WO2010084953A1 - Détecteur d'excitation plasmonique et procédé de test l'utilisant - Google Patents

Détecteur d'excitation plasmonique et procédé de test l'utilisant Download PDF

Info

Publication number
WO2010084953A1
WO2010084953A1 PCT/JP2010/050803 JP2010050803W WO2010084953A1 WO 2010084953 A1 WO2010084953 A1 WO 2010084953A1 JP 2010050803 W JP2010050803 W JP 2010050803W WO 2010084953 A1 WO2010084953 A1 WO 2010084953A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmon excitation
fluorescent dye
excitation sensor
thin film
ligand
Prior art date
Application number
PCT/JP2010/050803
Other languages
English (en)
Japanese (ja)
Inventor
英隆 二宮
高敏 彼谷
賢治 石田
法明 山本
Original Assignee
コニカミノルタホールディングス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2009011973A external-priority patent/JP5298876B2/ja
Priority claimed from JP2009011974A external-priority patent/JP5298877B2/ja
Priority claimed from JP2009025974A external-priority patent/JP5245125B2/ja
Application filed by コニカミノルタホールディングス株式会社 filed Critical コニカミノルタホールディングス株式会社
Publication of WO2010084953A1 publication Critical patent/WO2010084953A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention relates to a plasmon excitation sensor and an assay method using the same, an apparatus for the assay, and a kit for the assay. More specifically, the present invention is based on the principles of a plasmon excitation sensor in which a fluorescent dye and a ligand are immobilized on a metal thin film, and surface plasmon excitation enhanced fluorescence spectroscopy [SPFS: Surface Plasmon-field enhanced Fluorescence Spectroscopy]. The present invention relates to an assay method using the sensor, the assay device, and the assay kit.
  • the surface plasmon excitation enhanced fluorescence analysis method [SPFS] is performed by generating a dense wave (surface plasmon) on the metal thin film surface under the condition that the irradiated laser light attenuates total reflection [ATR] on the gold thin film surface.
  • the amount of photons in the laser light is increased to several tens to several hundreds times (electric field enhancement effect of surface plasmon), and by this, the fluorescent dye in the vicinity of the gold thin film is efficiently excited. It is a method that can detect an analyte.
  • Patent Document 1 discloses a surface plasmon in which a ligand (primary antibody) immobilization film using carboxymethyldextran is arranged on the surface of a metal substrate. A method for detecting a fluorescent dye associated with an antigen with an enhanced electric field is shown.
  • the amount of fluorescent dye in the conjugate associated with the antigen in the assay is also extremely small, which becomes a bottleneck in the amount of fluorescence generated, and therefore the plasmon electric field Even if enhancement is used, the amount of fluorescence signal does not increase, and it is difficult to improve assay sensitivity.
  • Patent Document 2 discloses hybrid probe particles including gold nanoparticles (diameter: 2 to 30 nm) having a high electric field enhancement effect.
  • the hybrid probe particles are disclosed wherein 1 to 100 antibody-type proteins are bound by gold-sulfur bonds on one of the gold nanoparticle surfaces, and on the other hand at least 10 fluorescent organic dyes are gold- Bonded by sulfur bond.
  • a fluorescent material is formed in a single layer or multiple layers on a surface in which flat silver particles having a cross-sectional particle diameter of 100 to 800 nm and a thickness of 30 to 50 nm are densely arranged as an island film on a substrate.
  • a spacer is provided on the metal particle surface.
  • Patent Document 4 examines signal amplification and non-specific reaction reduction by complexly combining an apoenzyme / holoenzyme reaction and an immune reaction on a sensor substrate.
  • extremely precise molecular orientation technology is premised. Therefore, when the apo / holoenzyme reaction is preferential or dominant over the immune reaction, the measurement system itself There is a high risk that
  • An object of the present invention is to provide a high-sensitivity and high-precision plasmon excitation sensor excellent in specificity that is indispensable for an immunoassay, an assay method using the same, an assay device, and an assay kit. .
  • the inventors of the present invention have problems with the above problems and the conventional sandwich immunoassay method (FIG. 1), that is, when detecting a very small amount of analyte, the conventional sandwich immunoassay method is used, and the conjugate itself is theoretical.
  • the conventional sandwich immunoassay method (FIG. 1) and the surface plasmon excitation enhanced fluorescence analysis method are combined, and a mechanism for quenching the fluorescence depending on the presence or absence of binding between the sensor and the analyte is provided, that is, light emission and quenching.
  • analyte for example, a target antigen
  • the plasmon excitation sensor of the present invention includes a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a dielectric formed on the other surface of the metal thin film that is not in contact with the substrate. And (V) a ligand is immobilized on a fluorescent dye layer formed on the other surface of the spacer layer that is not in contact with the metal thin film, or (W) A ligand labeled with a fluorescent dye is immobilized on the other surface of the spacer layer that is not in contact with the metal thin film.
  • the metal thin film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum.
  • the dielectric preferably contains silicon dioxide [SiO 2 ] or titanium dioxide [TiO 2 ].
  • the ligand may be an antibody that recognizes and binds to a tumor marker or carcinoembryonic antigen.
  • the first aspect of the plasmon excitation sensor of the present invention (hereinafter referred to as “plasmon excitation sensor (I)”) takes the form of (V) above, and the metal thin film is made of gold or silver. Is preferred.
  • the fluorescent dye layer is formed by applying a composition containing a fluorescent dye and a polymer to the other surface of the spacer layer that is not in contact with the metal thin film. Or formed by binding a fluorescent dye via a silane coupling agent.
  • the second aspect of the plasmon excitation sensor of the present invention (hereinafter referred to as “plasmon excitation sensor (II)”) takes the form of (W), and the metal thin film is preferably made of gold. .
  • the ligand is preferably immobilized on the spacer layer via a self-assembled monolayer [SAM] made of a silane coupling agent.
  • SAM self-assembled monolayer
  • the assay method of the present invention includes at least (X) the following steps (a1), (b1), (d) and (e), or (Y) the following steps (a2), (b1), (d) and Comprising (e) or (Z) comprising the following steps (a1), (b2), (c), (d) and (e);
  • Step (a1) a step of bringing a specimen into contact with the plasmon excitation sensor (I) of the present invention
  • Step (a2) a step of bringing a specimen into contact with the plasmon excitation sensor (II) of the present invention
  • the plasmon excitation sensor obtained through the step (a1) is further combined with a ligand / enzyme conjugate that may
  • Step (c) a step of reacting a quencher substrate with the plasmon excitation sensor obtained through the step (b2) to produce a quencher
  • the specimen may be at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.
  • the analyte may be a tumor marker or carcinoembryonic antigen.
  • the first aspect of the assay method of the present invention (hereinafter referred to as “assay method (X)”) and the second aspect (hereinafter referred to as “assay method (Y)”) are respectively the above (X) or ( In the case of Y), it is preferable to further use a ligand which is an analyte different from the above-mentioned analyte and to which an analyte competing with the above-mentioned analyte is bound in advance.
  • the enzyme is preferably ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase or glucose oxidase.
  • the assay device of the present invention comprises at least the plasmon excitation sensor, laser light source, optical filter, prism, cut filter, condensing lens, and surface plasmon excitation enhanced fluorescence obtained through the step (b1) or (c). It includes a detection unit, and is used in step (d) according to the assay method of the present invention.
  • the assay method when the assay method takes the form of (X), at least the transparent flat substrate, the metal thin film, the spacer layer composed of the dielectric, and the fluorescent dye layer are included.
  • the present invention is highly sensitive and accurate from a specimen containing an analyte (eg, target antigen) at a concentration of 10 ⁇ 18 mol (1 amol / L) to 10 ⁇ 12 mol (1 pmol / L) per liter.
  • analyte eg, target antigen
  • a plasmon excitation sensor capable of detecting the analyte can be provided.
  • the conventional sandwich immunoassay method when detecting a very small amount of analyte, the conventional sandwich immunoassay method has a small amount of fluorescence signal (fluorescence signal) and the S / N ratio is deteriorated.
  • the present invention provides a plasmon excitation sensor and a ligand (for example, secondary). Antibody) and a compound capable of quenching or absorbing fluorescence, and applied to the assay method of the present invention, the ratio of the target antigen is proportional to the compound capable of quenching or absorbing fluorescence. It is possible to provide a plasmon excitation sensor in which the ratio does not deteriorate.
  • the present invention can adjust the amount of fluorescence signal depending on the ability of a compound capable of quenching or absorbing fluorescence, it can provide a plasmon excitation sensor in which the assay method of the present invention can be carried out at an optimum S / N ratio. it can.
  • the present invention relates to an assay (X) and (Y) of the present invention, an analyte (target antigen), a secondary antibody (ligand) in which an antigen that competes with the analyte is previously bound, a quenching dye,
  • an analyte target antigen
  • a secondary antibody ligand
  • an antigen that competes with the analyte is previously bound
  • a quenching dye plasmon excitation sensors (I) and (II) that can make the fluorescence signal (fluorescence signal) amount and the target antigen amount proportional to each other.
  • FIG. 1 shows that in a conventional sandwich immunoassay method, a target antigen (3) contained in a specimen is bound to a primary antibody (2) immobilized on a substrate (1), and then a fluorescent dye (5) is used.
  • a state in which the labeled secondary antibody (4) is reacted is schematically shown.
  • FIG. 2 shows a prism (110); a transparent flat substrate in contact with the prism (110); a metal thin film formed on one surface of the substrate; and the metal thin film in contact with the substrate
  • a sensor chip comprising: a spacer layer made of a dielectric formed on the other surface; and a primary antibody immobilized on the other surface of the spacer layer not in contact with the metal thin film
  • FIG. 2 shows a schematic optical layout of the SPFS apparatus schematically showing a state in which the laser light is irradiated from the semiconductor laser (100) and the fluorescence amount is detected by the CCD (122).
  • the amount of light is adjusted by the ND filter (102), and 0.1 mW is incident on the sensor chip (111);
  • the fluorescent dye labeled on the secondary antibody contained in the flow path to which the sensor chip (111) is fixed is 0.4 nmol / L (measured antigen amount is 0.2 nmol / L of the secondary antibody). This corresponds to the labeling rate of fluorescent dye 2.
  • the “labeling rate” is the average number of labeling agents (eg, fluorescent dye, quenching dye, enzyme, etc.) per ligand (eg, antibody).
  • the effective plasmon region is present at a thickness of 100 nm; and (3) the molar extinction coefficient of the fluorescent dye is 250,000 and the fluorescence quantum yield is 0.47.
  • FIG. 3 shows an antibody that recognizes and binds to an antigen (7) other than the target antigen (analyte) contained in a sample by the type 1 (secondary antibody (4)) of the assay method (X) of the present invention. ) And type 2 (secondary antibody (4) is an antibody that recognizes and binds to target antigen (3) (analyte) contained in a specimen).
  • Step (b1) target antigen (2) bound to the primary antibody (2) by further reacting with the secondary antibody (4) on which the quenching dye (6) is immobilized.
  • the secondary antibody (4) binds to 3);
  • step (d) the fluorescent dye contained in the fluorescent dye layer (12) formed on one surface of the spacer layer made of a dielectric is excited by the surface plasmon. Then, the fluorescence (13) emitted from the fluorescent dye not quenched by the quenching dye (6) is measured.
