WO2010084953A1

WO2010084953A1 - Plasmon excitation sensor and method of assay using same

Info

Publication number: WO2010084953A1
Application number: PCT/JP2010/050803
Authority: WO
Inventors: 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: 2009-01-22
Filing date: 2010-01-22
Publication date: 2010-07-29

Abstract

A plasmon excitation sensor which has high sensitivity and high precision and has excellent specificity, which is essential for immunoassay; and a method of assay, an assay device, and an assay kit each using or including the sensor. The plasmon excitation sensor is characterized by comprising a transparent flat substrate, a thin metal film formed on one surface of the substrate, and a spacer layer constituted of a dielectric and formed on the surface of the thin metal film which is not in contact with the substrate. The sensor is further characterized in that (V) a ligand has been fixed to a fluorescent dye layer formed on the surface of the spacer layer which is not in contact with the thin metal film or that (W) a ligand labeled with a fluorescent dye has been fixed to the surface of the spacer layer which is not in contact with the thin metal film.

Description

Plasmon excitation sensor and assay method using the same

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.

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, in Patent Document 3, 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. 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.

Further, 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.
However, in order to establish such a measurement system, 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

Japanese Patent No. 3294605 Special table 2007-512522 gazette JP 2007-139540 A JP 2008-139245 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, 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. In order 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. As a result, 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. Thus, the present inventors have found that even a very small amount of analyte (for example, a target antigen) can achieve both fluorescence emission and specificity corresponding to the amount of photons, thereby completing the present invention.

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. 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.

In the plasmon excitation sensor (I), 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. .

In the plasmon excitation sensor (II), the ligand is preferably immobilized on the spacer layer via a self-assembled monolayer [SAM] made of a silane coupling agent.

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,
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. Reacting,
Step (c): a step of reacting a quencher substrate with the plasmon excitation sensor obtained through the step (b2) to produce a quencher,
Step (d): The plasmon excitation sensor obtained through the step (b1) or (c) is subjected to laser from the other surface of the transparent flat substrate on which the metal thin film is not formed via a prism. A step of irradiating light and measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (e): the amount of analyte contained in the specimen from the measurement result obtained in step (d) Calculating step.

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.

In the third aspect of the assay method of the present invention (hereinafter referred to as “assay method (Z)”), the enzyme is preferably β-galactosidase, β-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.

In the assay kit of the present invention, 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. Comprising a sensor and a quenching dye, and used in the assay method (X) of the present invention; when the assay method takes the embodiment of (Y), it comprises at least a transparent flat substrate, the metal thin film, and the dielectric. A sensor including a spacer layer, a fluorescent dye, and a quenching dye, and used in the assay method (Y) of the present invention; when the assay method takes the form of (Z), at least the transparent flat substrate and the metal thin film And a sensor comprising the spacer layer made of the dielectric and the fluorescent dye layer, the enzyme and the quencher substrate, and used in the assay method (Z) of the present invention.

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 the plasmon excitation sensor of the present invention is used in the assay method of the present invention, according to FIG. 2, it generates fluorescence commensurate with the electric field enhancement by using the fluorescent dye (signal amount = can be generated with the fluorescent dye). An example of an appropriate photon amount), and the fluorescence can be adjusted by a compound (quenching dye / quenching agent) that can quench or absorb the fluorescence according to the amount of the analyte (amount of noise = a compound that can quench or absorb the fluorescence) (Example of the amount of photons that can be extinguished by the above-mentioned method) Therefore, it is possible to assay with a high S / N ratio with less noise and more signal.

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). 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.

In addition, since 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.

Furthermore, 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, Thus, it is possible to provide 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).

Based on the assumptions of (1) to (3) below, “number of incident photons” using a semiconductor laser (100) as a light source, “number of excited photons” due to the electric field enhancement effect of surface plasmons excited by the light source, secondary antibody The “absorbed photon number” excited by the fluorescent dye labeled with the “fluorescent photon number” detected by the CCD (122) was calculated as follows.
(1) When irradiating laser light using the semiconductor laser (100), after P-polarized light, the amount of light is adjusted by the ND filter (102), and 0.1 mW is incident on the sensor chip (111);
(2) 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. In this specification, 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.

