JP2010181323A - Assay method using surface plasmon - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an assay method using surface plasmon having high sensitivity and high precision and superior in specificity indispensable to immunoassay by making an immunoreaction field and a detection field independent, respectively. <P>SOLUTION: The assay method is characterized by including the following steps (a)-(g): step (a) of bringing the particles fixed on the surface of a ligand into contact with a specimen, step (b) of reacting a conjugate of the ligand and enzyme with the particles, step (c) of further reacting a substrate with the particles to form a quenching agent, step (d) of isolating the quenching agent, step (e) of bringing the quenching agent into contact with the surface of the fluorescent coloring matter layer of a plasmon exciting sensor, step (f) of irradiating the plasmon exciting sensor with a laser beam through a prism and measuring the quantity of the fluorescence emitted from excited fluorescent coloring matter and step (g) of calculating the amount of the analyte contained in the specimen from the obtained measuring results. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an assay method using surface plasmon, the assay device, and the assay kit. More specifically, the present invention relates to an assay method utilizing surface plasmon, the assay device, and the assay kit based on the principle of surface plasmon-excited fluorescence spectroscopy (SPFS; Surface Plasmon-field enhanced Fluorescence Spectroscopy).

  With surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), irradiation is performed by generating a dense wave (surface plasmon) on the surface of the metal thin film under the condition that the irradiated laser light is attenuated by total reflection (ATR) on the gold thin film surface. The amount of photons that the laser beam has is increased to several tens to several hundreds times (electric field enhancement effect of surface plasmon), and by this, the fluorescent dye in the vicinity of the gold thin film is efficiently excited, so that a trace amount and / or extremely low concentration can be obtained. It is a method that can detect an analyte.

  As an example of a biosensor or biochip based on the principle of SPFS, Patent Document 1 discloses that a ligand (primary antibody) immobilization film using carboxymethyldextran is arranged on the surface of a metal substrate, and surface plasmon is used. A method of detecting a fluorescent dye associated with an antigen with an enhanced electric field is shown.

  However, in detecting very small amounts of analytes (such as 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. Even if electric field enhancement is used, the amount of fluorescence signal does not increase and it is difficult to improve assay sensitivity.

  Patent Document 2 examines signal amplification and non-specific reaction reduction by complexly combining a reaction with an apoenzyme or holoenzyme 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 JP 2008-139245 A

  The present invention relates to an assay method using a surface plasmon, which is highly sensitive and highly accurate by independence of an immune reaction field and a detection field, and has excellent specificity essential for an immunoassay, and the assay device And an assay kit.

As a result of diligent research to solve the above-mentioned problems, the present inventors can completely separate the immune reaction field and the detection field by using a secondary antibody labeled with an enzyme. The present invention has been completed by discovering that both the fluorescence emission corresponding to the amount of photons and the specificity can be compatible even with the target antigen of.

  That is, the assay method of the present invention includes the following steps (a) to (g).

Step (a): a step of contacting a specimen with particles having a ligand immobilized on the surface thereof,
Step (b): reacting a particle obtained through the step (a) with a conjugate of a ligand and an enzyme, which may be the same as or different from the ligand,
Step (c): a step of further reacting a substrate with the particles obtained through the step (b) to produce a quencher,
Step (d): a step of isolating the quencher obtained through the step (c)
Step (e): a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a spacer layer made of a dielectric formed on the other surface of the metal thin film not in contact with the substrate And a plasmon excitation sensor having at least the fluorescent dye layer formed on the other surface of the spacer layer not in contact with the metal thin film, obtained on the surface of the fluorescent dye layer through the step (d). Contacting with a quenched quencher,
Step (f): The plasmon excitation sensor obtained in step (e) was excited by being irradiated with laser light from the other surface of the substrate on which the thin film was not formed via a prism. A step of measuring the amount of fluorescence emitted from the fluorescent dye, and a step (g): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).

  It is preferable that the immune reaction field in the steps (a) to (d) and the detection field in the steps (e) to (g) are independent from each other.

  The ligand described in the above step (a) may be a primary antibody that recognizes and binds to a tumor marker or carcinoembryonic antigen.

  The specimen is preferably at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.

  The enzyme is preferably β-galactosidase, β-glucosidase, alkaline phosphatase or glucose oxidase.

  The metal thin film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum, and the metal is particularly preferably made of silver.

The dielectric preferably includes silicon dioxide (SiO ₄ ) or titanium dioxide (TiO ₂ ).

The fluorescent dye layer can also be 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,
It can also be formed by bonding to the other surface of the spacer layer not in contact with the metal thin film via a silane coupling agent.

  The apparatus of the present invention includes at least a plasmon excitation sensor obtained through the above step (e), a laser light source, an optical filter, a prism, a cut filter, a condensing lens, and a surface plasmon excitation enhanced fluorescence detection unit, It is used in the step (f).

  The kit of the present invention includes 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, the enzyme and the substrate, and is used for the assay method. And

  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.

In contrast, the assay method of the present invention comprises a sample 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, A plasmon excitation sensor capable of detecting the analyte with high sensitivity and high accuracy 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 amount of signal change deteriorates. However, the present invention provides a plasmon excitation sensor and a ligand (for example, a secondary antibody). ) And an enzyme that activates a quencher When applied to the assay method of the present invention, a quencher is proportional to the amount of target antigen, so that a plasmon excitation sensor that does not deteriorate the signal change amount is provided. can do.

  In addition, since the present invention can adjust the amount of fluorescence signal depending on the ability of the quencher, it is possible to provide a plasmon excitation sensor in which the assay method of the present invention can be carried out with an optimal amount of signal change.

  Furthermore, since the present invention can remove particles, that is, completely separate the immune reaction field and the detection field, the immune reaction conditions and the detection conditions can be optimized and is not easily affected by scattering noise. Furthermore, since it has three sensitivity amplification mechanisms, that is, immune reaction optimization, chemical amplification, and physical amplification, an extremely sensitive and highly accurate assay method can be provided.

