JP5652393B2 - Plasmon excitation sensor and assay method used for measurement method by SPFS-LPFS system - Google Patents

Description

  The present invention relates to a plasmon excitation sensor and an assay method used for a measurement method using an SPFS-LPFS system. More specifically, the present invention relates to a plasmon excitation sensor that is ultrasensitive by adding an electric field enhancement effect and an electric field enhancement area expansion effect by a metal colloid to the electric field enhancement effect by a metal thin film, and an assay method using the sensor About.

  SPR (Surface Plasmon Resonance) is a condition in which the irradiated laser light undergoes total reflection attenuation [ATR] on the surface of the gold thin film [ATR]. (Or refractive index) is a phenomenon in which when the wave number of the evanescent wave, which is easily affected by the difference in refractive index, coincides, the reflected light attenuates, and the interaction between the ligand and the analyte on the sensor surface A difference occurs in the dielectric constant (or refractive index) of the dielectric, and as a result, the surface plasmon resonance is changed, whereby the interaction between the ligand and the analyte can be quantified.

  As a biosensor using such SPR, for example, Patent Document 1 discloses a sensor unit used for SPR (surface plasmon resonance) analysis. As shown in FIG. A transparent plate 11 made of glass, plastic or other transparent material, a metal film 12 formed by sputtering or the like on one side of the plate, and a dielectric film attached to the metal film. This dielectric film is a dextran layer 14 to which a ligand is bound, the ligand having a bifunctional or multi-functional group having one active moiety consisting of anti-dextran and another part consisting of an antigen against the antibody to be detected. It is described that it may be a functional molecule 15.

  However, since such a sensor unit is based on a change in the average refractive index of the sensor surface, the response to small molecules is weak and the sensitivity is low, so there is room for improvement.

  Non-Patent Document 1 discloses an SPR sensor chip used for SPR analysis. As shown in FIG. 3, the SPR sensor chip includes a cover glass 6 and sputtering on one surface of the cover glass 6. The gold film 7 is formed, a gel 8 made of a molecular imprint polymer obtained by polymerization on the surface of the gold film 7, and gold nanoparticles 10 embedded in the gel 8. It is described that the gel 8 made of a molecularly imprinted polymer has a binding site 9 that recognizes a low molecule, and that it can be detected by SPR measurement even in a measurement targeting a relatively low molecule. ing.

  However, even in such a sensor unit, there is room for improvement in terms of sensitivity since it is based on the average refractive index change on the sensor surface.

Japanese Patent No. 3294605

Jun Matsui, Kensuke Akamatsu, Noriaki Hara, Daisuke Miyoshi, Hidemi Nawafune, Katsuyuki Tamaki, Naoki Sugimoto; SPR sensor chip for detection of small molecules using molecularly imprinted polymer with embedded gold nanoparticles, Anal. Chem., 2005, 77, p. .4282-4285

  An object of the present invention is to provide a highly sensitive and accurate plasmon excitation sensor capable of improving the fluorescence signal by SPFS, an assay method using the sensor, an analyzer for SPFS, and the assay kit.

  Note that SPFS (surface plasmon excitation enhanced fluorescence spectroscopy) means that a dense wave (surface plasmon) is applied to the surface of a metal thin film in contact with a dielectric under the condition that the irradiated laser light attenuates total reflection [ATR] on the gold thin film surface. By increasing the photon amount of the irradiated laser light by several tens to several hundreds (the surface plasmon electric field enhancement effect), the fluorescent dye in the vicinity of the gold thin film is efficiently excited, thereby making a trace amount. And / or a fluorometric method capable of detecting very low concentrations of analyte.

  As a result of diligent research to solve the above problems, the inventors of the present invention have found that in an assay system using SPFS, when a metal colloid is immobilized on a metal substrate in a state close to a dispersion and floating state and fluorescence measurement is performed, surface plasmon is obtained. In addition to the electric field enhancement effect by excitation, the local electric field enhancement effect by the metal colloid is also obtained at the same time, and it has been found that the intensity of the fluorescent signal emitted from the fluorescent dye is increased, and the present invention has been completed.

That is, the plasmon excitation sensor of the present invention includes a transparent flat substrate; a metal thin film formed on one surface of the substrate; and 2 formed on the other surface of the thin film that is not in contact with the substrate. SPFS [surface plasmon excitation enhanced fluorescence spectroscopy] -LPFS [localized surface plasmon excitation enhancement] comprising a layer composed of a polymer on which one or more metal colloids are immobilized; and an unreacted ligand immobilized on the polymer It is characterized in that it is subjected to a novel measurement method using a fluorescence spectroscopy system.

The thickness of the polymer layer of the plasmon excitation sensor of the present invention is preferably 10 nm to 1000 nm.
The density of the polymer in the plasmon excitation sensor of the present invention is preferably 10 ³ pieces / mm ² to 10 ¹⁵ pieces / mm ² .
Further, plasmon excitation sensor of the present invention preferably has a SAM [self-assembled monolayer] between the polymer layer which is fixed the metal thin film and the metal colloid.

  The metal colloid is preferably a colloid composed of at least one metal selected from the group consisting of gold, silver, platinum and alloys thereof, and particularly preferably a gold colloid.

  The particle size of the metal colloid is preferably 0.1 nm to 10 μm.

  The polymer is at least one polymer selected from the group consisting of proteins, nucleic acids, polysaccharides, natural rubber, synthetic resins, silicone resins, synthetic fibers, and synthetic rubbers, and is soluble in water. preferable.

  Moreover, the assay method of the present invention is characterized by including at least the following steps (a) to (d).

Step (a): a step of bringing a specimen into contact with the plasmon excitation sensor of the present invention,
Step (b): a step of reacting the plasmon excitation sensor obtained through the step (a) with a conjugate of a second ligand and a fluorescent dye,
Step (c): Laser light is irradiated via a prism from the surface opposite to the surface on which the metal thin film is formed on the transparent flat substrate of the plasmon excitation sensor obtained through the step (b). A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (d): calculating the amount of analyte contained in the sample from the measurement result obtained in step (c) Process.

  The analyzer for SPFS of the present invention includes at least the plasmon excitation sensor of the present invention, and is used for the assay method of the present invention.

The kit of the present invention comprises a transparent flat substrate; a metal thin film formed on one surface of the substrate; two or more formed on the other surface of the thin film that is not in contact with the substrate colloidal metal consists of a layer consisting of a fixed polymer and the plasmon excitation board sensor used for plasmon excitation sensor of the present invention, characterized in that it comprises at least.

