JP5298876B2 - Plasmon excitation sensor and assay method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plasmon excitation sensor that has high sensitivity and is precise, and is superior in specificity that is essential for immunoassay, to provide an assay method using the plasmon excitation sensor, and to provide a device for assay and a kit for assays. <P>SOLUTION: The plasmon excitation sensor includes a transparent planar substrate; a metal thin film formed on one surface of the substrate; a spacer layer, made of a dielectric formed on the other surface which does not abut against the substrate of the metal thin film; a fluorescent dye layer formed on the other surface which does not abut against the metal thin film of the spacer layer; and a ligand immobilized on the other surface which does not abut against the spacer layer of the fluorescent dye layer. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

The present invention relates to a plasmon excitation sensor and assay method using it. More specifically, the present invention is based on the principle 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). based about the assay method using the sensor.

  Surface plasmon excitation-enhanced fluorescence analysis (SPFS) is performed by generating a dense wave (surface plasmon) on the surface of a metal thin film under the condition that the irradiated laser light is attenuated by total reflection (ATR) on the surface of the gold thin film. The amount of photons that the laser beam has 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, so that a trace amount and / or extremely low concentration can be obtained. It is a method that can detect an analyte.

  As an example related to a biosensor or biochip based on the principle of SPFS, 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.

  However, in the detection of trace analytes (target antigens), 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.

  On the other hand, studies have been conducted to effectively enhance fluorescence intensity in enhancement of surface plasmon excitation. 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.

  Further, Patent Document 3 discloses that 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 size of 100 to 800 nm and a thickness of 30 to 50 nm are densely arranged on a substrate as an island film. In order to adjust the distance between the silver particle surface and the fluorescent material, a spacer is provided on the metal particle surface.

  In the inventions described in Patent Documents 2 and 3, there is a possibility that the use efficiency of photons is improved by arranging the fluorescent dye layer in the vicinity of the metal, and the efficiency of fluorescence generation may be improved. If there are few, it cannot respond. Also, fluorescent dyes are difficult to evolve into conjugate forms and are not specific to themselves because they are not related to the amount of analyte to be detected and cannot be used for immunoassays.

Japanese Patent No. 3294605 Special table 2007-512522 gazette JP 2007-139540 A

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 and an assay method using the same.

  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. As a result of diligent research to solve the problem that the amount of fluorescent probe that often corresponds to the enhanced electric field does not exist and is not satisfactory in terms of sensitivity, the conventional sandwich immunoassay method (FIG. 1) and In combination with surface plasmon excitation enhanced fluorescence analysis, a mechanism for quenching fluorescence by the presence or absence of binding between the sensor and the antigen is provided, that is, by sharing the functions of light emission and quenching, a very small amount of analyte ( For example, even if it is a target antigen), it has been found that both fluorescence emission and specificity commensurate with the amount of photons can be achieved. It has been completed.

  That is, 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. A spacer layer made of a body, 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.

The metal thin film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum, and gold is particularly preferable.
The dielectric preferably includes silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ).

  The fluorescent dye layer is formed by applying a composition containing a fluorescent dye and a polymer to the dielectric layer on the other surface of the spacer layer that is not in contact with the metal thin film, or the fluorescent dye layer. Can be bonded to the other surface of the spacer layer not in contact with the metal thin film via a silane coupling agent.

The ligand is preferably an antibody that recognizes and binds to a tumor marker or carcinoembryonic antigen.
The assay method of the present invention is characterized by comprising the following steps (a) to (d).

Step (a): a step of bringing a specimen into contact with the plasmon excitation sensor of the present invention,
Step (b): The plasmon excitation sensor obtained through the step (a) is further quenched or absorbed with a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor. Reacting a conjugate with the resulting compound;
Step (c): The plasmon excitation sensor obtained through the step (b) is irradiated with a laser beam via a prism from the other surface of the transparent flat substrate on which the metal thin film is not formed. A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and a step (d): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (c).

The conjugate is preferably bound to the analyte.
Moreover, it is preferable that the analyte which is different from the above-mentioned analyte and which competes with the above-mentioned analyte is bound to the conjugate in advance.

The specimen is preferably at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.
The analyte is preferably a tumor marker or carcinoembryonic antigen.

  Conventionally, in ultra-sensitive systems that measure extremely small amounts of analyte, there is a mismatch between the surface plasmon electric field enhancement (example of electric field-enhanced photons) and a minute amount of fluorescent dye (example of fluorescence photons). Occurs.

  On the other hand, when used in the assay method of the present invention, the plasmon excitation sensor of the present invention generates fluorescence commensurate with the electric field enhancement by providing a fluorescent dye layer (signal amount = generated in the dye layer). Example of possible photon amount), and fluorescence can be adjusted with a quencher according to the amount of analyte (noise amount = example of photon amount quenchable with quencher), so there is less noise , Assay with high signal-to-noise ratio and high signal / noise ratio is possible.

That is, 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. Thus, a plasmon excitation sensor capable of detecting the analyte can be provided.

  Further, 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. However, the present invention provides a plasmon excitation sensor and a ligand (for example, secondary). It is possible to provide a plasmon excitation sensor in which the S / N ratio does not deteriorate when applied to the assay method of the present invention using a conjugate of an antibody) and a quencher because the quencher is proportional to the target antigen amount. it can.

