JPWO2010123073A1 - Assay method using plasmon excitation sensor with stimulus-responsive polymer - Google Patents

Abstract

[PROBLEMS] To provide a high-sensitivity and high-accuracy assay method capable of improving not only the SPR signal but also the fluorescence signal by SPFS, the assay device, and the assay kit. And [Solution] An assay method comprising the following steps (a) to (e); step (a): contacting a specimen with a specific plasmon excitation sensor, step (b): step (a) A step of reacting a conjugate of a second ligand and a fluorescent dye to the sensor, step (c): a step of applying an external stimulus to the sensor of step (b), step (d): a sensor of step (c) Irradiating a laser beam from a surface opposite to the surface on which the metal thin film is formed of the transparent flat substrate having a laser beam via a prism and measuring the amount of fluorescence emitted from the excited fluorescent dye; and Step (e): A step of calculating the amount of analyte contained in the sample from the measurement result obtained in step (d).

Description

  The present invention relates to an assay method using a plasmon excitation sensor having a stimulus-responsive polymer. More particularly, the present invention relates to an assay using the sensor based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) and surface plasmon resonance [SPR]. The present invention relates to the assay device and the assay kit.

  SPR (Surface Plasmon Resonance) is a condition in which the applied laser beam is subjected to total reflection attenuation [ATR] on the surface of the metal 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 SPR, as shown in FIG. 2, a substrate 11 on which a metal film is formed, a stimulus-responsive polymer 13 such as poly-N-isopropylacrylamide bonded to the substrate, and the polymer A measurement chip for a biosensor having a biological substance recognition molecule (ligand) 14 bonded to is disclosed in Patent Document 1, and in addition to a change in refractive index caused by binding of an analyte to the polymer, It is described that the signal is enhanced by a change in refractive index due to a phase transition change of the polymer.

  However, the measurement chip for biosensor described in Patent Document 1 is inefficient in applying external stimuli, and there is room for improvement because it does not effectively use the phenomenon of phase transition.

  Further, in Patent Document 2, a dielectric sample that exhibits stimulus responsiveness due to heating, application of an electric field, or the like is brought into contact with one surface of a metal thin film and surface plasmon resonance is induced, and the state of the dielectric sample (crystal -Measurement devices capable of measuring liquid crystal phase transition, phase separation, orientation change, etc.) have been proposed.

  However, Patent Document 2 does not have a technical idea of effectively using the phase transition of the dielectric sample.

JP 2008-64465 A JP-A-6-167443

  An object of the present invention is to provide a high-sensitivity and high-accuracy assay method, the assay device, and the assay kit that can improve not only the fluorescence signal by SPFS but also the SPR signal. .

  Note that SPFS (surface plasmon excitation enhanced fluorescence spectroscopy) means that a dense wave (surface plasmon) is applied to the surface of the metal thin film in contact with the dielectric under the condition that the irradiated laser light is attenuated by total reflection [ATR] on the surface of the metal thin film. 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 method that can detect very low concentrations of analyte.

  As a result of earnest research to solve the above problems, the present inventors have effectively reduced the distance between the fluorescent dye to be measured and the metal thin film by efficiently applying an external stimulus to the stimulus-responsive polymer, The inventors have found that the fluorescence signals of SPFS and SPR are improved, and have completed the present invention.

That is, the assay method of the present invention includes at least the following steps (a) to (e).
Step (a): a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a layer containing a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate And contacting the analyte with a plasmon excitation sensor comprising a first ligand immobilized on the layer;
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): adding an external stimulus to which the stimulus-responsive polymer can respond to the plasmon excitation sensor obtained through the step (b),
Step (d): Laser light is irradiated 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 (c) via a prism. A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (e): calculating the amount of analyte contained in the sample from the measurement result obtained in step (d) Process.

  The stimuli-responsive polymer includes poly N-isopropylacrylamide, polymethacrylate, polyvinyl methyl ether, polyacrylic acid, polymethacrylic acid, poly (2-ethylacrylic acid), poly (2-propylacrylic acid), poly (2- It is preferably at least one polymer selected from the group consisting of (dimethylamino) methyl methacrylate, poly (2-diethylamino) methyl methacrylate and poly (2-methoxyaniline-5-sulfonate).

The external stimulus is preferably a temperature change and / or a pH change.
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 particularly preferably formed of gold.