  The present invention has an electric field enhancement effect and an electric field enhancement area expansion effect by the SPFS [surface plasmon excitation enhanced fluorescence spectroscopy] -LPFS [localized surface plasmon excitation enhanced fluorescence spectroscopy] system, that is, surface plasmon excitation and localized plasmon. By simultaneously inducing the electric field enhancement due to excitation, it is possible to further amplify the fluorescence signal, and in the SPFS system and the SPFS-LPFS system, the electric field enhancement area that is usually limited to the sensor electrode surface is three-dimensional. Therefore, it is possible to provide a plasmon excitation sensor having an effect of improving the stability of a fluorescent signal and used for an extremely sensitive and highly accurate assay method.

  In the present invention, as a factor that the fluorescence signal is dramatically amplified, (i) the electric field enhancement accompanying the resonance between the surface plasmon light and the localized surface plasmon light, and (ii) the obtained electric field enhancement is efficiently amplified. (Iii) expansion of the assay area in the height direction by arranging the metal colloids at appropriate intervals and adjusting the distance from the metal substrate.

FIG. 1 is a cross-sectional view schematically showing one embodiment of the plasmon excitation sensor of the present invention. FIG. 2 is a cross-sectional view schematically showing one aspect of the sensor unit described in Patent Document 1. As shown in FIG. FIG. 3 is a cross-sectional view schematically showing the SPR sensor chip described in Non-Patent Document 1.

  Hereinafter, the plasmon excitation sensor of the present invention will be specifically described.

<Plasmon excitation sensor>
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 metal colloid formed on the other surface of the thin film that is not in contact with the substrate. SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) -LPFS (Localized Surface-Pladson Plasma), comprising a polymer layer as a base point; and a ligand immobilized on the polymer. Fluorescence Spectroscopy (localized surface plasmon excitation enhanced fluorescence spectroscopy) system.

  In the plasmon excitation sensor of the present invention, a SAM (Self-Assembled Monolayer) is formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate, and the metal colloid is used as a base point. It is preferable to immobilize the polymer to be immobilized on the SAM.

  In addition, a spacer layer made of a dielectric may be appropriately formed for the purpose of preventing metal quenching of the fluorescent dye by the metal thin film. This spacer layer is preferably formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate.

(Transparent flat substrate)
In the present invention, a transparent flat substrate is used as a flat substrate that supports the structure of the plasmon excitation sensor. In the present invention, the transparent flat substrate is used as the flat substrate because light irradiation to a metal thin film described later is performed through the flat substrate.

  As for the transparent flat substrate used in the present invention, the material is not particularly limited as long as the object of the present invention is achieved. For example, the transparent flat substrate may be made of glass, or may be made of plastic such as polycarbonate [PC] or cycloolefin polymer [COP].

The refractive index at the d-line (588 nm) [n _d] is preferably from 1.40 to 2.20, is preferably thick 0.01 to 10 mm, more preferably if 0.5 to 5 mm, The size (vertical x horizontal) is not particularly limited.

Glass transparent flat substrates are commercially available products such as “BK7” (refractive index [n _d ] 1.52) and “LaSFN9” (refractive index [n _d ] 1.85) manufactured by Shot Japan Co., Ltd. “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 preferred from the viewpoint 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. In addition, this electric field serves as a group that enhances the local electric field between the metal thin film and the metal colloid, and also serves as a base for the local electric field between the metal colloid and the metal colloid, and from the fluorescent dye in the enhanced electric field. Further enhances luminescence.

  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 more preferably made of gold. preferable. These metals may be in the form of their alloys. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

  When a glass flat substrate is used as the transparent flat substrate, it is preferable to form a chromium, nickel chromium alloy or titanium thin film in advance in order to bond the glass and the metal thin film more firmly.

  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)
As the dielectric used for forming the spacer layer made of a dielectric, various optically transparent inorganic materials, natural or synthetic polymers can be used. Among them, it is preferable to contain silicon dioxide [SiO ₂ ] or titanium dioxide [TiO ₂ ] because it is excellent in chemical stability, production stability and optical transparency.

  The thickness of the spacer layer made of a dielectric is usually 10 nm to 1 mm, and preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. On the other hand, it is preferably 200 nm to 1 mm from the viewpoint of electric field enhancement, and more preferably 400 nm to 1,600 nm from the stability of the effect of electric field enhancement. In the assay method of the present invention, the thickness of the spacer layer is desirably 10 to 100 nm for the purpose of more effectively enhancing the electric field generated between the plasmon excitation sensor and the metal colloid during measurement.

  Examples of the method for forming a spacer layer made of a dielectric 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.

(SAM)
SAM (Self-Assembled Monolayer) is preferably formed on a surface of a metal thin film that is not in contact with a transparent flat substrate. In the plasmon excitation sensor of the present invention, an analyte and a fluorescent dye are used. Fluorescence is measured in a state of being captured by a metal thin film via a ligand. At this time, a polymer based on the metal colloid is fixed to the metal thin film via SAM. That is, the SAM has a role as a base when fixing a polymer based on a metal colloid to a metal thin film.

  In addition, when the “spacer layer made of a dielectric” is formed on the surface of the metal thin film that is not in contact with the transparent flat substrate, the SAM is preferably formed on the surface of the spacer layer that is not in contact with the metal thin film.

  When the SAM is directly formed on the surface of the metal thin film, the SAM contains usually a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.) Aminoalkanethiol, particularly preferably 10-carboxy-1-decanethiol, 10-amino-1-decanethiol (hereinafter also simply referred to as “carboxydecanethiol” or “aminodecanethiol”) is used. It is done. Carboxyalkanethiol and aminoalkanethiol having 4 to 20 carbon atoms have little optical influence of SAM formed using them, that is, properties such as high transparency, low refractive index, and thin film thickness. This is preferable.

  A method for forming such a SAM is not particularly limited, and a conventionally known method can be used. As a specific example, there is a method of immersing a transparent glass substrate on which a metal thin film is formed in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories). . In this way, the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the gold thin film to form a SAM.

  Further, when SAM is formed on the surface of the spacer layer made of a dielectric, a silane coupling agent is used as a single molecule included in SAM.

  The silane coupling agent is preferably a silane coupling agent having a reactive group. Examples of such a silane coupling agent include 3-aminopropyltriethoxysilane, 8-amino-octyltriethoxysilane, and 6-amino. -Hexyltriethoxysilane, 7-carboxy-heptyltriethoxysilane, 5-carboxy-pentyltriethoxysilane and the like.

  Such a silane coupling agent can also be bonded by, for example, amine coupling with alkanethiols containing a carboxy group or an amino group that can bond to a metal colloid.

  The method for forming the SAM composed of the silane coupling agent is not particularly limited, and a conventionally known method can be used.