In addition, since the present invention can adjust the amount of fluorescence signal depending on the ability of the quencher, it can provide a plasmon excitation sensor in which the assay method of the present invention can be carried out with an optimum S / N ratio.
Furthermore, in the assay method of the present invention, the present invention allows an analyte (target antigen) to compete with a conjugate of a quencher with a secondary antibody (ligand) that has previously bound an antigen that competes with the analyte. Thus, it is possible to provide a plasmon excitation sensor that can make the amount of fluorescent signal (fluorescent signal) proportional to the amount of target antigen.

FIG. 1 shows a secondary antibody 4 labeled with a fluorescent dye 5 after a target antigen 3 contained in a specimen is bound to a primary antibody 2 immobilized on a substrate 1 in a conventional sandwich immunoassay method. The state which is made to react is shown typically. FIG. 2 shows a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a spacer layer made of a dielectric formed on the other surface of the metal thin film not in contact with the substrate. The state where the sensor chip having the primary antibody immobilized on the other surface of the spacer layer not in contact with the metal thin film is irradiated with laser light and the amount of fluorescence is detected by the CCD. Optical arrangement diagram of SPFS apparatus schematically shown; and “number of incident photons” using Laser Diode (semiconductor laser) as light source, “number of excited photons” due to electric field enhancement effect of surface plasmon excited by the light source, 2 The flowchart in which the “absorption photon number” excited by the fluorescent dye labeled with the next antibody and the “fluorescence photon number” detected by the CCD are respectively calculated is shown. The numerical values are calculated based on the following assumptions (1) to (3): (1) When irradiating laser light using Laser Diode, the light quantity is adjusted with ND filter after P-polarization. Then, 0.1 mW is incident on the sensor chip. (2) The fluorescent dye labeled on the secondary antibody contained in the flow channel to which the sensor chip is fixed is 0.4 nmol / L (measured antigen amount is 0 2 nmol / L, corresponding to a secondary antibody fluorescent agent labeling rate of 2%.), Effective plasmon region is present at a thickness of 100 nm, (3) molar extinction coefficient of fluorescent dye is 250,000, fluorescence quantum yield The rate is 0.47; that is, when 0.1 mW laser light is incident and there is a 17-fold increase in the electric field on the gold substrate, the number of photons is about 15th power, but it is actually in the nmol / L level. Anti In the measurement, the number of photons generated through the fluorescent dye is reduced to the sixth power, most of the photons with the enhanced electric field are not used, and the detected signal is very small. Cannot be said to improve. FIG. 3 shows type 1 of the assay method of the present invention (secondary antibody 4 is an antibody that recognizes and binds antigen 7 other than the target antigen contained in the specimen), and type 2 (secondary antibody 4). Is an antibody that recognizes and binds to the target antigen 3 contained in the specimen.) After reacting the plasmon excitation sensor of the present invention with the specimen, the secondary with the quencher 6 immobilized thereon A state in which the antibody 4 is further reacted and the fluorescence 13 emitted from the fluorescent dye excited by the surface plasmon is represented is schematically shown.

Hereinafter, the present invention will be specifically described.
<Plasmon excitation sensor>
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. A spacer layer, a fluorescent dye layer formed on the other surface of the spacer layer not in contact with the metal thin film, and another surface of the fluorescent dye layer not in contact with the spacer layer. And an immobilized ligand.

  As the plasmon excitation sensor of the present invention, for example, a sensor chip having a gold thin film such as a sensor chip used in a Biacore system manufactured by GE Healthcare Biosciences can be used for the plasmon excitation sensor of the present invention. .

(Transparent flat substrate)
The transparent flat substrate used in the present invention may be made of glass or plastic such as polycarbonate (PC) or cycloolefin polymer (COP), and the refractive index [nd] is preferably 1.40. The size (length × width) is not particularly limited as long as it is ˜2.20 and the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.

  The transparent glass substrate made of glass is commercially available from BK7 (refractive index [nd] 1.52) and LaSFN9 (refractive index [nd] 1.85) manufactured by SCHOTT AG, manufactured by Sumita Optical Glass Co., Ltd. K-PSFn3 (refractive index [nd] 1.84), K-LaSFn17 (refractive index [nd] 1.88) and K-LaSFn22 (refractive index [nd] 1.90), manufactured by OHARA INC. -LAL10 (refractive index [nd] 1.72) or the like is preferable from the viewpoint of optical characteristics 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.

(Metal thin film)
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, more preferably gold. It is desirable to consist of these, and the alloy of these metals may be sufficient. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

  In addition, only when a glass flat substrate is used as the “transparent flat substrate”, the glass and the metal thin film can be bonded more firmly, so that a thin film of chromium, nickel chromium alloy or titanium is formed in advance. Is preferred.

  Examples of the 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.

  From the viewpoint of the electric field enhancing effect, gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm are more preferable, and chromium The thickness of the thin film is more preferably 1 to 3 nm.

  When the thickness of the metal thin film is within the above range, surface plasmons are easily generated, which is preferable. Moreover, if it is a metal thin film which has such thickness, a magnitude | size (length x width) will not be specifically limited.

(Spacer layer made of dielectric)
The “dielectric spacer layer” is formed on the other surface of the metal thin film not in contact with the “transparent flat substrate” for the purpose of preventing the metal quenching of the fluorescent dye by the “metal thin film”. As the dielectric, various optically transparent inorganic substances, natural or synthetic polymers can be used, but they are excellent in chemical stability, manufacturing stability and optical transparency. It is preferable to contain silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ).