The number of incident photons is Iph / S = 3.2 × 10 ¹⁴ / S.
The number of excitation photons is Exph / S = 5.4 × 10 ¹⁵ / S, and the electric field enhancement by plasmons is 17 times the number of incident photons.

The number of absorbed photons is Absph / S = excitation photon number × absorption efficiency≈excitation photon number × 2.3 × molar extinction coefficient × concentration × depth≈1.27 × 10 ⁷ / S.
The number of fluorescent photons is Emph / S = number of absorbed photons × quantum yield≈5.95 × 10 ⁶ / S.

That is, assuming that 0.1 mW laser light is incident and the electric field is increased 17 times on the gold substrate, the number of photons is about 15th. However, in the antigen measurement at the nmol / L level, the fluorescent dye Since the number of photons generated via the number decreases to the sixth power, most of the photons with an enhanced electric field are not used, and the amount of photons detected is extremely small, so it cannot be said that the accuracy of the assay is improved. .
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).

In the type 2 of the assay method (X) of the present invention, the target antigen (3) contained in the sample is primary by bringing the sample into contact with the sample (step a1) = plasmon excitation sensor (I) of the present invention. 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.

In type 2 of the assay method (Y) of the present invention, the target antigen (3) contained in the sample is primary by bringing the sample into contact with the sample (step ala2) = plasmon excitation sensor (II) of the present invention. 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)).

In the assay method (Z) of the present invention, the 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. To do.
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. .

Hereinafter, the present invention will be specifically described.
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).

In addition, 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.

[Transparent flat substrate]
In the present invention, a transparent flat substrate is used as a substrate that supports the structure of the plasmon excitation sensor. In the present invention, 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. For example, the transparent support may be made of glass, or may be made of plastic such as polycarbonate [PC] or cycloolefin polymer [COP].

Further, 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.

In addition, 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” (refractive index [n _d ] 1.84), “K-LaSFn17” (refractive index [n _d ] 1.88) and “K-LaSFn22” (refractive index) manufactured by Sumita Optical Glass Co., Ltd. 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.
[Metal thin film]
In the plasmon excitation sensor according to the present invention, 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.

When the plasmon excitation sensor of the present invention is used for assay methods (X) and (Y), 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.

In addition, it is preferable to form 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.

From the viewpoint of the electric field enhancement effect, 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, and alloys thereof: 10 to 70 nm are more preferable, and chromium The thickness of the thin film is more preferably 1 to 3 nm.

It is preferable that 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 | size (length x width) will not be specifically limited.

[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. As 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. 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 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.

[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 (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.

Examples of the “protein” include antibodies and the like, specifically, anti-α-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.

In the present invention, the term “antibody” includes a polyclonal antibody or a monoclonal antibody, an antibody obtained by gene recombination, and an antibody fragment.
(Analyte)
An analyte (target antigen) is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to a ligand immobilized on a plasmon excitation sensor. Examples of the “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. 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.

<Plasmon excitation sensor (I)>
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).
[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 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.

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 another polymerizable monomer, a fluorescent dye and a polymerization initiator and copolymerizing them.

In the case of (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.

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.
(Fluorescent dye)
In the present invention, 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. 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 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 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.

In the present invention, for example, 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.

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. 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).

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

(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. For example, 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.), and the like. Of these, aromatics, halogen-containing hydrocarbons, esters, and ketones are preferred 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.

Further, 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.

(Coating)
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.

[Ligand immobilization method]
As the method for immobilizing the ligand, in the case of the above (A) which is one embodiment of the formation of the fluorescent dye layer, functional groups such as hydroxyl group, amino group, carboxyl group and isocyanate group (preferably hydroxyl group, amino group and A method of forming a chemical bond by reacting with a carboxyl group) and a functional group possessed by a ligand (preferably combined with a functional group possessed by a polymer in consideration of the ability to promote immune reaction and suppress nonspecific adsorption reaction), Alternatively, a method of immobilizing the fluorescent dye layer via a compound having a bifunctional reactive group (for example, a silane coupling agent) can be used.

As a 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.

In addition to “a 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.

In the case of using 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. In this case, 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. 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, 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.