  • FIG. 4 shows type 1 (for example, Examples (2-8) to (2-14)) and type 2 (for example, Examples (2-1) to (2-) of the assay method (Y) of the present invention. It is the figure which showed 7)) typically.
  • Step (b1) target antigen (2) bound to the primary antibody (2) by further reacting with the secondary antibody (4) on which the quenching dye (6) is immobilized.
  • Step (d) fluorescent dye (5) immobilized on primary antibody (2) is excited by surface plasmon, and fluorescent dye (5) becomes a quenching dye. The fluorescence (13) emitted without being quenched by (6) is measured.
  • FIG. 5 is a diagram schematically showing one embodiment of the assay method (Z) of the present invention (in the case of the enzyme reaction (A)).
  • step (a1) the plasmon excitation sensor (I) of the present invention is brought into contact with the specimen, so that the target antigen (3) contained in the specimen is the primary antibody (2
  • Step (b2) the target antibody (3) bound to the primary antibody (2) is further reacted with the secondary antibody (4) having the enzyme (10) immobilized thereon.
  • Step (c) added quencher substrate (8) reacts with enzyme (10) to produce quencher (9);
  • step (d) consists of dielectric
  • the fluorescent dye contained in the fluorescent dye layer (12) formed on one surface of the spacer layer is excited by the surface plasmon, and the fluorescence (13) emitted by the fluorescent dye not quenched by the quencher (9) is measured.
  • FIG. 6 is a graph summarizing the blank signals and assay signals obtained in Examples (3-1) and (3-2) and Comparative Examples (3-1) and (3-2), respectively. .
  • the plasmon excitation sensor of the present invention comprises: a transparent flat substrate; a metal thin film formed on one surface of the substrate; a dielectric formed on the other surface of the metal thin film that is not in contact with the substrate (V) a ligand is immobilized on a fluorescent dye layer formed on the other surface of the spacer layer that is not in contact with the metal thin film, or (W) the spacer layer. A ligand labeled with a fluorescent dye is immobilized on the other surface of the layer not in contact with the metal thin film.
  • the plasmon excitation sensor of the present invention includes the plasmon excitation sensor (I) taking the form (V) and the plasmon excitation sensor (II) taking the form (W).
  • the plasmon excitation sensor of the present invention can be used for the plasmon excitation sensor of the present invention, such as a sensor chip used in a Biacore system manufactured by GE Healthcare Biosciences Co., Ltd. Can do.
  • a transparent flat substrate is used as a substrate that supports the structure of the plasmon excitation sensor.
  • the transparent flat substrate is used as the support because light irradiation to the metal thin film described later is performed through the transparent flat substrate.
  • the material for the transparent flat substrate used in the present invention is not particularly limited as long as the object of the present invention is achieved.
  • the transparent support may be made of glass, or may be made of plastic such as polycarbonate [PC] or cycloolefin polymer [COP].
  • the refractive index [n d ] at the d line (588 nm) is preferably 1.40 to 2.20, and the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.
  • the size (vertical x horizontal) is not particularly limited.
  • the transparent transparent substrate made of glass is “BK7” (refractive index [n d ] 1.52) and “LaSFN9” (refractive index [n d ] 1.85) manufactured by Shot Japan Co., Ltd. as commercially available products.
  • K-PSFn3 reffractive index [n d ] 1.84
  • K-LaSFn17 reffractive index [n d ] 1.88
  • K-LaSFn22 reffractive index
  • Ratio [n d ] 1.90) and “S-LAL10” (refractive index [n d ] 1.72) manufactured by OHARA INC. Are preferable from the viewpoints of optical properties and detergency.
  • the transparent flat substrate is preferably cleaned with acid and / or plasma before forming a metal thin film on the surface.
  • As the cleaning treatment with an acid it is preferable to immerse in 0.001 to 1N hydrochloric acid for 1 to 3 hours.
  • Examples of the plasma cleaning treatment include a method of immersing in a plasma dry cleaner (PDC200 manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.
  • a metal thin film is formed on one surface of the transparent flat substrate. This metal thin film has a role of generating surface plasmon excitation by light irradiated from a light source, generating an electric field, and causing emission of a fluorescent dye.
  • the metal thin film formed on one surface of the transparent flat substrate is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and is an alloy of these metals. Also good. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.
  • the metal thin film is preferably formed from gold that is most stable against oxidation, and when used for assay method (Z), it will be described later. Because of the use of fluorescent dyes such as Tb chelate, ECFP, 2-Me-4-OMe TG, 2-OMe-5-Me TG, 2-OMe TG, etc. Preferably it is formed from.
  • a thin film of chromium, nickel chromium alloy or titanium in advance because glass and a metal thin film can be more firmly bonded only when a transparent flat substrate made of glass is used as the transparent flat substrate. .
  • Examples of a method for forming a metal thin film on a transparent flat substrate include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and / or a metal thin film by sputtering or vapor deposition.
  • the thickness of the metal thin film is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm.
  • the thickness of the thin film is preferably 1 to 20 nm.
  • gold 20 to 70 nm
  • silver 20 to 70 nm
  • aluminum 10 to 50 nm
  • copper 20 to 70 nm
  • platinum 20 to 70 nm
  • alloys thereof 10 to 70 nm
  • chromium The thickness of the thin film is more preferably 1 to 3 nm.
  • the thickness of the metal thin film is within the above range because surface plasmons are easily generated. Moreover, if it is a metal thin film which has such thickness, a magnitude
  • Spacer layer made of dielectric A spacer layer made of a dielectric is formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate for the purpose of preventing metal quenching of the fluorescent dye by the metal thin film.
  • the dielectric various optically transparent inorganic substances, natural or synthetic polymers can be used, but silicon dioxide [SiO 2 ] because of its excellent chemical stability, production stability and optical transparency. or preferably contains titanium dioxide [TiO 2].
  • the thickness of the spacer layer is usually 10 nm to 1 mm, preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. Further, from the viewpoint of electric field enhancement, 200 nm to 1 mm is preferable, and from the viewpoint of stability of the electric field enhancement effect, 400 to 1,600 nm is preferable.
  • the thickness of the spacer layer included in the sensor will fluctuate. Since there is a possibility, the thickness of the spacer layer is particularly preferably 10 to 20 nm in order to ensure measurement stability.
  • Examples of the method for forming the spacer layer include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or an application by a spin coater.
  • a ligand is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding an analyte contained in a specimen.
  • a “molecule” or “molecular fragment” include, for example, , Nucleic acids (single stranded or double stranded DNA, RNA, polynucleotides, oligonucleotides, PNA [peptide nucleic acids] etc., or nucleosides, nucleotides and their modified molecules), proteins (polypeptides , Oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or their modified molecules, complexes, etc., are not particularly limited.
  • proteins examples include antibodies and the like, specifically, anti- ⁇ -fetoprotein [AFP] monoclonal antibody (available from Nippon Medical Laboratory, Inc.), anti-carcinoembryonic antigen [CEA Monoclonal antibodies, anti-CA19-9 monoclonal antibodies, anti-PSA monoclonal antibodies, and the like.
  • AFP anti- ⁇ -fetoprotein
  • CEA anti-carcinoembryonic antigen
  • an antibody includes a polyclonal antibody or a monoclonal antibody, an antibody obtained by gene recombination, and an antibody fragment.
  • An analyte is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to a ligand immobilized on a plasmon excitation sensor.
  • fragment include nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA [peptide nucleic acids] etc., which may be single-stranded or double-stranded, or nucleosides, nucleotides and modified molecules thereof.
  • Proteins polypeptides, oligopeptides, etc.
  • amino acids including modified amino acids
  • carbohydrates oligosaccharides, polysaccharides, sugar chains, etc.
  • lipids or their modified molecules, complexes, etc.
  • it may be a carcinoembryonic antigen such as AFP [ ⁇ -fetoprotein], a tumor marker, a signal transmitter, a hormone, etc. It is not particularly limited.
  • the plasmon excitation sensor (I) of the present invention is formed on a transparent flat substrate; a metal thin film formed on one surface of the substrate; and the other surface of the metal thin film not in contact with the substrate A spacer layer made of a dielectric; a fluorescent dye layer formed on the other surface of the spacer layer not in contact with the metal thin film; and the other of the fluorescent dye layer not in contact with the spacer layer And a ligand immobilized on the surface of the substrate.
  • the plasmon excitation sensor (I) of the present invention is used in the assay method (X) or (Z).
  • the fluorescent dye layer is a layer in which the fluorescent dye is immobilized on the other surface of the spacer layer made of the dielectric material that is not in contact with the metal thin film. Can be formed by coating the spacer layer on the spacer layer, or (B) by binding a fluorescent dye on the spacer layer via a silane coupling agent. it can.
  • the fluorescent dye and the polymer may or may not be chemically bonded, and (A ′) a silane coupling agent having a polymerizable group is bonded to the spacer layer.
  • a composition containing a fluorescent dye and a polymer can also be formed by adding another polymerizable monomer, a fluorescent dye and a polymerization initiator and copolymerizing them.
  • the fluorescent dye is bonded to the spacer layer.
  • (B) by binding the silane coupling agent having an amino group or a carboxyl group and a fluorescent dye introduced with a functional group that reacts with these groups and covalently binds, the fluorescent dye is bonded to the spacer layer. Can be immobilized.
  • the amount of the fluorescent dye that can be immobilized is large, and the strength of the resulting layer is high, which is preferable.
  • the fluorescent dye is a general term for substances that emit fluorescence by irradiating with predetermined excitation light or by using the electric field effect, and the “fluorescence” means various emission such as phosphorescence. Including.
  • the fluorescent dye used in the present invention is not particularly limited, and may be any known fluorescent dye.
  • fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred.
  • fluorescent dyes examples include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes. (Applied Biosystems Japan Co., Ltd.), Coumarin family fluorescent dye (Invitrogen Corp.), Rhodamine family fluorescent dye (GE Healthcare Bioscience Co., Ltd.), Cyanine family fluorescent dye, Indocarbocyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanide dye Millie's fluorescent dye, BODIPY® family fluorescent dye (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (Registered trademark) dye series (manufactured by Invitrogen Corp.) and the like, and further, U.S. Patent
  • Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.
  • terbium [Tb] chelate fluorescence wavelength: 490 nm
  • enhanced cyan fluorescent protein [ECFP] fluorescence wavelength: 475 nm
  • 2-Me TG and 2-Me-- Fluorescent dyes such as Tokyo Green [TG] including 4-OMe TG, 2-OMe-5-Me TG, 2-OMe TG, and the like can also be used.
  • fluorescent dyes have high water solubility, and in order to immobilize these fluorescent dyes as a fluorescent dye layer in a polymer by intermolecular interaction, a hydrophobic aromatic group is attached to the carboxyl group of the fluorescent dye. It is necessary to react with the amino group or alcohol of the aromatic ring to form a water-insoluble structure, or to bond chemically by reaction between the hydrophobic polymer and the active ester of the fluorescent dye. In the case where the polymer and the fluorescent dye do not have a chemical bond, it is preferable to modify the fluorescent dye so as to have a structure close to the solubility parameter of the polymer (SP).
  • SP solubility parameter of the polymer
  • polymer examples include polyacrylate, polymethacrylate, polystyrene-acrylate, polystyrene, polyvinyl butyral, and polyester.