(Metal colloid)
In the present invention, the metal colloid receives an electric field generated from a metal thin film excited by surface plasmon by light irradiation from a light source, and generates a surface plasmon excitation by this electric field, and a strong local electric field between the metal thin film and between the metal colloids. By generating the above, it plays a role of enhancing the light emission of the fluorescent dye captured by the plasmon excitation sensor. At this time, the electric field enhancement effect by the surface plasmon, and the electric field enhancement effect by the localized electric field generated between the metal colloid and the metal thin film and between the metal colloids are about 15 times and 70 times, respectively, as compared with the case where the metal colloid does not exist. It goes up to about twice. Although the cause is unknown, when the metal colloid does not exist, the emission intensity from the fluorescent dye at the time of fluorescence measurement by SPFS tends to quench with time, but when the metal colloid exists. The effect of reducing the degree of quenching with time in the emission intensity from the fluorescent dye appears with good reproducibility.

  The metal species constituting the metal colloid used in the present invention is not particularly limited, but has high chemical stability with respect to a sample and the like, and since plasmon resonance is efficiently generated by visible light, gold, silver, platinum At least one metal selected from the group consisting of noble metals such as these and their alloys is preferred, among which gold is particularly preferred. Further, the metal constituting the metal colloid may be the same as or different from the metal constituting the metal thin film.

  The particle size of the metal colloid is not particularly limited as long as it can maintain a dispersed state as a colloid in a colloid solution and can sufficiently induce localized surface plasmon resonance to such an extent that the action and effect of the present invention can be exhibited. . However, in general, the maximum absorption wavelength of the metal colloid varies depending on the constituent metal species, and even if the constituent metal species are the same, it tends to increase as the particle size of the metal colloid increases. Therefore, the particle diameter of the metal colloid is desirably such that the fluorescence from the fluorescent dye is not quenched by absorption by the metal colloid in relation to the maximum fluorescence wavelength of the fluorescent dye. Specifically, as a result of measuring the average value of the particle diameter using an electron microscope, the particle diameter is preferably in the range of 0.1 nm to 10 μm, and more preferably 1 nm to 100 nm.

  The shape of the metal colloid may be any shape, and examples thereof include those having a spherical shape, an elliptical shape, a cylindrical shape, and the like.

Such metal colloids are commercially available in the form of colloidal solutions of metal colloids having various particle sizes. Alternatively, such a metal colloid can be obtained by a conventionally known method, for example, by reducing a solution of a metal compound such as HAuCl ₄ , H ₂ PtCl ₄ or silver nitrate.

In addition, in order to improve the dispersibility of the metal colloid in the colloid solution and prevent non-specific adsorption, it is desirable to subject the surface of the metal colloid to a surface treatment such as polyethylene glycol [PEG] modification. The PEG-modified metal colloidal particles are prepared by mixing PEG having a mercapto group or a polyamine with metal fine particles as described in, for example, Japanese Patent Application Laid-Open Nos. 2001-200050 and 2005-328809. be able to. _{Alternatively, HAuCl 4, H 2 PtCl 4} , when the reduction of the solution of a metal compound such as silver nitrate, may be prepared in the coexistence of the PEG having such a functional group.

  What is marketed as a metal colloid used by this invention can also be used, for example, Tanaka Kikinzoku Kogyo Co., Ltd., Wine Red Chemical Co., Ltd., etc. are suitable.

(High molecular)
In the present invention, the polymer is used for capturing and immobilizing the colloidal gold in a state close to a dispersed floating state, and is inactive with respect to the ligand and the analyte and is soluble in water.

  As such a polymer, (i) the electric field enhancement associated with the resonance of the surface plasmon light and the localized surface plasmon light, and (ii) the fluorescent dye for efficiently linking the obtained electric field enhancement to the fluorescence signal amplification. (Iii) From the viewpoint of expanding the assay area in the height direction by arranging the metal colloids at appropriate intervals and adjusting the distance from the metal substrate, the following conditions (I) to (iii): Those satisfying (III) are preferred.

  Condition (I): A linear structure extending in a fibrous form, a continuous structure of spherical structures, and an XY plane (refers to the surface of a metal thin film, where the direction perpendicular to the surface is the Z direction) is limited. That form the structure.

  However, if there is a large amount between the metal colloid and the metal substrate, the enhanced electric field is shielded or attenuated. For example, gels that form a robust network structure by crosslinking, or layer structures such as high-density molded products and coatings Excludes polymers that form

  Even a polymer that forms a gel or a layer structure can be used depending on the patterning on the surface of the metal thin film.

  Condition (II): One having many reaction points for immobilizing the metal colloid and the ligand. Such a polymer is suitable because the ligand can be arranged in the vicinity of the metal colloid.

  Condition (III): Those having many hydratable functional groups such as hydroxyl groups and carboxy groups. Since the assay using the plasmon excitation sensor of the present invention is carried out in an aqueous solution, use of a polymer having a large number of hydrophobic functional groups tends to localize on the sensor surface and may cause nonspecific adsorption. Even a polymer having a large number of hydrophobic functional groups can be used as the polymer according to the present invention by applying hydrophilic processing.

  Specific examples of such a polymer include protein, nucleic acid, polysaccharide, natural rubber, synthetic resin, silicone resin, synthetic fiber, and synthetic rubber.

  Among these, nucleic acids (DNA and RNA); polysaccharides such as glucose, glycogen, starch, cellulose, dextran, dextrin, glucan, fructose; polymers with polysaccharides, polyethylene oxide structure-containing materials, acrylic acid derivatives, methacrylic Synthetic resins that can have a linear or polymer brush-like structure such as acid derivatives, polyvinyl alcohol, polyethylene glycol, polyimine, etc .; water-soluble silicone resins; water-soluble monomers such as acrylic acid, methacrylic acid, hydroxyethyl methacrylate, acrylamide, methacrylamide , Diacetone acrylamide, acrylate or methacrylate (which may contain an ethylene oxide structure), maleic acid, 2-methylpropanesulfonic acid or the like homopolymer or Such as the copolymer is preferred.

  In particular, dextran is preferred as the polysaccharide and polyacrylic acid is preferred as the synthetic resin. These macromolecules have a hydroxyl group [—OH] and a carboxyl group [—COOH] as functional groups highly effective in reducing non-specific adsorption, and prevent adsorption of non-specific proteins by the inclusion of water molecules by hydrogen bonding. Is preferable.

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

(Layer made of polymer based on metal colloid)
In the polymer based on metal colloid, one of the two ends of one molecule of the polymer is bonded to the metal colloid, and the other end is connected to the metal thin film (preferably The above SAM) or an embodiment in which the metal colloid is bound or released (in such an embodiment, since one end is necessarily bound to the metal colloid, “the metal colloid is a base point” Or an embodiment in which the metal colloid is bound to the side chain of the polymer.

  As a method for producing a polymer based on a metal colloid, for example, the carboxyl group of the polymer 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 dehydration reaction of the carboxyl group thus active esterified and the amino group of aminodecanethiol using water-soluble carbodiimide. Examples include a method of immobilizing a metal colloid to carboxymethyldextran via aminodecanethiol by reacting with a colloid solution containing the metal colloid after immobilization.