As the method for immobilizing the ligand, in the case of the above (B) in forming the fluorescent dye layer, a method of immobilizing a ligand (for example, a primary antibody) on the fluorescent dye layer by physical adsorption can be mentioned.
<Plasmon excitation sensor (II)>
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.
As 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.

As 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.

Further, 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.

<Assay method>
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. Reacting,
Step (c): a step of reacting a quencher substrate with the plasmon excitation sensor obtained through the step (b2) to produce a quencher,
Step (d): The plasmon excitation sensor obtained through the step (b1) or (c) is subjected to laser from the other surface of the transparent flat substrate on which the metal thin film is not formed via a prism. A step of irradiating light and measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (e): the amount of analyte contained in the specimen from the measurement result obtained in step (d) Calculating step.

That is, 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.
[Process (a1) / (a2)]
Steps (a1) and (a2) are steps of bringing the specimen into contact with the plasmon excitation sensors (I) and (II) of the present invention, respectively.

(Sample)
Examples of the specimen 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.

(contact)
In the state in which the specimen is contained in the liquid supply circulating in the flow path and only one surface on which the fluorescent dye and the ligand of the plasmon excitation sensor are immobilized is immersed in the liquid supply, the contact with the plasmon excitation sensor An embodiment in which a specimen is 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 (plasmon excitation sensor section) 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.

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 vertical and horizontal sections of the channel of the plasmon excitation sensor unit are independently about 100 nm to 1 mm.

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 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.

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 the 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 (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.

As the washing solution used in the washing step, 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.

Such 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-benzyl-1,4-dihydronicotinamide [BNAH], 9-anthracenecarbonitrile, 2-naphthol, 2-methoxynaphthalene, 1,4-naphthoquinone, 2,6-di -Tert-butyl 1,4-benzoquinone, 3,5-di-tert-butyl-1,2-benzoquinone, 1-naphthoic acid [1-NA], 2-naphthoic acid [2-NA], 1-pyrenebutyric acid [PyBA], Examples include 4-nitrobenzoic acid [PNBA], anthraquinone-2-carboxylic acid [AQCA], and pyrene. 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 quenching dyes, 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 Ltd. have a wide wavelength range. It is preferable as a dark quencher (a quenching dye that does not emit light itself).

Other 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).

Generally, energy transfer between a fluorescent dye and a quenching dye depends on a distance between the fluorescent dye and the quenching dye, that is, a critical transition distance. “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. When 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.

As a standard for the combination of a specific fluorescent dye and a quenching dye, 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 quenching dye; the fluorescence wavelength emitted by the quenching dye And the quantum yield of fluorescence emission by the quenching dye. In addition, when the quenching dye is also a fluorescent dye, it is preferable to combine the quenching dye and the fluorescent dye so that the fluorescence emitted by one can be easily distinguished from the fluorescence emitted by the other. For the selection of combinations of specific fluorescent dyes and quenching dyes, reference can be made to the general review of Klostermeier and Millar, 2002, Biopolymers 61: 159-179.

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 ligand and quenching dye)
The “conjugate of a ligand and a quenching dye, which may be the same as or different from the ligand contained in the plasmon excitation sensor”, uses the following embodiment (α) or (β) when a secondary antibody is used as the ligand: Is preferred.

Embodiment (α): An antibody capable of recognizing and binding to an analyte (target antigen) contained in a specimen is used as a secondary antibody. 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 the embodiment (α) competes with AFP contained in the specimen. A monoclonal or polyclonal antibody capable of recognizing and binding to the antigen of interest.

Aspect (α) 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.
Aspect (β): As a secondary antibody, an analyte competing with an analyte (target antigen) contained in a specimen (competitive antigen; provided that the primary antibody (ligand) is different from the target antigen) Are bound in advance). 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.

It is preferable to use the complex of the secondary antibody and the competitive antigen used in the embodiment (β) for the competitive immunoassay method. When 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. In addition, when a complex in which a competitive antigen is previously bound to a conjugate of a ligand and a quenching dye is used, noise due to nonspecific binding (adsorption) of the conjugate of the ligand and the quenching dye can be reduced, and The amount of fluorescent signal (fluorescent signal) and the amount of target antigen can be proportional to each other with higher accuracy.

