JP5853703B2 - Analyte detection probe, analyte detection reagent, and analyte detection method using the same - Google Patents

Description

  The present invention relates to a probe for detecting an analyte contained in a specimen or the like and an assay using the same.

  In the fields of medicine, biology, and the like, research and development of various analysis methods and probes used for the detection, tracking, and quantification of various biological substances (analytes) are being promoted. The so-called liquid-liquid (homogeneous) measuring system that can perform the operations such as bringing the analyte and the probe into contact with each other by simply mixing the solutions is such that the probe is immobilized on a glass substrate or the like. Compared to a so-called solid-liquid measurement system using a chip-like instrument, it has a superior aspect in analysis speed and simplicity.

  Non-Patent Document 1 describes a probe having a structure in which a fluorescent substance and gold nanoparticles (particle size of about 1.4 nm) are bound to molecules such as an antibody, a nucleic acid, and a receptor protein. However, this probe is intended to make it possible to observe, for example, a specific biological substance present in cells by both a fluorescence microscope (fluorescent substance) and an electron microscope (gold nanoparticles). The gold nanoparticles have a very small particle size and are effective in improving the permeability to cells and the like, but are insufficient to cause localized plasmon resonance on the surface.

  Patent Document 1 discloses that at least one organic probe molecule (such as an oligonucleotide) and at least 10 molecules having a light-emitting activity (such as a fluorescent organic dye) are formed on the surface of a gold nanoparticle having a particle size of 2 to 30 nm. Bound hybrid probes have been described. However, there is no specific description or suggestion of an embodiment in which such a probe is used in the analysis of a liquid-liquid system (for example, in FIG. 3, an oligo immobilized on a Sepharose ball on a substrate. Embodiments are disclosed in which nucleotides are labeled).

  On the other hand, a measurement system using FRET (excitation energy transfer due to resonance of a phosphor) that occurs only when a pair of phosphors called a donor and an acceptor are at a predetermined distance (about 1 to 10 nm) is known. is there. This measurement system is constructed so that, for example, FRET occurs when the ligand of the probe binds to the analyte in a predetermined manner (such as nucleic acid hybridization), and the fluorescence of the acceptor is measured. That is, the presence / absence and concentration of the analyte can be analyzed by the change in the fluorescence signal, so that there is an advantage that the operation of separating the probe that has not bound to the analyte is unnecessary.

Special table 2007-512522 gazette

NANOPROBES E-NEWS, Vol. 10, No.1, January 31, 2009.

  An object of the present invention is to provide an analysis method with improved sensitivity and a detection probe used therefor, taking advantage of the liquid-liquid system that allows rapid and simple analysis of analytes.

The present inventors have so that a region sandwiched between the two or more metal particles to produce localized plasmon resonance, i.e. a significant electric field enhancement effect is a donor and acceptor of FRET in a region obtained phosphor is disposed, before Symbol metal constructing a particle and the phosphor two or more measuring systems, such as to bind the detection probe to one target substance with, for example, supporting the previous SL phosphor ligand binding to the analyte, or before Symbol phosphor The above-mentioned problems can be solved by supporting a large number of the polymer on a linear polymer such as a polysaccharide, and preferably using a metal particle whose surface is coated with a layer made of a dielectric. As a result, the present invention has been completed.

That is, in one aspect, the present invention provides, as a detection probe for detecting an analyte, at least metal particles that cause localized plasmon resonance when irradiated with excitation light, and one or more bonded to the metal particles. And an analyte detection probe composed of a phosphor capable of being a donor or acceptor in one or more FRETs held on the metal particle, wherein two or more of the probes are analytes via the ligand. and at the time of forming the complex, in the region where the electric field enhancing effect is obtained by localized plasmon resonance resulting from two or more metal particles, as the phosphor can be disposed within a distance where FRET occurs, before Symbol phosphor There are retained on prior Symbol metal particles, the surface of the metal particles, characterized in that it is coated with a layer of a dielectric, analyzers To provide a door detection probe.