  It is preferable that 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 stimuli-responsive polymer is immobilized on the SAM.

The specimen is preferably at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.
The apparatus of the present invention includes at least the plasmon excitation sensor and is used in the assay method.

  The kit of the present invention also comprises a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate. And a layer for containing a plasmon excitation sensor, and is used in the assay method.

  The present invention not only improves the fluorescence signal in SPFS but also contributes to the improvement of the SPR signal by shortening the distance between the fluorescent dye to be measured and the metal thin film by applying an external stimulus to the stimulus-responsive polymer. An assay method, the assay device and the assay kit can be provided.

FIG. 1A schematically shows a cross-sectional view of the plasmon excitation sensor obtained in the step (b) of the assay method of the present invention, and FIG. 1B shows the step of the assay method of the present invention ( FIG. 3 schematically shows a cross-sectional view of the plasmon excitation sensor obtained in c). FIG. 2 schematically shows a cross-sectional view of the biosensor measurement chip described in Patent Document 1. As shown in FIG.

Hereinafter, the assay method of the present invention will be specifically described.
<Assay method>
The assay method of the present invention is characterized by including at least the following steps (a) to (e), and preferably further includes a washing step.

  Step (a): a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a layer containing a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate And contacting the analyte with a plasmon excitation sensor comprising a first ligand immobilized on the layer.

Step (b): A step of causing the plasmon excitation sensor obtained through the step (a) to react with a conjugate of a second ligand and a fluorescent dye.
Step (c): A step of applying an external stimulus to which the stimulus-responsive polymer can respond to the plasmon excitation sensor obtained through the step (b).

  Step (d): Laser light is irradiated 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 (c) via a prism. And measuring the amount of fluorescence emitted from the excited fluorescent dye.

Step (e): A step of calculating the amount of the analyte contained in the sample from the measurement result obtained in the step (d).
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) includes: a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate. A step of bringing the analyte into contact with a plasmon excitation sensor including a layer containing; and a first ligand immobilized on the layer.

(Plasmon excitation sensor)
The plasmon excitation sensor used in the present invention includes a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus responsiveness formed on the other surface of the thin film that is not in contact with the substrate A SAM [Self-Assembled Monolayer; self comprising a layer containing a polymer; a first ligand immobilized on the layer; and a metal thin film on the other surface not in contact with the transparent flat substrate. Preferably, the stimuli-responsive polymer can 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. The 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].

Further, the refractive index [n _d] is preferably from 1.40 to 2.20 at the d-line (589.3 nm), is preferably thick if 0.01 to 10 mm, more preferably 0.5~5mm For example, the size (vertical × horizontal) is not particularly limited.

In addition, the transparent transparent substrate made of glass is “BK7” (refractive index [n _d ] 1.52) and “LaSFN9” (refractive index [n _d ] 1.85) manufactured by Shot Japan Co., Ltd. as commercially available products. “K-PSFn3” (refractive index [n _d ] 1.84), “K-LaSFn17” (refractive index [n _d ] 1.88) and “K-LaSFn22” (refractive index) manufactured by Sumita Optical Glass Co., Ltd. Ratio [n _d ] 1.90) and “S-LAL10” (refractive index [n _d ] 1.72) manufactured by OHARA INC. Are 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.

  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 substances, 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.

  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 the surface of the metal thin film opposite to the transparent flat substrate. In the plasmon excitation sensor of the present invention, the fluorescence measurement is performed in a state where the specimen and the fluorescent dye are captured on the metal thin film via the ligand. At this time, the stimulus-responsive polymer is fixed to the metal thin film via the SAM. Is desirable. That is, the SAM has a role as a foundation when the stimulus-responsive polymer is fixed to the metal thin film.

  In the present invention, the SAM contained in the SAM is usually a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.), in particular. Preferably 10-carboxy-1-decanethiol is used. Carboxyalkanethiol having 4 to 20 carbon atoms has properties such as little optical influence of SAM formed using it, that is, high transparency, low refractive index, and thin film thickness. Therefore, it is preferable.

The method for forming the 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.

(Layer containing stimulus-responsive polymer)
As the stimulus-responsive polymer according to the present invention, a general stimulus-responsive polymer, that is, a polymer that changes a higher-order structure in response to external stimuli such as heat, light, and pH is used. Among them, it is preferable to use a temperature-responsive polymer or a pH-responsive polymer that can reversibly change the higher order structure by changing heat or pH.