Therefore, the embodiment (β) 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.

When the primary antibody is an anti-AFP monoclonal antibody, the secondary antibody of the embodiment (β) 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 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 A method of dehydrating a group with water-soluble carbodiimide and immobilizing it; a method of reacting and immobilizing a secondary antibody having an isothiocyanate and an amino group and a quenching dye; a secondary antibody having a sulfonyl halide and an amino group, respectively And a method of reacting and immobilizing a quenching dye; Doasetoamido and methods for immobilizing by reacting secondary antibody and quencher dyes each having a thiol group; and a method of immobilizing reacting a biotinylated quencher dye and streptavidinated by secondary antibodies.

The concentration of the conjugate of the ligand thus prepared and the quenching dye during feeding is preferably 0.001 to 10,000 μg / mL, more preferably 1 to 1,000) μg / 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 | cleaning process between a process (b2) and the following process (c) between a process (b1) and the following process (d).

[Step (b2)]
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.

(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 β-galactosidase, β-glucosidase, alkaline phosphatase and the like.
β-galactosidase catalyzes the reaction of eliminating βGal from the quencher substrate TG-βGal. In addition, β-glucosidase catalyzes a reaction for eliminating βGlu from TG-βGlu which is a quencher substrate. Since 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.

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).

In both (A) and (B), these enzymes can be used alone or in combination of two or more.
(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.

As a method for preparing a conjugate of a ligand and an enzyme, for example, 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]. Etc.) and N-hydroxysuccinimide [NHS], followed by dehydration reaction and immobilization of the active esterified carboxyl group and the amino group of the ligand using water-soluble carbodiimide; isothiocyanate And a method of immobilizing a ligand and an enzyme each having an amino group; a method of reacting and immobilizing a ligand and an enzyme each having a sulfonyl halide and an amino group; and a method of reacting and immobilizing a ligand and an enzyme each having an iodoacetamide and a thiol group, respectively. A method can be mentioned immobilizing reacting the ligand is an enzyme and streptavidin of which is biotinylated; method for immobilizing is.

The concentration of the ligand-enzyme conjugate thus prepared in the solution is preferably 0.001 to 10,000 μg / mL, more preferably 1 to 1,000) μg / mL.
The temperature, time, and flow rate at which the liquid is circulated are the same as those in step (a1) or (a2).

[Step (c)]
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 / Quencher)
Examples of the quencher substrate include TG-βGal, TG-βGlu, AttoPhos (registered trademark) substrate, glucose and the like as described above.

TG-βGal and TG-βGlu, which are quencher substrates used in the enzyme reaction (A), are compounds obtained by adding one molecule of β-galactose and β-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 β-galactosidase and β-glucosidase.

In the present invention, 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.

Examples of the quencher substrate used in the enzyme reaction (B) include glucose and oxygen which are substrates for glucose oxidase.
In the step (c) of the assay method of the present invention, 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 μg / mL, more preferably 1 to 1,000 μg / 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)]
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.

It is desirable that 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.

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 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.
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 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. 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 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. 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.

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)]
The 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).

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 (e), 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. 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)
<Assay device>
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).

That is, 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. In addition, 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) In addition, 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.) 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.
Examples of the “liquid feed pump” 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. For example, a tube pump may be difficult.

<Assay kit>
When the assay kit of the present invention takes the above-described embodiment (X), 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. Used in the assay method (X) of the present invention;
When the embodiment (Y) is adopted, the assay method of the present invention (Y) 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. );
When the embodiment of (Z) is taken, it 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.

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.

Specifically, 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 (β-galactosidase, β-glucosidase, alkaline phosphatase, glucose oxidase, etc.); quencher substrates (eg, TG-βGal, TG) -ΒGlu, AttoPhos (registered trademark) substrate, glucose, etc.); quenching dye for secondary antibody Or various reagents for immobilizing enzymes (for example, water-soluble carbodiimide (EDC, etc.), N-hydroxysuccinimide [NHS], etc.), which are necessary for carrying out the assay method of the present invention. Various devices or materials and the above assay devices can also be included.