  The thickness of the spacer layer is usually 10 nm to 1 mm, and is preferably 30 nm or less and more preferably 10 to 20 nm from the viewpoint of resonance angle stability. Moreover, 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. When the plasmon excitation sensor of the present invention is mass-produced in the future, it is assumed that 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 for the purpose of ensuring 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 a coating method using a spin coater.

(Fluorescent dye layer)
The “fluorescent dye layer” is a layer in which the fluorescent dye is immobilized on the other surface of the “spacer layer” that is not in contact with the “metal thin film”. Can be formed by coating the spacer layer with a composition containing a fluorescent dye, and (B) it can also be formed by bonding a fluorescent dye onto the spacer layer via a silane coupling agent. .

  In the case of (A), 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 and copolymerizing another polymerizable monomer, a fluorescent dye and a polymerization initiator.

  In the case of (B), the fluorescent dye is immobilized on the spacer layer by binding a silane coupling agent having an amino group or a carboxyl group and a ligand having a group that reacts with these groups and is covalently bonded. be able to.

Thus, in the case of (A) which forms a layer containing a fluorescent dye and a polymer, 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 predetermined excitation light in the present invention, or excited by using an electric field effect. Including luminescence.

  The “fluorescent dye” used in the present invention is not particularly limited and may be any known fluorescent dye. In general, 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.

  Examples of such “fluorescent dyes” 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), Coumarin family fluorescent dyes (Invitrogen), Rhodamine family fluorescent dyes (GE Healthcare Biosciences), cyanine family fluorescence Dyes, indocarbocyanine family fluorescent dyes, oxazine family fluorescent dyes, thiazine family fluorescent dyes, squaraine family fluorescent dyes, chelated lanthanide dyes 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 Nos. 6,406,297, 6,221,604, 5,994,063, The fluorescent dyes described in 5,808,044, 5,880,287, 5,556,959, and 5,135,717 can also be used in the present invention.

  Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.

Many of these 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. When 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 a solubility parameter (SP) of the polymer.

These fluorescent dyes may be used alone or in combination of two or more.
Examples of the “polymer” include polyacrylate, polymethacrylate, polystyrene-acrylate, polystyrene, polyvinyl butyral, polyester, and the like. Of these, 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.

In addition to the fluorescent dye and 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. For example, halogen-containing hydrocarbons (eg, dichloromethane, dichloroethane, tetrafluoropropane), alcohols (eg, 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 etc.), ketones (acetone, methyl ethyl ketone, etc.) etc. are mentioned. Of these, aromatics, halogen-containing hydrocarbons, esters, and ketones are preferable from the viewpoint of the dissolution stability of the polymer used.

  Examples of the “antioxidant” 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. When the fluorescent dye and the polymer have the above contents, the quenching efficiency is good.

  Moreover, 100-1,000 weight part is preferable with respect to 100 weight part of compositions, and 100-500 weight part is more preferable. 0.1-10 weight part is preferable with respect to 100 weight part of compositions, and 1-5 weight part is more preferable. 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 method of “coating” is not particularly limited, but for example, after applying by spin coating method, wire coating method, bar coating method, roll coating method, blade coating method, curtain coating method, screen printing method, etc. Dry at ~ 100 ° C for 5-30 minutes.

(Ligand)
A ligand is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding an analyte contained in a specimen. Examples of such a “molecule” or “molecular fragment” include, for example, , Nucleic acids (DNA that may be single-stranded or double-stranded, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., 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 modified molecules or complexes thereof, etc.

  Examples of the “protein” include antibodies and the like, specifically, anti-α-fetoprotein (AFP) monoclonal antibody (available from Japan Medical Laboratory), anti-carcinoembryonic antigen (CEA) ) Monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-PSA monoclonal antibody and the like.

In the present invention, the term “antibody” includes polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments.
As the method for immobilizing the ligand, in the case of (A) above, the functional group (preferably, hydroxyl group, amino group and carboxyl group) such as hydroxyl group, amino group, carboxyl group and isocyanate group possessed by the polymer and the functional group possessed by the ligand. (In consideration of immunoreaction promoting ability and nonspecific adsorption reaction inhibiting ability, it is preferable to combine with the functional group of the polymer as appropriate.) Or a compound having a 2-sensitive reactive group Examples thereof include a method of immobilizing the fluorescent dye layer via a silane coupling agent (for example, silane coupling agent).

  As a silane coupling agent having a 2-sensitive reactive group, it has an ethoxy group (or methoxy group) which gives a silanol group (Si-OH) by hydrolysis, and an amino group, a glycidyl group, a carboxyl group, etc. at the other end It will not specifically limit if it is a silane coupling agent which has a reactive group, A conventionally well-known silane coupling agent can be used.

  In addition to “a silane coupling agent having a 2-sensitive 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), biotin, avidin, streptavidin, protein A, protein G and the like are also suitable.

  In the case of using a silane coupling agent as the compound having a 2-sensitive 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 on one surface in order 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. In this case, drying is usually performed for 1 to 24 hours, preferably 10 hours, and at 100 ° C., usually for 10 minutes to 1 hour, preferably 30 minutes. Thereafter, the substrate is usually washed with water. At this point, 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.

  Next, 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). Then, the carboxyl group thus activated and the amino group of the silane coupling agent are dehydrated and immobilized using water-soluble carbodiimide.

  As a specific example of another preferable method, the carboxyl group of the polymer in the fluorescent dye layer is converted into a water-soluble carbodiimide (WSC) (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)). And N-hydroxysuccinimide (NHS), and the amino group of the ligand is dehydrated and immobilized using water-soluble carbodiimide.