  The concentration of the metal colloid contained in the colloid solution is preferably 0.0000001% to 0.1%, and more preferably 0.0000001% to 0.01%. It is preferable that the concentration of the metal colloid is in the above range because the effect of enhancing the local electric field by the metal colloid can be obtained effectively. When a commercially available colloid solution is used as the colloid solution, the concentration of the metal colloid in the colloid solution varies depending on the manufacturer and is usually about 0.03%. It is difficult.

  Of the methods exemplified as the above-mentioned “Method for producing polymer based on metal colloid”, the step of immobilizing metal colloid on carboxymethyldextran via aminodecanethiol will be described more specifically. In the present invention, as the step, the following step (I) or (II) can be appropriately selected.

[Step (I)]
First, a transparent flat substrate; a metal thin film formed on one surface of the substrate; and formed on the other surface (vertical: 2 mm, horizontal: 14 mm) of the thin film that is not in contact with the substrate, A substrate for a plasmon excitation sensor containing carboxymethyldextran having a weight average molecular weight of preferably 10,000 to 1,000,000 is fixed to a channel (height 0.5 mm, volume 1 mL).

  Next, 10 mL of a solution in which aminodecanethiol is preferably adjusted to a concentration of 0.001 to 100 mM is circulated through the channel, preferably for 0.5 to 2.0 hours. 10 mL of colloidal solution containing metal colloid preferably at a concentration of 0.0000001% to 0.1% is preferably circulated for 1 to 60 minutes (more preferably, after the colloidal solution is circulated for 1 to 60 minutes. The metal colloid can be immobilized on carboxymethyldextran via aminodecanethiol by circulating a phosphate buffer solution containing aminoethanol at a concentration of 1M for 30 to 60 minutes.

[Step (II)]
Aminodecanethiol is mixed in advance with a colloid solution containing a metal colloid preferably at a concentration of 0.0000001% to 0.1% so that the concentration is preferably 0.001 to 100 mM. Reacts with the metal colloid surface by reacting for 24 hours or longer.

  Then, the plasmon excitation sensor substrate is fixed to the flow path in the same manner as in the step (I).

  Next, 10 mL of a colloidal metal colloid solution (excluding unreacted metal colloid and aminodecanethiol) prepared in advance on the surface is preferably circulated in the channel for 24 hours or longer. Even in this manner, the metal colloid can be immobilized on carboxymethyldextran via aminodecanethiol.

  The polymer based on the metal colloid obtained by such a production method is bonded to the gold thin film 2 as shown in FIG. 1, for example, and the dextran 4 is linear between the gold colloids 3. It is also possible to connect to the above.

Such a polymer based on a metal colloid is preferably a metal thin film (preferably SAM) with a density of 10 ³ to 10 ¹⁵ pieces / mm ² , more preferably 10 ⁵ to 10 ⁹ pieces / mm ^2. Join by density. When the density of the polymer based on the metal colloid is within the above range, the balance between the density and the density is good, that is, the analyte in the channel and the ligand immobilized on the polymer can sufficiently contact each other. It is suitable because it is so dense that it is so coarse that substances other than the analyte in the flow path are not retained.

  In addition, the thickness of the layer made of the polymer based on the metal colloid (that is, the average length of the polymer based on the metal colloid) is preferably 10 to 1,000 nm, and more preferably 10 to 500 nm. When the thickness of such a layer is within the above range, it is preferable because a local electric field enhancement effect by a metal colloid and a three-dimensional spreading effect of a detection area can be obtained effectively.

(Ligand)
In the present invention, a ligand is bound to the polymer. This ligand is used for the purpose of fixing the analyte in the specimen to the plasmon excitation sensor. In the present invention, the ligand used in step (a) is referred to as “first ligand” in order to distinguish it from the ligand (“second ligand”) used in step (b) described later. .

  In the present invention, “ligand” refers to a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to an analyte contained in a specimen. Examples of such “molecules” or “molecular fragments” include nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), which may be single-stranded or double-stranded, etc., Alternatively, nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modifications thereof Examples include, but are not limited to, molecules and complexes.

  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.

  As a method for immobilizing this ligand, for example, a carboxyl group possessed by the silane coupling agent or the like that forms the SAM is converted into a water-soluble carbodiimide [WSC] (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Hydrochloric acid [EDC] etc.) and N-hydroxysuccinimide [NHS] for active esterification, dehydration reaction of the carboxyl group thus active esterified and the amino group of the ligand using water-soluble carbodiimide And immobilizing them.

  In order to prevent non-specific adsorption of a specimen or the like to be described later to the plasmon excitation sensor, the surface of the plasmon excitation sensor is blocked with bovine serum albumin [BSA] or aminoethanol after the ligand is immobilized. It is preferable to treat with an agent.

(Measurement method using SPFS-LPFS system)
In the present invention, the “measurement method by SPFS-LPFS system” using a metal colloid is novel, and the electric field enhancement effect of the metal thin film by surface plasmon excitation and the local electric field enhancement effect of the metal colloid can be obtained simultaneously. Note that the present invention can be used not only for the measurement method using the SPFS-LPFS system but also for the measurement method using the SPFS system.

  “Measurement method using SPFS-LPFS system” means irradiating laser light via a prism from the surface of the transparent flat substrate of the plasmon excitation sensor opposite to the surface on which the metal thin film is formed. Is a method for measuring the amount of fluorescence emitted from the excited fluorescent dye.

<Assay method>
The assay method of the present invention is characterized by including at least the following steps (a) to (d), and preferably includes a washing step as appropriate.

Step (a): a step of bringing a specimen into contact with the plasmon excitation sensor of the present invention,
Step (b): a step of reacting the plasmon excitation sensor obtained through the step (a) with a conjugate of a second ligand and a fluorescent dye,
Step (c): Laser light is irradiated via a prism from the surface opposite to the surface on which the metal thin film is formed on the transparent flat substrate of the plasmon excitation sensor obtained through the step (b). A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (d): calculating the amount of analyte contained in the sample from the measurement result obtained in step (c) Process.

  Cleaning step: a step of cleaning at least one of the surface of the plasmon excitation sensor obtained through the step (a) and the surface of the plasmon excitation sensor obtained through the step (b).

[Step (a)]
Step (a) is a plasmon excitation sensor of the present invention, that is, a “transparent flat substrate”; a “metal thin film” formed on one surface of the substrate; and the thin film is not in contact with the substrate This is a step of bringing a specimen into contact with a plasmon excitation sensor consisting of a “polymer based on a metal colloid” immobilized on the other surface; and a “first ligand” immobilized on the polymer.

  The “transparent flat substrate”, “metal thin film”, and “polymer based on metal colloid” are as described above.

(First ligand)
In the present invention, the ligand used in step (a) is referred to as “first ligand” in order to distinguish it from the ligand (“second ligand”) used in the following step (b).

