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WO2015194663A1 - Puce de détection pour utilisation en spectroscopie de fluorescence amplifiée par le champ du plasmon de surface (spfs), procédé de mesure par spfs, et trousse de spfs - Google Patents

Puce de détection pour utilisation en spectroscopie de fluorescence amplifiée par le champ du plasmon de surface (spfs), procédé de mesure par spfs, et trousse de spfs Download PDF

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Publication number
WO2015194663A1
WO2015194663A1 PCT/JP2015/067736 JP2015067736W WO2015194663A1 WO 2015194663 A1 WO2015194663 A1 WO 2015194663A1 JP 2015067736 W JP2015067736 W JP 2015067736W WO 2015194663 A1 WO2015194663 A1 WO 2015194663A1
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Prior art keywords
fading
solution
substance
sensor chip
measurement
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English (en)
Japanese (ja)
Inventor
幸司 宮崎
敬三 高野
井上　直哉
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Konica Minolta Inc
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Konica Minolta Inc
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Priority to JP2016529545A priority Critical patent/JP6500897B2/ja
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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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a sensor chip and a measurement method according to surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS). More specifically, the present invention relates to a sensor chip and a measuring method according to SPFS for the purpose of preventing fading of a fluorescent substance that labels a measurement target substance and preventing deterioration of a capture substance immobilized on the surface of the sensor chip. .
  • SPFS surface plasmon resonance excitation enhanced fluorescence spectroscopy
  • SPFS Surface Plasmon-field ⁇ enhanced Fluorescence Spectroscopy
  • the SPFS measurement method surface plasmons (dense waves) are generated on the surface of the metal thin film under conditions where excitation light such as laser light emitted from a light source attenuates total reflection (ATR) on the surface of the metal thin film.
  • excitation light such as laser light emitted from a light source attenuates total reflection (ATR) on the surface of the metal thin film.
  • ATR total reflection
  • the amount of photons contained in the excitation light emitted from the light source is increased by several tens to several hundreds, and the electric field enhancement effect of the surface plasmon light can be obtained.
  • a fluorescent substance bonded to a measurement target compound captured in the vicinity of the surface of the metal thin film is efficiently excited by this electric field enhancement effect, and the generated fluorescent emission is observed, whereby a very small amount of measurement target is measured. Compounds can also be measured.
  • FIG. 1 shows an example of a schematic configuration of an apparatus (hereinafter referred to as “SPFS apparatus”) for carrying out a surface plasmon resonance excitation enhanced fluorescence measurement method.
  • the SPFS device 100 includes a sensor chip loading unit 111, and the sensor chip 110 is loaded in the sensor chip loading unit 111.
  • the sensor chip 110 is formed at a predetermined position of a dielectric member 112, a metal thin film 113 formed on the main surface 112 a of the dielectric member 112, and a microchannel 117 on the metal thin film 113.
  • the sensor unit 116 is provided.
  • the sensor unit 116 is a region where a substance that captures the measurement target substance (hereinafter also referred to as a capture substance) is immobilized.
  • the fine channel 117 is formed on the main surface 112 a of the dielectric member 112 via the metal thin film 113 by the thin layer member 114 and the lid member 115.
  • the sensor chip 110 loaded in the sensor chip loading unit 111 of the SPFS device 100 is incident on the dielectric member 112 side from the incident surface 112i of the dielectric member 112, and a total reflection attenuation (ATR) occurs in the metal thin film 113.
  • ATR total reflection attenuation
  • a light detection means 130 that receives the fluorescence 131 emitted from the fluorescent substance that labels the measurement target substance captured by the sensor unit 116 is provided.
  • a light collecting member 126 for efficiently collecting the fluorescent light 131, and a wavelength selection for selectively transmitting only the fluorescent light 131 by removing light other than the fluorescent light 131.
  • a functional member 133 is provided.
  • Such an SPFS apparatus 100 is used as follows.
  • a sample liquid (measurement sample) containing a measurement target substance is caused to flow into the sensor unit 116 through the fine channel 117, and the measurement target substance is captured by a capture substance fixed to the sensor unit 116.
  • a substance that fluorescently labels the measurement target substance for example, a fluorescently labeled secondary antibody (hereinafter also referred to as a fluorescently labeled substance) is similarly allowed to flow through the microchannel 117 so that the sensor unit 116 is filled with the fluorescent substance. It is assumed that the labeled measurement target substance is captured.
  • the evanescent wave and the surface plasmon from the metal thin film 113 are irradiated from the light source 120 via the dielectric member 112 with the excitation light 121 at a predetermined incident angle ⁇ at which total reflection attenuation occurs in the metal thin film 113.
  • An electric field enhanced by resonance with the fluorescent substance 131 is generated, thereby efficiently exciting the fluorescence 131 by the fluorescent substance that labels the measurement target substance captured by the sensor unit 116.
  • Patent Document 1 International Publication WO2013 / 147081
  • a pathological section is immunostained and a fluorescent stained image is observed
  • the stained pathological section is used as a permanent specimen.
  • a method for preventing fading by improving the light resistance of a fluorescent material by blending an anti-fading agent into the encapsulant for the purpose is disclosed.
  • Patent Document 1 discloses a method for preventing the fading of a fluorescent substance in an SPFS measurement system in which processing such as fixation of a capture substance, capture of a measurement target substance, and binding of a fluorescently labeled secondary antibody is performed on the surface of a sensor chip. Not listed.
  • Patent Document 2 International Publication WO2011 / 111176
  • a blocking agent nonspecific adsorption protein such as BSA
  • BSA nonspecific adsorption protein
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2012-168165 describes a highly hydrophilic substance such as carboxymethyldextran.
  • the excitation light is irradiated to the fluorescent substance, and the sensor chip is irradiated with the excitation light before measuring the signal of the target substance labeled with the fluorescent substance.
  • the signal is measured after checking a predetermined incident angle at which total reflection attenuation (ATR) occurs in the upper metal thin film.
  • ATR total reflection attenuation
  • the detection value is positive even though the measurement target substance does not exist.
  • substances other than the measurement target substance contained in the specimen are adsorbed nonspecifically on the sensor chip and detected as signals.
  • the dissociation rate due to specific binding to the measurement target substance is different from the dissociation rate due to non-specific adsorption with substances other than the measurement target substance, and the latter dissociation progresses with time. It is known.
  • the signal obtained by the SPFS measurement is first measured as the first signal, the measurement is performed again after a certain amount of time has passed, and the obtained signal is designated as the second signal.
  • a method of detecting the detected first signal (measurement target substance) highly sensitively and quantitatively by comparing the signal decrease rates is conceivable. Even in such a case, since it is necessary to irradiate the excitation light a plurality of times, the deterioration of the fluorescent material is likely to proceed, which may affect the signal of the measurement target material.
  • the present invention can prevent the fading of the fluorescent substance that labels the measurement target substance captured by the sensor unit in the SPFS measurement method even under a severe situation in which the excitation light is irradiated a plurality of times as described above.
  • another object is to provide means for preventing deterioration of the trapping substance fixed to the sensor unit.
