • the reaction time and the dispersion time in the washing operation may be the same or different.
  • the time may be uniformly 10 minutes, or may be different from, for example, 5 minutes, 3 minutes, 1 minute, 30 seconds, and 10 seconds.
  • the sample liquid is prepared by sequentially reacting the first capture body and the second capture body, and is fixed on the metal film 30 of the analysis chip 10 by the third capture body.
  • the avidin-biotin reaction which is a strong affinity reaction, can be used, and is more effective from the viewpoint of shortening the reaction time and increasing the sensitivity of the fluorescence intensity.
  • FIG. 5 is a diagram schematically showing an intermediate state in another example of SPFS according to the present embodiment.
  • the control unit 160 supplies the fluorescent magnetic particles 400 from the container 131 containing the fluorescent magnetic particles 400 to the preparation container 131 (FIG. 5A), and then from the container 131 containing the sample stock solution.
  • the sample stock solution is supplied to the preparation container 131, and further, the biotinylated antibody 700 is supplied from the container 131 containing the biotinylated antibody 700 to the preparation container 131, and these are reacted for a certain time.
  • a second complex 650 is generated ((B) in FIG. 5).
  • control unit 160 places the magnet 137 in the vicinity of the peripheral wall of the preparation container 131, and draws the second composite 650 in the preparation container 131 to the wall surface of the container 131 ((C) of FIG. 5). ), The liquid in the container 131 is removed by suction. As the liquid is removed, the contaminants 600 and the excess biotinylated antibody 700 in the container 131 are removed from the container 131.
  • control part 160 repeats said washing
  • the control unit 160 supplies an appropriate amount of cleaning liquid to the preparation container 131. In this way, a sample solution for measurement is prepared.
  • control unit 160 transports the analysis chip 10 to the operation position of the sample solution preparation unit 130, and removes the moisturizing agent applied to the surface of the metal film 30 of the analysis chip 10 ((F) in FIG. 5). Then, the sample solution is supplied to the analysis chip 10 and allowed to react for a certain time, and the above-described washing operation is repeated, for example, five times for the analysis chip 10. By this operation, surplus fluorescent magnetic particles 400 are substantially removed from the analysis chip 10.
  • the control unit 160 injects a buffer solution into the analysis chip 10 to fill the flow path between the metal film 30 and the flow path lid 40 with the buffer solution, and the interface between the prism 20 and the metal film 30 in the analysis chip 10.
  • the excitation light ⁇ is incident on the metal film 30 at an incident angle greater than the total reflection angle to generate an enhanced electric field due to surface plasmons on the metal film 30, and the fluorescence of the fluorescent substance in the second complex excited by the enhanced electric field is generated. Detection is performed by the light receiving sensor 127 ((G) in FIG. 5).
  • preparing the sample liquid by simultaneously reacting the first capture body and the second capture body can further reduce the number of washing operations, and can reduce the cost. This is more effective from the viewpoint of further shortening the time required from sample preparation to highly sensitive fluorescence detection.
  • FIG. 6 is a diagram schematically showing a configuration of another example of an SPFS apparatus capable of performing SPFS according to the present embodiment.
  • the SPFS device 900 is a device for GC-SPFS.
  • the diffraction grating 37 is formed on the surface of the metal film 35 of the analysis chip 15 where the second complex is to be fixed, and the light source unit 111 of the excitation optical system unit 110 is diffracted. It is arranged on the surface side of the metal film 35 so as to irradiate the grating 37 with the excitation light ⁇ , and the light receiving sensor 127 of the light receiving optical system unit 120 is generated from the second complex fixed to the diffraction grating 37.
  • the light receiving optical system unit 120 is mainly composed of a light receiving sensor 127 and a fluorescent filter 128 for removing the excitation light ⁇ from the fluorescent ⁇ .
  • the configuration is the same as that of the SPFS device 100.
  • the analysis chip 15 includes a substrate 25 and a metal film 35 formed on the substrate 25.
  • a diffraction grating 37 is formed on the metal film 35.
  • a third capturing body is fixed to the diffraction grating 37.
  • the substrate 25 is a support member for the metal film 35.
  • the material of the substrate 25 is not particularly limited as long as it has mechanical strength capable of supporting the metal film 35.
  • Examples of the material of the substrate 25 include inorganic materials such as glass, quartz, and silicon, and resins such as polymethyl methacrylate, polycarbonate, polystyrene, and polyolefin.
  • the metal film 35 is configured in the same manner as the metal film 30 described above except that it has a diffraction grating 37.
  • the diffraction grating 37 is formed so as to generate an evanescent wave when the metal film 35 is irradiated with light.
  • the diffraction grating 37 may be a one-dimensional diffraction grating or a two-dimensional diffraction grating.
  • the diffraction grating 37 can be formed, for example, by imparting an uneven shape to the metal film 35 disposed on the flat substrate 25, or on the substrate 25 to which an uneven shape has been previously provided. It can be formed by forming the metal film 35.
  • the SPFS according to the present embodiment can be performed in the same manner as the method using the SPFS apparatus 100 described above except that the second complex is fixed on the diffraction grating 37 and the fluorescence ⁇ is detected.
  • the method using the SPFS apparatus 900 can detect fluorescence with high sensitivity in the same manner as the method using the SPFS apparatus 100.