  The first ligand is the same as the “ligand” described above.

(Sample)
In the present invention, “specimen” refers to various samples to be measured by the assay method of the present invention.

  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 in a desired solvent, buffer, 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 or more.

(contact)
In the present invention, “contact” means that a surface on which a ligand or the like of a plasmon excitation sensor is immobilized is immersed in the liquid supply, and an object contained in the liquid supply is referred to as the plasmon excitation sensor. To contact. In the step (a), the “contact” between the specimen and the plasmon excitation sensor means that the specimen is included in the liquid feed circulating in the flow path, and only the one surface on which the ligand of the plasmon excitation sensor is immobilized is the liquid feed. A mode in which the plasmon excitation sensor and the specimen are brought into contact with each other in a state of being immersed therein is preferable.

  The “channel” is a rectangular parallelepiped or a tube that can efficiently deliver a small amount of a chemical solution, and can change or circulate the solution feeding speed in order to promote the reaction. . As the shape of the flow path, 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 chemical solution is delivered preferably has a tubular shape.

  The material is a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material in the plasmon excitation sensor part; a light-transmitting material such as polyolefin such as polyethylene, and a silicone rubber, Teflon ( (Registered trademark), polyethylene, polypropylene, and other polymers are used.

  However, for the plasmon excitation sensor, all of the channel structure is not necessarily made of a light-transmitting material unless the channel is kept in a fixed shape during fluorescence measurement and the detection of fluorescence generated by plasmon excitation is not hindered. There is no need to consist only of. That is, of the flow path of the plasmon excitation sensor part, a part necessary for transmitting the fluorescence generated by plasmon excitation and guiding it to the detection part, specifically, a part including a transparent window necessary for collecting the fluorescence. However, other parts may be partially or entirely made of a chemically stable material other than the light transmissive material. When the flow path of the plasmon excitation sensor unit has a rectangular parallelepiped structure, when the surface of the plasmon excitation sensor on which the metal thin film (or preferably SAM) exists is defined as the bottom surface, for example, the surface is opposed to the bottom surface. The ceiling surface may be made of a light transmissive material, and the side surface may be made of a chemically stable material other than the light transmissive material.

  Here, the other parts, for example, the side surfaces are not necessarily rigid bodies as long as a certain shape is maintained at the time of fluorescence measurement, and may have an appropriate elasticity to ensure a sealing property. For example, regarding the flow path of the plasmon excitation sensor unit, the ceiling surface may be composed of polymethyl methacrylate [PMMA] and the side surface may be composed of silicone rubber.

  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.

  In the flow path, the position of the liquid feeding inlet for introducing the liquid feeding from the drug delivery section to the plasmon excitation sensor section and the position of the liquid feeding outlet for discharging the liquid feeding from the plasmon excitation sensor section are both obstructing the fluorescence measurement. Unless it becomes, it will not specifically limit. For example, when the flow path of the plasmon excitation sensor section has a rectangular parallelepiped structure, it is convenient to prepare the flow path for the liquid feed inlet and the liquid feed outlet on the ceiling surface. Either one or both of the liquid discharge ports may be provided on the side surface.

  The method for fixing the plasmon excitation sensor to the flow path is not particularly limited as long as the flow path is maintained in a certain shape and the fluorescence measurement is not hindered.

  As an example of such a fixing method, in a small lot (laboratory level), first, a silicone rubber sheet or O-ring having a certain thickness is formed on the surface on which the metal thin film of the plasmon excitation sensor is formed. Is formed, and then a light transmitting top plate (for example, a PMMA substrate) provided with a liquid feeding inlet and a liquid feeding outlet is disposed thereon. A method of forming a ceiling surface of the metal plate, and then crimping them to fix them with an appropriate fastener can be mentioned. At this time, if a silicone rubber sheet having an appropriate thickness and having a hole having an arbitrary shape and size is used as a material constituting the side surface structure, the inner periphery of the hole has a plasmon. Since it becomes the side structure of the flow path of an excitation sensor part, since the flow path which has a required shape and a size can be formed easily, it is preferable. For example, a perforated polydimethylsiloxane (PDMS) sheet having a flow path height of 0.5 mm is first formed on the surface on which the metal thin film of the plasmon excitation sensor is formed. Next, a PMMA substrate on which a liquid feeding inlet and a liquid feeding outlet are provided in advance is placed on the polydimethylsiloxane [PDMS] sheet, and then the PMMA substrate and the PMMA substrate are placed on the polydimethylsiloxane [PDMS] sheet. A method in which a polydimethylsiloxane [PDMS] sheet and the plasmon excitation sensor are pressure-bonded and fixed with a fastener such as a screw is preferred. In addition, when a silicone rubber sheet or O-ring and a light-transmitting top plate are pressure-bonded and fixed to the plasmon excitation sensor, an appropriate spacer made of a material such as silicone rubber or stainless steel is attached as necessary. You may use together.

  Also, in large lots (factory level) manufactured industrially, as a method of fixing the plasmon excitation sensor to the flow path, a gold substrate is directly formed on a plastic integrally molded product or a separately prepared gold substrate is fixed. Then, after forming a layer made of a polymer based on gold colloid (preferably SAM) and immobilizing the ligand on the gold surface, the lid is covered with an integrally molded plastic product corresponding to the top plate of the flow path. Can be manufactured. If necessary, the prism can be integrated into the flow path.

  Such “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]. 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 sample being sent may be 100 μg / mL to 0.0001 pg / mL.

  The total amount of liquid feeding, that is, the volume of the flow path is usually 0.0001 to 20 mL, preferably 0.01 to 1 mL.

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

[Washing process]
The cleaning step is a step of cleaning at least one of the surface of the plasmon excitation sensor obtained through the step (a) and the surface of the plasmon excitation sensor obtained through the step (b) described later. This washing step is preferably included in at least one of before and after step (b).

  As the washing solution used in the washing step, for example, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer solution used in the reaction of steps (a) and (b), What contains 00001-1 weight% is desirable.

  The temperature and flow rate at which the cleaning liquid is circulated are preferably the same as the “temperature and flow rate at which the liquid feed is circulated” in step (a).

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

[Step (b)]
The step (b) is a step of reacting the plasmon excitation sensor obtained through the step (a) with a conjugate of the second ligand and the fluorescent dye.

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

  The fluorescent dye used in the present invention is not particularly limited as long as it is not quenched due to light absorption by a metal colloid described later, 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.), hexachlorofluorescein family fluorescent dyes. (Applied Biosystems Japan), Coumarin family fluorescent dye (Invitrogen), Rhodamine family fluorescent dye (GE Healthcare Biosciences), Cyanine family fluorescent dye, Indocarbocyanine family fluorescent dye, Oxazine family fluorescent dye, Thiazine family fluorescent dye, Squalene family fluorescent dye, Chelated lanthanide family Fluorescent dyes, BODIPY (registered trademark) family fluorescent dyes (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, triphenylmethane family fluorescent dyes, 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.