As the method for immobilizing the ligand, in the case of (B) above, a method of immobilizing a ligand (for example, a primary antibody) on the fluorescent dye layer by physical adsorption can be mentioned.
Examples of the “specimen” include blood (serum / plasma), urine, nasal fluid, saliva, feces, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc., and appropriately diluted in a desired solvent, buffer solution, etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred.

  “Analyte (target antigen)” is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to a ligand immobilized on the “fluorescent dye layer”. “Molecules” or “molecular fragments” include, for example, 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 modified molecules and complexes thereof. Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signal transmitter, a hormone, etc. It is not particularly limited.

<Assay method>
The assay method of the present invention is characterized by comprising the following steps (a) to (d).

Step (a): a step of bringing a specimen into contact with the plasmon excitation sensor of the present invention,
Step (b): The plasmon excitation sensor obtained through the step (a) is further quenched or absorbed with a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor. Reacting a conjugate with the resulting compound;
Step (c): The plasmon excitation sensor obtained through the step (b) is irradiated with a laser beam via a prism from the other surface of the transparent flat substrate on which the metal thin film is not formed. A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and a step (d): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (c).

(Process (a))
Step (a) is a step of bringing the specimen into contact with the plasmon excitation sensor of the present invention.

  “Contact” means that the specimen is contained in the liquid supply circulating in the flow path, and the plasmon excitation is performed when only one surface on which the fluorescent dye and the ligand of the plasmon excitation sensor are immobilized is immersed in the liquid supply. An embodiment in which the sensor and the specimen are brought into contact is preferable.

  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.

  As the material, the plasmon excitation sensor part is composed of a homopolymer or copolymer, polyethylene, polyolefin, etc. containing methyl methacrylate, styrene or the like as a raw material, and the chemical solution delivery part is made of silicon rubber, Teflon (registered trademark), polyethylene, polypropylene. Etc. are used.

  In the plasmon excitation sensor unit, from the viewpoint of increasing the contact efficiency with the specimen and shortening the diffusion distance, it is preferable that the cross section of the channel of the plasmon excitation sensor unit is independently about 100 nm to 1 mm in length and width.

  As 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 a 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.

  In large lots (factory level) manufactured industrially, as a method of fixing the plasmon excitation sensor to the flow path, 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.

  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 μg / 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 liquid feeding is usually 1 to 2,000 μL / min, preferably 5 to 500 μL / min.

(Washing process)
The washing step is preferably included before and / or after the following step (b), and the particles obtained in the step (a) or the surface of the plasmon excitation sensor obtained in the following step (b) are washed. It is a process.

  As the washing solution used in the step, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer used in the reaction of steps (a) and (b), preferably 0.00001 to Those containing 1% by weight are desirable.

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 (a).
The time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

(Process (b))
The step (b) may be the same as or different from the plasmon excitation sensor obtained through the step (a), 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 a compound capable of quenching or absorbing fluorescence.

  “Compound capable of quenching or absorbing fluorescence”, that is, “quenching agent (quenching dye)” or “quencher” is a compound having an appropriate energy level capable of absorbing the excited energy of the “fluorescent dye”. If an appropriate quencher is added to a certain fluorescent dye, the fluorescence disappears.

  “Quenchers” 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 Dyes, oxazine family quenching dyes, thiazine family quenching dyes, squarain family quenching dyes, chelated lanthanide family quenching dyes, BODIPY® 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) (Biosearch) technology Japan BTJ Co., Ltd.), Iowa Black (registered trademark) (manufactured by Integrated DNA Technologies), DABCYL (4- (4′-dimethylaminophenylazo) benzoic acid) (manufactured by Integrated DNA Technologies), TAMRA (N, N, N ′, N′-tetramethyl-6-carboxyrhodamine) (manufactured by Invitrogen), Cy3 (registered trademark) (manufactured by GE Healthcare Biosciences), Cy5 (registered trademark) (GE Healthcare) Bioscience Co., Ltd.), 1-benzyl-1,4-dihydronicotinamide (BNAH), 9-anthracenecarbonitrile, 2-naphthol, 2-methoxynaphthalene, 1,4-naphthoquinone, 2,6-di- tert-butyl-1,4-be Zoquinone, 3,5-di-tert-butyl-1,2-benzoquinone, 1-naphthoic acid (1-NA), 2-naphthoic acid (2-NA), 1-pyrenebutyric acid (PyBA), 4-nitrobenzoic acid An acid (PNBA), anthraquinone-2-carboxylic acid (AQCA), pyrene, etc. are mentioned. Further, the present invention uses the quencher described in US Pat. Nos. 6,399,392, 6,348,596, 6,080,068, and 5,707,813. You can also.

  Among these quenchers, BHQ-1 (maximum wavelength 534 nm), BHQ-2 (maximum wavelength 579 nm), BHQ-3 (maximum wavelength 672 nm), etc., manufactured by Biosearch Technologies Japan BTJ, Inc. have a wide wavelength range. Is preferable as a dark quencher (quenching agent that does not emit light itself).

  Other than the above-mentioned "quenching agents", tetracyanoquinodimethanes, aminiums, diimmoniums, hydrazines, hydrazides, hydroxylamines, hydroquinones, tetrasubstituted boron anions, or nickels And metal complexes such as heavy metal complexes of azo dyes, formazan heavy metal complexes, dipyrromethene metal complexes, porphyrin heavy metal complexes, heavy metal phthalocyanines, heavy metal naphthalocyanines, metallocenes, and the like.