  • polyacrylates and polymethacrylates, polystyrene, and polyvinyl butyral have excellent compatibility with fluorescent dyes and nonspecific adsorption (eg, proteins (albumin, fibrinogen, immunoglobulin), lipids, saccharides (glucose)). Can be suppressed, which is preferable.
  • composition in addition to the fluorescent dye and the polymer, the composition can also contain a solvent and, if necessary, additives such as an antioxidant.
  • the “solvent” is not particularly limited as long as it has high volatility.
  • halogen-containing hydrocarbons for example, dichloromethane, dichloroethane, tetrafluoropropane, etc.
  • alcohols for example, methanol, ethanol, propanol, butanol, Tertiary butanol, tetrafluoropropanol
  • aromatics eg, toluene, xylene, etc.
  • ethers eg, diethyl ether, diethylene glycol monomethyl ether, etc.
  • esters eg, ethyl acetate, butyl acetate, etc.
  • glycols for example, ethylene glycol
  • ketones acetone, methyl ethyl ketone, etc.
  • aromatics, halogen-containing hydrocarbons, esters, and ketones are preferred from the viewpoint of the dissolution stability of the polymer used.
  • antioxidant examples include pentaerythrityl tetrakis [3- (3,5-di-tert-butyl-4-hydroxyphenyl)] propionate, 2,6-di-tert-butyl-4-methylphenol. 2,2′-dioxy-3,3′-di-t-butyl-5,5′-dimethyldiphenylmethane, tetrakis [methylene-3- (3,5-di-t-butyl-4-hydroxyphenyl) propionate ] Methane etc. are mentioned.
  • the fluorescent dye is preferably 1 to 75% by weight, more preferably 30 to 70% by weight, and the polymer is preferably 25 to 99% by weight, more preferably 70 to 30% by weight, based on the total amount of the composition (100% by weight). preferable.
  • the quenching efficiency is good.
  • the solvent is preferably 100 to 1,000 parts by weight, more preferably 100 to 500 parts by weight with respect to 100 parts by weight of the composition.
  • the additive is preferably from 0.1 to 10 parts by weight, more preferably from 1 to 5 parts by weight, based on 100 parts by weight of the composition. It is preferable that the solvent or additive has the above blending amount because the coating property is good and the fluorescence quantum yield is not lowered.
  • the coating method is not particularly limited. For example, it is usually 20 to 100 after application by spin coating, wire coating, bar coating, roll coating, blade coating, curtain coating, screen printing, or the like. Dry at 30 ° C. for 5-30 minutes.
  • silane coupling agent having a bifunctional reactive group it has an ethoxy group (or methoxy group) that gives a silanol group [Si-OH] by hydrolysis, and an amino group, a glycidyl group, a carboxyl group, etc. at the other end.
  • Any silane coupling agent having a reactive group may be used. Specific examples include 3-aminopropyltriethoxysilane, 8-amino-octyltriethoxysilane, 6-amino-hexyltriethoxysilane, and 7-carboxy-heptyltriethoxy. Examples thereof include silane and 5-carboxy-pentyltriethoxysilane, but the present invention is not limited to these, and conventionally known silane coupling agents can also be used.
  • silane coupling agent having a bifunctional reactive group since it has excellent ligand immobilization ability, for example, carboxymethyl dextran, polyethylene glycol, iminodiacetic acid derivatives ((N-5-amino-1-carboxypentyl) iminodiacetic acid, etc.), biotin, avidin, streptavidin, protein A, protein G and the like are also suitable.
  • a silane coupling agent as the compound having a bifunctional reactive group in (A), as a specific example of a method for immobilizing a ligand, first, a thin gold film, a spacer layer made of a dielectric, and a fluorescent dye layer are:
  • the transparent flat substrate formed in order on one surface thereof is immersed in an aqueous solution containing a silane coupling agent in a concentration of usually 0.1 to 10%, preferably 0.5%, for 30 minutes to 2 hours, and then at room temperature.
  • drying is usually performed for 1 to 24 hours, preferably 10 hours, and at 100 ° C.
  • drying is usually performed for 10 minutes to 1 hour, preferably 30 minutes, and then the substrate is usually washed with water.
  • a monomolecular film is formed in which silanol groups [Si—OH] obtained by hydrolysis of one end of the silane coupling agent are arranged on the fluorescent dye layer side.
  • the amino group and carboxyl group of the silane coupling agent are exposed on the outside of the monomolecular film made of the silane coupling agent.
  • the carboxyl group of the ligand is converted into water-soluble carbodiimide [WSC] (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC]) and N-hydroxysuccinimide [NHS].
  • WSC water-soluble carbodiimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • a carboxyl group of a polymer in a fluorescent dye layer is converted into a water-soluble carbodiimide [WSC] (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC] etc.) And N-hydroxysuccinimide [NHS], and the amino group of the ligand is dehydrated and immobilized using water-soluble carbodiimide.
  • WSC water-soluble carbodiimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • the plasmon excitation sensor (II) of the present invention comprises a transparent flat substrate; a metal thin film formed on the surface of the substrate; a dielectric film formed on the other surface of the metal thin film that is not in contact with the substrate A spacer layer comprising a body; and a ligand labeled with a fluorescent dye, which is immobilized on the other surface of the spacer layer that is not in contact with the metal thin film.
  • the plasmon excitation sensor (II) of the present invention is used in the assay method (Y) of the present invention.
  • the spacer layer made of the transparent flat substrate, the metal thin film, and the dielectric used in the plasmon excitation sensor (II) of the present invention the same spacer layer as that of the plasmon excitation sensor (I) of the present invention can be used.
  • the method is the same as that of the plasmon excitation sensor (I) of the present invention.
  • Ligand labeled with fluorescent dye As a method for labeling the above-mentioned fluorescent dye to the above-mentioned ligand, for example, an active ester of the fluorescent dye is prepared and further amine-coupled with the ligand.
  • Various functional groups such as thiocyanate group, sulfonyl chloride group, mercapto group, iodoacetamide group and the like can be introduced, and a method for forming a chemical bond under a condition in which the reactive group and the functional group of the ligand can react And so on.
  • a method for immobilizing a ligand labeled with a fluorescent dye on a spacer layer made of the above-mentioned dielectric a method for immobilizing on a spacer layer via a SAM (self-assembled monolayer) made of a silane coupling agent Is preferred.
  • the method for immobilizing a ligand labeled with a fluorescent dye is the same as in the case of the plasmon excitation sensor (I) of the present invention.
  • a method of labeling a fluorescent dye on the ligand immobilized on the spacer layer can be used. After immobilizing the ligand with a silane coupling agent having a functional group capable of reacting with the ligand, the reactive group is further added. It is also possible to produce an immobilized ligand labeled with a fluorescent dye by reacting the fluorescent dye with the fluorescent dye.
  • the assay method of the present invention comprises at least (X) includes the following steps (a1), (b1), (d) and (e), (Y) includes the following steps (a2), (b1), (d) and (e), or (Z) includes the following steps (a1), (b2), (c), (d) and (e) It is characterized by including.
  • Step (a1) a step of bringing a specimen into contact with the plasmon excitation sensor (I) of the present invention
  • Step (a2) a step of bringing a specimen into contact with the plasmon excitation sensor (II) of the present invention
  • Step (b1) the plasmon excitation sensor obtained through the step (a1) or (a2), and a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor, and a quenching dye Reacting the conjugate with Step (b2):
  • the plasmon excitation sensor obtained through the step (a1) is further combined with a ligand / enzyme conjugate that may be the same as or different from the ligand contained in the plasmon excitation sensor.
  • Step (c) a step of reacting a quencher substrate with the plasmon excitation sensor obtained through the step (b2) to produce a quencher
  • the assay method of the present invention includes the assay method (X) taking the embodiment of (X), the assay method (Y) taking the embodiment of (Y), and the assay method (Z) taking the embodiment of (Z). Is included.
  • the assay method of the present invention preferably further includes a washing step as appropriate.
  • the assay method of the present invention is preferably carried out while maintaining a constant temperature.
  • Steps (a1) / (a2) are steps of bringing the specimen into contact with the plasmon excitation sensors (I) and (II) of the present invention, respectively.
  • specimen examples include blood (serum / plasma), urine, nasal fluid, saliva, feces, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc. It may be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred.
  • the “flow channel” is a rectangular parallelepiped or a tube that can efficiently deliver a small amount of a chemical solution and can change the liquid feeding speed or circulate in order to promote the reaction.
  • the vicinity of the place where the plasmon excitation sensor is installed preferably has a rectangular parallelepiped structure, and the vicinity of the place where the drug solution is delivered preferably has a tubular shape.
  • the materials include homopolymers or copolymers, polyethylene, polyolefin, etc. containing methyl methacrylate, styrene, etc. as raw materials in the plasmon excitation sensor part, and silicon rubber, Teflon (registered trademark), polyethylene, polypropylene in the chemical solution delivery part. Etc. are used.
  • the vertical and horizontal sections of the channel of the plasmon excitation sensor unit are independently about 100 nm to 1 mm.
  • a method of fixing the plasmon excitation sensor to the flow path in a small-scale lot (laboratory level), first, on the surface on which the metal thin film of the plasmon excitation sensor is formed, A dimethylsiloxane [PDMS] sheet is pressure-bonded so as to surround the portion where the metal thin film of the plasmon excitation sensor is formed, and then the polydimethylsiloxane [PDMS] sheet and the plasmon excitation sensor are closed with screws or the like.
  • a method of fixing with a tool is preferred.
  • a gold substrate is formed on a plastic integrally molded product, or a separately manufactured gold substrate is fixed, and the gold surface is fixed. Further, after the dielectric layer, the fluorescent dye layer, and the ligand are immobilized, it can be manufactured by covering with a plastic integrally formed product corresponding to the top plate of the flow path. If necessary, the prism can be integrated into the flow path.
  • the “liquid feeding” is preferably the same as the solvent or buffer in which the specimen is diluted, and examples thereof include phosphate buffered saline (PBS) and Tris buffered saline (TBS), but are not particularly limited. It is not something.
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • the temperature and time for circulating the liquid supply vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. ⁇ 1 to 60 minutes, preferably 37 ° C. ⁇ 5 to 15 minutes.
  • the initial concentration of the analyte contained in the specimen being sent may be 100 ⁇ g / mL to 0.001 pg / mL.
  • the total amount of liquid feeding, that is, the volume of the flow path is usually 0.001 to 20 mL, preferably 0.1 to 1 mL.
  • the flow rate of the liquid feeding is usually 1 to 2,000 ⁇ L / min, preferably 5 to 500 ⁇ L / min.
  • the washing step is preferably included before and / or after the following step (b1) or (b2), and the plasmon obtained in the above step (a1) or (a2) or the following step (b1) or (b2) This is a step of cleaning the surface of the excitation sensor.
  • a surfactant or a surfactant such as Tween 20 or Triton X100 is used in the same solvent or buffer as used in the reaction of the above step (a1) or (a2) or the following step (b1) or (b2). It is desirable that it is dissolved in a liquid and preferably contains 0.00001 to 1% by weight.
  • the temperature and flow rate at which the cleaning liquid is circulated are preferably equal to the “temperature and flow rate at which the liquid feed is circulated” in step (a1) or (a2).
  • the time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.
  • Step (b1) The step (b1) is different from the step (a1) or (a2), preferably the plasmon excitation sensor obtained through the washing step, even if the ligand contained in the plasmon excitation sensor is the same.