  • the present inventors use a solution containing an anti-fading agent for a fluorescent substance at the time of manufacturing a sensor chip for SPFS or measuring SPFS using the sensor chip. It was found that the measured value can be stabilized by preventing fading. In particular, when an antioxidant is used as an anti-fading agent during the manufacture of a sensor chip, not only can the fluorescent material be prevented from fading, but also the degradation of the captured substance fixed on the surface of the sensor chip can be prevented. It has also been found that the reactivity with a substance can be improved, and the present invention has been completed.
  • a dielectric member, a metal thin film formed on a main surface of the dielectric member, and a substance to be measured formed on a part of the metal thin film are specifically specified.
  • a sensor chip for a measurement method based on surface plasmon resonance excitation enhanced spectroscopy which has a region where a capture substance to be captured is fixed, and contains an anti-fading agent for the region where the capture substance is fixed
  • a sensor chip for SPFS which is subjected to anti-fading treatment by bringing a solution into contact therewith.
  • a dielectric member, a metal thin film formed on a main surface of the dielectric member, and a substance to be measured formed on a part of the metal thin film are specified.
  • a measurement method based on surface plasmon resonance excitation enhanced spectroscopy using a sensor chip having a region to which a capture substance to be captured is fixed, in the vicinity of a fluorescent labeling agent bound to the measurement target substance captured by the capture substance
  • an SPFS measurement method including a step of measuring fluorescence by irradiating excitation light in the presence of an antifading agent.
  • the present invention provides a dielectric member, a metal thin film formed on a main surface of the dielectric member, and a capture specifically capturing a measurement target substance formed on a part of the metal thin film.
  • a sensor chip for a measurement method based on surface plasmon resonance excitation enhanced spectroscopy having a region to which a substance is fixed, and contacting a solution containing an anti-fading agent to the region to which the capture substance is fixed
  • a kit including at least one that has undergone fading prevention treatment.
  • the dielectric member, the metal thin film formed on the main surface of the dielectric member, and the capture substance that specifically captures the measurement target substance formed on a part of the metal thin film are fixed.
  • a sensor chip for a measurement method based on surface plasmon resonance excitation enhanced spectroscopy and a kit containing at least an anti-fading solution or an anti-fading agent and a solvent for preparing the same.
  • the fluorescent substance is prevented from fading and the fluorescence intensity measurement value is stabilized, and preferably the measurement is performed by preventing the capture substance fixed on the sensor part from being deteriorated.
  • the binding property with the target substance can be maintained, and the reliability with respect to the quantitativeness of the measurement target substance can be increased.
  • such a sensor chip fabrication method and an SPFS measurement method with the same effects can be efficiently performed using the same system as the conventional sensor chip fabrication and SPFS measurement. Can do.
  • the anti-fading effect of the present invention is excellent, so that even if the excitation light is irradiated multiple times or the output of the excitation light is high, the fluorescent material is not easily faded and the measured value of the fluorescence intensity is stable. It is possible to improve the reliability with respect to the quantitativeness of the measurement target substance.
  • FIG. 1 is a schematic diagram for explaining a general configuration of a surface plasmon resonance excitation enhanced fluorescence measuring apparatus.
  • FIG. 2 is a schematic diagram illustrating one embodiment of a sensor chip of the present invention having one sensor unit.
  • FIG. 3 is a schematic view illustrating an embodiment of the sensor chip of the present invention having one sensor portion and having a flow path formed therein.
  • FIG. 4 is a schematic diagram for explaining an embodiment of a sensor chip of the present invention having three sensor portions.
  • FIG. 5 is a schematic diagram for explaining an embodiment of a process for performing a fading prevention process for producing the sensor chip of the present invention.
  • FIG. 6 is a graph showing changes in the S / N ratio with the passage of time, measured in the examples and comparative examples. The horizontal axis represents time (minutes), and the vertical axis represents the ratio of the S / N ratio at each time when the S / N ratio at 0 minutes is 1.
  • the SPFS sensor chip of the present invention specifically captures a dielectric member, a metal thin film formed on the main surface of the dielectric member, and a substance to be measured formed on a part of the metal thin film.
  • a sensor chip for a measurement method (SPFS measurement method) based on surface plasmon resonance excitation enhanced spectroscopy, which has a region (hereinafter referred to as “sensor unit”) to which the capture material to be immobilized is fixed.
  • the anti-fading treatment is carried out by bringing a solution containing the anti-fading agent (hereinafter referred to as “fading prevention treatment liquid”) into contact with the region including the region where is fixed.
  • fading prevention treatment liquid a solution containing the anti-fading agent
  • FIG. 2 shows an example of a sensor chip having one sensor part.
  • FIG. 3 shows an example of using a sensor chip having one sensor unit as shown in FIG.
  • FIG. 4 is an example of a sensor chip having three sensor portions. In the case where a sensor chip having a plurality of sensor portions as shown in FIG. 4 is used, a channel can be formed in the upper portion as in FIG.
  • the sensor chip 200 includes a dielectric member 201, a metal thin film 202 formed on the main surface 201 a of the dielectric member 201, and a sensor unit 203 provided on the metal thin film 202. And have.
  • the entire region on the metal thin film 202 does not need to be a sensor portion, and one or a plurality of sensor portions may be formed in a partial region on the metal thin film 202. It is only necessary that the region including the sensor unit 203 be subjected to the fading prevention treatment with the fading prevention treatment liquid, and it is not necessary to perform the fading prevention processing on all the regions on the metal thin film 202.
  • a measurement sample or the like flows by installing a thin layer member 204 on the metal thin film 202 on which the sensor unit 203 is formed and installing a lid member 205 on the thin layer member 204.
  • a flow path 206 can be formed.
  • a measurement sample or the like is introduced into the flow path 206 from the inflow / discharge port 207 using a pipette or the like, and the introduced measurement sample is accumulated in the liquid reservoir 208.
  • the material of the thin layer member 204 is, for example, an acrylic adhesive sheet, and the thickness thereof may be determined according to the height of the target flow path, and is, for example, 20 to 1000 ⁇ m.
  • the material of the lid member 205 is, for example, a resin material similar to a dielectric member described later.
  • the shape of the inflow / discharge port 207 and the liquid reservoir 208 is such that the measurement sample collected in the liquid reservoir 208 can be easily stirred by repeatedly sucking and injecting the measurement sample with a pipette or the like at the inflow / discharge port 207. It can be set as appropriate.
  • the sensor chip 200 is used in combination with the thin layer member 204 and the lid member 205 as described above to form a flow path communicating with the inflow / discharge port 207 and the liquid reservoir 208, and the bottom surface of the flow path.
  • the present invention is not limited to the positioning of the sensor unit 203.
  • a well having no opening other than the upper end is formed, a sensor part 203 is positioned on the bottom of the well, a measurement sample is injected into the well, and after the reaction It may be used such as sucking.
  • the anti-fading agent is held on the surface or inside of the sensor part.
  • the anti-fading agent is considered to be immobilized by interaction with the SAM. .
  • the amount of the anti-fading agent present in the sensor portion is not particularly limited, and can be appropriately adjusted in consideration of the anti-fading effect.