  • the syringe 134 need not be cleaned. For this reason, it is preferable from the viewpoint of suppressing the influence of impurities and the like. In the case where the syringe 134 is not configured to be replaceable, replacement of the syringe 134 becomes unnecessary by further including a configuration or operation for cleaning the inside of the syringe 134. For this reason, it is preferable from a viewpoint of suppressing consumption of the syringe 134.
  • the sample solution preparation unit 130 further includes a device that detects the position of the tip of the syringe 134, and adjusts the relative height between the syringe 134 and the analysis chip 10 to a constant level in the analysis chip 10. It is preferable from the viewpoint of managing the remaining liquid amount at a constant level.
  • the liquid storage part that stores the liquid to be supplied to the analysis chip 10 is protected by the multilayer film, and the syringe 134 passes through the multilayer film for supplying the liquid. It is preferable that the analysis chip 10 and the syringe 134 are configured so as to seal the liquid container.
  • the SPFS irradiates the excitation light toward the metal film on which the detection target substance labeled with the fluorescent substance is fixed, and the excitation light is applied to the metal film.
  • a surface plasmon enhanced fluorescence measurement method for measuring the detected substance by detecting fluorescence of the fluorescent substance excited by an enhanced electric field generated by surface plasmon generated by irradiation of A first step of allowing the fluorescent magnetic particles containing the fluorescent substance to carry the substance to be detected in the sample stock solution in a container disposed independently; and generating a magnetic field in the container to generate the fluorescent magnetic particles.
  • a second step of preparing a sample solution for measurement by removing impurities in the sample stock solution in the container while fixing the sample and a second step of fixing the substance to be detected in the sample solution to the metal film 3 mechanics And, including the.
  • the relative fall of the sensitivity of the to-be-detected substance which the noise signal by a foreign material brings about can be suppressed. Therefore, since the measurement result of the substance to be detected by SPFS unique to high sensitivity is not buried in the noise signal, the above-mentioned SPFS can obtain the measurement result of the substance to be detected more sensitively than the conventional SPFS.
  • the fluorescent magnetic particles have a first capturing body that specifically binds to the detected substance on the surface thereof, and the detected substance is used as the first capturing body in the first step.
  • the substance to be detected is supported on the fluorescent magnetic particles by specifically binding, and further, before the third step, the substance to be detected carried on the fluorescent magnetic particles is specific to the substance to be detected.
  • a fourth step of binding a second capture body that binds automatically, and the metal film has a third capture body for capturing the second capture body on the surface thereof, and the third In the step, the second capturing body is captured by the third capturing body to fix the detected substance to the metal film, thereby reducing noise signals due to contaminants and the detected substance.
  • highly sensitive measurement results It is more effective and more from the viewpoint of easily performing under mild conditions.
  • the SPFS devices 100 and 900 used for the SPFS are both labeled with a light source for irradiating excitation light toward a metal film on which a target substance labeled with a fluorescent substance is to be fixed, and a fluorescent substance.
  • a detector for detecting fluorescence generated by irradiating the metal film with the excitation light when the detected substance to be detected is fixed, and a sample containing the substance to be detected to be fixed to the metal film
  • any of the SPFS apparatuses 100 and 900 the substance to be detected can be fixed in the container by the magnetic field generator by supporting the substance to be detected on the aforementioned fluorescent magnetic particles. Therefore, each of the SPFS apparatuses 100 and 900 can perform SPFS in which the relative decrease in the sensitivity of the target substance caused by the noise signal due to the contaminants is suppressed.
  • the metal film is disposed on a part of the surface of a prism made of a dielectric, and the light source is disposed so as to irradiate the excitation light toward the metal film through the prism. Therefore, it is possible to implement PC-SPFS that achieves the above effects.
  • the metal film has a diffraction grating on the surface thereof, and the light source is arranged so as to irradiate the excitation light toward the diffraction grating. It is possible to implement GC-SPFS.
  • the surface plasmon enhanced fluorescence measurement method according to the present invention can measure a substance to be detected with high sensitivity because noise signals due to contaminants are greatly reduced. Therefore, the measurement method is expected to be used in a technical field where higher sensitivity measurement is required, such as research and development of biomolecules, and to contribute to the development of the technical field.

Abstract

Dans un récipient (131), une substance devant être détectée (500) est amenée à se combiner avec des particules magnétiques fluorescentes (400) ayant une substance fluorescente. Lorsque le liquide dans le récipient (131) est évacué, le premier composite (450) obtenu par la combinaison est fixé à la paroi interne du récipient (131) par un aimant (137). L'utilisation d'un liquide de nettoyage pour le récipient (131), la fixation du premier composite (450) par l'aimant (137) et l'évacuation du liquide sont répétés pour éliminer des impuretés de l'échantillon brut liquide. Un échantillon liquide ainsi obtenu est fixé sur le film métallique d'une puce analytique (10) pour la spectroscopie par fluorescence renforcée par plasmon de surface (SPFS) et soumis à une spectroscopie par fluorescence renforcée par plasmon de surface.
PCT/JP2015/059358 2014-05-23 2015-03-26 Procédé de spectroscopie par fluorescence renforcée par plasmon de surface et dispositif pour spectroscopie par fluorescence renforcée par plasmon de surface WO2015178099A1 (fr)

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