  • This is a step of reacting a conjugate of an optional ligand and a quenching dye.
  • quenching dye The quenching dye (or quencher) used in step (b1), that is, assay method (X) or (Y) is included in a compound capable of quenching or absorbing fluorescence, and the fluorescent dye is excited. If the compound has an appropriate energy level capable of absorbing the energy and an appropriate quenching dye is added to a certain fluorescent dye, the fluorescence disappears.
  • quenching dyes include fluorescein family quenching dyes, polyhalofluorescein family quenching dyes, hexachlorofluorescein family quenching dyes, coumarin family quenching dyes, rhodamine family quenching dyes, cyanine family quenching dyes.
  • Quenching dyes oxazine family quenching dyes, thiazine family quenching dyes, squarain family quenching dyes, chelated lanthanide family quenching dyes, BODIPY (registered trademark) family quenching dyes, and more Specifically, for example, BHQ (registered trademark) family dyes (including quenchers described in WO 01/86001: BHQ-1, BHQ-2 and BHQ-3) (Bio Searchte Nonology Japan BTJ, Iowa Black (registered trademark) (Integrated DNA Technologies), DABCYL (4- (4'-dimethylaminophenylazo) benzoic acid) (Integrated DNA Technologies), TAMRAN , N, N ′, N′-tetramethyl-6-carboxyrhodamine) (manufactured by Invitrogen), Cy3 (registered trademark) (manufactured by GE Healthcare Biosciences), Cy5 (registered trademark) (GE Health) Care Biosciences), 1-benz
  • BHQ-1 maximum wavelength 534 nm
  • BHQ-2 maximum wavelength 579 nm
  • BHQ-3 maximum wavelength 672 nm
  • a dark quencher a quenching dye that does not emit light itself
  • quenching dye systems usually include tetracyanoquinodimethanes, aminiums, diimmoniums, hydrazines, hydrazides, hydroxylamines, hydroquinones, tetrasubstituted boron anions, nickels, azo Examples include heavy metal complexes of dyes, formazan heavy metal complexes, dipyrromethene metal complexes, porphyrin heavy metal complexes, heavy metal phthalocyanines, heavy metal naphthalocyanines, and metallocenes.
  • the photons emitted by the fluorescent dye excited by the surface plasmon are considered to be quenched by energy transfer to the quenching dye in an electronically excited state. That is, it is considered that a quenching dye that has absorbed energy in an electronically excited state emits energy as photons or heat having different wavelengths. Therefore, in some cases, a compound exemplified as a fluorescent dye such as TG can be used as a quenching dye (or quencher).
  • a critical transition distance is a distance at which the electronic excitation level (usually singlet) of a fluorescent dye can interact with the lowest empty orbit of the quenching dye.
  • the quenching dye is an organic substance, it is usually a distance of about 10 nm, In the case of a metal-containing material, the distance is usually about 30 nm.
  • the critical transition distance between a specific fluorescent dye and a quenching dye is well known in the art, for example, Wu and Brand, 1994, Anal. Biochem. 218: 1-3 You can refer to the distance.
  • the quenching dye is also a fluorescent dye
  • An exemplary combination of a fluorescent dye and a quenching dye used in the present invention includes 6-carboxyfluorescein [FAM] as the fluorescent dye and Cy5 (registered trademark) as the quenching dye; Alexa Fluor (registered trademark) 647 ⁇ fluorescent dye> BHQ-3 ⁇ quenching dye>; FAM, TET, JOE, HEX and Oregon Green ⁇ fluorescent dye> and BHQ-1 ⁇ quenching dye>; FAM, TAMRA, ROX, Cy3, Cy3.5, CAL Red and Red 640 ⁇ fluorescence Dye> and BHQ-2 ⁇ quenching dye>; Cy5 and Cy5.5 ⁇ fluorescent dye> and BHQ-3 ⁇ quenching dye>, combinations described in US Pat. No. 6,245,514, listed in Table 2 However, the present invention is not limited to these combinations.
  • Molecules that can be used as both fluorescent and quenching dyes include, for example, fluorescein, 6-carboxyfluorescein, 2 ′, 7′-dimethoxy-4 ′, 5′-dichloro-6-carboxyfluorescein, rhodamine, 6-carboxyl And rhodamine, 6-carboxy-X-rhodamine, 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid [EDANS] and the like.
  • conjugate of a ligand and quenching dye uses the following embodiment ( ⁇ ) or ( ⁇ ) when a secondary antibody is used as the ligand: Is preferred.
  • the secondary antibody may be a monoclonal antibody or a polyclonal antibody.
  • the secondary antibody is preferably a monoclonal antibody that recognizes an epitope that the primary antibody does not recognize, or a polyclonal antibody.
  • the primary antibody used as a ligand immobilized on the plasmon excitation sensor of the present invention is, for example, an AFP monoclonal antibody
  • the secondary antibody of the embodiment ( ⁇ ) competes with AFP contained in the specimen.
  • Aspect ( ⁇ ) is preferable because the amount of fluorescence signal can be adjusted depending on the ability of the quencher, and therefore the assay method of the present invention can be carried out at an optimal S / N ratio.
  • Such a secondary antibody may be a monoclonal antibody or a polyclonal antibody as long as it binds to a competitive antigen without binding to a target antigen.
  • the complex of the secondary antibody and the competitive antigen used in the embodiment ( ⁇ ) is preferable to use the complex of the secondary antibody and the competitive antigen used in the embodiment ( ⁇ ) for the competitive immunoassay method.
  • the competitive immunoassay method is applied to, for example, the assay method (X) or (Y) of the present invention, that is, in the assay method (X) or (Y) of the present invention, instead of the conjugate of the ligand and the quenching dye.
  • the embodiment ( ⁇ ) is preferable because the amount of fluorescent signal (fluorescent signal) and the amount of target antigen can be proportional.
  • the complex of the secondary antibody and the competitive antigen is brought into contact with the plasmon excitation sensor in excess after the step (b1), preferably after the washing step.
  • the secondary antibody of the embodiment ( ⁇ ) is an anti-AFP polyclonal antibody or an anti-AFP monoclonal antibody that can recognize and bind to an epitope that the anti-AFP monoclonal antibody does not recognize. Requires antibody.
  • a secondary antibody As a method for preparing a conjugate of a ligand and a quenching dye, when a secondary antibody is used as the ligand, for example, first, a carboxyl group is added to the quenching dye, and the carboxyl group is converted into a water-soluble carbodiimide [WSC] (for example, 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC] and the like) and N-hydroxysuccinimide [NHS], and then the active esterified carboxyl group and the amino acid contained in the secondary antibody
  • WSC water-soluble carbodiimide
  • EDC EDC] and the like
  • NHS N-hydroxysuccinimide
  • the concentration of the conjugate of the ligand thus prepared and the quenching dye during feeding is preferably 0.001 to 10,000 ⁇ g / mL, more preferably 1 to 1,000) ⁇ g / mL.
  • the temperature, time and flow rate at which the liquid is circulated are the same as those in step (a1) or (a2). Moreover, it is preferable to include the said washing
  • the step (b2) may be the same as or different from the plasmon excitation sensor obtained through the step (a1), preferably the washing step, and the ligand contained in the plasmon excitation sensor. This is a step of reacting a conjugate of a ligand and an enzyme.
  • the enzyme used in the step (b2), that is, the assay method (Z) is for generating a “quenching agent” capable of quenching the fluorescence emitted from the fluorescent dye from a predetermined “quenching substrate”. More specifically, the enzyme activates the quencher by, for example, (A) removing the protecting group by enzymatic reaction from the following “quencher substrate” blocked by the protecting group, or (B) It is used for lowering the fluorescence intensity by lowering the pH around the fluorescent dye with a quencher activated by an enzymatic reaction using a specific “quencher substrate” described later.
  • Examples of the “enzyme” used in the enzyme reaction (A) include ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase and the like.
  • ⁇ -galactosidase catalyzes the reaction of eliminating ⁇ Gal from the quencher substrate TG- ⁇ Gal.
  • ⁇ -glucosidase catalyzes a reaction for eliminating ⁇ Glu from TG- ⁇ Glu which is a quencher substrate.
  • free TG has an excitation wavelength of 490 nm and a fluorescence dye having a fluorescence wavelength of 475 to 495 nm and Fluorescence Resonance Energy Transfer (FRET; fluorescence resonance energy transfer), fluorescence of terbium [Tb] chelate (fluorescence wavelength: 495 nm) ) Or enhanced cyan fluorescent protein (Enhanced Cyan Fluorescence Protein; ECFP) (fluorescence wavelength: 475 nm) can be quenched.
  • FRET Fluorescence Resonance Energy Transfer
  • Alkaline phosphatase catalyzes a reaction of hydrolyzing an AttoPhos (registered trademark) substrate to produce BBT [2 ′-[2-benzthiazoyl] -6′-hydroxyl-benzthiazole], which is a fluorescent substance.
  • the produced BBT is a fluorescent substance having an excitation wavelength of 482 nm, and, like the above-described TG, can cause terbium [Tb] chelate or ECFP and FRET to quench them.
  • Examples of the “enzyme” used in the enzyme reaction (B) include glucose oxidase.
  • Glucose oxidase produces gluconolactone and hydrogen peroxide by an enzyme reaction using glucose as a quencher substrate.
  • Fluorescence intensity such as 2-Me-4-OMe TG, 2-OMe-5-Me TG or 2-OMe TG used as a fluorescent dye as the pH of the water is lowered by hydrogen peroxide dissolved in the water becomes smaller (ie, extinguished).
  • conjugate of ligand and enzyme “The conjugate of a ligand and an enzyme, which may be the same as or different from the ligand contained in the plasmon excitation sensor” is a ligand labeled with the enzyme, and the ligand is the same as the ligand Or different.
  • a carboxyl group possessed by an enzyme is first converted into a water-soluble carbodiimide [WSC] (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC].
  • WSC water-soluble carbodiimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • the concentration of the ligand-enzyme conjugate thus prepared in the solution is preferably 0.001 to 10,000 ⁇ g / mL, more preferably 1 to 1,000) ⁇ g / mL.
  • the temperature, time, and flow rate at which the liquid is circulated are the same as those in step (a1) or (a2).
  • the step (c) is a step in which a quencher substrate is reacted with the plasmon excitation sensor obtained through the step (b2), preferably the washing step, to generate a quencher.
  • quencher substrate examples include TG- ⁇ Gal, TG- ⁇ Glu, AttoPhos (registered trademark) substrate, glucose and the like as described above.
  • TG- ⁇ Gal and TG- ⁇ Glu which are quencher substrates used in the enzyme reaction (A) are compounds obtained by adding one molecule of ⁇ -galactose and ⁇ -glucose as protective groups to the fluorescent dye, TokyoGreen [TG], respectively. In this state, almost no fluorescence is observed, but strong light is emitted by the removal of the protecting group by ⁇ -galactosidase and ⁇ -glucosidase.
  • TG includes 2-Me TG and 2-Me-4-OMe TG represented by the above formula.
  • the AttoPhos (registered trademark) substrate hardly emits fluorescence even in a solution at pH 9.5, but is converted to BBT as a result of the enzymatic reaction with alkaline phosphatase, and emits strong fluorescence.
  • the quencher produced by the enzyme reaction (A) is included in a compound that can quench or absorb fluorescence in the same manner as the quenching dye, and can be used in an appropriate energy level capable of absorbing the excited energy of the fluorescent dye. When a suitable quenching dye is added to a certain fluorescent dye, the fluorescence disappears.
  • quencher substrate used in the enzyme reaction (B) examples include glucose and oxygen which are substrates for glucose oxidase.
  • preferable combinations of an enzyme, a quencher substrate, a quencher and a fluorescent dye include those shown in the following table.
  • the concentration of such a quencher substrate during feeding is preferably 0.001 to 10,000 ⁇ g / mL, more preferably 1 to 1,000 ⁇ g / mL.
  • the temperature, time, and flow rate at which the liquid is circulated are the same as those in step (a1) or (a2).
  • Step (d) refers to the step (b1), preferably the plasmon excitation sensor obtained through the cleaning step or the step (c), from one side of the transparent flat substrate on which the metal thin film is not formed. This is a step of measuring the amount of fluorescence emitted from the excited fluorescent dye by irradiating laser light through a prism.