FIG. 1 shows a “primary antibody as a ligand” 2 in which “analyte (target antigen) 3 contained in a specimen” 3 is immobilized on the surface of “particle” 1 and “enzyme” in an immune reaction field. 7 is bound to the “secondary antibody as a ligand” 4, and a “quencher substrate” 8 is further added to produce a “quencher” 9. A “transparent flat substrate having a gold thin film formed on its surface” 5 as a detection field, and a “fluorescent dye layer” 6 formed on the other surface of the gold thin film that is not in contact with the substrate 5. The “plasma excitation sensor” having the “fluorescent dye layer” 6 is brought into contact with the surface, and laser light is irradiated from the other surface of the substrate 5 on which the gold thin film is not formed via a prism. (Not shown), the fluorescent dye contained in the “fluorescent dye layer” 6 is surface plasmo It is excited and emits light by a schematic view of the assay of the present invention. FIG. 2 shows steps (a) to (c): “analyte (target antigen) contained in specimen” 3 in “test tube” 12 which is an immune reaction field, and “particle” 1 having magnetism. The “primary antibody as a ligand” 2 immobilized on the surface of the enzyme and the “secondary antibody as a ligand” 4 labeled with the “enzyme” 7 bind to each other, and a quencher substrate (not shown) is further added. Addition produces “quencher” 9; step (d): “quencher” 9 is isolated by bringing “magnet” 11 close to the outside of “test tube” 12; steps (e) and (F): “Transparent flat substrate having a gold thin film formed on its surface” 5 as a detection field, and “Fluorescent dye layer” formed on the other surface of the gold thin film not in contact with the substrate 5 The “fluorescent dye layer” 6 side surface of the “plasmon excitation sensor” having The other surface where the gold thin film is not formed is irradiated with laser light via a prism (not shown), and the fluorescent dye contained in the “fluorescent dye layer” 6 is excited by the surface plasmon. FIG. 2 shows a schematic diagram of the assay method of the present invention, emitting light. FIG. 3 is a graph summarizing the blank signals and assay signals obtained in Examples 1 and 2 and Comparative Examples 1 and 2, respectively.

  Hereinafter, the present invention will be specifically described.

<Assay method>
The assay method of the present invention comprises the following steps (a) to (g), and preferably further comprises a washing step.

Step (a): a step of contacting a specimen with particles having a ligand immobilized on the surface thereof,
Step (b): reacting a particle obtained through the step (a) with a conjugate of a ligand and an enzyme, which may be the same as or different from the ligand,
Step (c): a step of further reacting a substrate with the particles obtained through the step (b) to produce a quencher,
Step (d): a step of isolating the quencher obtained through the step (c)
Step (e): a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a spacer layer made of a dielectric formed on the other surface of the metal thin film not in contact with the substrate And a plasmon excitation sensor having at least a fluorescent dye layer formed on the other surface of the spacer layer not in contact with the metal thin film, obtained on the thin film surface through the step (d). Contacting the quencher,
Step (f): The plasmon excitation sensor obtained in step (e) was excited by being irradiated with laser light from the other surface of the substrate on which the thin film was not formed via a prism. A step of measuring the amount of fluorescence emitted from the fluorescent dye, and a step (g): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).

  The assay method of the present invention is preferably carried out while maintaining a constant temperature.

[Step (a)]
The step (a) is a step of bringing the specimen having the ligand immobilized on the surface thereof into contact with the specimen.

(particle)
A “particle” is a water-insoluble carrier used in the present invention to detect an analyte contained in a specimen. The material that forms water-insoluble particles may be insoluble in water. The term “water-insoluble” specifically means a solid phase that does not dissolve in water or any other aqueous solution. The particles may be any known support or matrix that is currently widely used and proposed for applications such as fixation and separation.

  The material forming the water-insoluble particles includes an inorganic compound, a metal, a metal oxide, an organic compound, or a composite material combining these. If the ligand immobilized on the surface of the particle can bind the analyte contained in the specimen, the material, shape and size of the particle are not particularly limited, but preferably the amount of ligand immobilized is increased. From the viewpoint, it is a material that can provide a large surface area.

The material used as particles is not particularly limited, but generally synthetic organic polymers such as polystyrene, polypropylene, polyacrylate, polymethyl methacrylate, polyethylene, polyamide, latex; glass, silica, silicon dioxide, nitriding It may be an inorganic substance such as silicon, zirconium oxide, aluminum oxide, sodium oxide, calcium oxide, magnesium oxide, zinc oxide, iron oxide or chromium oxide; or a metal such as stainless steel or zirconia. These materials generally have a porous irregular surface, and may be, for example, fibers, webs, sintered bodies, porous bodies, and the like.

  Examples of the shape of the particles include a sphere shape, an ellipsoid shape, a cone shape, a cube shape, and a rectangular parallelepiped shape. Of these, spherical particles are preferable because they are easy to produce and easy to rotate and agitate the particles during use.

The particle size, that is, the average particle diameter is preferably 0.5 to 10 μm, and 2 to 6 μm.
m is more preferable. When the average particle size is less than 0.5 μm, when the particles are magnetic particles,
Sufficient magnetic responsiveness is not exhibited, it takes a considerably long time to separate the magnetic particles, and a very large magnetic force is required to separate them. On the other hand, when the average particle diameter exceeds 10 μm, the particles are likely to settle in the aqueous solution, and thus an operation of stirring the medium is required when contacting the specimen. Further, since the surface area of the particle body is small, it may be difficult to capture the analyte contained in the specimen.

  In addition to the aspect in which the entire particle including the surface is composed of the same material, it may be composed of a hybrid body composed of a plurality of materials as required. For example, in order to be able to cope with the automation of analysis, the core portion is made of a magnetically responsive material such as iron oxide or chromium oxide, and the surface thereof is coated with an organic synthetic polymer.