As the first aspect of the previous SL analyte detection probe, at least, the metal particles to produce a plasmon resonance, and one or more ligands bound to the metal particles, the donor in one or more FRET bound to the ligand or The thing comprised with the fluorescent substance which can become an acceptor is mentioned.

Further, a second aspect of prior SL analyte detection probe, at least, the metal particles cause localized plasmon resonance when irradiated with excitation light, and one or more ligands bound to the metal particles, the metal Examples include those composed of one or more linear polymers bonded to particles and a phosphor that can be a donor or acceptor in one or more FRET bonded to the linear polymers.

  In the present invention, a substance (mainly a biological substance) to be subjected to fluorescence analysis using a detection probe is referred to as “analyte”, and a substance that specifically binds to the analyte is referred to as “ligand”.

The metal particles are preferably particles made of a metal selected from the group consisting of gold, silver, copper, aluminum, platinum, and alloys of two or more of these. The metal particles preferably have a volume average particle size of 10 to 100 nm. Further, a layer made of pre Ki誘 collector, for example, preferably contains a layer consisting of SiO _2, the thickness of the dielectric layer is 1~20nm is preferred. On the other hand, as the ligand, for example, an antibody or a Fab fragment or F (ab ′) ₂ fragment thereof can be used.

  The analyte detection probe of the present invention as described above is an analyte detection probe having an analyte detection probe (hereinafter referred to as “probe (D)”) having a phosphor serving as a donor in FRET and a fluorescent molecule serving as an acceptor. A probe (hereinafter referred to as “probe (A)”) can be used in combination.

That is, each of the present invention one aspect, a analyte detection reagent containing pre Symbol probe (D) and a probe (A), the probe (D) and probe (A) is at least, the excitation light , A metal particle that causes localized plasmon resonance when irradiated with light, one or more ligands bound on the metal particle, and a phosphor that can be a donor or acceptor in one or more FRET held on the metal particle An electric field enhancement effect due to localized plasmon resonance generated from two or more metal particles when two or more of the probes form a complex with the analyte via a ligand. in the region to be obtained, so that FRET phosphor can be disposed occur within a distance, wherein the phosphor is held on the metal particles It characterized the door, to provide analyte detection reagent.

Further, in the present invention one aspect, a probe (D) and probe (A) and contacting the analyte step of forming their complex (1) contained in the prior SL analyte detection reagent, And a step (2) of measuring a fluorescence signal generated when the complex is irradiated with excitation light.

  In the step (2), the excitation light is irradiated from the back surface of the metal thin film in a state where the complex is located in an upper region of the metal thin film formed on the dielectric member by using an SPFS measuring device. It is also suitable to be performed.

  By using the detection probe of the present invention, the fluorescence signal emitted in the presence of the analyte is much higher than in the prior art (relatively reduced background noise), so that the analyte signal is better than in the conventional analysis. The detection limit value is lowered, and even a very small amount of analyte can be quantified with high accuracy. Moreover, since the detection probe of the present invention utilizes FRET, B / F separation is unnecessary, and analysis can be performed quickly and easily in a liquid-liquid system.

The schematic diagram which shows a state when the detection probe of the 1st aspect of this invention forms a complex with an analyte and FRET occurs. The schematic diagram which shows a state when the detection probe of the 2nd aspect of this invention forms a complex with an analyte and FRET occurs. Schematic which shows the aspect shown to the measurement example 1 etc. of the measuring method of this invention. The schematic of the measuring apparatus (system) for SPFS used in the aspect etc. which were shown in the measurement example 2 of the measuring method of this invention.