  The layer containing the stimulus-responsive polymer preferably contains the stimulus-responsive polymer and a polymer such as carboxydextran for immobilizing the ligand. Examples of such a polymer include carboxymethyldextran. Such as dextran, glycogen, starch (amylose, amylopectin), cellulose, glucan (β1,3-glucan) and the like, and polysaccharides obtained by polymerizing glucose. As long as it has a reactive functional group for immobilizing, there is no particular limitation.

  As shown in FIG. 1, the layer 3 containing the stimulus-responsive polymer causes a volume change due to a phase transition from (A) to (B) at the lower critical solution temperature [LCST] or a specific pH. Therefore, the fluorescent dye included in the conjugate 4 of the captured fluorescent dye and the second ligand is efficiently excited by being closer to the metal thin film. The “volume change” of the polymer in the present invention means shrinkage.

  As the temperature-responsive polymer, poly (N-substituted acrylamide), poly (N-substituted methacrylamide), poly (N, N-disubstituted acrylamide), polyvinyl ethers, and the like can be used. Specifically, poly (N-ethylacrylamide), poly (N-cyclopropylacrylamide), poly (N-isopropylacrylamide), poly (Nn-propylacrylamide), poly (N-cyclopropylmethacrylamide), Poly (N-isopropylmethacrylamide), poly (Nn-propylmethacrylamide), N-methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide, N , N-dimethylacrylamide, polyvinyl methyl ether and the like. Of these, poly N-isopropylacrylamide is preferred because the phase transition temperature is relatively close to body temperature, around 30 ° C.

  Examples of the pH-responsive polymer include polyacrylic acid, polymethacrylic acid, poly (2-ethylacrylic acid), poly (2-propylacrylic acid), poly (2-dimethylamino) methyl methacrylate, and poly (2-diethylamino). ) Methyl methacrylate, poly (2-methoxyaniline-5-sulfonate) and the like. Of these, polyacrylic acid is preferred because the phase transition pH is relatively close to the pH of a living body, around pH 7.4.

Moreover, not only these single polymers but the copolymer which combined 2 or more types of the monomer unit which comprises these polymers, and the copolymer with another polymerizable monomer are also contained.
These stimuli-responsive polymers may be used alone or in combination of two or more.

  The weight average molecular weight of the stimulus-responsive polymer is preferably 1,000 to 1,000,000. When the weight average molecular weight is within the above range, when the stimulus-responsive polymer is subjected to an external stimulus, the distance between the fluorescent dye captured by the polymer and the metal thin film is preferably within about 200 nm.

  The stimulus-responsive polymer in the present invention can be obtained by a known polymerization method by dissolving a monomer constituting the stimulus-responsive polymer in a solvent. Specifically, N-isopropylacrylamide is copolymerized using an initiator. As the initiator, in radical polymerization, azobisisobutyronitrile [AIBN], 2,2-azobis (2-amidinopropane) dihydrochloride [ABAH], 2- [N- (2-hydroxyethyl) carbamoyl] propyl It is preferable to use 2-dithiobenzoate.

  In addition, when manufacturing gel using the obtained poly N-isopropylacrylamide, it is preferable to use a crosslinking agent. Specific examples of the crosslinking agent include N, N′-methylenebisacrylamide, ethylene glycol dimethacrylate, and the like.

Such stimuli-responsive polymer, thin metal film (preferably SAM), preferably at a density of 1-100 ng / mm ^2, more preferably binds at a density of 1-10 ng / mm ^2. When the density of the stimulus-responsive polymer is within the above range, the balance between the density and the density is good, i.e., the analyte in the channel and the ligand immobilized on the stimulus-responsive polymer are sufficiently dense to contact each other. It is preferable because it is so coarse that substances other than the analyte in the flow path are not retained.

  Examples of the method for forming the layer containing the stimulus-responsive polymer 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 spin coater coating.