  • the amount of anti-fading agent in such a range can be held in the sensor unit by applying the amount of anti-fading agent used or the processing time in the first or second embodiment of anti-fading processing described later. Is possible.
  • the sensor chip 200 is used by being loaded into an SPFS device.
  • a measurement sample or the like is injected from the inlet / outlet 207.
  • a measurement sample is injected from the inflow / discharge port 207 with a pipette or the like and reacted for a predetermined time, whereby the measurement target substance is captured by the capture substance fixed to the sensor unit 203.
  • the measurement sample is discharged from the inlet / outlet 207 by a pipette or the like.
  • a cleaning liquid for example, PBS in which a surfactant is dissolved
  • PBS in which a surfactant is dissolved
  • the cleaning liquid is sucked from the inlet / outlet 207.
  • a solution of the fluorescent substance is injected from the inflow / exhaust port 207 and reacted for a predetermined time as in the case of the measurement sample. -Suction from the outlet 207.
  • the cleaning liquid is again injected from the inlet / outlet 207 to wash away the fluorescent substance solution remaining in the flow path 206 and the non-specifically adsorbed fluorescent substance, and then the cleaning liquid in which they are dissolved is sucked from the inlet / outlet outlet 207. To do.
  • a measurement liquid for example, PBS
  • PBS surface plasmon excitation enhanced fluorescence spectroscopy
  • the substance to be measured in the present invention refers to a substance that specifically binds to a capture substance immobilized on the surface of a sensor chip (sensor part), which is detected or quantified by SPFS. Sugars, nucleic acids, cells, and other biological materials.
  • the measurement sample in the present invention refers to a liquid sample that is provided to the sensor chip of the present invention for SPFS measurement for quantifying the measurement target substance.
  • the measurement sample is typically a biological sample, that is, a sample collected from a human or animal and a sample containing a substance derived from the biological sample, but the measurement prepared as a model of such a biological sample. A solution containing the target substance is also included.
  • the dielectric member 201 is formed in a hexahedral shape with a trapezoidal cross section.
  • the upper surface is a main surface 201a, and one side surface of the hexahedron is an incident surface 201i that is an incident surface for excitation light.
  • the shape of the dielectric member 201 is not limited to the hexahedral shape as described above. It has at least a main surface 201a where the sensor unit 203 is formed and an incident surface 201i on which excitation light is incident. The excitation light incident from the incident surface 201i passes through the dielectric member 201 and is totally reflected.
  • the sensor unit 203 may be configured to irradiate with a predetermined incident angle ⁇ as a condition.
  • the shape may be a cone shape, a pyramid shape such as a triangular pyramid or a quadrangular pyramid, or a kamaboko shape. Good. It is also possible to form two or more incident surfaces 201 i on the dielectric member 201.
  • the material of the dielectric member is not particularly limited as long as it is formed of a material that is optically transparent at least with respect to excitation light. However, in order to provide a sensor chip that is inexpensive and excellent in handleability, it is formed of, for example, a resin material.
  • the dielectric member is formed from a resin material
  • a resin material for example, polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate, polyolefins such as polyethylene (PE) and polypropylene (PP), cyclic olefin copolymer (COC), cyclic Polycyclic olefins such as olefin polymer (COP), vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polystyrene, polyether ether ketone (PEEK), polysulfone (PSF), polyether sulfone (PES), polycarbonate ( PC), polyamide, polyimide, acrylic resin such as polymethyl methacrylate resin (PMMA), triacetyl cellulose (TAC), or the like can be used.
  • PET polyethylene terephthalate
  • PP polypropylene
  • COC cyclic olefin copolymer
  • COP cyclic Polycycl
  • the formation method of the dielectric member is not particularly limited.
  • the resin material as described above it can be formed by injection molding.
  • the metal thin film 202 can use the same metal as the metal thin film which comprises the sensor chip used for a general SPFS apparatus. That is, the metal thin film is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and more preferably gold. About these metals, the form of the alloy may be sufficient and the thing which laminated
  • a conventional method may be used.
  • an electron beam heating vacuum deposition method a resistance heating vacuum deposition method, a magnetron sputtering method, a plasma assisted sputtering method may be used.
  • a metal thin film can be formed on the main surface of the dielectric member by a vacuum film formation method such as an ion assisted deposition method or an ion plating method.
  • the thickness of the metal thin film is preferably 5 to 500 nm for gold, 5 to 500 nm for silver, 5 to 500 nm for aluminum, 5 to 500 nm for copper, 5 to 500 nm for platinum, and 5 to 500 nm for these alloys.
  • gold is preferably 20 to 70 nm
  • silver is 20 to 70 nm
  • aluminum is 10 to 50 nm
  • copper is 20 to 70 nm
  • platinum is 20 to 70 nm
  • these alloys are more preferably 10 to 70 nm.
  • the sensor part 203 is provided in the one part area
  • a plurality of sensor units may be provided, and different capture substances may be fixed to each sensor unit (see FIG. 4).
  • the capture substance is a substance that specifically captures the measurement target substance (protein, lipid, sugar, nucleic acid, or other substance).
  • the capture substance include an antibody against an antigen, an enzyme against a substrate / coenzyme, a receptor against a hormone, protein A / protein G against an antibody, avidin against biotin, calmodulin against calcium, a lectin against a sugar, and the like.
  • the substance to be measured is a nucleic acid
  • a nucleic acid having a sequence that specifically binds to it can also be used as a capture substance.
  • a conventional method may be used. For example, a modification group that generates a specific bond is introduced on the surface of the metal thin film, and this capture group is supported by this modification group.
  • the trapping substance can be fixed on the metal thin film by introducing the reactive group and bonding the modifying group and the reactive group.
  • the surface of the metal thin film is treated with a silane coupling agent having an amino group at the terminal and modified with an amino group, Subsequently, it is treated with NHS (N-hydroxysuccinimide) -PEG4-biotin, biotin is bound to the amino group, avidin is reacted with biotin, and then a biotinylated capture substance (eg, antibody) is reacted. As a result, the trapping substance can be fixed on the metal thin film.
  • a silane coupling agent having an amino group at the terminal and modified with an amino group
  • the surface of the metal thin film is treated with a silane coupling agent having a carboxyl group at the end and modified with a carboxyl group, followed by treatment with EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and NHS.
  • EDC 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride
  • NHS N-(2-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride)
  • the capture substance can also be immobilized on the metal thin film by reacting a capture substance (for example, an antibody) having an amino group after the carboxyl group has been converted into an active ester.
  • a SAM Self-Assembled Monolayer
  • the SAM serves as a foundation for fixing the trapping substance on the metal thin film.
  • a carboxyalkanethiol having about 4 to 20 carbon atoms for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.
  • Carboxyalkanethiol having 4 to 20 carbon atoms has properties such as little optical influence of SAM formed using it, that is, high transparency, low refractive index, and thin film thickness. Therefore, it is preferable.
  • the SAM formation method is not particularly limited, and a conventionally known method can be used.
  • a specific example is a method of immersing a metal thin film in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories).