  In the present invention, “binding” between the ligand and the metal particle is not limited to the case where the original (unmodified) ligand is directly reacted and bonded to the metal particle, but as a so-called linker molecule or spacer molecule, When they are bonded via a predetermined molecule that functions to link two substances that cannot originally bind, or a specific coating layer (shell) is formed on the surface of the metal particle (core), and the ligand Including the case of bonding to the coating layer (bonding to the metal particles through the coating layer), regardless of whether the bonding is direct or indirect. Similarly, whether the phosphor and the ligand, the linear polymer and the metal particle, or the phosphor and the linear polymer are “bonded”, it does not matter whether the binding is direct or indirect. However, as for the linear polymer binding to the metal particle, the linear polymer binds to the ligand and the ligand binds to the metal particle, that is, the linear polymer is indirectly bound via the ligand. The mode of binding to metal particles is excluded from the above “bonding”.

− Detection probe −
As shown in FIG. 1, the detection probe 1 and the detection probe 2 according to the first aspect of the present invention include at least a metal particle 10 that causes localized plasmon resonance when irradiated with excitation light, and 1 bonded to the metal particle 10. It includes one or more ligands 20 and one or more phosphors (donor 31 or acceptor 32) bonded to the ligands 20 as constituent elements, and further includes a dielectric layer 11 formed on the surface of the metal particle 10. Also good.

  As shown in FIG. 2, the detection probe 1 and the detection probe 2 of the second aspect of the present invention include at least a metal particle 10 that generates localized plasmon resonance when irradiated with excitation light, and a 1 bonded to the metal particle 10. One or more ligands 20, one or more linear polymers 30 bonded to the metal particles 10, and one or more phosphors (donor 31 or acceptor 32) bonded to the linear polymers 30. As a constituent element, and may further include a dielectric layer 11 formed on the surface of the metal particle 10.

  In both the first aspect and the second aspect, when the detection probe 1 and the detection probe 2 and the analyte 40 form the complex 50 and are irradiated with excitation light, the donor 31 that is close within a predetermined distance. And FRET occurs between the acceptor 32 and the acceptor 32.

Metal particle The detection probe of the present invention includes a metal particle that generates localized plasmon resonance when irradiated with excitation light as one of its constituent elements.

  The type of metal is not particularly limited as long as particles capable of generating plasmon resonance can be prepared, but gold, silver, copper, aluminum, platinum, or an alloy of two or more of these is preferable. The particle diameter of the metal particles is not particularly limited as long as localized plasmon resonance occurs, and can be appropriately set by those skilled in the art, but is preferably 10 to 100 nm, and the average particle diameter is It is preferable to use a group of metal particles in such a range. Metal particles (its colloidal solution) can be prepared according to known methods, and various commercial products are available. The average particle size of the metal particles in the present invention is “volume average particle size”, and can be measured by using a dynamic light scattering nanotrack particle size analyzer or the like.

-Dielectric layer In order to prevent metal quenching of the phosphor, the metal particles of the detection probe of the present invention may be partially or entirely covered with a layer made of a dielectric.

As a dielectric constituting such a dielectric layer, for example, SiO ₂ , titanium oxide, and a synthetic polymer are suitable, and other known dielectrics can also be used. In the present invention, it is preferable to include a layer made of SiO ₂ as the dielectric layer. In the detection probe of the second aspect, a linear polymer such as dextran used therein can correspond to the dielectric layer, but if necessary, other dielectric layer (for example, a layer made of SiO ₂ ) is provided. Further, it may be provided on the metal surface.

Metal particles having a dielectric layer formed on the surface can be prepared according to a known method. For example, metal particles having a dielectric layer made of SiO ₂ can be prepared by a method such as adding metal particles to an acidic silicic acid solution. In addition, when forming a dielectric layer made of dextran or the like on metal particles, a method described in “Linear polymer” described later can be applied. The thickness of the dielectric layer (for example, made of SiO ₂ ) is preferably 1 to 20 nm, and the thickness can be adjusted by adjusting reaction conditions when forming the dielectric layer according to the material of the dielectric layer. Can be within the above range.

-Ligand The detection probe of the present invention includes a ligand for forming a complex with an analyte as one of its constituent elements.