  • the “laser light” adjusts the energy and the amount of photons immediately before entering the prism through the optical filter. Irradiation with laser light generates surface plasmons on the surface of the metal thin film under the total reflection attenuation condition [ATR]. Due to the electric field enhancement effect of surface plasmons, the fluorescent dye is excited by photons that are increased by several tens to several hundred times the amount of photons irradiated. The increase in photons due to the electric field enhancement effect depends on the refractive index of the glass serving as the substrate, the metal species and the film thickness of the metal thin film, but is usually about 10 to 20 times the increase in gold.
  • the fluorescent dye In the fluorescent dye, the electrons in the molecule are excited by light absorption, move to the first electronic excited state in a short time, and when returning from this state (level) to the ground state, the fluorescent dye has a wavelength corresponding to the energy difference. To emit.
  • quenching if there is a quencher in the vicinity that can absorb energy corresponding to the energy required for transition from the ground state of the fluorescent dye to the first electronic excited state, energy is transferred from the fluorescent dye to the quenching dye or the quencher.
  • the fluorescent dye moves and returns from the first electron excited state to the ground state without generating fluorescence. This phenomenon is called quenching.
  • the fluorescence that has not been quenched by the quenching dye / quenching agent is incident on the SPFS detector through the cut filter by the condenser lens, and the count value of the incident light is measured.
  • a light source of “laser light” for example, an LED capable of irradiating laser light having a wavelength of 400 to 840 nm and an incident light amount of about 1 mW, a wavelength of 230 to 800 nm (resonance wavelength is determined by the metal type used in the metal thin film), 0.
  • Examples thereof include a semiconductor laser [LD] capable of irradiating a laser beam of 01 to 100 mW.
  • both LED and LD can be used in SPR, but SPFS requires high energy to excite the fluorescent dye, and LD is preferable from the viewpoint of high sensitivity.
  • the “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon excitation sensor, and the refractive index is preferably the same as that of the “transparent flat substrate”.
  • various prisms for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape.
  • a 60-degree dispersion prism may be used.
  • Examples of such commercially available prisms include those similar to the above-mentioned commercially available “glass-made transparent flat substrate”.
  • optical filter examples include a neutral density [ND] filter and a diaphragm lens.
  • the “darkening [ND] filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.
  • the “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons.
  • Cut filters are: external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattering light (excitation light originated from plasmon A filter that removes various noise light such as scattered light generated due to the influence of structures or deposits on the surface of the excitation sensor), autofluorescence of the enzyme fluorescent substrate, fluorescence emitted by the quenching dye / quenching agent by FRET, For example, an interference filter, a color filter, etc. are mentioned.
  • the “condensing lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system.
  • a simple condensing system a commercially available objective lens (for example, manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be used.
  • the magnification of the objective lens is preferably 10 to 100 times.
  • the “SPFS detector” is preferably a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics) from the viewpoint of ultra-high sensitivity. Also, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed.
  • Step (e) is a step of calculating the amount of the analyte contained in the specimen from the measurement result obtained in the step (d).
  • a calibration curve is created by performing measurement with an analyte having a known concentration, and the analyte (target antigen) in the sample to be measured is calculated from the measurement signal based on the created calibration curve. It is a process.
  • the blank fluorescent signal measured before the step (d), the assay fluorescent signal obtained in the step (d), and a metal substrate not modified at all are fixed to the channel.
  • the assay S / N ratio represented by the following formula can be calculated.
  • the assay device of the present invention comprises at least the plasmon excitation sensor, laser light source, optical filter, prism, cut filter, condensing lens, and surface plasmon excitation enhanced fluorescence obtained through the step (b1) or (c). It includes a detector and is used in the step (d).
  • the assay device of the present invention is for carrying out the assay method (X), (Y) or (Z) of the present invention using the plasmon excitation sensor (I) or (II) of the present invention. is there.
  • the “apparatus” includes at least a light source, an optical filter, a prism, a flow path, a plasmon excitation sensor, a liquid feed pump, a cut filter, a condenser lens, and an SPFS detection unit.
  • the surface plasmon resonance [SPR] detector that is, the angle variable unit for adjusting the optimum angle of the photodiode, SPR and SPFS as a light receiving sensor dedicated to SPR (in order to obtain the total reflection attenuation [ATR] condition by the servomotor)
  • the angle between 30 ° and 85 ° is changed in synchronization with the photodiode and the light source.
  • the resolution is preferably 0.01 ° or more.
  • liquid feed pump examples include a micro pump suitable for a small amount of liquid feed, a syringe pump with high feed accuracy and low pulsation, which is preferable but cannot be circulated, and a simple and excellent handleability but a small amount of liquid feed.
  • a tube pump may be difficult.
  • the sensor includes at least a transparent flat substrate, the metal thin film, the spacer layer made of the dielectric, and the fluorescent dye layer, and the quenching dye.
  • the assay method of the present invention includes at least a sensor including a transparent flat substrate, the metal thin film, and a spacer layer made of the dielectric, the fluorescent dye, and the quenching dye.
  • the embodiment of (Z) includes at least a sensor including a transparent flat substrate, the metal thin film, the spacer layer made of the dielectric, and the fluorescent dye layer, and the enzyme and quencher substrate. It is used for the assay method (Z) of the invention.
  • the assay kit of the present invention includes everything necessary for performing the assay method (X), (Y) or (Z) of the present invention in addition to the specimen, the primary antibody and the secondary antibody. It is preferable.
  • the kit of the present invention blood as a specimen, and an antibody against a specific tumor marker, the content of the specific tumor marker can be detected with high sensitivity and high accuracy. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.
  • such a “kit” includes a metal thin film and a dielectric spacer layer (and a fluorescent dye layer) formed in this order on a transparent flat substrate; reagents for immobilizing a ligand (For example, silane coupling agent, water-soluble carbodiimide (EDC, etc.), N-hydroxysuccinimide [NHS], etc.); solution or dilution solution for dissolving or diluting the sample; reaction between the plasmon excitation sensor and the sample Various reaction reagents and washing reagents; quenching dyes (eg, BHQ-3); enzymes ( ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, glucose oxidase, etc.); quencher substrates (eg, TG- ⁇ Gal, TG) - ⁇ Glu, AttoPhos (registered trademark) substrate, glucose, etc.); quenching dye for secondary antibody Or various reagents for immobilizing enzymes (
  • kit element a standard material for preparing a calibration curve, a manual, a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of samples may be included.
  • Examples (1-1) to (1-7) and (2-1) to (2-7) and Comparative Examples (1-1), (1-2) and (2-1), (2 -2) carried out the sandwich immunoassay method.
  • Examples (1-8) to (1-14) and (2-8) to (2-14) and Comparative Examples (1-3) and (1-4 ), (2-3), and (2-4) were subjected to competitive immunoassay.
  • Preparation Example (1-1) (Preparation of conjugate of secondary antibody and quenching dye) BHQ-2, BHQ-3 (manufactured by Biosearch Technologies Japan BTJ Co., Ltd./manufactured by Nippon Bioservice Co., Ltd.) or DABCYL (manufactured by Integrated DNA Technologies) are mixed with anti- ⁇ -fetoprotein [AFP] monoclonal antibody (1D5; 2. 5 mg / mL, immobilized on Japan Medical Clinical Laboratory Laboratories).
  • BHQ-2, BHQ-3 manufactured by Biosearch Technologies Japan BTJ Co., Ltd./manufactured by Nippon Bioservice Co., Ltd.
  • DABCYL manufactured by Integrated DNA Technologies
  • Example (1-1) An excess amount of AFP was mixed and complexed with the obtained conjugate of the secondary antibody and the quenching dye, and then purified using centrifugation and chromatography.
  • the assay was performed using SPFS in a region with a small amount of AFP (antigen) (pmol / L to fmol / L).
  • a spacer layer made of silicon dioxide [SiO 2 ] as a dielectric was formed by sputtering on one side of the gold thin film that was not in contact with the chromium thin film.
  • the spacer layer had a thickness of 15 nm.
  • aqueous solution containing 5% by weight of 3-aminopropyltriethoxysilane was applied onto the fluorescent dye layer of the substrate thus obtained with a spin coater, allowed to dry naturally at room temperature for 2 hours, and then at 50 ° C. for 10 minutes. Heated.
  • the surface of the fluorescent dye layer treated with the silane coupling agent is provided with a spacer made of polydimethylsiloxane [PDMS] having a hole of 2 mm ⁇ 10 mm, an outer shape of 20 mm ⁇ 20 mm, and a thickness of 0.5 mm.
  • the substrate was placed in the flow path so that is inside the flow path.
  • a polymethyl methacrylate plate having a thickness of 4 mm and the same outer shape as that of the PDMS spacer was put on the substrate so as to cover the substrate from the outside of the flow channel, and the flow channel and the polymethyl methacrylate plate were fixed with screws.
  • Ultrapure water was circulated for 10 minutes and then PBS was circulated for 30 minutes by a peristaltic pump at 30 ° C. and a flow rate of 500 ⁇ L / min.
  • the total volume of liquid delivery is 15 mL.
  • a semiconductor laser [LD] is used as a light source, a laser beam with a wavelength of 633 nm is irradiated, a photon amount is adjusted using a neutral density filter as an optical filter, and Sigma Kogyo Co., Ltd.
  • the surface plasmon measurement was started by irradiating the plasmon excitation sensor before immobilization of the ligand fixed to the flow path through the 60-degree prism manufactured.
  • the resonance angle shift was measured with surface plasmons to confirm the immobilization of the ligand.
  • the immobilization amount was 3 ng / mm 2 .
  • non-specific adsorption prevention treatment was performed by circulating and feeding in PBS buffered saline containing 1% by weight of bovine serum albumin [BSA] for 30 minutes.
  • Step (a1) The solution was replaced with PBS, 5 mL of PBS containing 10 ng / mL of AFP was added and circulated for 30 minutes.
  • Washing step Washing was performed by circulating 10 minutes using PBS containing 0.05% by weight of Tween 20 as a solution.
  • the plasmon excitation sensor fixed to the flow path using an LD is irradiated, and the cut filter manufactured by Nippon Vacuum Optics Co., Ltd. is 10 times as a condenser lens.
  • an objective lens manufactured by Nikon Co., Ltd.
  • fluorescence by SPFS was detected through a CCD image sensor (manufactured by Texas Instruments Japan Ltd.) to obtain a “blank fluorescence signal”.
  • Step (b1) 5 mL of PBS containing 1,000 ng / mL of the conjugate of the secondary antibody obtained in Preparation Example (1-1) and a quenching dye was added and circulated for 30 minutes. Washing step: Washing was performed by circulating PBS containing 0.05% by weight of Tween 20 for 20 minutes.
  • the other flow path not modified on the gold substrate was separately installed in the SPFS, and the resonance angle was reset based on the surface plasmon measurement while flowing ultrapure water, and the SPFS was measured.
  • the signal was defined as “initial noise”.
  • the assay S / N ratio was evaluated.
  • the assay S / N ratio indicates that the reliability of the assay signal is high when the absolute value of the numerical value of the fluorescent signal that changes with the amount of conjugate proportional to the amount of antigen is large and sufficiently large relative to the initial noise. means.
  • Example (1-2) A plasmon excitation sensor of the present invention was produced and assayed in the same manner as in Example (1-1) except that the fluorescent dye was changed to Alexa Fluor (registered trademark) 647 in Example (1-1). . Table 4 shows the obtained results.