  • the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the gold thin film to form a SAM.
  • the method for immobilizing the capture substance on the formed SAM is not particularly limited, and a conventionally known method can be used. For example, the above-described method of treating with EDC and NHS can be used.
  • the shape and area of the region where the trapping substance is fixed on the metal thin film, that is, the sensor portion is not particularly limited, but is preferably equal to or larger than the area of the irradiation region irradiated with the incident excitation light.
  • the shape of the sensor unit is preferably the same as the region irradiated with the excitation light.
  • a member such as a solution storage member described later is provided on the metal thin film according to the shape of the region to fix the capture substance.
  • the reagent used in the method for fixing the capture substance on the metal thin film may be added to this member.
  • the region including the sensor unit 203 is subjected to a fading prevention process for preventing the fading of the fluorescent labeling substance, particularly the fluorescent material molecule.
  • the anti-fading agent used for the anti-fading treatment is not particularly limited as long as it is a substance having an effect of preventing the fading of the fluorescent labeling substance used in the SPFS measurement system.
  • An antioxidant (antioxidant) that can effectively prevent the oxidation of the labeling substance is suitable. Further, the antioxidant may prevent oxidation of a capture substance immobilized on the sensor unit 203, for example, a protein such as an antibody or a lectin, or other biological substance during storage or contact with a specimen such as blood. It is also suitable in that it can be done.
  • the antioxidant also functions as a protective agent for the capture substance.
  • the anti-fading treatment liquid to be brought into contact with the sensor unit 203 is preferably prepared using water or a buffer solution as a solvent so that the function of the capture substance fixed to the sensor unit 203 is not hindered. It is preferably water-soluble.
  • the water-soluble antioxidant can be selected from various antioxidants used in the technical field to which the present invention belongs and other technical fields.
  • Typical antioxidants include phenolic antioxidants, amine antioxidants, phosphorus antioxidants, sulfur antioxidants and unsaturated hydrocarbon antioxidants.
  • phenolic antioxidants include natural products-derived phenols and hindered phenols.
  • phenols derived from natural products various flavonoids having a phenolic hydroxyl group, specifically, flavonols having a phenolic hydroxyl group such as quercetin, rutin, myricetin, myricitrin, fisetin, and morin. And glycosides thereof: hesperetin, hesperidin, methyl hesperidin, naringenin, naringin, etc., flavanones having a phenolic hydroxyl group and glycosides thereof; flavones having a phenolic hydroxyl group, such as apigenin, luteolin; phenols such as taxifolin, etc.
  • Flavonols having a functional hydroxyl group having a functional hydroxyl group
  • catechins such as catechin, gallocatechin, gallocatechin gallate and epigallocatechin gallate
  • isofines such as genistein and daidzein (4 ′, 7-dihydroxyisoflavone) Labones
  • anthocyanidins having a phenolic hydroxyl group such as cyanidin, delphinidin, malvidin, pelargonidin, and peonidin.
  • phenols derived from natural products include polyphenols such as gallic acid and gallic acid esters such as propyl gallate. Tannin, tocopherol, tocotrienol and the like can also be used as a phenolic antioxidant.
  • hindered phenols examples include 2,6-di-tert-butyl-4-methylphenol, p-phenylazophenol, 4-nitroaniline, 2,6-di-tert-butyl-4-hydroxymethylphenol, N , N'-disalicylical-1,2-propanediamine, triethylene glycol-bis [3- (3-tert-butyl-5-methyl-4-hydroxyphenyl) propionate], 1,6-hexanediol-bis [3 -(3,5-di-tert-butyl-4-hydroxyphenyl) propionate], 2,4-bis- (n-octylthio) -6- (4-hydroxy-3,5-di-tert-butylanilino)- 1,3,5-triazine, pentaerythrityl tetrakis [3- (3,5-di-tert-butyl-4-hydroxyphenyl) propio , 2, ⁇ ⁇ 2-thio-diethylenebis [
  • amine-based antioxidants examples include tertiary amines such as 1,4-diazabicyclo [2.2.2] octane (DABCO: registered trademark, Air Products & Chemicals, Inc.); phenothiazine, phenyl- ⁇ -naphthylamine, p , P′-dioctyldiphenylamine, aromatic amines such as p-phenylenediamine; bis (2,2,6,6-tetramethyl-4-piperidyl) sebacate, 2,2,6,6-tetramethylpiperidine, etc. Hindered amines; and alkaloids such as caffeine.
  • an aromatic amine is mentioned as a suitable amine-based antioxidant, and among them, phenothiazine is mentioned as a particularly suitable amine-based antioxidant.
  • phosphorus antioxidants examples include 2-mercaptobenzimidazole, triphenyl phosphite, tris (2-carboxyethyl) phosphine hydrochloride (TCEP HCl), diisodecylpentaerythritol diphosphite, 9,10-dihydro-9- And oxa-10-phosphaphenanthrene-10-oxide.
  • sulfur-based antioxidants include sulfides such as didodecyl 3,3-thiodipropionate and 2,2′-thiodiethanol, dibenzyl disulfide, DL- ⁇ lipoic acid (thioctic acid), 3,6-dithia- Examples thereof include disulfides such as 1,8-octanediol, and thiols such as dithiothreitol and octanediol.
  • unsaturated hydrocarbon antioxidants include carotenes such as lutein, lycopene, astaxanthin, canthaxanthin, capsanthin, myxoxanthophyll, zeaxanthin, carotene, retinoic acid, carotenoids, xanthophylls, ascorbic acid (L-ascorbic acid) , Isoascorbic acid (erythorbic acid) or salts thereof such as Na), tocotrienol, unsaturated fatty acids and the like.
  • carotenes such as lutein, lycopene, astaxanthin, canthaxanthin, capsanthin, myxoxanthophyll, zeaxanthin, carotene, retinoic acid, carotenoids, xanthophylls, ascorbic acid (L-ascorbic acid) , Isoascorbic acid (erythorbic acid) or salts thereof such as Na), tocotrienol,
  • phenol-based antioxidants and amine-based antioxidants such as 1,4-diazabicyclo [2.2. 2] Octane (DABCO), p-phenylenediamine and the like are preferable.
  • the unsaturated hydrocarbon antioxidants such as ascorbic acid are also suitable antioxidants.
  • ascorbic acid is a natural product (natural product), it has a low influence (influence) on measurement values and is inexpensive, and thus is preferable.
  • Some phenolic antioxidants may contain an alcoholic hydroxyl group in addition to the phenolic hydroxyl group. In this case, it is preferable to have two or more phenolic hydroxyl groups and alcoholic hydroxyl groups.
  • Antioxidants may be used alone or in combination of two or more. Further, when a sensor chip having a plurality of sensor parts is used, the antioxidant used for each sensor part may be changed as necessary.
  • the antioxidant preferably has no absorption at an absorption wavelength of 450 to 600 nm, and preferably has no emission at an emission wavelength of 500 to 700 nm, so as not to adversely affect the measured fluorescence intensity emitted by the fluorescent labeling substance. Absorption may lead to a decrease in measured fluorescence intensity. In addition, if there is light emission, noise may increase when measuring the fluorescence intensity.