(Ligand)
In the present invention, the first ligand is immobilized on the layer containing the stimulus-responsive polymer. This ligand is used for the purpose of immobilizing (capturing) the analyte in the specimen on 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 polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments.
As a method for immobilizing this ligand, for example, a carboxyl group possessed by a polymer having a reactive functional group such as carboxymethyldextran, preferably contained in a layer containing a stimulus-responsive polymer, is converted into a water-soluble carbodiimide [WSC] ( For example, active esterification with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC] and the like and N-hydroxysuccinimide [NHS], and thus the active esterified carboxyl group, A method of dehydrating and immobilizing an amino group possessed by the ligand using a water-soluble carbodiimide; a carboxyl group possessed by a silane coupling agent or the like that forms the SAM, and an amino group possessed by the ligand as described above Examples include a method of dehydrating and immobilizing.

  In order to prevent non-specific adsorption of a specimen or the like, which will be described later, to the plasmon excitation sensor, after immobilizing the ligand, the surface of the plasmon excitation sensor is treated with a blocking agent such as bovine serum albumin [BSA]. It is preferable to do.

(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.), etc., 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 above-mentioned “flow path” is a rectangular tube or cylinder (tube) that can efficiently deliver a small amount of drug solution and can change or circulate the solution feeding speed in order to promote the reaction. ). Further, as the shape of this flow path, the vicinity of the place where the plasmon excitation sensor is installed preferably has a rectangular tube structure, and the vicinity of the place where the drug solution is delivered preferably has a cylindrical (tube) 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 by placing a light-transmitting top plate (for example, a PMMA substrate) provided with a liquid feeding inlet and a liquid feeding outlet thereon. Examples include a method of forming a ceiling surface of a road, and then crimping and fixing them with an appropriate fastener. 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 the metal thin film, 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 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 thin film is small when performing fluorescence measurement described later. For example, when gold is used as the metal thin film, 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 thin film. 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 thin film, 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.

  In addition, a complex in which a second analyte that competes with an analyte (target antigen) contained in a specimen (competitive antigen; however, different from the target antigen) and a 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)]
The step (c) is a step of applying an external stimulus to which the stimulus-responsive polymer can respond to the plasmon excitation sensor obtained through the step (b).

(External stimulus)
In the present invention, the external stimulus is appropriately selected depending on the type of stimulus-responsive polymer used. For example, when poly N-isopropylacrylamide [NIPAM], which is a temperature-responsive polymer, is used at room temperature (about 25 ° C.), the phase transition temperature of NIPAM is around 30 ° C. Is equivalent to raising the temperature to At that time, the temperature of the liquid feeding is controlled using a plasmon excitation sensor and a temperature control device attached to the flow path. In addition, when polyacrylic acid, which is a pH-responsive polymer, is used at a pH of about 7.4, the phase transition pH of polyacrylic acid is around pH 4, so the pH is lowered to about 3.0 or less as an external stimulus. It corresponds to.

[Step (d)]
Step (d) 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 (c) 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 a metal thin film, but in terms of unity of wavelength distribution and intensity of light energy, laser light is used. Is preferably used as the 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 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.

  As the “laser light”, for example, an LD having a wavelength of 200 to 900 nm, an LD 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 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 for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape. For example, a 60-degree dispersion prism may be used. Examples of such commercially available prisms include those similar to the above-mentioned commercially available “glass-made transparent flat substrate”.

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

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

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

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

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

(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 an oncofetal antigen such as AFP [α-fetoprotein], a tumor marker, a signal transmitter, a hormone, etc. There.

(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)).

<Device>
The apparatus of the present invention includes at least the plasmon excitation sensor and is used for the assay method, and is for carrying out the assay method of the present invention.

  In addition to the plasmon excitation sensor, the “apparatus” includes, for example, a light source, various optical filters, a prism, a cut filter, a condenser lens, a surface plasmon excitation enhanced fluorescence detection unit, a temperature change unit (heater), 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.

  In addition, the surface plasmon resonance [SPR] detector, that is, the angle variable unit for adjusting the optimum angle of the photodiode, SPR and SPFS as a light receiving sensor dedicated to SPR (in order to obtain the total reflection attenuation [ATR] condition by the servomotor) 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.

Preferred embodiments of the light source, the optical filter, the cut filter, the condensing lens, and the SPFS detection unit are the same as those described above.
As the “liquid feed pump”, for example, a micro pump suitable for a small amount of liquid feed, a syringe pump with high feed accuracy and low pulsation, which is preferable but cannot be circulated, a simple and excellent handleability but a small amount of liquid feed For example, a tube pump may be difficult.