A suitable ligand may be selected according to the analyte to be analyzed, and is not particularly limited. For example, if the analyte is a protein, an antibody (immunoglobulin) that binds to a specific structural unit (epitope) can be used as a ligand. In this case, as the antibody of the probe (D) having a donor fluorophore and the antibody of the probe (A) having an acceptor fluorophore, a polyclonal antibody or a monoclonal antibody that recognizes different epitopes is used. When the same monoclonal antibody is used, the probes (D) and (A) compete with each other, and both probes cannot bind to one molecule of protein, and FRET does not occur. On the other hand, when a virus, bacteria, or the like is used as an analyte, the same monoclonal antibody can be used as a ligand for the probes (D) and (A) because a plurality of epitopes of the same type usually exist. Further, not an antibody (entirely including the Fab region and Fc region), but an antibody fragment having an antigen recognition site such as Fab or F (ab ′) ₂ may be used.

The ligand can be bound to the metal particle or the coating layer formed on the surface thereof according to a known method. For example, a molecule having a functional group (thiol group, silanol group, etc.) bonded to a metal particle or a coating layer at one end and a functional group (amino group, carboxyl group, hydroxyl group, etc.) capable of binding to a ligand at the other end ( SAM, silane coupling agent, etc.) can be used to bind the ligand to the metal particles or the coating layer. Examples of such SAM reagents include 10-carboxy-1-decanethiol, 7-carboxy-1-heptanethiol, 5-carboxy-1-pentanethiol, thiosemicarbazide, thiourea, thioacetamide, thiocarbazide (thiocarbohydrazide). ), 11-amino-1-undecanethiol, 8-amino-1-octanethiol, 6-amino-1-hexanethiol. Examples of the silane coupling agent include (Me) ₂ SiCl— (CH ₂ ) _m —CO—NHS, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, N-2- (aminoethyl) -3-aminopropylmethyldimethoxysilane, N-2- (aminoethyl) -3-amino Examples include propyltrimethoxysilane, N-2- (aminoethyl) -3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropyltriethoxysilane. The molecule as described above can be reacted with a ligand first, and the obtained ligand derivative can be reacted with a metal particle. Alternatively, the molecule can be reacted with a metal particle first, and the surface of the metal particle is modified with such a molecule. Can also be reacted. It is also possible to increase or decrease the number (density) of ligands bound to the metal particles by adjusting the reaction conditions.

-Linear Polymer The detection probe of the present invention includes a linear polymer for supporting a phosphor as one of the constituent elements.

  The linear polymer is a linear polymer having a functional group for bonding to the metal particles or the coating layer formed on the surface thereof, and usually a plurality of reactive functional groups for bonding to the ligand. A polymer having a structure (which may have a branched structure but does not have a crosslinked structure) can be used.

  Since analysis of biological materials is usually performed in an aqueous solvent, linear polymers have many hydrophilic groups such as hydroxyl groups and carboxyl groups and have high affinity with aqueous solvents (water-soluble It is preferable that Examples of such a linear polymer include polysaccharides such as dextran, glycogen, starch (amylose, amylopectin), cellulose, glucan (β1,3-glucan) (these are all polymers of glucose). Or a functional group (such as a carboxymethyl group) for enhancing water solubility, a site for linking a ligand, or the like. In particular, carboxymethyl dextran (a compound in which a part of the hydroxyl group of the glucose unit of dextran is substituted with a carboxymethyl group) having a small branched structure and high solubility in cold water is preferable.

  The molecular weight of the linear polymer may be adjusted within an appropriate range in consideration of the number of phosphors to be bonded. For example, in the case of carboxymethyl dextran, those having an average molecular weight in the range of about 3000 to 100,000 are preferred, and usually several phosphors can be bound per 10,000 molecular weight.