  Photons emitted by fluorescent dyes excited by surface plasmons are believed to be quenched by energy transfer to the quencher in the electronically excited state. That is, it is considered that a quencher that absorbs energy in an electronically excited state releases energy as photons or heat having different wavelengths. Thus, the quencher may be a fluorescent dye.

  In general, the energy transfer between the fluorescent dye and the quencher depends on the distance between the fluorescent dye and the quencher, that is, the critical transition distance. “Critical transition distance” is the distance at which the electronic excitation level (usually singlet) of the fluorescent dye can interact with the lowest unoccupied orbital of the quencher. When the quencher is an organic substance, the distance is usually around 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 quencher is well known in the art and is described, for example, in Wu and Brand, 1994, Anal. Biochem. 218: 1-3 pages. Can be referred to.

  The standard of the combination of a specific fluorescent dye and a quencher includes, for example, the quantum yield of fluorescence emission by the fluorescent dye; the fluorescence wavelength emitted by the fluorescent dye; the extinction coefficient of the quencher; the fluorescence wavelength emitted by the quencher And the quantum yield of fluorescence emission by the quencher. When the quencher is also a fluorescent dye, the quencher and the fluorescent dye are preferably combined so that the fluorescence emitted by one can be easily distinguished from the fluorescence emitted by the other. Reference can be made to the general review of Klostermeier and Millar, 2002, Biopolymers 61: 159-179 for the selection of specific fluorescent dye and quencher combinations.

  Exemplary combinations of fluorescent dyes and quenchers used in the present invention include 6-carboxyfluorescein (FAM) as the fluorescent dye and Cy5® as the quencher; Alexa Fluor® 647 (fluorescent dye) BHQ-3 (quenching agent); FAM, TET, JOE, HEX and Oregon Green (fluorescent dye) and BHQ-1 (quenching agent); FAM, TAMRA, ROX, Cy3, Cy3.5, CAL Red and Red 640 (fluorescence) Dye) and BHQ-2 (quenching agent); Cy5 and Cy5.5 (fluorescent dye) and BHQ-3 (quenching agent), 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 dyes and quenchers include, for example, fluorescein, 6-carboxyfluorescein, 2 ′, 7′-dimethoxy-4 ′, 5′-dichloro-6-carboxyfluorescein, rhodamine, 6-carboxyl. Rhodamine, 6-carboxy-X-rhodamine, 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) and the like can be mentioned.

  “Conjugation of a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor and a compound that can quench or absorb fluorescence” refers to the following embodiment (when a secondary antibody is used as the ligand: I) or (II) is preferred.

  Aspect (I): The secondary antibody is an antibody capable of recognizing and binding to an analyte (target antigen) contained in a specimen. However, when the primary antibody used as a ligand immobilized on the plasmon excitation sensor of the present invention is a polyclonal antibody, the secondary antibody may be a monoclonal antibody or a polyclonal antibody. When the secondary antibody is a monoclonal antibody, the secondary antibody is preferably a monoclonal antibody that recognizes an epitope that the primary antibody does not recognize, or a polyclonal antibody.

  When 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 embodiment (I) competes with AFP contained in the specimen. A monoclonal or polyclonal antibody capable of recognizing and binding to the antigen of interest.

Aspect (I) 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 optimum S / N ratio.
Aspect (II): The secondary antibody is an antibody in which an analyte competing with the analyte (target antigen) contained in the specimen (competitive antigen; however, different from the target antigen) is bound in advance. . Since the competing antigens contained in the specimen are zero or one or more, the secondary antibody itself is not required, or one or more secondary antibodies are required.

Aspect (II) is preferable because the amount of fluorescent signal (fluorescent signal) and the amount of target antigen can be proportional.
When the primary antibody is an anti-AFP monoclonal antibody, the secondary antibody of embodiment (II) 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.

  As a production method of “a conjugate of a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor and a compound capable of quenching or absorbing fluorescence”, the “secondary antibody” is used as the ligand. When used, for example, a carboxyl group is first imparted to the quencher, and the carboxyl group is converted into a water-soluble carbodiimide (WSC) (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or the like). And N-hydroxysuccinimide (NHS), which is then active esterified, and then the active esterified carboxyl group and the amino group of the secondary antibody are dehydrated and immobilized using water-soluble carbodiimide; isothiocyanate and Reacting and immobilizing a secondary antibody and a quencher each having an amino group Method: Method of reacting and immobilizing a secondary antibody and a quencher having a sulfonyl halide and an amino group, respectively; Method of reacting and immobilizing a secondary antibody and a quencher having an iodoacetamide and a thiol group, respectively; Biotinylated Examples thereof include a method of reacting and immobilizing a quencher with a streptavidinated secondary antibody.

  The concentration of the thus-prepared “conjugate of a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor and a compound capable of quenching or absorbing fluorescence” in the solution is 0 0.001 to 10,000 μg / mL is preferable, and 1 to 1,000) μg / mL is more preferable.

The temperature, time, and flow rate at which the liquid is circulated are the same as in step (a).
Moreover, it is preferable to include the said washing | cleaning process between a process (b) and the following process (c).

(Process (c))
The step (c) means that the plasmon excitation sensor obtained through the above step (b), preferably the above washing step, is subjected to laser from one side of the transparent flat substrate on which the metal thin film is not formed via a prism. This is a step of measuring the amount of fluorescence emitted from the excited fluorescent dye by irradiating light.