The fluorescent dye is not limited to the organic fluorescent dye. For example, rare earth complex fluorescent dyes such as Eu and Tb can also be used as fluorescent dyes in the present invention. In general, rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there. An example of a commercially available rare earth complex-based fluorescent dye is ATBTA-Eu ³⁺ . In addition, blue fluorescent protein [BFP], cyan fluorescent protein [CFP], green fluorescent protein [GFP], yellow fluorescent protein [YFP], red fluorescent protein [DsRed], or allophycocyanin [APC] (LyoFlogen (registered trademark)), etc. Representative fluorescent proteins, fluorescent fine particles such as latex and silica, and the like can also serve as fluorescent dyes used in the present invention.

  In the present invention, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength in a wavelength region where light absorption by the metal contained in the metal colloid is small when performing fluorescence measurement described later. For example, when gold is used as the metal colloid, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 600 nm or more in order to minimize the influence of light absorption by the gold colloid. Therefore, in this case, it is particularly desirable to use a fluorescent dye having a maximum fluorescence wavelength in the near-infrared region, such as Cy5, Alexa Fluor (registered trademark) 647. The use of a fluorescent dye having the maximum fluorescence wavelength in the near-infrared region can minimize the influence of light absorption by iron derived from blood cell components in the blood. Is also useful. On the other hand, when silver is used as the metal colloid, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more.

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

(Conjugate of second ligand and fluorescent dye)
The “conjugate comprising the second ligand and the fluorescent dye” is preferably an antibody capable of recognizing and binding to the analyte (target antigen) contained in the specimen when a secondary antibody is used as the ligand.

  In the assay method of the present invention, the second ligand is a ligand used for the purpose of labeling the analyte with a fluorescent dye, and may be the same as or different from the first ligand. However, when the primary antibody used as the first ligand is a polyclonal antibody, the secondary antibody used as the second ligand may be a monoclonal antibody or a polyclonal antibody, but the primary antibody is monoclonal. In the case of an antibody, the secondary antibody is preferably a monoclonal antibody that recognizes an epitope that the primary antibody does not recognize, or a polyclonal antibody.

  Further, a complex in which a second analyte that competes with the analyte (target antigen) contained in the specimen (competitive antigen; however, different from the target antigen) and the secondary antibody are bound in advance. The embodiment to be used is also preferable. Such an embodiment is preferable because the amount of fluorescent signal (fluorescent signal) and the amount of target antigen can be proportional.

  As a method for producing a “conjugate of a second ligand and a fluorescent dye”, when a secondary antibody is used as the second ligand, for example, a carboxyl group is first given to the fluorescent dye, and the carboxyl group is converted into a water-soluble substance. Carboxyimide [WSC] (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC] etc.) and N-hydroxysuccinimide [NHS] and then esterified carboxyl A method of dehydrating and immobilizing a group and an amino group of a secondary antibody using water-soluble carbodiimide; a method of reacting and immobilizing a secondary antibody and a fluorescent dye each having an isothiocyanate and an amino group; a sulfonyl halide and Reacting with secondary antibody and fluorescent dye each having amino group A method of reacting and immobilizing a secondary antibody and a fluorescent dye each having iodoacetamide and a thiol group; a biotinylated fluorescent dye and a streptavidinized secondary antibody (or streptavidinated fluorescence) And a method of immobilizing the dye by reacting with a biotinylated secondary antibody).

  The concentration of the thus-prepared “conjugate of the second ligand and the fluorescent dye” during feeding is preferably 0.001 to 10,000 μg / mL, and more preferably 1 to 1,000 μg / mL.

  The temperature, time, and flow rate at which the liquid is circulated are the same as in step (a).

[Step (c)]
Step (c) refers to laser light from the surface opposite to the surface on which the metal thin film is formed of the transparent flat substrate of the plasmon excitation sensor obtained through the step (b) via a prism. , And the amount of fluorescence emitted from the excited fluorescent dye is measured.

(Optical system)
The light source used in the assay method of the present invention is not particularly limited as long as it can cause plasmon excitation in the metal thin film and the metal colloidal particles, but the unity of wavelength distribution and the intensity of light energy Thus, it is preferable to use laser light as a light source. It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through the optical filter.

  Irradiation with laser light generates surface plasmons on the surface of the metal thin film under the total reflection attenuation condition [ATR]. Due to the electric field enhancement effect of the surface plasmon, the fluorescent dye is excited by photons increased to several tens to several hundred times the amount of photons irradiated. In addition, although the photon increase amount by this electric field enhancement effect depends on the refractive index of the transparent flat substrate, the metal species and the film thickness of the metal thin film, the increase amount is usually about 10 to 20 times in gold. In the assay method of the present invention, in addition to surface plasmon generation in the metal thin film, surface plasmon generation in the metal colloid and local electric field enhancement generated between the metal thin film and the metal colloid also occur, so further electric field enhancement is induced. As a result, photons are increased and the fluorescent dye is excited more strongly.

  In the fluorescent dye, electrons in the molecule are excited by light absorption, move to the first electron excited state in a short time, and when returning from this state (level) to the ground state, the wavelength of the wavelength corresponding to the energy difference Fluoresce.

  Examples of the “laser light” include an LD having a wavelength of 200 to 900 nm, an LD having a wavelength of 0.001 to 1,000 mW, a wavelength of 230 to 800 nm (resonance wavelength is determined by the metal species used in the metal thin film), and a semiconductor having a wavelength of 0.01 to 100 mW. A laser etc. are mentioned.

  The “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon excitation sensor, and preferably has the same refractive index as that of the transparent flat substrate. In the present invention, various prisms capable of setting the total reflection condition can be selected as appropriate, and therefore, the angle and the shape are not particularly limited. 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 filter” means external light (illumination light outside the SPFS analyzer), excitation light (excitation light transmission component), stray light (excitation light scattering component), plasmon scattering light (excitation light source) And a filter that removes various noise light such as autofluorescence of the enzyme fluorescent substrate, such as an interference filter and a color filter. Is mentioned.

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

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

[Step (d)]
Step (d) is a step of calculating the amount of analyte contained in the sample from the measurement result obtained in step (c).

  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)
The analyte is a molecule or molecular fragment capable of specifically recognizing (or recognizing and binding) the first ligand immobilized on the polymer, and the “molecule” or “molecular fragment” Examples of nucleic acids include, for example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., which may be single-stranded or double-stranded, or nucleosides, nucleotides and modified molecules thereof). , Proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or their modified molecules, complexes, etc. Specifically, it may be a carcinoembryonic antigen such as AFP [α-fetoprotein], a tumor marker, a signaling substance, a hormone, etc., and is not particularly limited. .