Furthermore, as a 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.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. 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-α-fetoprotein [AFP] monoclonal antibody (1D5; 2. 5 mg / mL, immobilized on Japan Medical Clinical Laboratory Laboratories).

Specifically, the carboxyl group of the quenching dye and the amino group of the antibody were immobilized by an amino coupling method.
[Preparation Example (1-2)] (Preparation of conjugate of secondary antibody and quenching dye complexed 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 quenching dye, and then purified using centrifugation and chromatography.
[Example (1-1)]
The assay was performed using SPFS in a region with a small amount of AFP (antigen) (pmol / L to fmol / L).

(Production 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. Was formed by sputtering, and then 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 reaction product of Cy5 (registered trademark) as a fluorescent dye and “BL-S” (polyvinyl) manufactured by Sekisui Chemical Co., Ltd. as a polymer with respect to one side of the spacer layer not in contact with the gold thin film A composition containing 5 parts by weight of butyral) and 25 parts by weight of methyl ethyl ketone as a solvent was applied by a spin coating method and dried in the dark at 50 ° C. for 10 minutes 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 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. 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, 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.

Further, 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 Clinical Laboratory, Inc.) was circulated for 2 hours to produce plasmon excitation sensor (I).

The resonance angle shift was measured with surface plasmons to confirm the immobilization of the ligand. The immobilization amount was 3 ng / mm ² .
Further, 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.

(Execution of assay method (X))
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. Here, after measuring the surface plasmon and fixing it to the optimum angle, 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. Using 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.

Step (d): The SPFS signal value when 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 (e): “blank fluorescence signal” before starting assay measurement in the plasmon excitation sensor obtained by making, “blank signal before starting assay of comparative 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)
Table 4 shows the obtained results.

[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. Was formed by sputtering, and then 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 at 50 ° C. for 10 minutes. Heated.

Prepare 5 mL of PBS containing 50 mM N-hydroxysuccinimide [NHS], 100 mM water-soluble carbodiimide [WSC] and 50 mM Alexa Fluor (Registered Trade Mark) 647, apply with a spin coater (50 ° C. × 10 minutes) ) To produce a plasmon excitation sensor having a fluorescent dye layer containing no polymer.

(Execution of assay method (X))
Assay method (X) was carried out in the same manner as in Example (1-1). Table 4 shows the obtained results.

[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)]
In Example (1-4), 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.

[Comparative Example (1-1)]
(Manufacture of plasmon excitation sensor)
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. Was formed by sputtering, and then 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 of 10-carboxy-1-decanethiol for 24 hours or more to form 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, followed by 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 the solution 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 Alexa Fluor (registered trademark) 647-labeled secondary antibody (PBS solution prepared to be 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 (1-1). Table 4 shows the obtained results.

[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.

From Table 4, 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 used in the plasmon excitation sensor of the present invention is provided and the quenching dye is bound to the secondary antibody, both have higher fluorescent signal values than those of Comparative Examples (1-1) and (1-2) It was found that the signal is carried by more fluorescent dyes per unit antigen and the numerical value is highly reliable. 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.

[Preparation Example (1-3)] (Preparation of conjugate of secondary antibody and fluorescent dye complexed with competitive antigen)
In Preparation Example (1-2), in place of the quenching dye, 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.

[Comparative Example (1-3)]
In the implementation of the assay method of Comparative Example (1-1), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), it was obtained in Preparation Example (1-3). The competitive immunoassay method was carried out in the same manner as in Comparative Example (1-1) except that the “conjugate of secondary antibody complexed with competitive antigen and Alexa Fluor (registered trademark) 647” was used. The results obtained are shown in Table 5.

[Comparative Example (1-4)]
In the execution of the assay method of Comparative Example (1-2), instead of the “conjugate of secondary antibody and quenching dye” obtained in Preparation Example (1-1), it was obtained in Preparation Example (1-3). A competitive immunoassay was carried out in the same manner as in Comparative Example (1-2) except that the obtained “conjugate of secondary antibody and Cy3 complexed with competitive antigen” was used. The results obtained are shown in Table 5.

From 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. 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. 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).