It is desirable to adjust the energy and photon amount immediately before the laser light enters 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 the surface plasmon, the fluorescent dye is excited by photons increased to several tens to several hundred times the amount of photons irradiated. The amount of 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 that of gold.

  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.

  However, if there is a quencher in the vicinity that can absorb the energy corresponding to the energy required for transition from the ground state of the fluorescent dye to the first electronic excited state, the energy is transferred from the fluorescent dye to the quencher, The fluorescent dye 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 quencher enters the SPFS detector through the cut filter by the condenser lens, and measures the count value of the incident light.
As 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 a metal type used for the metal thin film) Examples thereof include a semiconductor laser (LD) that can irradiate a laser beam of 01 to 100 mW. Among these light sources, 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”. In the present invention, 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. For example, 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”.

Examples of the “optical filter” 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 types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor), autofluorescence of the enzyme fluorescent substrate, and examples thereof include interference filters and color filters. It is done.

  The “collecting lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system. As 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.

  As the “SPFS detection unit”, a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics Co., Ltd.) is preferable from the viewpoint of ultrahigh 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.

(Process (d))
The step (d) is a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (c).

  More specifically, 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.

Further, in the step (d), the blank fluorescence signal measured before the step (c), the assay fluorescence signal obtained in the step (c), and the gold substrate not modified at all are fixed to the channel. When the signal obtained by measuring SPFS while flowing ultrapure water is used as initial noise, the assay S / N ratio represented by the following formula can be calculated.
Assay S / N ratio = | (assay fluorescence signal) − (blank fluorescence signal) | / (initial noise)

<Device>
The apparatus of the present invention is used in the step (c).
That is, the apparatus of the present invention is for carrying out the assay method of the present invention using the plasmon excitation sensor of the present invention.

  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. In addition, a surface plasmon resonance (SPR) detection unit, that is, a photodiode as a light receiving sensor dedicated to SPR, an angle variable unit for adjusting the optimum angle of SPR and SPFS (to determine total reflection attenuation (ATR) conditions with a servomotor) In addition, the photodiode and the light source are synchronized with each other and the angle is changed from 30 ° to 85 °. The resolution is preferably 0.01 ° or more.), Including a computer for processing information input to the SPFS detector But you can.

Preferred embodiments of the light source, the optical filter, the cut filter, the condensing lens, and the SPFS detection unit are the same as those described above.
As the “liquid feed pump”, for example, 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, a simple and excellent handleability but a small amount of liquid feed For example, a tube pump may be difficult.

<Kit>
The kit of the present invention is characterized in that it is used in the assay method of the present invention, and everything necessary other than the specimen, the primary antibody and the secondary antibody in carrying out the assay method of the present invention. Are preferably included.

  For example, by using 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.

  As such a “kit”, specifically, a metal thin film, 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), N-hydroxysuccinimide (NHS), etc.); dissolved or diluted solution for dissolving or diluting the sample; various types for reacting the plasmon excitation sensor with the sample Reaction reagent and washing reagent; compound capable of quenching or absorbing fluorescence (for example, BHQ-3); various reagents for immobilizing a compound capable of quenching or absorbing fluorescence on secondary antibody (for example, water-soluble carbodiimide) (EDC), N-hydroxysuccinimide (NHS), etc.) and are required to carry out the assay method of the invention It is also possible to include a seed equipment or materials and the "device".

  Further, the kit element may include a standard material for preparing a calibration curve, instructions, a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of samples, and the like.

  Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these. In Examples 1 to 7 and Comparative Examples 1 and 2, sandwich immunoassay was performed, and in Examples 8 to 14 and Comparative Examples 3 and 4, competitive immunoassay was performed.

[Preparation Example 1] (Preparation of conjugate of secondary antibody and quencher)
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-alpha fetoprotein (AFP) monoclonal antibody (1D5, 2. 5 mg / mL, immobilized on Japan Medical Clinical Laboratory Laboratories).
Specifically, the carboxyl group of the quencher and the amino group of the antibody were immobilized by an amino coupling method.

[Preparation Example 2] (Preparation of conjugate of secondary antibody and quencher combined with competitive antigen)
First, 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-α fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Nippon Medical Laboratory Co., Ltd.) by an amino coupling method.
Next, an excess amount of AFP was mixed and complexed with the obtained conjugate of the secondary antibody and the quencher, and then purified using centrifugation and chromatography.

[Example 1]
The assay was performed using SPFS in a region with a small amount of AFP (antigen) (pmol / L to fmol / L).

(Production of plasma excitation sensor)
A glass transparent flat substrate (BK7 manufactured by SCHOTT AG) having a refractive index [nd] of 1.52, a thickness of 1 mm and an outer shape of 20 mm × 20 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by a sputtering method. After that, a thin gold film was formed on the surface by sputtering. The chromium thin film had a thickness of 1 nm, and the gold thin film had a thickness of 50 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 spacer layer had a thickness of 15 nm.

  5 parts by weight of a phenethylamine reactant of Cy5 (registered trademark) as a fluorescent dye and BL-S (polyvinyl butyral) manufactured by Sekisui Chemical Co., Ltd. as a polymer with respect to one side of the spacer layer that is not in contact with the gold thin film A composition containing 5 parts by weight and 25 parts by weight of methyl ethyl ketone as a solvent was applied by a spin coater 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.

  An aqueous solution containing 5% by weight of 3-aminopropyltriethoxysilane was applied on the fluorescent dye layer of the substrate thus obtained with a spin coater, allowed to air dry at room temperature for 2 hours, and then 10 ° C. at 50 ° C. Heated for minutes.