  The linear polymer can be bonded to metal particles or a coating layer formed on the surface thereof according to a known method. For example, a functional group (amino group having a functional group (thiol group, silanol group, etc.) that binds to a metal particle or coating layer at one end and a aldehyde group at the reducing end of a linear polymer at the other end. The linear polymer and the metal particles or the coating layer can be bonded using a molecule having a group or the like (so-called silane coupling agent). The above molecule can be reacted with a linear polymer first, and the resulting linear polymer derivative can be reacted with a metal particle. Alternatively, the surface can be modified with such a molecule. It is also possible to react a linear polymer with the metal particles. It is also possible to increase or decrease the number (density) of linear polymers bonded to the metal particles by adjusting the reaction conditions.

-Fluorescent substance The detection probe of the present invention includes a fluorescent substance for fluorescently labeling an analyte as one of the constituent elements.

  More specifically, the phosphor may be a phosphor serving as a FRET donor (hereinafter also simply referred to as “donor”) and a phosphor serving as an acceptor (hereinafter simply referred to as “acceptor”). ), That is, a phosphor having an appropriate relationship as a donor / acceptor in which the fluorescence spectrum of one phosphor greatly overlaps with the excitation spectrum of the other phosphor is used.

The combination of the FRET pair (donor / acceptor) is not particularly limited, and examples thereof include the following:
Alexa Fluor647 / Cy5, HiLyte Fluor647 / Cy5, fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), R-phycoerythrin (R-PE) / allophycocyanin (APC), fluorescein / Cy5, Naphthalene ¹⁴ / Dansyl, Dansyl ⁹⁵ / FITC, Dansyl ¹⁴ / ODR, ε-A ¹⁴ / NBD, IAF ¹⁴ / TMR, Pyrene ¹⁴ / Coumarin, FITC ¹⁴ / TMRI, AEDANS ¹⁴ / FITC, IAEDANS ¹⁴ / IAF, IAF ¹⁴ / EIA, CF / TR Bodipy ²⁵ / Bodipy, BPE ¹⁴ / Cy5, Terbium ⁹⁶ / Rhodamine, Europium ⁹⁴ / Cy5, Europium ⁹⁷ / APC; CFP / YFP, GFP / RFP for fluorescent proteins.

  A combination used in LANCE TR-FRET using a europium (Eu) complex as a donor and allophycocyanin as an acceptor is also suitable. Rare earth complexes such as europium (Eu) and terbium (Tb) generally have a large wavelength difference between the excitation wavelength (about 310 to 340 nm) and the emission wavelength (about 615 nm for the Eu complex and about 545 nm for the Tb complex) It has a feature that the fluorescence lifetime is as long as several hundred microseconds (and therefore, the time-resolved fluorescence measurement can eliminate various short-lived background fluorescence and enables highly sensitive measurement).

  Various products are available as the above phosphor, and an appropriate one may be selected from known phosphors in consideration of the excitation / fluorescence spectrum and the absorption spectrum of metal particles.

  In the present invention, when two or more detection probes form a complex with an analyte, the electric field enhancement effect in which the donor phosphor (D) and the acceptor phosphor (A) are sandwiched between metal particles within a distance where FRET occurs. To be efficiently located in a high area. Therefore, in the first embodiment, these phosphors are not bonded to the metal particles separately from the ligand, but are bonded (supported) to the ligand, and in the second embodiment, they are bonded (supported) to the linear polymer. The

  In the first embodiment, the phosphor can be bound to various ligands according to known methods. For example, when an antibody (protein) is used as a ligand, an amino group, a carboxyl group, a thiol group, etc. appearing in the side chain or terminal of an amino acid constituting the antibody can be used. By preparing a phosphor having a functional group capable of binding to these functional groups or a phosphor derivative having such a functional group introduced, and reacting the phosphor or the phosphor derivative with a ligand by a predetermined method, Can be combined. Moreover, you may couple | bond a ligand and fluorescent substance through the molecule | numerator called what is called a linker like a C3-C20 alkylene group or a polyethyleneglycol chain. Furthermore, after the ligand is biotinylated and reacted with avidin (including streptavidin and the like), 1 to 4 phosphors can be bound per avidin molecule by reacting the biotinylated phosphor. . The ligand may be bound to the metal particles in advance before the phosphor is bound as described above, or the ligand bound to the phosphor may be bound to the metal particles.