[Preparation Example (2-3)] (Preparation of a ligand labeled with a fluorescent dye)
By using a fluorescent dye (Cy3 (registered trademark), Cy5 (registered trademark), Alexa Fluor (registered trademark) 633, 647 or TRITC) as a ligand, an anti-α-fetoprotein [AFP] monoclonal antibody (6D2; 2.5 mg) as a primary antibody / ML, manufactured by Nippon Medical Laboratory, Ltd.) was labeled by the amino coupling method. In order to remove the unlabeled primary antibody, it was further purified using centrifugation and chromatography.

[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.

(Manufacture of plasmon excitation sensor (II))
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. Was formed by sputtering, and then a thin gold film was formed on the surface by sputtering. The chromium thin film had a thickness of 2 nm, and 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. . As a result, a SAM made of a silane coupling agent was formed on the spacer layer.

Provided with 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. A board | substrate is arrange | positioned so that the surface may become an inner side of a flow path. Next, 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 μL / min. The total volume of liquid delivery is 15 mL.
Here, 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.

Further, 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 the ligand (primary antibody labeled with Cy5 (registered trademark) as a fluorescent dye) obtained in Preparation Example 3 was circulated for 2 hours to produce a plasmon excitation sensor (II). The resonance angle shift was measured with surface plasmons to confirm the immobilization of the ligand. The immobilization amount was 400 ng / cm ² . Furthermore, non-specific adsorption prevention treatment was performed by circulating the solution in PBS buffered saline containing 1% bovine serum albumin [BSA] for 30 minutes.

(Execution of assay method (Y))
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.

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.

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

Step (e): The blank fluorescence signal before the start of assay measurement in the obtained plasmon excitation sensor (II) and the blank fluorescence signal before the start of the assay of the comparative sample are defined as “blank fluorescence signal”, and “assay fluorescence signal” From the relationship with the “blank fluorescence signal”, the S / N ratio was evaluated by the following formula. That is, the reliability of the assay signal is high when the absolute value of the value of the assay fluorescence signal, which varies with the amount of secondary antibody proportional to the amount of antigen, is large and sufficiently large numerically with respect to the initial noise.

Assay S / N ratio = | (assay fluorescence signal) − (blank fluorescence signal) | / (initial noise)
The obtained results are shown in Table 6.

[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 (2-4)]
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)]
In Example (2-4), 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.

[Comparative Example (2-1)]
(Manufacture of plasmon excitation sensor)
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. Was formed by sputtering, and then a thin gold film was formed on the surface by sputtering. The chromium thin film had a thickness of 2 nm, and the gold thin film had a thickness of 48 nm.

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 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 2.5 mL of the primary antibody solution. Was circulated for 30 minutes to immobilize the ligand on the SAM. Non-specific adsorption prevention treatment was performed by circulating and feeding in PBS buffered saline containing 1% bovine serum albumin [BSA] 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 (PBS solution prepared to be 1,000 ng / mL) labeled with Alexa Fluor (registered trademark) 647 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 with the optimum resonance angle was measured and used as the assay fluorescence signal. The SPFS measurement signal when AFP was 0 ng / mL was used as a blank fluorescence signal. The assay was evaluated by calculating the same assay S / N ratio as in Example (2-1). The obtained results are shown in Table 6.

[Comparative Example (2-2)]
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.

From 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. 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 Examples where the primary antibody labeled with a fluorescent dye is immobilized and the quenching dye is bound to the secondary antibody, both are compared with Comparative Examples (2-1) and (2-2). It has a high fluorescence signal value, and it is found that the signal is carried by more fluorescent dyes per unit antigen and the numerical reliability is high. It was also found that the measurement sensitivity can be set higher because the assay S / N ratio is orders of magnitude higher than that of the comparative example.

[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.

[Comparative Example (2-4)]
Competing in the same manner as in Comparative Example (2-2), except that the plasmon excitation sensor produced in Comparative Example (2-2) was used and a conjugate of Cy3-labeled secondary antibody complexed with AFP was used. An immunoassay was performed. The results obtained are shown in Table 7.

From 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. In addition, as described above, the 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 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.

[Preparation Example (3-1)] (Preparation of conjugate of secondary antibody and β-galactosidase)
β-galactosidase was immobilized on an anti-α-fetoprotein [AFP] monoclonal antibody (6D2; 2.5 mg / mL, manufactured by Japan Medical Laboratory).