  • the absence of antioxidant absorption means that when the xylene solution of 1 mg / mL concentration of antioxidant is prepared and the absorbance is measured in a 10 mm cell, the absorbance at 450 nm and 600 nm is both 0.5 or less. Say something.
  • substances that can be used as anti-fading agents other than the above-mentioned antioxidants include, for example, “Slow® Fade” (registered trademark, Thermo Fisher Scientific), “Perma® Fluor” known as an anti-fading encapsulant. (Trademark, Thermo Fisher Scientific) and other products.
  • the anti-fading treatment using the anti-fading agent described above is to prepare an anti-fading treatment solution in which the anti-fading agent is dissolved, and the treatment solution is brought into contact with the region including the sensor part of the metal thin film. And by holding for a predetermined time.
  • Examples of such fading prevention processing include the following embodiments.
  • the first embodiment of the anti-fading process of the present invention is a process for forming a region where a trapping substance is fixed on a part of a metal thin film when manufacturing a sensor chip. It is performed in conjunction with. For example, as shown in FIG. 5, such an embodiment can be performed using a predetermined instrument in the manufacturing stage of the sensor chip (that is, before loading the sensor chip into the SPFS device), and only for the sensor unit. Therefore, it is suitable for efficient fading prevention treatment.
  • a cylindrical solution storage member 304 having the same inner diameter as the circular sensor unit 303 is placed on the metal thin film 302 so that the inner periphery thereof overlaps the circumference of the sensor unit 303.
  • the material of the solution storage member 304 is not particularly limited as long as it can store the blocking agent solution, and examples thereof include resins such as polystyrene (PS), polypropylene (PP), and polymethyl methacrylate resin (PMMA).
  • PS polystyrene
  • PP polypropylene
  • PMMA polymethyl methacrylate resin
  • the anti-fading solution 305 is added to the region surrounded by the solution storage member 304 by a pipette or the like so that the entire sensor unit 303 is covered, and held at a predetermined temperature and time, for example, room temperature for 1 hour.
  • the solution storage member 304 and the metal thin film 302 are prevented from leaking out from the gap between the solution storage member 304 and the metal thin film 302. It is appropriate to seal the gap.
  • This sealing is performed by, for example, a sealing member 306 wound around the lower part of the solution storage member 304.
  • the material of the seal member 306 is rubber, for example.
  • the solution storage member 304 is fixed on the solution storage member 304 by, for example, a method of applying force by sandwiching the solution storage member 304 and the dielectric member 301 between appropriate plate members (not shown). It is preferable that the sealing member 306 is brought into close contact with the surface of the metal thin film 302 by applying force. If a through hole is provided in this plate member, the anti-fading treatment liquid 305 can be added to the region surrounded by the solution storage member 304 therefrom.
  • the anti-fading treatment liquid is removed from the solution storage member 304 with a pipette or the like, and after removing the solution storage member 304, the sensor chip 300 is placed in a thermostat and dried.
  • the dried sensor chip 300 is preferably stored in a sealed state until it is subjected to SPFS measurement.
  • the shape of the sensor portion is not limited to a circle.
  • a solution storage member corresponding to the shape may be installed.
  • the above-described fading prevention process may be performed on each of the plurality of sensor units.
  • the first embodiment of the anti-fading process as described above is accompanied by a process for forming a region (that is, the sensor unit 303) in which the trapping substance is fixed on a part of the metal thin film when the sensor chip is manufactured. It can be carried out. For example, by forming a SAM having a carboxyl group at the terminal on the surface of the metal thin film 302, and subsequently treating with EDC and NHS to convert the carboxyl group into an active ester group, an antibody as a capture substance is reacted. , And can be immobilized on the sensor unit 305 via the amino group of the antibody.
  • a treatment for inactivating the unreacted active ester group to which the antibody has not bound using Tris (trishydroxymethylaminomethane) buffer is performed.
  • the fading prevention treatment can be performed simultaneously with the inactivation treatment.
  • a process for immobilizing the trapping substance such as applying an anti-fading treatment solution to an area including the sensor unit 303 after performing an inactivation treatment using a Tris buffer solution containing no anti-fading agent.
  • a process for preventing discoloration may be performed as a separate process.
  • the concentration of the anti-fading agent in the anti-fading treatment solution is not particularly limited and can be adjusted as appropriate in consideration of the anti-fading effect. Generally, the higher the concentration, the more easily the anti-fading effect is exhibited. It is appropriate to make the concentration sufficiently high according to the anti-fading agent used. Also, the time for the anti-fading treatment is not particularly limited and can be appropriately adjusted in consideration of the anti-fading effect. Generally, the longer the processing time, the more easily the anti-fading effect is exhibited. It is appropriate to make the time sufficiently long according to the inhibitor.
  • the second embodiment of the anti-fading treatment of the present invention supplies a predetermined solution to an area where a capture substance is fixed when performing a SPFS measurement method using a sensor chip. This is performed in association with the process.
  • the SPFS measurement is performed after loading the SPFS device. Therefore, the process can be carried out as an integral or continuous process, and the anti-fading treatment can be efficiently performed.
  • an anti-fading agent is added to and dissolved in a predetermined solution used in the conventional SPFS, that is, a solution such as a measurement sample (specimen dilution solution), a fluorescent labeling solution, a cleaning solution, or a fluorescent measurement solution. deep.
  • a solution such as a measurement sample (specimen dilution solution), a fluorescent labeling solution, a cleaning solution, or a fluorescent measurement solution.
  • a flow path is used to react the capture substance fixed to the sensor unit with the measurement target substance in the measurement sample in the first step of SPFS.
  • the anti-fading process is simultaneously performed on the sensor unit.
  • the fading prevention treatment may be performed in the baseline signal measurement step. It is preferable to perform the fading prevention treatment in the fluorescence measurement step, since no further liquid feeding is performed after that, and the possibility that the immobilized antifading agent will flow down is the lowest.
  • the amount of anti-fading agent added to the measurement sample (specimen diluent), fluorescent labeling solution, or washing solution is not particularly limited and can be adjusted as appropriate in consideration of the anti-fading effect, but the concentration is generally high. Since the anti-fading effect is more likely to be exhibited, it is appropriate to set the concentration sufficiently high according to the anti-fading agent used.
  • the time of the measurement sample reaction process, the fluorescent labeling process or the washing process is not particularly limited, and is approximately the same as the time of the normal measurement sample reaction process, the fluorescent labeling process or the washing process, or Although it can be adjusted as appropriate considering the anti-fading effect as necessary, generally the longer the processing time, the more easily the anti-fading effect is exhibited, so the time should be sufficiently long according to the anti-fading agent used. Is appropriate.
  • ⁇ Blocking treatment and stabilization treatment> For the region including the sensor unit 203, impurities (proteins other than the measurement target substance, lipids, sugars, etc.) and fluorescent labeling substances are adsorbed non-specifically on the sensor unit, as necessary. You may perform the blocking process for preventing combining.