  In the second embodiment, the phosphor can be bound to a linear polymer (polymer polysaccharide) according to a known method. For example, a hydroxyl group of a linear polymer, an aldehyde group formed by oxidative cleavage of a part of a 1,2-diol part contained in a constituent monosaccharide with metaperiodic acid or the like, and other polymer polysaccharides An introduced reactive functional group (for example, a carboxyl group possessed by carboxymethyldextran) can be used. A phosphor having a functional group capable of reacting with these functional groups, or a phosphor derivative having such a functional group introduced therein, is prepared, and the phosphor or the phosphor derivative and the linear polymer are combined by a predetermined method. By making it react, both can be couple | bonded. Alternatively, the linear polymer and the phosphor may be bonded via a so-called linker molecule such as an alkylene group having about 3 to 20 carbon atoms or a polyethylene glycol chain. Furthermore, 1 to 4 biotinylated phosphors per molecule of avidin may be bound by converting a linear polymer into avidin (including streptavidin and the like), biotinylating the phosphor and then reacting them. it can. The linear polymer may be bonded to the metal particles in advance before bonding the phosphor as described above, and the linear polymer bonded to the phosphor is bonded to the metal particles. You may do it.

  By changing the reactive functional group, reaction conditions, etc., it is possible to adjust the position and number of phosphors bound to the ligand or linear polymer. Since FRET can achieve high energy transfer efficiency when the donor and acceptor are at appropriate distances in the range of approximately 1-10 nm, it is designed to increase the likelihood of the donor and acceptor being located at such locations. It is desirable to do. For example, when an antibody or antibody fragment is used as a ligand, a synthetic protein in which a fluorescent substance (fluorescent protein) is bound to its Fab region (N-terminal) may be used.

-Analyte detection reagent The analyte detection reagent of this invention contains both the probe (D) which has the fluorescent substance used as a donor, and the probe (A) which has the fluorescent substance used as an acceptor. It suffices that at least one donor / acceptor combination is contained, and two or more donor / acceptor combinations may be contained for the purpose of analyzing a plurality of analytes at the same time.

  Even if the analyte detection reagent is prepared as a one-component reagent containing all the probes (D) and (A), it is a two-component or multi-component in which the probes (D) and (A) are packaged. It may be prepared as a type reagent.

  Such an analyte detection reagent may be made into a kit in combination with various members for improving the convenience of analysis. For example, a container for preparing a mixed solution such as a sample or a reagent and mounting it on an analyzer, or a standard solution containing an analyte for preparing a calibration curve at a predetermined concentration may be used as a component of the kit. it can.

− Measurement method −
Fluorescence analysis using the detection probe of the present invention is basically performed by a procedure similar to that of fluorescence analysis using conventional FRET, ie, contacting probe (D) and probe (A) with an analyte. The analyte can be detected qualitatively or quantitatively through a step (1) of forming a complex and a step (2) of measuring a fluorescent signal generated when the complex is irradiated with excitation light. it can.

  In order to bring the probe (D) and probe (A) into contact with the analyte in step (1), the solution (analyte detection reagent) in which the probe (D) and probe (A) are dissolved and the analyte are usually used. The dissolved solution (sample) may be mixed, and the mode of contact or mixing is not particularly limited. If the analyte detection reagent is a one-component type, the reagent is usually added to the sample and mixed. If the analyte detection reagent is a two-component type, the solution is usually added to the sample simultaneously or Mix by adding sequentially.

  When measuring fluorescence in the step (2), the liquid mixture prepared by the above operation is irradiated with light including the excitation wavelength of the donor phosphor (D) (hereinafter simply referred to as “excitation light”). To do. However, the excitation light must further include an absorption wavelength of the metal particles, that is, a wavelength at which localized plasmon resonance is generated and an electric field enhancement effect is obtained. By this electric field enhancement effect, the excitation wavelength in the region sandwiched between the metal particles, the fluorescence wavelength of the donor phosphor (D), and the intensity of the fluorescence wavelength of the final acceptor phosphor (A) can be dramatically improved. It becomes possible. The excitation light may be emitted from an appropriate light source (laser diode, mercury lamp, etc.) and transmitted through an appropriate member (filter, dichroic mirror, etc.) for adjusting the wavelength spectrum as necessary. There may be.