  As such a “particle”, the surface immobilization density of the following ligand can be easily adjusted, and from the viewpoint of easy B / F separation and further solid-liquid separation, magnetic particles are preferable, and the step (d) shown in FIG. The magnetic particles contain a magnetic material such as a paramagnetic material, a strong paramagnetic material, or a ferromagnetic material in that the magnetic particles can be easily (solid-liquid) separated and recovered by the magnetic force of the magnet. What is formed is preferable, and what contains a paramagnetic substance and / or a strong paramagnetic substance is more preferable. In particular, it is preferable to use a strong paramagnetic substance in that there is no or little residual magnetization.

Specific examples of such a “magnetic material” include triiron tetroxide (Fe ₃ O ₄ ), γ-heavy sesquioxide (γ-Fe ₂ O ₃ ), various ferrites, iron, manganese, cobalt, chromium, and the like. And various alloys such as cobalt, nickel, and manganese, and among these, triiron tetroxide is particularly preferable. Cobalt and nickel have an affinity for the histidine tag.

  The “magnetic material” used for the particles is a bead made of particles having a small particle diameter, has excellent magnetic separation (ie, ability to separate in a short time by magnetism), and is operated by a gentle up-and-down shaking operation. It is preferable that it can be redispersed.

  The content ratio of the magnetic substance in the magnetic particles is 70% by weight or less, preferably 20 to 70% by weight, more preferably 30 to 30% because the content ratio of the non-magnetic organic substance or the like is 30% by weight or more. 70% by weight is desirable. When such a content is less than 20% by weight, sufficient magnetic responsiveness is not exhibited, and it may be difficult to separate particles in a short time by a required magnetic force. On the other hand, when the content exceeds 70% by weight, the amount of the magnetic substance exposed on the surface of the particle main body increases, so that elution of constituent components of the magnetic substance, for example, iron ions occurs. The material may be adversely affected, and the particle body may become brittle and a practical strength may not be obtained.

Commercially available products can be used as such magnetic particles, for example, Dynabeads.
Series (manufactured by Dynal Biotech ASA) and the like.

(Ligand)
A “ligand” is a molecule or molecular fragment that can specifically recognize (or be recognized) and bind to an analyte contained in a specimen, and as such a “molecule” or “molecular fragment” For example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), or nucleosides, nucleotides and their modified molecules, which may be single-stranded or double-stranded), proteins ( Polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules and complexes thereof are not particularly limited.

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

  In the present invention, the term “antibody” includes polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments.

  As a method for immobilizing a ligand on the particle surface, an optimum crosslinking method can be selected in accordance with the particle surface terminal functional group. In addition, depending on the properties of the particle surface, a method of directly adsorbing directly to the surface can also be mentioned as an effective means.

(Sample)
Examples of the “specimen” include blood, serum, plasma, urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.) and the like, and appropriately diluted with a desired solvent, buffer solution, etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred. These may be used alone or in combination of two.

(contact)
As a condition for contacting the specimen with the ligand immobilized on the surface of the ligand, the temperature is usually 4 to 50 ° C., preferably 10 to 40 ° C., and the time is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

(Washing process)
The washing step is preferably included before and after the following step (b), and is a step of washing the surface of the particles obtained in the step (a) and the surface of the particles obtained in the following step (b). .

  As the washing solution used in the step, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer used in the reaction of steps (a) and (b), preferably 0.00001 to Those containing 1% by weight or those containing 150 to 500 mM of a salt such as sodium chloride or potassium chloride are desirable. Alternatively, it may be a low pH buffer solution such as 10 mM Glycine HCl having a pH of 1.5 to 4.0.

[Step (b)]
Step (b) refers to a ligand and an enzyme that may be the same as or different from the ligand used in step (a) on the particles obtained through step (a), preferably the washing step. And a conjugate reaction.

(Ligand)
The “ligand” used in step (b) may be the same as or different from the ligand used in step (a). However, when the “ligand” immobilized on the particle surface (step (a)) is a monoclonal antibody, the “ligand” used in step (a) is recognized by the “ligand” used in step (b). It is preferable to recognize parts other than the part to be performed.

(enzyme)
The “enzyme” is activated as a quencher by an enzyme reaction when the following “substrate” is a (A) quencher substrate blocked by a protecting group, or (B) in the case of a “substrate” other than (A), Used to lower the pH by enzymatic reaction.

  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 TG-βGal as a quencher substrate. In addition, β-glucosidase catalyzes a reaction for eliminating βGlu from TG-βGlu as a quencher substrate. Note that free TG has an excitation wavelength of 490 nm and causes fluorescence dye having a fluorescence wavelength of 475 nm to 495 nm and Fluorescence Resonance Energy Transfer (FRET; fluorescence resonance energy transfer), and thus fluorescence of terbium (Tb) chelate (fluorescence wavelength: 495 nm) or enhanced cyan fluorescent protein (ECFP) (fluorescence wavelength: 475 nm) can be quenched.

  Alkaline phosphatase catalyzes a reaction in which the substrate, AttoPhos® substrate, produces a strong fluorescent substance. The fluorescent substance BBT (2 ′-[2-benzthiazoyl] -6′-hydroxy-benzthiazole) having an excitation wavelength of 482 nm generated here is terbium (Tb) chelate or enhanced cyan fluorescent protein (ECFP) and FRET in the same manner as TG described above. Can be quenched.

  Examples of the “enzyme” used in the enzyme reaction (B) include glucose oxidase (hereinafter also referred to as “GOD”).

  GOD generates gluconolactone and hydrogen peroxide by an enzyme reaction using glucose as a substrate (see the following reaction formula). In addition, as the pH of the water decreases due to hydrogen peroxide dissolved in the water, the fluorescence intensity of 2-Me-4-OMe TG used as the fluorescent dye decreases.