  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. Then, 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 μL / min. The total volume of liquid delivery is 15 mL.
Here, an LD laser 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 manufactured by Sigma Kogyo Co., Ltd. is used. 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.

  Furthermore, 5 mL of PBS containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was added (final concentrations were NHS: 50 mM and WSC: 100 mM, respectively) and circulated for 20 minutes. Later, 40 μL of anti-α-fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Laboratory) was circulated for 2 hours to produce a plasmon excitation sensor.

The resonance angle shift was measured with surface plasmons to confirm the immobilization of the ligand. The immobilization amount was 3 ng / mm ² .
Moreover, the nonspecific adsorption | suction prevention process was performed by circulating for 30 minutes in the PBS buffer physiological saline containing 1 weight% bovine serum albumin (BSA).

(Implementation of assay method)
Step (a): 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 PBS for 10 minutes using PBS containing 0.05% by weight of Tween 20 as a solution. Here, after measuring the surface plasmon and fixing it at an optimum angle, it is irradiated with a plasmon excitation sensor fixed to the flow path using an LD laser, and a cut filter manufactured by Nippon Vacuum Optics Co., Ltd. Using a double objective lens (manufactured by Nikon Corporation), fluorescence by SPFS was detected through a CCD image sensor (manufactured by Texas Instruments Japan Ltd.) to obtain a “blank fluorescence signal”.

Step (b): 5 mL of PBS containing 1,000 ng / mL of the conjugate of the secondary antibody and the quencher obtained in Preparation Example 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.

  Step (c): The SPFS signal value observed from the CCD 20 minutes after the start of washing was measured and used as an “assay signal”. In addition, 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”.

Step (d): “blank fluorescence signal” before starting assay measurement in the plasmon excitation sensor obtained by making, “blank signal before starting assay of comparison sample” as “initial signal”, and from the assay fluorescence signal, 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.
Assay S / N ratio = | (assay fluorescence signal) − (blank fluorescence signal) | / (initial noise)
The obtained results are shown in Table 3.

[Example 2]
In Example 1, except that the fluorescent dye was changed to Alexa Fluor (registered trademark) 647, the plasmon excitation sensor of the present invention was produced and assayed in the same manner as in Example 1. The obtained results are shown in Table 3.

[Example 3]
(Production of plasma excitation sensor)
A glass transparent flat substrate (BK7 manufactured by SCHOTT AG) having a refractive index [nd] of 1.52, a thickness of 1 mm and an outer shape of 20 mm × 20 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by a sputtering method. After that, a thin gold film was formed on the surface by sputtering. The chromium thin film had a thickness of 1 nm, and 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.

  An 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 10 ° C. at 50 ° C. Heated for minutes.

  5 mL of PBS containing 50 mM of N-hydroxysuccinimide (NHS), 100 mM of water-soluble carbodiimide (WSC) and 50 mM of Alexa Fluor (registered trademark) 647 was prepared and applied with a spin coater (50 ° C. × 10 Plasmon excitation sensor having a fluorescent dye layer containing no polymer.

(Implementation of assay method)
The assay was carried out in the same manner as in Example 1. The obtained results are shown in Table 3.
[Example 4]
In Example 1, the plasmon excitation sensor of the present invention was prepared in the same manner as in Example 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. Made and assayed. The obtained results are shown in Table 3.

[Example 5]
In Example 4, a plasmon excitation sensor of the present invention was prepared and assayed in the same manner as in Example 4 except that the metal species was aluminum and the thickness of the metal thin film was 15 nm. The obtained results are shown in Table 3.

[Example 6]
In Example 5, a plasmon excitation sensor of the present invention was produced and assayed in the same manner as in Example 5 except that the thickness of the metal thin film was 20 nm. The obtained results are shown in Table 3.

[Example 7]
In Example 6, except that the fluorescent dye was changed to Cy3, a plasmon excitation sensor of the present invention was prepared and assayed in the same manner as in Example 6. The obtained results are shown in Table 3.

[Comparative Example 1]
(Production of plasma excitation sensor)
A glass transparent flat substrate (BK7 manufactured by SCHOTT AG) having a refractive index [nd] of 1.52, a thickness of 1 mm and an outer shape of 20 mm × 20 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by a sputtering method. After that, a thin gold film was formed on the surface by sputtering. The chromium thin film had a thickness of 1 nm, and the gold thin film had a thickness of 50 nm.

  The obtained substrate was immersed in an ethanol solution containing 1 mM 10-carboxy-1-decanethiol for 24 hours or more to form a SAM (Self Assembled Monolayer) 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 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 μL / min to equilibrate the surface.

  Subsequently, 5 mL of PBS containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was fed and circulated for 20 minutes, and then anti-α-fetoprotein (AFP) monoclonal. The primary antibody was solid-phased on the SAM by circulating 2.5 mL of an antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Clinical Laboratory Laboratories) solution for 30 minutes. 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 to produce a plasmon excitation sensor.

(Implementation of assay method)
Instead of PBS, 5 mL of a PBS solution containing 10 ng / mL of AFP was added and circulated for 30 minutes.

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 not labeled with a quencher (PBS solution adjusted to 1,000 ng / mL) was added and circulated for 30 minutes.

Then, it was cleaned by circulating TBS containing 0.05% by weight of Tween 20 for 20 minutes.
The signal value when observed from the CCD image sensor with the optimum resonance angle was measured and designated as “assay signal”. The SPFS measurement signal when AFP was 0 ng / mL was defined as “blank signal”. The assay was evaluated by calculating the same assay S / N ratio as in Example 1. The obtained results are shown in Table 3.