  • the blocking agent used for the blocking treatment is not particularly limited, and a normal agent may be used.
  • blocking agents for example, skim milk, fish gelatin, bovine serum albumin (BSA), surfactants, casein, protamine, polyethylene glycol, trehalose, dextran, etc. are known, and are appropriate according to the measurement sample and the substance to be measured. Choose the right one. Of these, bovine serum albumin, casein, gelatin, and skim milk are more commonly used. Any one type of blocking agent may be used alone, or a plurality of types may be used in combination.
  • BSA bovine serum albumin
  • Any one type of blocking agent may be used alone, or a plurality of types may be used in combination.
  • a stabilization treatment with a stabilizer such as a saccharide may be performed together. Since the stabilization treatment has the effect of stably protecting the structure of the capture substance, particularly a protein capture substance such as an antibody, and preventing the capture effect of the measurement target substance from decreasing over time. It is preferably combined with the first embodiment of the anti-fading process that is sometimes performed.
  • Saccharides that can be used as a stabilizer include monosaccharides (eg, glucose, fructose, etc.), disaccharides (sucrose, maltose, etc.), and oligosaccharides composed of 3 to 10 monosaccharides (raffinose, panose, etc.) And at least one sugar selected from the group consisting of:
  • the blocking treatment and the stabilization treatment can be performed simultaneously by adding a stabilizer to the blocking agent solution.
  • the amount of saccharide added to the blocking agent solution is preferably 1 to 20% by weight, and more preferably 5 to 12% by weight.
  • the blocking treatment as described above may be performed by preparing a blocking agent solution and bringing it into contact with the region containing the sensor part of the metal thin film and holding it for a predetermined time. For example, before or after the anti-fading treatment step, using the same solution storage member as that of the first embodiment of the anti-fading treatment described above, blocking containing a blocking agent and, if necessary, a stabilizer (saccharide).
  • the treatment liquid can be brought into contact with the region including the sensor unit.
  • the SPFS measurement method of the present invention includes a step of measuring fluorescence by irradiating excitation light in the presence of an anti-fading agent in the vicinity of a fluorescent labeling agent bound to a measurement target substance captured by a capture substance. It is.
  • the “near” here is not particularly limited as long as it is a distance at which the anti-fading agent has an effect of preventing the fading of the phosphor contained in the fluorescent labeling agent.
  • the sensor unit The distance from the surface of the metal thin film on which the sapphire is formed to the phosphor, that is, a distance within a range of about several hundred nanometers where the evanescent wave enhanced by the surface plasmon resonance reaches is preferable. Examples of such an SPFS measurement method include the following embodiments.
  • fluorescence is emitted by irradiating excitation light in a state where an anti-fading agent is present in a region (sensor part) where a capture substance is fixed. Measure.
  • the first embodiment of such an SPFS measurement method includes both cases (A) and (B) below:
  • a measurement sample to which an anti-fading agent is added, a cleaning solution, a fluorescent labeling solution, and the like are supplied to the sensor unit, so that the capture substance is fixed in the region.
  • the anti-fading agent present in the region where the capture substance is immobilized is compared with the region including the region where the capture substance is immobilized before the sensor chip is used for the SPFS measurement method. And contained in a solution for anti-fading treatment.
  • the anti-fading agent present in the region where the capture substance is fixed is added to the sample solution, washing solution or fluorescent labeling solution used in the SPFS measurement method.
  • the embodiment includes a step of supplying a measurement sample, a washing solution, a fluorescent labeling solution, etc., to which an antifading agent is added, to a region where a capture substance is fixed, before the step of measuring fluorescence by irradiating excitation light. It will be. Such measurement sample reaction step, washing step, and fluorescent labeling step will be further described later.
  • the anti-fading agent is present in a fluorescence measurement liquid used in the SPFS measurement method, and is irradiated with excitation light.
  • a fluorescence measuring solution to which an anti-fading agent is added is supplied to the region fixed to the capture substance. That is, the fluorescence is measured by irradiating the excitation light in the state where the anti-fading agent is present in the fluorescence measuring solution filling the periphery of the fluorescent labeling agent.
  • a fluorescence measuring solution to which an anti-fading agent is added is supplied to the region fixed to the capture substance. That is, the fluorescence is measured by irradiating the excitation light in the state where the anti-fading agent is present in the fluorescence measuring solution filling the periphery of the fluorescent labeling agent.
  • Steps included in the SPFS measurement method When starting the SPFS measurement method, the sensor chip is usually in a state where a flow path is formed in combination with a thin layer member and a frame member, or in a state where a well is formed in combination with a plate. , Loaded into the SPFS device.
  • a conventionally known SPFS apparatus can be used.
  • the SPFS measurement method of the present invention is basically the same as a conventionally known measurement method except for matters relating to anti-fading, and can be carried out, for example, according to the following steps: (1) Measurement sample reaction process: supply a measurement sample to a flow path or a well, and bring the measurement sample into contact with a sensor part formed on a part of a metal thin film that hits the bottom surface of the measurement substance, so that the captured substance and the measurement target are contacted.
  • Fluorescent labeling step a step of reacting the measurement target substance captured by the capture substance with the fluorescent labeling agent by bringing the solution containing the fluorescent labeling agent into contact with the sensor part that has undergone the measurement sample contacting step;
  • Fluorescence measurement step After filling the flow path or well with a fluorescence measurement solution, the sensor unit that has passed through the fluorescence labeling step is irradiated with excitation light corresponding to the phosphor contained in the fluorescence labeling agent, and is generated based on SPFS. Measuring the intensity of the fluorescence (assay signal).
  • the fluorescence intensity (blank signal) is measured in the same manner as above except that a blank (for example, only a buffer solution) is used instead of the measurement sample.
  • a step of measuring the intensity of fluorescence (baseline signal) in the same manner as the fluorescence measurement step may be further performed.
  • a washing step for washing away the remaining solution used in the previous step, if necessary, May be provided.
  • fluorescence labeling step In the measurement sample reaction step, fluorescence labeling step, fluorescence measurement step, and washing step, fill the flow path or well with the measurement sample, fluorescence labeling solution, fluorescence measurement solution, and washing solution, respectively, and remove them when finishing each step To do.
  • the fluorescence measurement solution is water that does not contain a fluorescent labeling agent or a cleaning agent, a buffer solution, or other solvent.
  • the buffer solution itself used for the preparation of a fluorescent standard solution or a cleaning solution can be used as the fluorescence measurement solution. .
  • the same buffer solution can also be used as the specimen diluent used as necessary when preparing the measurement sample.
  • a fluorescence measurement step is performed by adding an anti-fading agent to a measurement sample (specimen diluent), a fluorescence labeling solution or a washing solution used in a measurement sample reaction step, a fluorescence labeling step or a washing step. It is possible to measure fluorescence by forming a sensor part that has been subjected to anti-fading treatment earlier and irradiating excitation light in a state where the anti-fading agent is present in the region where the capture substance is fixed.
  • the amount of anti-fading agent added to the measurement sample (specimen diluent), fluorescent labeling solution or washing solution, and the time of the measurement sample reaction step, fluorescent labeling step or washing step (ie anti-fading treatment) are the second implementation of the anti-fading treatment. This is the same as described above in relation to the form.