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

(Conjugate of ligand and enzyme)
A “conjugate of a ligand and an enzyme” is a ligand labeled with an enzyme. As a method for labeling an enzyme with a ligand, for example, first, a carboxyl group of the enzyme is converted into a water-soluble carbodiimide (WSC) (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)). And N-hydroxysuccinimide (NHS), and then the esterification of the carboxyl group and the amino group of the ligand by dehydration reaction using water-soluble carbodiimide; isothiocyanate and amino group A method of reacting and immobilizing a ligand and an enzyme each having a sulfonyl group; a method of reacting and immobilizing a ligand and an enzyme each having a sulfonyl halide and an amino group; and reacting and immobilizing a ligand and an enzyme each having an iodoacetamide and a thiol group Law; and a method of immobilizing reacted biotinylated enzyme and streptoavidin of ligand and the like.

  The concentration of the “ligand labeled with an enzyme” thus prepared is preferably 0.001 to 10,000 μg / mL, more preferably 1 to 1,000 μg / mL.

  As reaction conditions, the temperature and time may be the same as those in the above step (a).

[Step (c)]
The step (c) is a step in which a quencher is produced by further reacting the substrate with the particles obtained through the step (b), preferably the washing step.

(Substrate)
As described above, the “substrate” includes, in the case of (A): a quencher substrate blocked by a protecting group, and in the case of (B): a “substrate” other than (A).

  Examples of the quencher substrate in (A) include TG-βGal, TG-βGlu, AttoPhos (registered trademark) substrate, and the like.

  In TG-βGal and TG-βGlu, one molecule of β-galactose and β-glucose are added as protective groups to the fluorescent dye TokyoGreen (TG), respectively. In this state, almost no fluorescence is observed. -Strong fluorescence is emitted when a protecting group is eliminated by galactosidase and β-glucosidase.

  In the present invention, TG includes 2-Me TG represented by the following formula (i), 2-Me-4-OMe TG represented by the following formula (ii), and the like.

  The AttoPhos® substrate emits weak fluorescence in a solution at pH 9.5, but emits strong fluorescence as a result of the enzymatic reaction with alkaline phosphatase.

  Examples of the “substrate” in (B) include glucose and oxygen which are substrates for glucose oxidase.

  In the present invention, preferable combinations of an enzyme, a substrate and the following “fluorescent dye” include those shown in Table 1.

  The concentration of such a quencher substrate during feeding is preferably 0.001 to 10,000 μg / mL, and more preferably 1 to 1,000 μg / mL.

(Quenching agent)
The “quencher” is produced by the reaction of the enzyme labeled with the above-mentioned ligand and the above “substrate”. In the case of (A), for example, TokyoGreen (TG), AttoPhos (registered trademark) In the case of (B): for example, hydrogen peroxide, etc. are mentioned.

  In the case of (A): TG or AttoPhos (registered trademark) substrate causes terbium (Tb) and FRET used as fluorescent dyes to quench the fluorescence of terbium (Tb) (fluorescence wavelength: 495 nm) In the case of (B): Hydrogen peroxide dissolved in water lowers the pH of the water, whereby 2-Me-4-OMe TG used as a fluorescent dye can be quenched.

[Step (d)]
Step (d) is a step of isolating the quencher obtained through the above step (c).

  As a method of isolating the quencher, for example, when the particle is a magnetic particle, a method of solid-liquid separation of the solution containing the quencher and the particle by magnetic force or centrifugal separation is exemplified, and the particle is sintered. In the case of a body, a porous body or a substrate, the solution and particles containing the quencher can be isolated without using the solid-liquid separation method.

[Step (e)]
The step (e) includes a “transparent flat substrate”, a “metal thin film” formed on one surface of the substrate, and the other surface of the metal thin film that is not in contact with the substrate. Surface of the thin film of the “plasmon excitation sensor” having at least a “spacer layer made of a dielectric” and a “fluorescent dye layer” formed on the other surface of the spacer layer not in contact with the metal thin film And a step of contacting the quencher obtained through the step (d).

  Such a “plasmon excitation sensor” is, for example, a sensor chip having a substrate and a gold thin film, such as a sensor chip used in a Biacore system manufactured by GE Healthcare Bioscience Co., Ltd. Also includes those in which a layer is formed.

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

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

  The transparent flat substrate is preferably cleaned with acid and / or plasma before forming a metal thin film on the surface.

  As the cleaning treatment with an acid, it is preferable to immerse in 0.001 to 1N hydrochloric acid for 1 to 3 hours.

  Examples of the plasma cleaning treatment include a method of immersing in a plasma dry cleaner (PDC200 manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.

(Metal thin film)
The metal thin film formed on one surface of the “transparent flat substrate” is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably gold. It is desirable to consist of these, and the alloy of these metals may be sufficient. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

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

  Examples of the method for forming a metal thin film on a transparent flat substrate include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and / or a metal thin film by sputtering or vapor deposition.

  The thickness of the metal thin film is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm. The thickness of the thin film is preferably 1 to 20 nm.

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

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

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

The thickness of the spacer layer is usually 10 nm to 1 mm, and is preferably 30 nm or less and more preferably 10 to 20 nm from the viewpoint of resonance angle stability. 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 1,60.
0 nm is preferable. When the plasmon excitation sensor of the present invention is mass-produced in the future, it is assumed that the thickness of the spacer layer included in the sensor will fluctuate. Since there is a possibility, the thickness of the spacer layer is particularly preferably 10 to 20 nm for the purpose of ensuring measurement stability.

  Examples of the method for forming the spacer layer include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or a coating method using a spin coater.

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

  In the case of (I), the fluorescent dye and the polymer may or may not be chemically bonded, and (I ′) a silane coupling agent having a polymerizable group is bonded to the spacer layer. A composition containing a fluorescent dye and a polymer can also be formed by adding and copolymerizing another polymerizable monomer, a fluorescent dye and a polymerization initiator.

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

  Thus, in the case of (I) which forms a layer comprising a fluorescent dye and a polymer, the amount of the fluorescent dye that can be immobilized is large, and the strength of the resulting layer is high, which is preferable.