<Kit>
The kit of the present invention comprises at least a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate. And a plasmon excitation sensor substrate including the layer to be used. The substrate is used in the assay method.

  In carrying out the assay method of the present invention, the kit of the present invention, except for the substrate for the plasmon excitation sensor, except for the first ligand, the second ligand and the sample, for example, 1 All necessary substances except ligands such as secondary antibodies and antigens (that is, the analyte contained in the sample is not limited to antigens and may be antibodies), and samples and secondary antibodies. It is preferable to include.

  For example, by using the 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 a “kit” includes the plasmon excitation sensor substrate; a carboxyalkanethiol having about 4 to 20 carbon atoms for forming a SAM; a fluorescent dye; and a dissolution for dissolving or diluting a specimen. Liquid or diluent; various reaction reagents and washing reagents for reacting a plasmon excitation sensor with a specimen, and various equipment or materials necessary for carrying out the assay method of the present invention and the above-mentioned “device” It 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 (6D2; 2.5 mg / mL, manufactured by Nippon Medical Laboratory), 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.

[Production Example 2] (Production of plasmon excitation sensor in which the stimulus-responsive polymer is a temperature-responsive polymer)
First, the gold thin film is deposited by vapor-depositing gold on one surface of “BK7” (manufactured by Shot Japan Co., Ltd.), which is an optical glass, as a transparent flat substrate so that the thickness of the metal thin film made of gold becomes 50 nm. Formed.

  The obtained gold substrate was set on a spin coater so that the surface on which the gold thin film was formed was exposed, and 100 μL of poly N-isopropylacrylamide ethanol solution (1 mg / mL) was dropped near the center of the substrate, and 1,000 rpm Rotate for 1 minute.

  Further, 1 mL of an acetone solution (2 mg / mL) of carboxymethyl dextran (manufactured by Meito Sangyo Co., Ltd .; molecular weight 1 million) is dropped from above and rotated at 1,000 rpm for 1 minute. By sufficiently drying at room temperature, a layer containing a stimulus-responsive polymer (temperature-responsive polymer) is formed on the other surface of the gold thin film that is not in contact with the transparent flat substrate.

  Next, the obtained gold substrate was fixed to the flow path, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; manufactured by Dojin Chemical Laboratory) 400 mM, and N-hydroxysuccinic acid An active esterification of carboxymethyldextran is performed by flowing a mixed solution with 100 mM of imide (NHS; manufactured by Thermo Scientific) at 10 μL / min for 10 minutes.

  Subsequently, an anti-α-fetoprotein [AFP] monoclonal antibody (1D5; 2.5 mg / mL, manufactured by Japan Medical Laboratory) was diluted to 20 μg / mL with 10 mM acetate buffer (pH 5.0). The resulting solution is flowed at 10 μL / min for 10 minutes to immobilize the antibody on carboxymethyldextran.

  Finally, a plasmon excitation sensor using a temperature-responsive polymer as a stimulus-responsive polymer was subjected to blocking treatment by flowing an aqueous solution of 1M ethanolamine hydrochloride (manufactured by SIGMA; pH 8.5) at 10 μL / min for 10 minutes. Is made.

[Production Example 3] (Production of plasmon excitation sensor in which the stimulus-responsive polymer is a pH-responsive polymer)
In Preparation Example 2, a plasmon excitation sensor was prepared in the same manner as in Preparation Example 2, except that an ethyl methyl ketone solution (1 mg / mL) of methyl polyacrylate was used instead of an ethanol solution (1 mg / mL) of poly N-isopropylacrylamide. Is made.

The plasmon excitation sensor obtained in Production Example 3 uses a pH responsive polymer as a stimulus responsive polymer.
[Example 1]
As a step (a), α-fetoprotein (AFP; 2.0 mg / mL, manufactured by Acris Antibodies GmbH) was added to the plasmon excitation sensor obtained in Production Example 2 to 10 ng / mL with PBS buffer (pH 7.4). The diluted solution is contacted by flowing at 10 μL / min for 20 minutes.

  As a step (b), a diluted solution prepared by adjusting the labeled secondary antibody obtained in Preparation Example 1 to 2.5 μg / mL with 1% BSA PBS buffer (pH 7.4) was further added at 10 μL / min. React by flowing for 20 minutes.