  • the second embodiment of the SPFS measurement method by adding an anti-fading agent to the fluorescence measurement solution, a solution around the fluorescent labeling agent bound to the measurement target substance captured by the capture substance (that is, the fluorescence measurement solution) ) And fluorescence can be measured by irradiating with excitation light in the presence of an anti-fading agent.
  • the addition amount of the anti-fading agent with respect to the fluorescence measurement solution is not particularly limited, and can be appropriately adjusted in consideration of the anti-fading effect as described above.
  • the measurement sample is typically a specimen collected from a human or an animal, and typical examples thereof include blood (including serum and plasma) and urine.
  • a suspension prepared using cells collected or cultured according to a predetermined method can also be used as a measurement sample.
  • the collected specimen is subjected to anticoagulation, centrifugation, extraction, and other necessary treatments as necessary, diluted to an appropriate concentration with a diluent (buffer solution, etc.), and used as a measurement sample. Also good.
  • the substance to be measured is a protein, lipid, sugar, nucleic acid, or other biological substance to be detected or quantified from a measurement sample.
  • substances to be measured when the measurement sample is blood include myocardial markers such as troponin I, NT-ProBNP, and D-Dimer.
  • a fluorescent labeling agent is a complex containing a substance that specifically binds to a substance to be measured and a fluorescent substance that can emit fluorescence when irradiated with predetermined excitation light.
  • a fluorescent labeling agent can be prepared by a known technique, and a fluorescent labeling agent for a specific measurement target substance is also commercially available.
  • the phosphor used for the fluorescent labeling agent in the SPFS measurement method of the present invention may be the same as the fluorescent labeling agent in the known SPFS measurement method, and various known phosphors can be used. Examples of such phosphors include fluorescent materials.
  • fluorescent substances include rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules.
  • Alexa Fluor registered trademark, manufactured by Invitrogen
  • BODIPY registered trademark, manufactured by Invitrogen
  • Cy registered trademark, manufactured by GE Healthcare
  • Dy registered trademark, manufactured by DYOMICS
  • HiLyte registered trademark, manufactured by Anaspec
  • dye molecule DyLight (registered trademark, manufactured by Thermo Scientific)
  • dye molecule ATTO (registered trademark, manufactured by ATTO-TEC) Molecules, MFP (registered trademark, manufactured by Mobitec) -based dye molecules, and the like can be used.
  • kit of the present invention is used for producing the sensor chip of the present invention as described above or for carrying out the SPFS measurement method of the present invention as described above.
  • kit embodiments include the following embodiments.
  • the first embodiment of the kit of the present invention corresponds to (A) of the first embodiment of the fading prevention treatment and the first embodiment of the SPFS measurement method described above, and at least captures It includes an SPFS sensor chip that has been subjected to anti-fading treatment by bringing a solution containing the anti-fading agent into contact with the region where the substance is fixed.
  • This kit also includes reagents and instruments suitable for the SPFS measurement method performed using the above-mentioned SPFS sensor chip, such as a sample diluent, a cleaning liquid (or a cleaning agent and a solvent for preparing the same, and a preparation).
  • kit may further include instructions for (B) of the first embodiment of the anti-fading treatment or the first embodiment of the SPFS measurement method.
  • the second embodiment of the kit of the present invention corresponds to (B) of the second embodiment of the anti-fading process and the first embodiment of the SPFS measurement method described above, and at least SPFS.
  • Sensor chip (not subjected to anti-fading treatment by bringing a solution containing an anti-fading agent into contact with the area where the capture substance is fixed) and anti-fading treatment liquid (or to prepare it) Antifading agents and solvents).
  • the anti-fading treatment solution is prepared by adding an anti-fading agent to a sample solution, a cleaning solution, a fluorescent labeling solution, or other solution used in a step performed before the fluorescence measurement step.
  • a sample solution with an anti-fading agent as an anti-fading solution
  • the agent may be dissolved to form one pack, and when performing the SPFS measurement method, the contents of such a pack may be mixed with the specimen or the processed product to prepare a sample solution.
  • a cleaning solution with an anti-fading agent or a fluorescent labeling solution with an anti-fading agent is used as the anti-fading treatment solution.
  • This kit also includes reagents and instruments suitable for the SPFS measurement method performed using the above-mentioned SPFS sensor chip, such as a sample diluent, a cleaning solution (or a cleaning agent and a solvent for preparing the same, and a preparation). Instruments), fluorescent labeling solutions (or secondary antibodies, phosphors, reaction reagents and solvents and preparation instruments for preparing them), fluorescence measurement solutions, or various solutions including the above-described anti-fading treatment solution.
  • a reagent container that can be loaded into the SPFS device and used can be included.
  • the kit may further include instructions for (B) of the second embodiment of the anti-fading treatment or the first embodiment of the SPFS measurement method.
  • the third embodiment of the kit of the present invention corresponds to the second embodiment of the SPFS measurement method described above, and at least the sensor chip for SPFS (with respect to the region where the capture substance is fixed) And an anti-fading treatment solution by contact with a solution containing the anti-fading agent) and an anti-fading treatment liquid (or an anti-fading agent and a solvent for preparing the anti-fading treatment solution).
  • the SPFS sensor chip may not be subjected to the anti-fading treatment by bringing a solution containing the anti-fading agent into contact with the region where the trapping substance is fixed.
  • such fading prevention processing may be performed.
  • the anti-fading treatment solution is prepared by adding an anti-fading agent to the fluorescence measurement solution.
  • the anti-fading agent solution and the fluorescence measuring solution may be separately made into two packs, or the anti-fading agent may be dissolved in the fluorescent measuring solution from the beginning to make one pack.
  • This kit also includes reagents and instruments suitable for the SPFS measurement method performed using the above-mentioned SPFS sensor chip, such as a sample diluent, a cleaning solution (or a cleaning agent and a solvent for preparing the same, and a preparation). Instruments), fluorescent labeling solutions (or secondary antibodies, phosphors, reaction reagents and solvents and preparation instruments for preparing them), or various solutions including the above-described anti-fading treatment solution, and sealed in advance.
  • a reagent container that can be used by being loaded into the SPFS apparatus may be included.
  • the kit may further include instructions for the second embodiment of the SPFS measurement method.
  • Example 1 Manufacture of sensor chip Step (a): Main surface of a prism member having a substantially trapezoidal cross-sectional shape created by using cycloolefin polymer resin (ZEON Corporation) and ZEONEX (registered trademark) as a material of a dielectric member. First, a chromium thin film was formed by sputtering, and further a gold thin film was formed on the surface by sputtering. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.
  • cycloolefin polymer resin ZEON Corporation
  • ZEONEX registered trademark
  • the prism was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.
  • the solution storage member and the prism were sandwiched from above and below using two stainless steel plates and fixed with screws (the upper stainless steel plate was loaded with a reagent or the like on the solution storage member) An opening for taking in and out is provided).