  The “fluorescent dye” is a general term for substances that emit fluorescence by irradiating predetermined excitation light in the present invention, or excited by using an electric field effect. Including luminescence.

  Examples of the “fluorescent dye” used in the present invention include terbium (Tb) chelate (fluorescence wavelength: 490 nm), ECFP protein (fluorescence wavelength: 475 nm), 2-Me-4-OMe TG represented by the following formula, 2-OMe-5-Me TG, 2-OMe TG, and the like.

  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 an amino group or an alcohol contained in the aromatic ring to form an unnecessary structure in water, or to chemically bond it by a reaction between a hydrophobic polymer and an active ester of a fluorescent dye. When the polymer and the fluorescent dye do not have a chemical bond, it is preferable to modify the fluorescent dye so as to have a structure close to the solubility parameter of the polymer.

  These fluorescent dyes may be used alone or in combination of two or more.

Examples of the “polymer” include polyacrylate, polymethacrylate, polystyrene-acrylate, polystyrene, polyvinyl butyral, polyester, and the like. Of these, polyacrylates and polymethacrylates, polystyrene, and polyvinyl butyral have excellent compatibility with fluorescent dyes and nonspecific adsorption (eg, proteins (albumin, fibrinogen, immunoglobulin), lipids, saccharides (glucose)) Can be suppressed, which is preferable.

  In addition to the fluorescent dye and polymer, the “composition” can also contain a solvent and, if necessary, additives such as an antioxidant.

  The “solvent” is not particularly limited as long as it has high volatility. For example, halogen-containing hydrocarbons (eg, dichloromethane, dichloroethane, tetrafluoropropane), alcohols (eg, methanol, ethanol, propanol, butanol, Tertiary butanol, tetrafluoropropanol), aromatics (eg, toluene, xylene, etc.), ethers (eg, diethyl ether, diethylene glycol monomethyl ether, etc.), esters (eg, ethyl acetate, butyl acetate, etc.), glycols (For example, ethylene glycol etc.), ketones (acetone, methyl ethyl ketone, etc.) etc. are mentioned. Of these, aromatics, halogen-containing hydrocarbons, esters, and ketones are preferable from the viewpoint of the dissolution stability of the polymer used.

Examples of the “antioxidant” include pentaerythrityl tetrakis [3- (3,5-di-t-butyl-4-hydroxyphenyl)] propionate, 2,6-di-t-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 and the like.

  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.

The solvent is preferably 100 to 1,000 parts by weight with respect to 100 parts by weight of the composition,
100-500 weight part is more preferable. 0.1-10 weight part is preferable with respect to 100 weight part of compositions, and 1-5 weight part is more preferable. It is preferable that the solvent or additive has the above blending amount because the coating property is good and the fluorescence quantum yield is not lowered.

  The method of “coating” is not particularly limited, but for example, after applying by spin coating method, wire coating method, bar coating method, roll coating method, blade coating method, curtain coating method, screen printing method, etc. Dry at ~ 100 ° C for 5-30 minutes.

(contact)
Examples of the method of bringing the quencher obtained through the step (d) into contact with the spacer layer side surface of such a “plasmon excitation sensor” include dropping, spraying, and coating of a solution containing the quencher. A method is mentioned. Further, there is a method in which the following “flow path” is formed on the plasmon excitation sensor, and the solution containing the quencher is brought into contact with the surface of the plasmon excitation sensor.

  The “flow channel” is a rectangular parallelepiped or a tube that can efficiently deliver a small amount of a chemical solution and can change the liquid feeding speed or circulate in order to promote the reaction. The vicinity of the place where the plasmon excitation sensor is installed preferably has a rectangular parallelepiped structure, and the vicinity of the place where the drug solution is delivered preferably has a tubular shape.

  The material includes a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material in the plasmon excitation sensor unit; a polyolefin such as polyethylene, and a silicon rubber, Teflon (registered trademark), polyethylene, A polymer such as polypropylene is used.

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

  As a method of fixing the plasmon excitation sensor to the flow path, in a small-scale lot (laboratory level), first, on the surface on which the metal thin film of the plasmon excitation sensor is formed, A dimethylsiloxane (PDMS) sheet is pressure-bonded so as to surround a portion where the metal thin film of the plasmon excitation sensor is formed, and then the polydimethylsiloxane (PDMS) sheet and the plasmon excitation sensor are closed with screws or the like. A method of fixing with a tool is preferred.

  In large lots (factory level) manufactured industrially, as a method of fixing the plasmon excitation sensor to the flow path, a gold substrate is formed on a plastic integrally molded product, or a separately manufactured gold substrate is fixed, and the gold surface is fixed. Further, after the dielectric layer, the fluorescent dye layer, and the ligand are immobilized, it can be manufactured by covering with a plastic integrally formed product corresponding to the top plate of the flow path. If necessary, the prism can be integrated into the flow path.

  The “liquid feeding” is preferably the same as the solvent or buffer in which the specimen is diluted. For example, phosphate buffered saline (PBS), Tris buffered saline (TBS), Hepes buffered saline (HBS) However, it is not particularly limited.

  The temperature and time for circulating the liquid supply vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. × 1 to 60 minutes, preferably 37 ° C. × 5 to 15 minutes.

  The initial concentration of the analyte contained in the specimen being sent may be 100 μg / mL to 0.001 pg / mL.

  The total amount of liquid feeding, that is, the volume of the flow path is usually 0.001 to 20 mL, preferably 0.1 to 1 mL.

The flow rate of liquid feeding is usually 1 to 2,000 μL / min, preferably 5 to 500 μL / min.
It is.

[Step (f)]
The step (f) means that the plasmon excitation sensor obtained in the step (e) is passed through a prism from the other surface of the “transparent flat substrate” where 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 with laser light.

  It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through an optical filter and a polarizing filter.

  Irradiation with laser light generates surface plasmons on the surface of the metal thin film under the total reflection attenuation condition (ATR). Due to the electric field enhancement effect of the surface plasmon, the fluorescent dye is excited by photons increased to several tens to several hundred times the amount of photons irradiated. In addition, although the photon increase amount by 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, the increase amount is usually about 40 to 100 times for silver.