Specifically, the carboxyl group of the enzyme and the amino group of the antibody were immobilized by an amino coupling method.
[Preparation Example (3-2)] (Preparation of conjugate of secondary antibody and glucose oxidase)
Following the same procedure as in Preparation Example (3-1), glucose oxidase was immobilized on an anti-α-fetoprotein [AFP] monoclonal antibody (6D2; 2.5 mg / mL, manufactured by Japan Medical Clinical Laboratory, Inc.).

[Preparation Example (3-3)] (Preparation of conjugate of secondary antibody and dark quencher)
In accordance with the same procedure as in Preparation Example (3-1), Eclipse (registered trademark) (manufactured by Epoch Biosciences), which is a dark quencher, was added to anti-α fetoprotein [AFP] monoclonal antibody (6D2; 2.5 mg / mL, strain ) Immobilized in Japan Medical Clinical Laboratory.

[Preparation Example (3-4)] (Preparation of Alexa Fluor (registered trademark) 647-labeled secondary antibody)
A solution of biotinylated anti-AFP monoclonal antibody and a streptavidin-labeled Alexa Fluor (registered trademark) 647 (Molecular Probes) solution were mixed and reacted by stirring at 4 ° C. for 60 minutes.

Next, 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. Was formed by sputtering, and a silver 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.

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 And 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.

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, 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.

(Execution of assay method (Z))
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. Here, using blank fluorescence, an LD laser as a light source, a laser beam having a wavelength of 340 nm, an amount of photons is adjusted by an optical filter: (Sigma Kogyo Co., Ltd.), and a prism: (“Ohara Co., Ltd. S-LAL10 ”(refractive index [n _d ] = 1.72) is applied to the plasmon excitation sensor fixed to the flow path, and is used as a cut filter (Sigma Kogyo Co., Ltd.) and as a condenser lens. 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 β-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 (c): An enzyme quenching substrate (TG-bGal) solution adjusted with TBS was introduced into the plasmon excitation sensor obtained through the washing step and allowed to react.
Step (d): The signal value observed from the CCD 20 minutes after the introduction of the enzyme quenching substrate solution was measured and used as an assay signal. The SPFS measurement signal when AFP was 0 ng / mL was used as a blank signal.

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 = | (assay fluorescence signal) − (blank signal) |
The obtained results are shown in Table 8 and FIG.

[Example (3-2)]
(Manufacture of plasmon excitation sensor (I))
The same procedure as in Example (3-1) was performed, except that 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.

(Execution of assay method (Z))
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.

The obtained results are shown in Table 8 and FIG.
[Comparative Example (3-1)]
(Manufacture of plasmon excitation sensor)
A plasmon excitation sensor was manufactured in the same manner as in Example (3-1).

(Implementation 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 obtained results are shown in Table 8 and FIG.
[Comparative Example (3-2)]
(Manufacture of plasmon excitation sensor)
A glass transparent flat substrate (“S-LAL 10” manufactured by OHARA INC.) Having a refractive index [n _d ] of 1.72 and a thickness of 1 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by sputtering. After the formation, a gold thin film was further formed on the surface by a sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

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.

(Implementation of assay method)
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 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, followed by 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. In addition, the nonspecific adsorption | suction prevention process was performed by circulating 30 minutes by PBS buffer physiological saline containing 1 weight% bovine serum albumin [BSA].

Instead of PBS, 0.5 mL of a PBS solution containing 1 ng / mL of AFP was added and circulated for 25 minutes.
Washing was carried out by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes.

2.5 mL of secondary antibody (PBS solution prepared to be 1,000 ng / mL) labeled with Alexa Fluor (registered trademark) 647 prepared in Preparation Example (3-4) was added and circulated for 20 minutes. .

Then, it was cleaned by circulating TBS containing 0.05% by weight of Tween 20 for 20 minutes.
The signal value observed from the CCD was measured and used as an assay signal. The SPFS measurement signal when AFP was 0 ng / mL was used as a blank signal. As assay evaluation, it evaluated by calculating the assay signal variation | change_quantity similar to Example (3-1).

The obtained results are shown in Table 8 and FIG.

Since 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).