  • 5610 Nito Denko Corporation
  • a 10 mm thick polymethylmethacrylate plate having an inlet / outlet 207 and a liquid reservoir 208 is adhered, and a flow path 206 is formed.
  • a sensor chip To form a sensor chip.
  • Baseline signal measurement step Fluorescence (baseline signal) treated as a blank with a flow path filled with TBS containing 0.05% by weight of Tween 20 using a laser light source as a light source and an output of 1 mW or less (0.2 -0.8 mW), 635 nm wavelength laser light is adjusted by an optical filter (Sigma Kogyo Co., Ltd.), irradiated onto the metal thin film of the sensor chip, and cuts wavelengths other than fluorescent components as a cut filter Detection was performed with a CCD image sensor (manufactured by Texas Instruments Co., Ltd.) using a cut filter and an objective lens (20 ⁇ ).
  • Fluorescent labeling step anti-troponin I [TnI] monoclonal antibody (primary) obtained by aspirating and removing TBS containing 0.05% by weight of Tween 20 and fluorescently labeled using Alexa Fluor (trademark) 647 protein labeling kit (Invitrogen) PBS buffered saline containing a clone different from the antibody) was injected into the channel and allowed to react for 15 minutes to form an immune complex.
  • the PBS buffered saline which is a solution containing the fluorescently labeled antibody, was removed by suction, and then the operation of injecting and removing TBS containing 0.05% by weight of Tween 20 was repeated several times to perform the washing operation.
  • Assay signal measurement step A TBS containing 0.05% by weight of Tween 20 was added 0.1% by weight of L-ascorbic acid Na as an anti-fading agent to obtain a fluorescence measuring solution to which the anti-fading agent was added. In a state where the flow path was filled with this fluorescence measurement solution, fluorescence was measured in the same manner as the baseline signal measurement, and the fluorescence signal derived from the immune complex was measured. Laser light was continuously emitted for at least 10 minutes from the start of the assay signal measurement step, and the fluorescence signal was measured during that time.
  • Blank signal measurement step The measurement of the blank signal derived from non-specific adsorption of the labeled antibody is performed by using PBS containing 1 wt% bovine serum albumin [BSA] that does not contain troponin I as an antigen in the measurement sample reaction step.
  • BSA bovine serum albumin
  • a similar sensor chip was used in the same process except injecting buffered saline.
  • S / N was calculated from the signal obtained by using the solution containing the noise component (baseline signal, blank signal) and the antigen obtained in the above measurement according to the following formula.
  • S / N
  • Example 2 (1) Production of sensor chip In the same manner as in Example 1, a sensor chip subjected to the fading prevention treatment was produced.
  • TBS itself containing 0.05% by weight of Tween 20 to which no anti-fading agent is added is measured by fluorescence.
  • Baseline signals, assay signals, and blank signals were measured in the same manner as in Example 1 except that they were used as solutions, that is, the anti-fading treatment was not performed in the assay signal measurement step.
  • Example 3 (1) Preparation of sensor chip In step (e), no anti-fading agent was added to 0.2 mL of 50 mM Tris (pH 7.4), and no anti-fading treatment was performed on the sensor part (unreacted activated ester). A sensor chip was produced in the same manner as in Example 1 except that only the group was inactivated.
  • Example 4 (1) Preparation of sensor chip In step (e), no anti-fading agent was added to 0.2 mL of 50 mM Tris (pH 7.4), and no anti-fading treatment was performed on the sensor part (unreacted activated ester). A sensor chip was produced in the same manner as in Example 1 except that only the group was inactivated.
  • TBS itself containing 0.05% by weight of Tween 20 to which no anti-fading agent is added is measured by fluorescence.
  • the fluorescence measurement liquid to which the anti-fading agent is added is used, that is, the baseline signal measurement process is performed instead of performing the anti-fading process in the assay signal measurement process.
  • the baseline signal, the assay signal, and the blank signal were measured in the same manner as in Example 1 except that the anti-fading treatment was performed.
  • Example 5 (1) Preparation of sensor chip In step (e), no anti-fading agent was added to 0.2 mL of 50 mM Tris (pH 7.4), and no anti-fading treatment was performed on the sensor part (unreacted activated ester). A sensor chip was produced in the same manner as in Example 1 except that only the group was inactivated.
  • Example 1 The results of Examples 1 to 4 and Comparative Example 1 are shown in Table 1 and FIG.
  • the numerical value of the S / N ratio represents each irradiation time (2, 4, 6, 8 or 10 minutes) when the value of the excitation light irradiation time in the assay signal measurement step is 0 minutes (immediately after irradiation) is 1. Is the ratio of the values of.
  • the S / N ratio decreases more slowly with the irradiation time than in Comparative Example 1 in which the anti-fading process is not performed.
  • the anti-fading effect is shown.
  • Example 1 in which the anti-fading treatment was performed both in the sensor chip production stage and in the measurement stage (assay signal measurement process) showed the slowest S / N ratio decrease with irradiation time and the most excellent anti-fading effect. .
  • Example 5 The results of Example 5 and Comparative Example 2 are shown in Table 2.
  • the numerical value of the S / N ratio is the ratio of the number of times of irradiation (1 to 5 times) when the value when the number of times of excitation light irradiation is 1 is 1 in the assay signal measurement step of Example 5 It is.
  • the S / N ratio decreases more slowly with the number of times of irradiation than in Comparative Example 2 where the anti-fading treatment is not performed. There is an anti-fading effect.
  • Example 5 in the measurement conditions of Example 5 and Comparative Example 2, the output of the excitation light irradiated to the fluorescent material is 30 mW, which is less than 1 mW (0) of the excitation light irradiated in Examples 1 to 4 and Comparative Example 1 .2 to 0.8 mW), it causes fading just by irradiating the fluorescent material. It can be said that Example 5 has a more excellent anti-fading effect as compared to Example 1.

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Abstract

　La présente invention vise à fournir un moyen pour une utilisation dans la prévention de l'atténuation d'une substance fluorescente utilisée en SPFS pour marquer une substance à mesurer, qui est piégée dans une unité de détection, et de préférence aussi pour empêcher une dégradation d'une substance de piégeage qui a été immobilisée dans l'unité de détection. Ce procédé de détection se fonde sur une spectroscopie de fluorescence amplifiée par le champ du plasmon de surface, qui utilise une puce de détection qui possède un élément diélectrique, une couche mince métallique formée sur une surface principale de l'élément diélectrique, et une zone formée par-dessus une portion de la couche mince métallique, une substance de piégeage destinée à piéger spécifiquement une substance à mesurer immobilisée dans cette zone, le procédé comprenant une étape d'irradiation, par une lumière d'excitation en présence d'un agent anti-altération, de la région entourant un marqueur fluorescent lié à la substance à mesurer, qui a été piégé par la substance de piégeage, et de mesure de la fluorescence.
PCT/JP2015/067736 2014-06-20 2015-06-19 Puce de détection pour utilisation en spectroscopie de fluorescence amplifiée par le champ du plasmon de surface (spfs), procédé de mesure par spfs, et trousse de spfs Ceased WO2015194663A1 (fr)

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