  • Example (1-3) Manufacture of plasmon excitation sensor (I)
  • a glass transparent flat substrate having a refractive index [n d ] of 1.52 and a thickness of 1 mm and an outer shape of 20 mm ⁇ 20 mm (“BK7” manufactured by Shot Japan Co., Ltd.) is plasma-cleaned, and a chromium thin film is formed on one side of the substrate.
  • a chromium thin film was formed on the surface by sputtering.
  • the chromium thin film had a thickness of 1 nm
  • the gold thin film had a thickness of 50 nm.
  • a spacer layer made of TiO 2 as a dielectric was formed by sputtering on one side of the gold thin film that was not in contact with the chromium thin film.
  • the spacer layer had a thickness of 15 nm.
  • aqueous solution containing 5% by weight of 3-aminopropyltriethoxysilane was applied to one side of the spacer layer not in contact with the gold thin film with a spin coater, allowed to dry naturally at room temperature for 2 hours, and then at 50 ° C. for 10 minutes. Heated.
  • Example (1-4) In Example (1-1), the same as Example (1-1) except that the metal species was silver, the thickness of the metal thin film was 45 nm, the fluorescent dye was TRITC, and the wavelength of the laser beam was 532 nm. Thus, a plasmon excitation sensor of the present invention was prepared and assay method (X) was performed. Table 4 shows the obtained results.
  • Example (1-5) the plasmon excitation sensor (I) of the present invention was produced in the same manner as in Example (1-4) except that the metal species was aluminum and the thickness of the metal thin film was 15 nm. Assay method (X) was performed. Table 4 shows the obtained results.
  • Example (1-6) In Example (1-5), the plasmon excitation sensor (I) of the present invention was prepared in the same manner as in Example (1-5) except that the thickness of the metal thin film was changed to 20 nm. Carried out. Table 4 shows the obtained results.
  • Example (1-7) In Example (1-6), except that the fluorescent dye was changed to Cy3, the plasmon excitation sensor (I) of the present invention was produced in the same manner as in Example (1-6), and assay method (X) was performed. . Table 4 shows the obtained results.
  • the obtained substrate was immersed in an ethanol solution containing 1 mM of 10-carboxy-1-decanethiol for 24 hours or more to form SAM (Self Assembled Monolayer) on one side of the gold thin film.
  • SAM Self Assembled Monolayer
  • a polydimethylsiloxane [PDMS] sheet having a flow path height of 0.5 mm was provided on the surface of the SAM, and a polymethyl methacrylate top plate was further disposed.
  • Ultrapure water was fed as a liquid for 10 minutes, and then PBS was circulated for 20 minutes with a peristaltic pump at room temperature and a flow rate of 500 ⁇ L / min to equilibrate the surface.
  • Washing was carried out by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes. 2.5 mL of Alexa Fluor (registered trademark) 647-labeled secondary antibody (PBS solution prepared to be 1,000 ng / mL) was added and circulated for 30 minutes.
  • Alexa Fluor registered trademark 647-labeled secondary antibody
  • Comparative Example (1-2) In Comparative Example (1-1), a plasmon excitation sensor was prepared in the same manner as Comparative Example (1-1) except that the metal species was changed to aluminum and the thickness of the metal thin film was changed to 20 nm. The assay method was carried out in the same manner as in Comparative Example (1-1) except that it was changed to. Table 4 shows the obtained results.
  • the assay S / N ratio obtains the measured fluorescence amount changed with respect to the original fluorescence amount, and shows the reliable dynamic range of the numerical value in the measurement, and further divides by the noise level of the base substrate.
  • the reliability limit including the noise level can be obtained. That is, the higher the assay S / N ratio, the more accurate numerical value is provided for the measured antigen amount.
  • Preparation Example (1-3) (Preparation of conjugate of secondary antibody and fluorescent dye complexed with competitive antigen)
  • a fluorescent dye Alexa Fluor (registered trademark) 647 or Cy3 was used in the same manner as in Preparation Example (1-2).
  • the conjugate of secondary antibody and Alexa Fluor (registered trademark) 647 ”and“ conjugate of secondary antibody and Cy3 complexed with competitive antigen ” were prepared.
  • Example (1-8) In the implementation of the assay method (X) of Example (1-1), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-1) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • Example (1-9) In the implementation of the assay method (X) of Example (1-2), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-2) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • Example (1-10) In carrying out the assay method (X) of Example (1-3), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-3) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • Example (1-11) In carrying out the assay method (X) of Example (1-4), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-4) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • Example (1-12) In the implementation of the assay method (X) of Example (1-5), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-5) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • Example (1-13) In the implementation of the assay method (X) of Example (1-6), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-6) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • Example (1-14) In carrying out the assay method (X) of Example (1-7), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), Preparation Example (1-2 The competitive immunoassay was carried out in the same manner as in Example (1-7) except that the “conjugate of secondary antibody and quenching dye complexed with competitive antigen” obtained in (1) was used. The results obtained are shown in Table 5.
  • the fluorescence signal value of the present invention is sufficiently high, indicating that the abundance of the antigen can be measured with sufficient accuracy. Moreover, the above-mentioned assay S / N ratio also showed a high value, and it was confirmed that the reliability was high.
  • the competitive assay of the comparative example since it is a competitive system, the fluorescence signal value shows a high value and there is a sufficient amount of fluorescence with respect to the antigen abundance, but the assay S / N ratio is a measured value that is lower than that of the present invention. It was suggested that the reliability accuracy of was low. Presuming from this result, it is shown that the quenching dye-labeled secondary antibody measurement system of the present invention has a wider dynamic range on the fluorescence amount change than the assay measurement system of the fluorescent dye alone.
  • Preparation Example (2-1) (Preparation of secondary antibody with immobilized quenching dye) In the same manner as in Preparation Example (1-1), a conjugate of anti-AFP monoclonal antibody and BHQ-2, BHQ-3 or DABCYL was prepared.
  • Preparation Example (2-2) (Preparation of secondary antibody with immobilized quenching dye and complexed with competitive antigen) In the same manner as in Preparation Example (1-2), a complex was prepared by previously binding AFP to the conjugate obtained in Preparation Example (2-1).
  • Example (2-1) The assay is performed using SPFS in a region (pmol / L to fmol / L) where the amount of AFP (antigen) is very small.
  • a glass transparent flat substrate having a refractive index [n d ] of 1.52 and a thickness of 1 mm and an outer shape of 20 mm ⁇ 20 mm (“BK7” manufactured by Shot Japan Co., Ltd.) is plasma-cleaned, and a chromium thin film is formed on one side of the substrate.
  • a chromium thin film was formed on one side of the substrate.
  • the chromium thin film had a thickness of 2 nm
  • the gold thin film had a thickness of 48 nm.
  • a spacer layer made of silicon dioxide [SiO 2 ] as a dielectric was formed by sputtering on one side of the gold thin film that was not in contact with the chromium thin film.
  • the thickness of the spacer layer was 10 nm.
  • the substrate thus obtained was immersed in a 50% ethanol aqueous solution containing 5% by weight of 7-carboxy-heptyltriethoxysilane, reacted at 30 ° C. for 30 minutes, and then dried at 100 ° C. for 30 minutes. .
  • a SAM made of a silane coupling agent was formed on the spacer layer.
  • a spacer made of polydimethylsiloxane [PDMS] having an outer shape of 20 mm ⁇ 20 mm and a thickness of 0.5 mm having a flow path of 2 mm ⁇ 10 mm on the surface of the spacer layer treated with a silane coupling agent.
  • substrate is arrange
  • a 4 mm thick polymethyl methacrylate plate having the same outer shape with two through-holes for taking in and out of the liquid is put on the substrate so as to cover the substrate from the outside of the flow channel, and the flow channel and the polymethyl methacrylate plate are bonded with screws. Fixed.
  • Ultrapure water was circulated for 10 minutes and then PBS was circulated for 30 minutes by a peristaltic pump at 30 ° C. and a flow rate of 500 ⁇ L / min.
  • the total volume of liquid delivery is 15 mL.
  • an LD is used as a light source, a laser beam having a wavelength of 633 nm is irradiated, a photon amount is adjusted using an attenuating filter (neutral density filter) as an optical filter, and 60 degrees made by Sigma Kogyo Co., Ltd.
  • the surface plasmon measurement was started by irradiating the plasmon excitation sensor before immobilization of the ligand fixed to the flow path through the prism.
  • Step (a2) The solution was replaced with PBS, 5 mL of PBS containing 20 ng / mL of AFP was added and circulated for 30 minutes.
  • Washing step Washing was performed by circulating 10 minutes using PBS containing 0.05% by weight of Tween 20 as a solution. After measuring the surface plasmon and fixing it to the optimum angle, the plasmon excitation sensor (II) fixed to the flow path is irradiated with an LD laser and used as a cut filter (manufactured by Nippon Vacuum Optical Co., Ltd.). The fluorescence by SPFS was detected through a CCD image sensor (manufactured by Texas Instruments) using a 20 ⁇ objective lens (manufactured by Nikon Corporation) to obtain a blank fluorescence.
  • CCD image sensor manufactured by Texas Instruments
  • 20 ⁇ objective lens manufactured by Nikon Corporation
  • Step (b1) 5 mL of PBS containing 1,000 ng / mL of the secondary antibody obtained in Preparation Example (2-1) was added and circulated for 30 minutes. Washing step: Washing was performed by circulating PBS containing 0.05% by weight of Tween 20 for 20 minutes.
  • the other flow path not modified on the gold substrate was separately installed on the SPFS, and the resonance angle was reset based on the surface plasmon measurement while flowing ultrapure water, and the SPFS was measured.
  • the signal was defined as “initial noise”.
  • Example (2-2) The plasmon excitation sensor (II) of the present invention was produced in the same manner as in Example (2-1) except that the fluorescent dye was changed to Alexa Fluor (registered trademark) 647 in Example (2-1), and assay method (Y) was carried out. The obtained results are shown in Table 6.
  • Example (2-3) The plasmon excitation sensor (II) of the present invention was produced in the same manner as in Example (2-1) except that the fluorescent dye was changed to Alexa Fluor (registered trademark) 633 in Example (2-1), and assay method (Y) was carried out. The obtained results are shown in Table 6.
  • Example (2-1) is the same as Example (2-1) except that the metal species is silver, the thickness of the metal thin film is 45 nm, the fluorescent dye is TRITC, and the wavelength of the laser beam is 532 nm.
  • the plasmon excitation sensor (II) of the present invention was manufactured, and the assay method (Y) was performed. The obtained results are shown in Table 6.
  • Example (2-5) the plasmon excitation sensor (II) of the present invention was produced in the same manner as in Example (2-4) except that the metal species was aluminum and the thickness of the metal thin film was 15 nm. Assay method (Y) was performed. The obtained results are shown in Table 6.
  • Example (2-6) In Example (2-5), the plasmon excitation sensor (II) of the present invention was produced in the same manner as in Example (2-5) except that the thickness of the metal thin film was changed to 20 nm. Carried out. The obtained results are shown in Table 6.
  • Example (2-7) A plasmon excitation sensor (II) of the present invention was produced in the same manner as in Example (2-6) except that the fluorescent dye was changed to Cy3 (registered trademark) in Example (2-6), and assay method (Y ). The obtained results are shown in Table 6.
  • Such a substrate was immersed in an ethanol solution containing 1 mM of 10-carboxy-1-decanethiol for 24 hours or more to form a SAM on one side of the gold thin film.