[Comparative Example 2]
Comparative Example 1 except that the plasmon excitation sensor was prepared in the same manner as in Comparative Example 1 except that the metal species was changed to aluminum and the thickness of the metal thin film was changed to 20 nm, and the fluorescent dye was changed to Cy3. The assay was performed as described above. The obtained results are shown in Table 3.

From Table 3, 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. Thus, 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.

  In the sample in which the fluorescent dye layer of the present invention was provided and the quencher was bound to the secondary antibody, both had higher fluorescent signal values than those in Comparative Examples 1 and 2, and more per unit antigen. It was found that the signal is carried by the fluorescent dye and the numerical reliability is high. Moreover, since the assay S / N ratio was significantly higher than that of the comparative example, it was found that the measurement sensitivity could be set higher.

[Example 8]
In the implementation of the assay method of Example 1, instead of the “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, the “secondary antibody complexed with the competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Example 1 except that the “conjugate of antibody and quencher” was used. Table 4 shows the obtained results.

[Example 9]
In the implementation of the assay method of Example 2, instead of the “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, the “secondary antibody complexed with the competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was performed in the same manner as in Example 2 except that the conjugate of antibody and quencher was used. Table 4 shows the obtained results.

[Example 10]
In the implementation of the assay method of Example 3, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Example 3 except that the antibody-quencher conjugate was used. Table 4 shows the obtained results.

[Example 11]
In the execution of the assay method of Example 4, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Example 4 except that the “conjugate of antibody and quencher” was used. Table 4 shows the obtained results.

[Example 12]
In the execution of the assay method of Example 5, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Example 5 except that the conjugate of antibody and quencher was used. Table 4 shows the obtained results.

[Example 13]
In the execution of the assay method of Example 6, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Example 6 except that the conjugate of antibody and quencher was used. Table 4 shows the obtained results.

[Example 14]
In the implementation of the assay method of Example 7, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Example 7 except that the antibody-quencher conjugate was used. Table 4 shows the obtained results.

[Comparative Example 3]
In the execution of the assay method of Comparative Example 1, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 was used. A competitive immunoassay was carried out in the same manner as in Comparative Example 1 except that the “conjugate of antibody and quencher” was used. Table 4 shows the obtained results.

[Comparative Example 4]
In the execution of the assay method of Comparative Example 2, instead of “conjugate of secondary antibody and quencher” obtained in Preparation Example 1, “secondary antibody complexed with competitive antigen” obtained in Preparation Example 2 A competitive immunoassay was carried out in the same manner as in Comparative Example 2 except that the “conjugate of antibody and quencher” was used. Table 4 shows the obtained results.

From Table 4, 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. On the other hand, in 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. Assuming from this result, it is shown that the quencher-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.

  Since 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.

DESCRIPTION OF SYMBOLS 1 ... Substrate 2 ... Primary antibody 3 ... Quenching agent (target antigen) contained in specimen
4 ... Secondary antibody 5 ... Conjugate of secondary antibody and fluorescent dye 6 ... Conjugate of secondary antibody and quencher 7 ... Different from target antigen contained in specimen , An antigen that competes with the target antigen (competitive antigen)
DESCRIPTION OF SYMBOLS 11 ... Metal thin film 12 formed on one surface of transparent flat substrate 12 ... Fluorescent dye layer formed on one surface of spacer layer made of dielectric 13 ... Fluorescent dye excited by surface plasmon Fluorescence emitted from

Claims (12)

A transparent planar substrate;
A metal thin film formed on one surface of the substrate;
A spacer layer made of a dielectric formed on the other surface of the metal thin film not in contact with the substrate;
A fluorescent dye layer formed on the other surface of the spacer layer not in contact with the metal thin film;
A plasmon excitation sensor comprising: a ligand immobilized on the other surface of the fluorescent dye layer not in contact with the spacer layer.   The plasmon excitation sensor according to claim 1, wherein the metal thin film is formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum.   The plasmon excitation sensor according to claim 2, wherein the metal is made of gold. The plasmon excitation sensor according to claim 1, wherein the dielectric includes silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ).   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. The plasmon excitation sensor according to any one of the above.   The said fluorescent pigment layer is formed by couple | bonding through the silane coupling agent with the other surface which is not in contact with the said metal thin film of the said spacer layer. Plasmon excitation sensor.   The plasmon excitation sensor according to any one of claims 1 to 6, wherein the ligand is an antibody that recognizes and binds to a tumor marker or carcinoembryonic antigen. An assay method comprising the following steps (a) to (d):
Step (a): contacting the specimen with the plasmon excitation sensor according to any one of claims 1 to 7,
Step (b): The plasmon excitation sensor obtained through the step (a) is further quenched or absorbed with a ligand that may be the same as or different from the ligand contained in the plasmon excitation sensor. Reacting a conjugate with the resulting compound;
Step (c): The plasmon excitation sensor obtained through the step (b) is irradiated with a laser beam via a prism from the other surface of the transparent flat substrate on which the metal thin film is not formed. A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and a step (d): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (c).   The assay method according to claim 8, wherein the conjugate binds to the analyte.   The assay method according to claim 8, wherein an analyte different from the analyte and competing with the analyte is previously bound to the conjugate.   The assay method according to any one of claims 8 to 10, wherein the specimen is at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.   The assay method according to claim 8, wherein the analyte is a tumor marker or carcinoembryonic antigen.