  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.

  As the “laser light”, an LD laser having a wavelength of 200 to 900 nm and 0.001 to 1,000 mW, or a semiconductor laser having a wavelength of 230 to 800 nm and 0.01 to 100 mW is preferable.

  The “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon excitation sensor, and the refractive index is preferably the same as that of the “transparent flat substrate”. In the present invention, various prisms for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape. For example, a 60-degree dispersion prism may be used. Examples of such commercially available prisms include those similar to the above-mentioned commercially available “glass-made transparent flat substrate”.

  Examples of the “optical filter” include a neutral density (ND) filter and a diaphragm lens.

The “darkening (ND) filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.

  The “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons.

  “Cut filters” are external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattering light (excitation light originated from plasmon A filter that removes various types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor), autofluorescence of the enzyme fluorescent substrate, and examples thereof include interference filters and color filters. It is done.

  The “collecting lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system. As a simple condensing system, a commercially available objective lens (manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be diverted. The magnification of the objective lens is preferably 10 to 100 times.

  As the “SPFS detection unit”, a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics Co., Ltd.) is preferable from the viewpoint of ultrahigh sensitivity. Also, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed.

[Step (g)]
The step (g) is a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).

  More specifically, a calibration curve is created by performing measurement with a target antigen or target antibody at a known concentration, and the target antigen amount or target antibody amount in the sample to be measured is measured based on the created calibration curve. This is a step of calculating from the signal.

(Analyte)
“Analyte” is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to a ligand immobilized on the “fluorescent dye layer”, wherein such “molecule” or “ “Molecular fragment” includes, for example, nucleic acid (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., which may be single-stranded or double-stranded, or nucleoside, nucleotide and modifications thereof. Molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules or complexes thereof. Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, etc. It is not.

(Assay signal change)
Furthermore, in the step (g), when the signal measured before the step (d) is “blank signal”, the amount of assay signal change represented by the following formula can be calculated.

Signal change amount = | (assay fluorescence signal) − (blank fluorescence signal) |
<Device>
The apparatus of the present invention includes at least a plasmon excitation sensor obtained through the above step (e), a laser light source, an optical filter, a prism, a cut filter, a condensing lens, and a surface plasmon excitation enhanced fluorescence detection unit, It is used in the step (f).

  That is, the apparatus of the present invention is for carrying out the assay method of the present invention using the plasmon excitation sensor.

  The “apparatus” includes at least a light source, various optical filters, a prism, a cut filter, a condenser lens, and an SPFS detection unit. It is preferable to have a liquid feeding system combined with a plasmon excitation sensor when handling a sample liquid, a washing liquid, a labeled antibody liquid, or the like. As the liquid feeding system, for example, a microchannel device connected to a liquid feeding pump may be used.

  In addition, a surface plasmon resonance (SPR) detection unit, that is, a photodiode as a light receiving sensor dedicated to SPR, an angle variable unit for adjusting the optimum angle of SPR and SPFS (to determine total reflection attenuation (ATR) conditions with a servomotor) The angle of 45 to 85 ° can be changed by synchronizing the photodiode and the light source with a resolution of preferably 0.01 ° or more.), A computer for processing information input to the SPFS detector May also be included.

  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 that is suitable when the amount of the liquid is small, a syringe pump that is high in feed accuracy and less pulsating, but is not circulated, and simple and excellent in handleability, For example, a tube pump may be difficult.

<Kit>
The kit of the present invention includes at least a sensor including the transparent flat substrate, the metal thin film, the spacer layer made of the dielectric, and the fluorescent dye layer, the enzyme and the substrate, and is used for the assay method of the present invention. In conducting the assay method of the present invention, it is preferable to include everything necessary other than a primary antibody, a ligand such as an antigen, a specimen, and a secondary antibody.

  For example, the content of a specific tumor marker can be detected with high sensitivity and high accuracy by using the kit of the present invention, blood, plasma or serum as a specimen, and an antibody against the specific tumor marker. 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 plasmon excitation sensor in which a metal thin film is formed on one surface of a transparent flat substrate; a solution or dilution liquid for dissolving or diluting the specimen; a plasmon excitation sensor and a specimen; These include various reaction reagents and washing reagents for reacting, and can also include various equipment or materials required for carrying out the assay method of the present invention and the above-mentioned “device”.

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

  Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.

[Preparation Example 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 Nippon Medical Laboratory, Inc.).

  Specifically, the carboxyl group of the enzyme and the amino group of the antibody were immobilized by an amino coupling method.

[Preparation Example 2] (Preparation of conjugate of secondary antibody and glucose oxidase)
Glucose oxidase was immobilized on an anti-α fetoprotein (AFP) monoclonal antibody (6D2, 2.5 mg / mL, manufactured by Nippon Medical Laboratory, Inc.) in the same manner as in Preparation Example 1.

[Preparation Example 3] (Preparation of secondary antibody labeled with alkaline phosphatase)
An anti-α-fetoprotein (AFP) monoclonal antibody (available from Nippon Medical Laboratory, Inc.) as a secondary antibody was biotinylated using a biotinylation kit (manufactured by Dojindo Laboratories). The procedure followed the protocol attached to the kit.

  First, the obtained biotinylated anti-AFP monoclonal antibody solution and a streptavidin-labeled alkaline phosphatase (ALP) solution (Phosphatase-labeled streptavidin (manufactured by KPL)) are mixed and stirred at 4 ° C. for 60 minutes. It was made to react.

  Next, the unreacted antibody and the unreacted enzyme were purified using a molecular weight cut filter (manufactured by Nippon Millipore) to obtain an alkaline phosphatase-labeled anti-AFP monoclonal antibody solution. The obtained antibody solution was stored at 4 ° C. after protein quantification.