As a washing step, a TBS solution (pH 7.4) containing 0.005% Tween 20 is flowed at 10 μL / min for 10 minutes.
As a step (c), an external stimulus is applied by changing the temperature setting from 25 ° C. to 37 ° C. by the temperature control device attached to the plasmon excitation sensor and the flow path.

  In steps (d) and (e), laser light is irradiated via a prism, and a fluorescence signal is measured from a CCD camera attached to the apparatus. In addition, a fluorescence signal measures SPFS signal value when it observes from CCD 1 minute after adding external stimulus.

The predicted results are shown in Table 2.
[Example 2]
In Example 1, the plasmon excitation sensor used in step (a) was obtained in Preparation Example 3 instead of that obtained in Preparation Example 2, and 10 mM glycine was used as an external stimulus in Step (c) instead of temperature change. The assay method is carried out in the same manner as in Example 1 except that a pH change of flowing an aqueous hydrochloric acid solution (pH 3.0) at 10 μL / min for 10 minutes is performed, and the fluorescence signal is measured.

The predicted results are shown in Table 2.
[Comparative Example 1]
In Example 1, the step (c) is not carried out, that is, the assay method is carried out in the same manner as in Example 1 except that all the steps of Example 1 are carried out at 25 ° C., and the fluorescence signal is measured.

The predicted results are shown in Table 2.
[Comparative Example 2]
In Example 1, the assay method is carried out in the same manner as in Example 1 except that step (c) is not carried out and all steps are carried out at 37 ° C., and the fluorescence signal is measured.

The predicted results are shown in Table 2.
[Comparative Example 3]
The assay method is performed in the same manner as in Example 2 except that step (c) is not performed in Example 2, that is, all steps of Example 2 are performed at pH 7.4, and the fluorescence signal is measured.

The predicted results are shown in Table 2.
[Comparative Example 4]
In Example 2, the assay method is carried out in the same manner as in Example 2 except that step (c) is not carried out and all steps are carried out at pH 3.0, and the fluorescence signal is measured.

  The predicted results are shown in Table 2.

  From Table 2, it can be seen that by adding an external stimulus to the assay process, the phase transition can be used efficiently and the sensitivity enhancement of the plasmon excitation sensor is excellent.

  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.

1 .... Transparent flat substrate 2 .... Metal thin film 3 .... Layer containing stimuli-responsive polymer 4 .... Conjugate of fluorescent dye and second ligand 11 .... Substrate 13 with metal film formed ... Stimulus responsive polymer 14 .... Biological substance recognition molecule

Claims (9)

An assay method comprising at least the following steps (a) to (e);
Step (a): a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a layer containing a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate And contacting the analyte with a plasmon excitation sensor comprising a first ligand immobilized on the layer;
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): adding an external stimulus to which the stimulus-responsive polymer can respond to the plasmon excitation sensor obtained through the step (b),
Step (d): Laser light is irradiated 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 (c) via a prism. A step of measuring the amount of fluorescence emitted from the excited fluorescent dye, and step (e): calculating the amount of analyte contained in the sample from the measurement result obtained in step (d) Process.   The stimuli-responsive polymer is poly N-isopropylacrylamide, polymethacrylate, polyvinyl methyl ether, polyacrylic acid, polymethacrylic acid, poly (2-ethylacrylic acid), poly (2-propylacrylic acid), poly (2- The assay method according to claim 1, which is at least one polymer selected from the group consisting of (dimethylamino) methyl methacrylate, poly (2-diethylamino) methyl methacrylate and poly (2-methoxyaniline-5-sulfonate).   The assay method according to claim 1 or 2, wherein the external stimulus is a temperature change and / or a pH change.   The assay method according to claim 1, wherein the metal thin film is formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum.   The assay method according to claim 4, wherein the metal is gold.   The SAM (self-assembled monomolecular film) is formed on the other surface of the metal thin film not in contact with the transparent flat substrate, and the stimulus-responsive polymer is immobilized on the SAM. The assay method according to -5.   The assay method according to any one of claims 1 to 6, wherein the specimen contains at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.   An apparatus comprising at least the plasmon excitation sensor and used in the assay method according to claim 1.   Plasmon excitation comprising: a transparent planar substrate; a metal thin film formed on one surface of the substrate; and a layer containing a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate A kit comprising at least a sensor substrate and used in the assay method according to claim 1.