(Assay S / N ratio)
Further, in the step (d), the “blank fluorescent signal” measured before the step (c), the “assay fluorescent signal” obtained in the step (c), and the gold substrate not modified at all are flowed. Assuming that the signal obtained by measuring SPFS while flowing ultrapure water while being fixed to the road as “initial noise”, the assay S / N ratio represented by the following formula (1a) can be calculated:
Assay S / N ratio = | Ia / Io | / In (1a)
(In the above formula (1a), Ia is the assay fluorescence signal, Io is the blank fluorescence signal, and In is the initial noise).

However, in calculating the assay S / N ratio, instead of the above formula (1a), the following formula is used on the basis of “assay noise signal” when the concentration of the analyte contained in the sample is 0. You may calculate according to (1b):
Assay S / N ratio = | Ia | / | Ian | (1b)
(In the above formula (1b), Ian is an assay noise signal, and Ia is an assay fluorescence signal as in the case of the above formula (1a)).

<SPFS analyzer>
The analyzer for SPFS of the present invention includes at least the plasmon excitation sensor of the present invention and is used in the assay method of the present invention, and can be diverted to measurement by an SPR system.

  Such an “SPFS analyzer” can include, for example, a light source, various optical filters, a prism, a cut filter, a condensing lens, a surface plasmon excitation enhanced fluorescence detector, and the like. It is preferable to have a liquid feeding system combined with a plasmon excitation sensor when handling sample liquid, washing liquid, labeled antibody liquid and the like. The type of liquid feeding system is not limited as long as the object of the present invention can be achieved. For example, a microchannel device connected to a liquid pump may be used.

  Also, a surface plasmon resonance [SPR] detector, 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 the total reflection attenuation [ATR] condition with a servo motor) The angle of 45 to 85 ° can be changed by synchronizing the photodiode and the light source, and the resolution is preferably 0.01 ° or more.) The information input to the surface plasmon excitation enhanced fluorescence detection unit is processed. A computer for the purpose may also be included.

  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>
The assay kit of the present invention comprises: a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a metal colloid formed on the other surface of the thin film that is not in contact with the substrate. It is composed of a polymer layer as a base point, and includes at least a plasmon excitation sensor substrate used in the plasmon excitation sensor of the present invention.

  Such an “assay kit” is necessary for carrying out the assay method of the present invention except for the first ligand, the second ligand, and the specimen, such as a primary antibody, an antigen, etc. It is preferable to include all of those required except for the ligand (that is, the analyte contained in the sample is not limited to an antigen but may be an antibody), the sample, and the secondary antibody.

  For example, by using the assay kit of the present invention, blood or serum 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 an “assay kit” includes a plasmon excitation sensor substrate; a carboxyalkanethiol having about 4 to 20 carbon atoms for forming SAM; a fluorescent dye; and a sample for dissolving or diluting a specimen. Dissolved solution or diluted solution; various reaction reagents and washing reagents for reacting a plasmon excitation sensor with a specimen, and various devices or materials required for carrying out the assay method of the present invention or the above-mentioned “for SPFS” An "analyzer" can also be included.

  Further, the kit element may include 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, 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 Alexa Fluor (registered trademark) 647-labeled secondary antibody)
As a secondary antibody, an anti-α-fetoprotein [AFP] monoclonal antibody (1D5; 2.5 mg / mL, manufactured by Nippon Medical Laboratory, Inc.), a commercially available biotinylation kit (manufactured by Dojindo Laboratories) Was biotinylated. The procedure followed the protocol attached to the kit.

  Next, the solution of the obtained biotinylated anti-AFP monoclonal antibody and the streptavidin-labeled Alexa Fluor (registered trademark) 647 (Molecular Probes) solution are mixed and reacted by stirring at 4 ° C. for 60 minutes. It was.

  Finally, 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 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 was immersed in 10 mL of 10-amino-1-decanethiol ethanol solution prepared to 1 mM for 24 hours to form a SAM on one side of the gold thin film. The substrate was taken out from the ethanol solution, washed with ethanol and isopropanol, and then dried using an air gun.

  Subsequently, in phosphate buffered saline [PBS] containing 2 mg / mL of carboxymethyldextran having a molecular weight of 500,000, 50 mM of N-hydroxysuccinimide [NHS], and 100 mM of water-soluble carbodiimide [WSC]. The substrate on which the SAM was formed was immersed for 1 hour, and carboxymethyldextran was immobilized on the SAM.

  Next, on the surface of the SAM thus formed on the substrate, a sheet made of polydimethylsiloxane [PDMS] having a flow path height of 0.5 mm and having an appropriate shape and size is provided. Furthermore, a silicone rubber spacer was disposed around the PDMS sheet (the silicone rubber spacer is not in contact with the liquid feed). On the PDMS sheet and the silicone rubber spacer, a PMMA substrate in which a hole for introducing a liquid supply and a hole for discharging a liquid are formed in advance are placed inside the region surrounded by the sheet made of PDMS. (In this case, the PMMA substrate is arranged so that the SAM surface is inside the flow path). These were pressure-bonded from the outside of the flow path, and the PMMA substrate, the flow path sheet (that is, the PDMS sheet) and the plasmon excitation sensor (that is, the substrate on which the SAM was formed) were fixed with screws.

  Subsequently, 5 mL of phosphate buffered saline [PBS] containing 50 mM of N-hydroxysuccinimide [NHS] and 100 mM of water-soluble carbodiimide [WSC] was fed and circulated for 20 minutes. Primary antibody against carboxymethyldextran by circulating 2.5 ml of anti-α-fetoprotein [AFP] monoclonal antibody (1D5; 2.5 mg / mL, manufactured by Nippon Medical Laboratory) for 30 minutes Was immobilized.

  Furthermore, 10 mL of a solution prepared by adjusting aminodecanethiol to a concentration of 1 mM was circulated for 1 hour. A colloidal solution containing gold colloid having an average particle diameter of 20 nm at a concentration of 0.00075% was circulated for 10 minutes and then left for 12 hours to immobilize the gold colloid on carboxymethyldextran via aminodecanethiol. .

  In addition, the nonspecific adsorption | suction prevention process was performed by circulating for 30 minutes in PBS containing 1 weight% bovine serum albumin [BSA] and 1M aminoethanol.

[Comparative Example 1] (Production of plasmon excitation sensor)
In Example 1, when SAM was formed, 10-carboxy-1-decanethiol was used instead of 10-amino-1-decanethiol, gold colloid and dextran were not used, and N-hydroxysuccinimide [ After 5 mL of phosphate buffered saline [PBS] containing 50 mM NHS] and 100 mM water-soluble carbodiimide [WSC] was circulated and circulated for 20 minutes, anti-α fetoprotein [AFP] monoclonal antibody ( 1D5; 2.5 mg / mL (manufactured by Japan Medical Laboratory) Co., Ltd., except that the primary antibody was immobilized on SAM by circulating 2.5 mL of the solution for 30 minutes. Similarly, a plasmon excitation sensor was produced.