[Preparation Example 4] (Preparation of Alexa Fluor (registered trademark) 647-labeled secondary antibody)
First, a biotinylated anti-AFP monoclonal antibody solution and a streptavidin-labeled Alexa Fluor (registered trademark) 647 (manufactured by 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 1]
(Production of plasma excitation sensor)
A glass transparent flat substrate (BK7 manufactured by SCHOTT AG) having a refractive index [nd] of 1.52, a thickness of 1 mm and an outer shape of 20 mm × 20 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by a sputtering method. After that, a 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 that was 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 parts by weight of BL-S (polyvinyl butyral) manufactured by Sekisui Chemical Co., Ltd. as a polymer, with respect to one side of the spacer layer not in contact with the silver thin film, And the composition containing 25 weight part of methyl ethyl ketone as a solvent was apply | coated by the spin coater method, it was made to dry at 50 degreeC for 10 minutes in the dark place, and the solvent was volatilized. The thickness of the obtained fluorescent dye layer was 10 nm.

(Implementation of assay method)
As step (a), first, anti-α-fetoprotein (AFP) monoclonal antibody (obtained from Nippon Medical Laboratory) is immobilized on Dynabeads (manufactured by Dynal Biotech ASA) as magnetic particles. Turned into. The immobilization method was in accordance with the protocol attached to Dynabeads.

  A specimen containing AFP (prepared in a 1 ng / mL TBS solution) as a target antigen is brought into contact with 100 μL of magnetic particles (prepared in a 0.015 wt% TBS solution) on which the anti-AFP monoclonal antibody is immobilized. The reaction was allowed for 10 minutes.

As the washing step, the particles obtained through the step (a) were collected with a magnet for solid-liquid separation, and only the liquid of the reaction solution after the step (a) was discarded. To the remaining particles, 300 μL of TBS containing 0.05% by weight of Tween 20 was dispensed, stirred for 1 minute, and collected by a magnet. Such a washing process was repeated three times.
As the step (b), the particles obtained through the washing step were added to the β-galactosidase-labeled anti-AFP monoclonal antibody (TBS prepared to 1,000 ng / mL) obtained in Preparation Example 1.
200 μL of (solution) was added and allowed to react for 10 minutes.

As the washing step, the particles obtained through the step (b) were collected with a magnet for solid-liquid separation, and only the liquid of the reaction solution after the step (b) was discarded. To the remaining particles, 300 μL of TBS containing 0.05% by weight of Tween 20 was dispensed and stirred for 1 minute, and then the particles were collected by a magnet. Such a washing process was repeated three times.

  In step (c), 100 μL of enzyme quenching substrate solution (TG-bGal) adjusted with TBS was dispensed to the particles obtained through the washing step, and the mixture was reacted for 5 minutes after stirring.

  As the step (d), the reaction solution obtained through the above step (c) was subjected to solid-liquid separation by collecting particles with a magnet and isolated as a fluorescent dye quenching solution.

As the step (e), the fluorescent dye quenching solution obtained through the step (d) was brought into contact with the surface of the plasma excitation sensor obtained in the above (production of the plasma excitation sensor).

  As the step (f), the prism (Sigma Kogyo Co., Ltd. product) is applied to the plasmon excitation sensor obtained in the step (e) from the other surface of the glass transparent flat substrate where the silver thin film is not formed. ) Was irradiated with laser light (340 nm, 40 μW), and the signal value when the amount of fluorescence emitted from the excited fluorescent dye was observed from the CCD was measured and used as an “assay signal”.

The SPFS measurement signal when AFP was 0 ng / mL was defined as “blank signal”.
As a process (g), the assay signal change amount was evaluated by the following formula from the measurement result obtained in the above process (f).

Signal change amount = | (assay fluorescence signal) − (blank fluorescence signal) |
The obtained results are shown in Table 2 and FIG.

[Example 2]
(Production of plasma excitation sensor)
The same procedure as in Example 1 was performed except that 2-Me-4-OMe TG was used as the fluorescent dye instead of terbium chelate.

(Implementation of assay method)
The same procedure as in Example 1 was performed, except that glucose and oxygen were used as the secondary antibody obtained in Preparation Example 2, the enzyme quenching substrate solution, and laser light having an excitation wavelength of 490 nm was used.

  The obtained results are shown in Table 2 and FIG.

[Comparative Example 1]
(Production of plasma excitation sensor)
A glass transparent flat substrate (S-LAL 10 manufactured by OHARA INC.) Having a refractive index [nd] 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. A gold thin film was further formed on the surface by sputtering. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

  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 arranged 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)
As step (a), first, anti-α-fetoprotein (AFP) monoclonal antibody (obtained from Nippon Medical Laboratory) is immobilized on Dynabeads (manufactured by Dynal Biotech ASA) as magnetic particles. Turned into. The immobilization method was in accordance with the protocol attached to Dynabeads.

  A specimen containing AFP (prepared in a 1 ng / mL TBS solution) as a target antigen is brought into contact with 100 μL of magnetic particles (prepared in a 0.015 wt% TBS solution) on which the anti-AFP monoclonal antibody is immobilized. The reaction was allowed for 10 minutes.

As the washing step, the particles obtained through the step (a) were collected with a magnet for solid-liquid separation, and only the liquid of the reaction solution after the step (a) was discarded. To the remaining particles, 300 μL of TBS containing 0.05% by weight of Tween 20 was dispensed, stirred for 1 minute, and collected by a magnet. Such a washing process was repeated three times.

In step (b), 200 μL of the alkaline phosphatase-labeled anti-AFP monoclonal antibody (TBS solution prepared at 1,000 ng / mL) obtained in Preparation Example 3 was added to the particles obtained through the washing step. The reaction was allowed for 10 minutes.

As the washing step, the particles obtained through the step (b) were collected with a magnet for solid-liquid separation, and only the liquid of the reaction solution after the step (b) was discarded. To the remaining particles, 300 μL of TBS containing 0.05% by weight of Tween 20 was dispensed and stirred for 1 minute, and then the particles were collected by a magnet. Such a washing process was repeated three times.