  • the substrate was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.
  • a similar plasmon excitation sensor was prepared by providing a polydimethylsiloxane [PDMS] sheet having a flow path height of 0.5 mm on the surface of the SAM and further placing a polymethylmethacrylate relay top plate. Ultrapure water was fed as a liquid for 10 minutes, and then PBS was circulated for 20 minutes with a peristaltic pump at room temperature and a flow rate of 500 ⁇ L / min to equilibrate the surface.
  • PDMS polydimethylsiloxane
  • Washing was carried out by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes. 2.5 mL of a secondary antibody (PBS solution prepared to be 1,000 ng / mL) labeled with Alexa Fluor (registered trademark) 647 was added and circulated for 30 minutes.
  • a secondary antibody PBS solution prepared to be 1,000 ng / mL labeled with Alexa Fluor (registered trademark) 647 was added and circulated for 30 minutes.
  • Comparative Example (2-1) In Comparative Example (2-1), the assay method was the same as Comparative Example (2-1) except that the metal species was aluminum, the thickness of the metal thin film was 20 nm, and the fluorescent dye was changed to Cy3 (registered trademark). Carried out. The obtained results are shown in Table 6.
  • the assay S / N ratio obtains the measured fluorescence amount changed with respect to the original fluorescence amount, and shows the reliable dynamic range of the numerical value in the measurement, and further divides by the noise level of the base substrate.
  • the reliability limit including the noise level can be obtained. That is, the higher the assay S / N ratio, the more accurate numerical value is provided for the measured antigen amount.
  • Example (2-8) Using the plasmon excitation sensor (II) prepared in Example (2-1), a conjugate obtained by complexing the secondary antibody labeled with the quenching dye obtained in Preparation Example (2-2) with AFP A competitive immunoassay was performed in the same manner as in Example (2-1) except that it was used. The results obtained are shown in Table 7.
  • Example (2-9) Using the plasmon excitation sensor (II) prepared in Example (2-2), a conjugate obtained by complexing the quencher-labeled secondary antibody obtained in Preparation Example (2-2) with AFP A competitive immunoassay was performed in the same manner as in Example (2-2) except that it was used. The results obtained are shown in Table 7.
  • Example (2-10) Using the plasmon excitation sensor (II) prepared in Example (2-3), a conjugate obtained by complexing the secondary antibody labeled with the quenching dye obtained in Preparation Example (2-2) with AFP A competitive immunoassay was carried out in the same manner as in Example (2-3) except that it was used. The results obtained are shown in Table 7.
  • Example (2-11) Using the plasmon excitation sensor (II) prepared in Example (2-4), a conjugate obtained by complexing the secondary antibody labeled with the quenching dye obtained in Preparation Example (2-2) with AFP A competitive immunoassay was carried out in the same manner as in Example (2-4) except that it was used. The results obtained are shown in Table 7.
  • Example (2-12) Using the plasmon excitation sensor (II) produced in Example (2-5), a conjugate obtained by complexing the secondary antibody labeled with the quenching dye obtained in Preparation Example (2-2) with AFP A competitive immunoassay was carried out in the same manner as in Example (2-5) except that it was used. The results obtained are shown in Table 7.
  • Example (2-13) Using the plasmon excitation sensor (II) produced in Example (2-6), the conjugate complexed with the secondary antibody labeled with the quenching dye obtained in Production Example (2-2) was used. A competitive immunoassay was performed in the same manner as in Example (2-6) except for the above. The results obtained are shown in Table 7.
  • Example (2-14) Using the plasmon excitation sensor (II) produced in Example (2-7), a conjugate obtained by complexing the secondary antibody labeled with the quenching dye obtained in Production Example (2-2) with AFP A competitive immunoassay was carried out in the same manner as in Example (2-7) except that it was used. The results obtained are shown in Table 7.
  • Comparative Example (2-3) Comparative Example (2-1) except that the plasmon excitation sensor produced in Comparative Example (2-1) was used and a conjugate of Alexa Fluor (registered trademark) 647-labeled secondary antibody complexed with AFP was used.
  • the competitive immunoassay was carried out in the same manner as described above. The results obtained are shown in Table 7.
  • the fluorescence signal values in the examples are sufficiently high, indicating that the abundance of the antigen can be measured with sufficient accuracy.
  • the assay S / N ratio also showed a high value, and it was confirmed that the reliability was high.
  • the competitive assay of the comparative example since it is a competitive system, the fluorescence signal value shows a high value and there is a sufficient amount of fluorescence with respect to the antigen abundance, but the assay S / N ratio is lower than the example and the measured value It was suggested that the reliability accuracy of was low. Assuming from this result, it is shown that the quencher-labeled secondary antibody measurement system of the example has a wider dynamic range on the fluorescence amount change than the assay measurement system of the fluorescent dye alone.
  • the unreacted antibody and the unreacted enzyme were purified using a molecular weight cut filter (manufactured by Nippon Millipore) to obtain an Alexa Fluor (registered trademark) 647-labeled anti-AFP monoclonal antibody solution.
  • the obtained antibody solution was stored at 4 ° C. after protein quantification.
  • Example (3-1) Manufacture of plasmon excitation sensor (I)
  • a glass transparent flat substrate having a refractive index [n d ] of 1.52, a thickness of 1 mm and an outer shape of 20 mm ⁇ 20 mm (“BK7” manufactured by Shot Japan Co., Ltd.) is plasma-cleaned, and a chromium thin film is formed on one side of the substrate.
  • a chromium thin film was formed on the surface by sputtering.
  • the thickness of the chromium thin film was 1 nm, and the thickness of the silver thin film was 45 nm.
  • a spacer layer made of silicon dioxide [SiO 2 ] as a dielectric was formed by sputtering on one side of the silver thin film not in contact with the chromium thin film.
  • the spacer layer had a thickness of 15 nm.
  • terbium [Tb] chelate 5 parts by weight of terbium [Tb] chelate as a fluorescent dye and 5% by weight of “BL-S” (polyvinyl butyral) manufactured by Sekisui Chemical Co., Ltd. as a fluorescent dye with respect to one side of the spacer layer not in contact with the silver thin film
  • BL-S polyvinyl butyral
  • a composition containing 25 parts by weight of methyl ethyl ketone as a solvent was applied by a spin coating method and dried at 50 ° C. for 10 minutes in the dark to volatilize the solvent.
  • the thickness of the obtained fluorescent dye layer was 10 nm.
  • aqueous solution containing 5% by weight of 3-aminopropyltriethoxysilane was applied onto the fluorescent dye layer of the substrate thus obtained with a spin coater, allowed to dry naturally at room temperature for 2 hours, and then at 50 ° C. for 10 minutes. Heated.
  • the surface of the fluorescent dye layer treated with the silane coupling agent is provided with a spacer made of polydimethylsiloxane [PDMS] having a hole of 2 mm ⁇ 10 mm, an outer shape of 20 mm ⁇ 20 mm, and a thickness of 0.5 mm.
  • the substrate was placed in the flow path so that is inside the flow path.
  • a polymethyl methacrylate plate having a thickness of 4 mm and the same outer shape as that of the PDMS spacer was put on the substrate so as to cover the substrate from the outside of the flow channel, and the flow channel and the polymethyl methacrylate plate were fixed with screws.
  • Ultrapure water was circulated for 10 minutes and then PBS was circulated for 30 minutes by a peristaltic pump at 30 ° C. and a flow rate of 500 ⁇ L / min.
  • the total volume of liquid delivery is 15 mL.
  • a laser beam having a wavelength of 340 nm is irradiated using an LD laser as a light source, and a photon amount is adjusted using a neutral density filter as an optical filter.
  • the surface plasmon measurement was started by irradiating the plasmon excitation sensor before immobilization of the ligand fixed to the flow path through the degree prism.
  • the resonance angle shift was measured with surface plasmons to confirm the immobilization of the ligand.
  • the immobilization amount was 3 ng / mm 2 .
  • non-specific adsorption prevention treatment was performed by circulating and feeding in PBS buffered saline containing 1% by weight of bovine serum albumin [BSA] for 30 minutes.
  • Step (a1) The solution was replaced with PBS, 0.5 mL of PBS containing 1 ng / mL of AFP was added and circulated for 25 minutes.
  • Washing step Washing was performed by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes.
  • an LD laser as a light source
  • an amount of photons is adjusted by an optical filter: (Sigma Kogyo Co., Ltd.)
  • Detection was performed by a CCD image sensor (manufactured by Texas Instruments Co., Ltd.) using a double objective lens (manufactured by Nikon Corporation).
  • Step (b2) 5 mL of PBS containing 1,000 ng / mL of the ⁇ -galactosidase-modified secondary antibody obtained in Preparation Example (3-1) was added and circulated for 20 minutes. Washing step: Washing was performed by circulating TBS containing 0.05% by weight of Tween 20 for 20 minutes.
  • Step (e): The amount of assay signal change in the plasmon excitation sensor of the present invention was evaluated by the following equation. Signal change
  • the obtained results are shown in Table 8 and FIG.
  • Example (3-2) Manufacture of plasmon excitation sensor (I)
  • 2-Me-4-OMe TG was used instead of the terbium chelate as a fluorescent dye, and laser light having a wavelength of 490 nm was used.
  • Example (3-1) The same procedure as in Example (3-1) except that glucose oxidase-modified secondary antibody obtained in Preparation Example (3-2), glucose and oxygen as the enzyme quenching substrate solution, and laser light having a wavelength of 490 nm were used. Went in the way.
  • Examplementation of assay method The same procedure as in Example (3-1) was performed except that the dark quencher-modified secondary antibody obtained in Preparation Example (3-3) was used and step (c) was not performed.
  • the substrate thus obtained is immersed in an ethanol solution containing 1 mM of 10-carboxy-1-decanethiol for 24 hours or more to form a SAM (Self Assembled Monolayer) on one surface of a gold thin film. did.
  • the substrate was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.
  • a polydimethylsiloxane [PDMS] sheet having a flow path height of 0.5 mm is provided on the surface of the SAM, and the substrate is disposed so that the SAM surface is inside the flow path (however, the silicon rubber spacer is used for liquid feeding).
  • the pressure-sensitive adhesive sheet was pressed from the outside of the flow path, and the flow path sheet and the plasmon excitation sensor were fixed with screws.
  • the obtained plasmon excitation sensor was fixed to the flow path, and ultrapure water was supplied as a liquid for 10 minutes, and then PBS was circulated for 20 minutes with a peristaltic pump at room temperature and a flow rate of 500 ⁇ L / min to equilibrate the surface.
  • the fluorescent dye layer is formed on the substrate on the plasmon excitation sensor of the example, an extremely high fluorescence signal is obtained in the blank state, which is compared with the conventional fluorescence labeled SPFS measurement of the comparative example (3-2). It was found that an extremely sensitive measurement with an order of magnitude is possible. Further, it was found that by providing a quencher enzyme amplification mechanism, a higher signal change amount can be achieved as compared to the case where the quencher is directly modified to the secondary antibody of Comparative Example (3-1).
  • the plasmon excitation sensor of the present invention has high sensitivity and high accuracy, it can be directly applied to, for example, a selective biosensor or bioprobe using a molecular recognition reaction of a biomolecule such as carcinoembryonic antigen or tumor marker.
  • the assay method of the present invention using the plasmon excitation sensor of the present invention is a method that can be detected with high sensitivity and high accuracy, for example, even a very small amount of tumor marker contained in blood is detected. From this result, it is also possible to predict with high accuracy the presence of a preclinical non-invasive cancer (carcinoma in situ) that cannot be detected by palpation or the like.