[Comparative Example 2] (Production of plasmon excitation sensor)
In Example 1, a plasmon excitation sensor was produced in the same manner as in Example 1 except that no gold colloid was used.

  The configuration of the plasmon excitation sensor manufactured in each of Example 1 and Comparative Examples 1 and 2 is summarized in the following table.

[Example 2] (Execution of assay method)
As step (a), first, 0.5 mL of a PBS solution containing 1 ng / mL of AFP as a target antigen was added to the plasmon excitation sensor obtained in Example 1 and circulated for 25 minutes.

  As a step (b), after washing by circulating 10 minutes using Tris-buffered saline [TBS] containing 0.05% by weight of Tween 20 as a solution, Alexa Fluor (registered trademark) 647 obtained in Preparation Example 2 was used. 2.5 mL of labeled secondary antibody (PBS solution prepared to be 1,000 ng / mL) was added and circulated for 20 minutes.

  As the step (c), first, washing was performed by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes. The plasmon excitation sensor is irradiated with laser light (640 nm, 40 μW) via a prism (manufactured by Sigma Koki Co., Ltd.) from the other surface of the glass transparent flat substrate on which the gold thin film is not formed. 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 signal when AFP was 0 ng / mL was defined as “assay noise signal”.

  As the step (d), the assay S / N ratio is calculated from the measurement results obtained in the above step (c) by using the following formula, and the measurement results without using colloidal gold for the sensitivity under each condition (comparison) Evaluation was made based on standard values (LP enhancement rate) based on Example 1).

Assay S / N ratio = | (assay fluorescence signal) | / | (assay noise signal) |
That is, if the standard value (LP enhancement rate) is large, it means that the assay S / N ratio is improved, and it can be seen that the reliability of the immunoassay measurement is high.

  The obtained results are shown in Table 3.

[Example 3] (Execution of assay method)
In step (c) of Example 2, after washing by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes, 0.5 mL, 0.00075% colloidal gold solution (gold colloid average particle diameter) The assay S / N ratio was calculated in the same manner as in Example 2 except that the colloidal gold was brought into contact with the plasmon excitation sensor by feeding 20 mL) at 0.5 mL / min.

  The obtained results are shown in Table 3.

[Comparative Example 3] (Implementation of assay method)
In Example 2, the assay S / N ratio was calculated in the same manner as in Example 2 except that the plasmon excitation sensor obtained in Comparative Example 1 was used instead of the plasmon excitation sensor obtained in Example 1.

  The obtained results are shown in Table 3.

[Comparative Example 4] (Implementation of assay method)
In Example 2, the assay S / N ratio was calculated in the same manner as in Example 2 except that the plasmon excitation sensor obtained in Comparative Example 2 was used instead of the plasmon excitation sensor obtained in Example 1.

  The obtained results are shown in Table 3.

  From Table 3, it can be seen that the sensitivity is significantly improved in the system using the colloidal metal colloid (Examples 2 and 3) as compared with the case using surface plasmon enhanced fluorescence (Comparative Examples 2 and 3). it is obvious.

  In the present invention, since the metal colloid is immobilized in a state close to dispersion in the polymer layer, the metal colloid density is extremely uniform. Therefore, high reproducibility is expected as compared with a method using a localized plasmon as reported conventionally, and it can be seen that the present invention is a highly sensitive and highly accurate measurement method.

  Since the assay method 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 can be detected. From this result, it is possible to predict with high accuracy the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like.

1 .... Transparent flat substrate 2 .... Gold thin film 3 .... Gold colloid 4 .... Dextran 5 .... Antibody 6 ....・ Cover glass 7 ・ ・ ・ ・ ・ ・ Gold film 8 ・ ・ ・ ・ ・ ・ Gel composed of molecular imprint polymer 9 ・ ・ ・ ・ ・ ・ Binding site 10 ・ ・ ・ ・ ・ ・ Gold nanoparticle 11 ・ ・ ・ ・.. Transparent plate 12... Metal film 14... Dextran layer 15... One active part consisting of anti-dextran and another part consisting of antigen to antibody to be detected Bifunctional or polyfunctional molecules having

Claims (11)

A transparent planar substrate;
A metal thin film formed on one surface of the substrate;
A layer made of a polymer fixing two or more metal colloids formed on the other surface of the thin film not in contact with the substrate;
An unreacted ligand immobilized on the polymer,
A plasmon excitation sensor characterized by being subjected to a measurement method using an SPFS [surface plasmon excitation enhanced fluorescence spectroscopy] -LPFS [localized surface plasmon excitation enhanced fluorescence spectroscopy] system.   The plasmon excitation sensor according to claim 1, wherein the polymer layer has a thickness of 10 nm to 1000 nm. The plasmon excitation sensor according to claim 1 or 2, wherein the polymer has a density of 10 ³ pieces / mm ² to 10 ¹⁵ pieces / mm ² .   The plasmon excitation sensor according to any one of claims 1 to 3, further comprising a SAM (self-assembled monolayer) between the metal thin film and a layer made of a polymer to which the metal colloid is fixed.   The plasmon excitation sensor according to any one of claims 1 to 4, wherein the metal colloid is a colloid composed of at least one metal selected from the group consisting of gold, silver, platinum, and alloys thereof.   The plasmon excitation sensor according to claim 1, wherein the metal colloid has a particle size of 0.1 nm to 10 μm.   The polymer is at least one polymer selected from the group consisting of proteins, nucleic acids, polysaccharides, natural rubber, synthetic resins, silicone resins, synthetic fibers and synthetic rubbers, and is soluble in water. The plasmon excitation sensor according to any one of 1 to 6.   The plasmon excitation sensor according to claim 7, wherein the polymer is a polysaccharide. An assay method comprising at least the following steps (a) to (d);
Step (a): a step of bringing a specimen into contact with the plasmon excitation sensor according to any one of claims 1 to 8,
Step (b): a step of reacting the plasmon excitation sensor obtained through the step (a) with a conjugate of a second ligand and a fluorescent dye,
Step (c): Laser light is irradiated via a prism from the surface opposite to the surface on which the metal thin film is formed on the transparent flat substrate of the plasmon excitation sensor obtained through the step (b). A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (d): calculating the amount of analyte contained in the sample from the measurement result obtained in step (c) Process.   An analyzer for SPFS, comprising at least the plasmon excitation sensor according to any one of claims 1 to 8, and used in the assay method according to claim 9.   A transparent flat substrate, a metal thin film formed on one surface of the substrate, and a polymer on which two or more metal colloids formed on the other surface of the thin film not in contact with the substrate are fixed An assay kit comprising at least a plasmon excitation sensor substrate used in the plasmon excitation sensor according to any one of claims 1 to 8.