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.

Since 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.

1 .... Substrate 2 .... Primary antibody 3 .... Target antigen contained in specimen 4 .... Secondary antibody 5 .... Fluorescent dye 6 .... Quenching dye 7 .... An antigen competing with the target antigen, which is different from the target antigen contained in the specimen (competitive antigen)
8 .... Quencher substrate 9 .... Quencher 10 .... Enzyme 11 .... Metal thin film formed on one surface of transparent flat substrate 12 .... ... Fluorescent dye layer formed on one surface of spacer layer made of dielectric 13... Fluorescence emitted by fluorescent dye excited by surface plasmon 100... Semiconductor laser [ LD]
101... Diaphragm lens 102... Neutral density filter (Neutral density [ND] filter)
103... Polarizing filter 104... Photodiode 110... Prism 111... Sensor chip 120. -Cut filter and adapter 122-CCD image sensor

Claims

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, and (V) the other of the spacer layer not in contact with the metal thin film. The ligand is immobilized on the fluorescent dye layer formed on the surface, or (W) the ligand labeled with the fluorescent dye is immobilized on the other surface of the spacer layer that is not in contact with the metal thin film. A plasmon excitation sensor characterized by comprising:
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 1 or 2, wherein the dielectric includes silicon dioxide [SiO 2 ] or titanium dioxide [TiO 2 ].
The plasmon excitation sensor according to any one of claims 1 to 3, wherein the ligand is an antibody that recognizes and binds to a tumor marker or carcinoembryonic antigen.
The plasmon excitation sensor according to any one of claims 1 to 4, which takes the form (V).
The plasmon excitation sensor according to claim 5, wherein the metal thin film is made of gold or silver.
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 silane coupling. The plasmon excitation sensor according to claim 5 or 6, which is formed by binding a fluorescent dye via an agent.
The plasmon excitation sensor according to any one of claims 1 to 4, which takes the form (W).
The plasmon excitation sensor according to claim 8, wherein the metal thin film is made of gold.
The plasmon excitation sensor according to claim 8 or 9, wherein the ligand is immobilized on the spacer layer via a self-assembled monolayer [SAM] made of a silane coupling agent.
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) An assay characterized by comprising;
Step (a1): contacting the specimen with the plasmon excitation sensor according to any one of claims 5 to 7,
Step (a2): contacting the specimen with the plasmon excitation sensor according to any one of claims 8 to 10,
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. Reacting,
Step (c): a step of reacting a quencher substrate with the plasmon excitation sensor obtained through the step (b2) to produce a quencher,
Step (d): The plasmon excitation sensor obtained through the step (b1) or (c) is subjected to laser from the other surface of the transparent flat substrate on which the metal thin film is not formed via a prism. A step of irradiating light and measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (e): the amount of analyte contained in the specimen from the measurement result obtained in step (d) Calculating step.
The assay method according to claim 11, 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 11 or 12, wherein the analyte is a tumor marker or carcinoembryonic antigen.
When taking the embodiment of (X) or (Y), an analyte that is different from the analyte and that competes with the analyte is previously bound to the conjugate. An assay method according to any of the above.
The assay method according to any one of claims 11 to 13, wherein the enzyme is β-galactosidase, β-glucosidase, alkaline phosphatase or glucose oxidase.
12. At least a plasmon excitation sensor obtained through the step (b1) or (c), a laser light source, an optical filter, a prism, a cut filter, a condensing lens, and a surface plasmon excitation enhanced fluorescence detection unit. An assay device, which is used in the step (d) according to any one of to 15.
When the assay method takes the embodiment of (X) above,
At least a sensor including a transparent flat substrate, the metal thin film, a spacer layer made of the dielectric, and the fluorescent dye layer and a quenching dye;
When the assay method takes the embodiment (Y) above,
Including at least a sensor including a transparent flat substrate, the metal thin film, and a spacer layer made of the dielectric, a fluorescent dye, and a quenching dye;
When the assay method takes the embodiment of (Z) above,
A sensor comprising at least a transparent flat substrate, the metal thin film, a spacer layer made of the dielectric, and the fluorescent dye layer, the enzyme, and a quencher substrate. A kit for the assay described in 1.