Abstract

La présente invention concerne un détecteur d'excitation plasmonique qui présente une grande sensibilité et une grande précision ainsi qu'une excellente spécificité, qui est essentiel pour un test immunologique ; ainsi qu'un procédé de test, un dispositif de test, et un kit de test utilisant ou comprenant le détecteur. Le détecteur d'excitation plasmonique est caractérisé en ce qu'il comprend un substrat plat transparent, un film métallique fin formé sur une surface du substrat, et une couche de séparation constituée par un diélectrique et formée sur la surface du film métallique fin qui n'est pas en contact avec le substrat. Le détecteur est en outre caractérisé en ce que (V) un ligand a été fixé à une couche de colorant fluorescent formée sur la surface de la couche de séparation qui n'est pas en contact avec le film métallique fin ou en ce que (W) un ligand marqué avec un colorant fluorescent a été fixé à la surface de la couche de séparation qui n'est pas en contact avec le film métallique fin.
PCT/JP2010/050803 2009-01-22 2010-01-22 Détecteur d'excitation plasmonique et procédé de test l'utilisant WO2010084953A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP2009011973A JP5298876B2 (ja) 2009-01-22 2009-01-22 プラズモン励起センサおよびそれを用いたアッセイ法
JP2009-011973 2009-01-22
JP2009-011974 2009-01-22
JP2009011974A JP5298877B2 (ja) 2009-01-22 2009-01-22 プラズモン励起センサを用いたアッセイ法
JP2009-025974 2009-02-06
JP2009025974A JP5245125B2 (ja) 2009-02-06 2009-02-06 表面プラズモンを利用したアッセイ法

Publications (1)

Publication Number Publication Date
WO2010084953A1 true WO2010084953A1 (fr) 2010-07-29

Family

ID=42356003

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/050803 WO2010084953A1 (fr) 2009-01-22 2010-01-22 Détecteur d'excitation plasmonique et procédé de test l'utilisant

Country Status (1)

Country Link
WO (1) WO2010084953A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012042233A (ja) * 2010-08-13 2012-03-01 Konica Minolta Holdings Inc Ｓｐｆｓ（表面プラズモン励起増強蛍光分光法）により測定される蛍光シグナルの補正方法およびこれを用いたアッセイ方法、並びにこれらの方法に用いられる構造体および表面プラズモン共鳴センサー

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006208069A (ja) * 2005-01-26 2006-08-10 National Institute Of Advanced Industrial & Technology プラズモン共鳴蛍光を用いた生体分子相互作用検出装置及び検出方法
WO2007137060A2 (fr) * 2006-05-16 2007-11-29 Applied Biosystems Systèmes, procédés, et appareil destinés au séquençage d'une molécule unique
JP2008224561A (ja) * 2007-03-15 2008-09-25 Fujifilm Corp 表面プラズモン増強蛍光センサ

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006208069A (ja) * 2005-01-26 2006-08-10 National Institute Of Advanced Industrial & Technology プラズモン共鳴蛍光を用いた生体分子相互作用検出装置及び検出方法
WO2007137060A2 (fr) * 2006-05-16 2007-11-29 Applied Biosystems Systèmes, procédés, et appareil destinés au séquençage d'une molécule unique
JP2008224561A (ja) * 2007-03-15 2008-09-25 Fujifilm Corp 表面プラズモン増強蛍光センサ

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KNOLL W ET AL.: "Surface Plasmon Fluorescence Immunoassay of Free Prostate-Specific Antigen in Human Plasma at the Femtomolar Level", ANAL CHEM, vol. 76, no. 22, 15 November 2004 (2004-11-15), pages 6765 - 6770 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012042233A (ja) * 2010-08-13 2012-03-01 Konica Minolta Holdings Inc Ｓｐｆｓ（表面プラズモン励起増強蛍光分光法）により測定される蛍光シグナルの補正方法およびこれを用いたアッセイ方法、並びにこれらの方法に用いられる構造体および表面プラズモン共鳴センサー

Similar Documents

Publication Publication Date Title
JP5652393B2 (ja) Ｓｐｆｓ−ｌｐｆｓ系による測定方法に供するプラズモン励起センサおよびアッセイ法
JP5720248B2 (ja) 表面プラズモンおよび蛍光共鳴エネルギー転移を利用した免疫アッセイ
JP5428322B2 (ja) プラズモン励起センサを用いたアッセイ法
JP5994890B2 (ja) アナライト検出プローブ
JP5958339B2 (ja) 近接場増強蛍光センサチップ
JP5772612B2 (ja) 表面プラズモン励起増強蛍光分光法を利用する蛍光測定装置用センサチップを用いたアッセイ方法、およびアッセイ用キット
JP2008102117A (ja) 表面プラズモン増強蛍光センサおよび蛍光検出方法
JP2009080011A (ja) 蛍光検出方法
JP5660035B2 (ja) 融合タンパク質含有集合体、その製造方法及び該集合体を用いたアッセイ法
JP5516198B2 (ja) プラズモン励起センサチップおよびこれを用いたプラズモン励起センサ、並びにアナライトの検出方法
WO2010041736A1 (fr) Procédé de dosage utilisant un plasmon de surface
WO2010084953A1 (fr) Détecteur d'excitation plasmonique et procédé de test l'utilisant
EP2607888B1 (fr) Capteur de spectroscopie par fluorescence renforcée par champ de plasmon de surface équipé d'un mécanisme de purification du type à adsorption non spécifique
JP5541003B2 (ja) プラズモン励起センサチップおよびこれを用いたアッセイ法
JP5245125B2 (ja) 表面プラズモンを利用したアッセイ法
JP5298877B2 (ja) プラズモン励起センサを用いたアッセイ法
JP5298876B2 (ja) プラズモン励起センサおよびそれを用いたアッセイ法
JP5754309B2 (ja) 表面プラズモン励起増強蛍光分光法を利用して蛍光量を測定する定量分析方法ならびにそれに用いられる定量分析用キットおよびアナライト解離抑制剤
JP2011127991A (ja) プラズモン励起センサおよび該センサを用いたアッセイ法
JP5169891B2 (ja) 表面プラズモンを利用したアッセイ法
JP2012037477A (ja) センサチップおよびそれを用いたアッセイ法
JP2010091527A (ja) 表面プラズモンを利用したアッセイ法
JP2011169609A (ja) プラズモン励起センサを用いたアッセイ法
JP5516197B2 (ja) プラズモン励起センサおよび該センサを用いたアッセイ法

Legal Events

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

Ref document number: 10733560

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10733560

Country of ref document: EP

Kind code of ref document: A1