  As the step (c), the particle obtained through the washing step is added to an enzyme fluorescent substrate solution (1,3-diculo-9,9-dimethyl-acid-2-one-7-yl phosphate; DDAO prepared by TBS. 100 μL of phosphate (manufactured by Molecular Probes) was dispensed and allowed to react for 5 minutes after stirring.

  As the step (d), the reaction solution obtained through the step (c) was subjected to solid-liquid separation by collecting particles with a magnet and isolated as a fluorescent dye solution.

  As the step (e), the fluorescent dye solution obtained through the step (d) was brought into contact with the surface of the plasma excitation sensor obtained in Production Example 1 by liquid feeding.

  As the step (f), the prism (Sigma Kogyo Co., Ltd. product) is applied to the plasmon excitation sensor obtained in the step (e) from the other surface of the glass transparent flat substrate on which the gold thin film is not formed. ) Was irradiated with laser light (640 nm, 40 μW), and the signal value when the amount of fluorescence emitted from the excited fluorescent dye was observed from the CCD was measured and used as an “assay signal”.

The SPFS measurement signal when AFP was 0 ng / mL was defined as “blank signal”.
As the step (g), the signal change amount was calculated from the measurement result obtained in the above step (f) by the following formula.

Signal change amount = | (assay fluorescence signal) − (blank signal) |
The obtained results are shown in Table 2 and FIG.

[Comparative Example 2]
(Production of plasma excitation sensor)
It was produced in the same manner as Comparative Example 1.

(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. By circulating 2.5 mL of antibody (1D5, 2.5 mg / mL, manufactured by Nippon Medical Laboratory) for 30 minutes, SAM
The primary antibody was immobilized on the top. In addition, the nonspecific adsorption | suction prevention process was performed by circulating 30 minutes by PBS buffer physiological saline containing 1weight% 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 the secondary antibody (PBS solution prepared to be 1,000 ng / mL) labeled with Alexa Fluor (registered trademark) 647 prepared in Preparation Example 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 1. FIG.

  The obtained results are shown in Table 2 and FIG.

Since the fluorescent dye layer is formed on the substrate on the plasmon excitation sensor of the present invention, an extremely high fluorescence signal is obtained in the blank state, which is an order of magnitude higher than that of the conventional fluorescence-labeled SPFS measurement in Comparative Example 2. It was found that measurement with extremely high sensitivity is possible. Furthermore, compared with the case of the fluorescent dye enzyme amplification system of Comparative Example 1, it was found that a high signal change amount can be achieved in the region of a high fluorescent signal.

  Since the assay method of the present invention can be detected with high sensitivity and high accuracy, for example, even a very small amount of tumor marker contained in blood can be detected. The presence of pre-clinical non-invasive cancer (carcinoma in situ) that cannot be detected by, for example, can be predicted with high accuracy.

DESCRIPTION OF SYMBOLS 1 ... Particle 2 ... Primary antibody as a ligand 3 ... Analyte (target antigen) contained in specimen
4 ... Secondary antibody as ligand 5 ... Transparent flat substrate with gold thin film formed on its surface 6 ... Fluorescent dye layer 7 ... Enzyme 8 ... Quencher substrate 9 ... Quenching Agent 10 ... Fluorescence 11 excited by surface plasmon ... Magnet 12 ... Test tube

Claims (12)

An assay method comprising the following steps (a) to (g):
Step (a): a step of contacting a specimen with particles having a ligand immobilized on the surface thereof,
Step (b): reacting a particle obtained through the step (a) with a conjugate of a ligand and an enzyme, which may be the same as or different from the ligand,
Step (c): a step of further reacting a substrate with the particles obtained through the step (b) to produce a quencher,
Step (d): a step of isolating the quencher obtained through the step (c)
Step (e): a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a spacer layer made of a dielectric formed on the other surface of the metal thin film not in contact with the substrate And a plasmon excitation sensor having at least a fluorescent dye layer formed on the other surface of the spacer layer not in contact with the metal thin film, obtained on the surface of the fluorescent dye layer through the step (d). Contacting with a quenched quencher,
Step (f): The plasmon excitation sensor obtained in step (e) was excited by being irradiated with laser light from the other surface of the substrate on which the thin film was not formed via a prism. A step of measuring the amount of fluorescence emitted from the fluorescent dye, and a step (g): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).   The assay method according to claim 1, wherein the immunoreaction field in steps (a) to (d) and the detection field in steps (e) to (g) are independent of each other.   The assay method according to claim 1 or 2, wherein the ligand described in step (a) is a primary antibody that recognizes and binds to a tumor marker or carcinoembryonic antigen.   The assay method according to any one of claims 1 to 3, 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 1, wherein the enzyme is β-galactosidase, β-glucosidase, alkaline phosphatase, or glucose oxidase.   The assay method according to any one of claims 1 to 5, 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 assay method according to claim 6, wherein the metal comprises silver. The assay method according to claim 1, wherein the dielectric contains silicon dioxide (SiO ₄ ) or titanium dioxide (TiO ₂ ).   The said fluorescent pigment | dye layer is formed by applying the composition containing a fluorescent pigment | dye and a polymer to the other surface which is not in contact with the said metal thin film of the said spacer layer. The assay method according to any one of the above.   The said fluorescent pigment layer is formed by couple | bonding through the silane coupling agent with the other surface which is not in contact with the said metal thin film of the said spacer layer. Assay method. The process according to claim 1, comprising at least a plasmon excitation sensor obtained through the step (e), a laser light source, an optical filter, a prism, a cut filter, a condensing lens, and a surface plasmon excitation enhanced fluorescence detection unit. A device used in (f).   The sensor comprising at least a transparent flat substrate, the metal thin film, the spacer layer made of the dielectric, and the fluorescent dye layer, the enzyme and the substrate, and used in the assay method according to claim 1. Kit characterized by being able to.