  The absorption wavelength spectrum (maximum wavelength) of the metal particles varies depending on the metal type and particle size of the metal particles, the modification state of the surface of the metal particles, etc., but slightly longer from the absorption wavelength spectrum (maximum wavelength). It is appropriate to design the metal particles and the donor (D) so that a relationship exists in which the spectrum (maximum wavelength) of the excitation wavelength of the donor (D) exists.

  When the complex of the analyte and the detection probe is not formed in the mixed solution when the excitation light is irradiated, FRET does not occur except between the adjacent phosphors by chance. D) only. On the other hand, when a complex of an analyte and a detection probe is formed in the mixed solution, the fluorescence of the donor (D) is weakened and the fluorescence of the acceptor (A) is observed by FRET. If the wavelength and intensity of the fluorescence are measured before and after mixing, it can be observed whether such a change has occurred. The fluorescence may be detected using an appropriate detector after passing through an appropriate filter as necessary.

  Separately, use one or more standard samples with known analyte concentrations and, if necessary, standard samples that do not contain analyte (for background noise), measure fluorescence intensity in the same manner as above, and measure the fluorescence intensity. If a calibration curve is prepared from the results, the concentration of the analyte in the sample can be quantified from the fluorescence intensity measured for each sample.

  The step (2) can be performed using a measuring device such as a fluorescence plate reader as in the case of performing fluorescence observation using general FRET. As one embodiment of the present invention, the SPFS (Surface Plasmon- Field enhanced Fluorescence Spectroscopy (Surface plasmon excitation enhanced fluorescence spectroscopy) may be used. The principle and basic mode of SPFS are known, for example, from Japanese Patent No. 3294605 and Japanese Patent Application Laid-Open No. 2006-218169.

  In SPFS, when a metal thin film formed on a dielectric member (prism or transparent flat substrate) is irradiated with excitation light at an angle that causes total reflection attenuation (ATR) from the dielectric member side, the metal thin film is transmitted. The fluorescence emitted from the phosphor is measured by utilizing the effect that the evanescent wave resonates with the surface plasmon generated in the metal thin film and is enhanced several tens to several hundreds.

  Therefore, in the step (2), the irradiation light is irradiated from the back surface of the metal thin film in a state where the complex is located in the upper region of the metal thin film formed on the dielectric member using the SPFS measuring device. In this case, the effect of irradiating the complex of the analyte and the detection probe with excitation light (evanescent wave) stronger than usual is obtained. In addition, it is an effect also seen in LPFS (Localized Surface Plasmon-field enhanced Fluorescence Spectroscopy) that can be used in combination with the SPFS. When is located, plasmon (localized plasmon) is also generated on the surface of the metal particle, and the effect of enhancing the evanescent wave also by resonance with the localized plasmon is obtained. By adding such an effect to the original effect of the present invention in which excitation light is enhanced in a region sandwiched between two or more metal particles, the sensitivity is much higher than in an embodiment that does not use an SPFS measuring device. The concentration of the analyte in the sample can be quantified.

  The SPFS measuring device (detection system) used in the above-described aspect basically includes a light source, a prism, a photodetector, and the like, and usually further includes a condenser lens, a cut filter, and the like (see FIG. 3), an apparatus used in a known SPFS measurement method can be used. Means (liquid feed pump, etc.) for feeding various fluids to a predetermined area at a predetermined flow velocity, timing, etc., a computer for controlling various operations and information processing, etc. are integrated in the measuring device. Also good.

  The metal thin film may be formed directly on the horizontal plane of the prism. However, in consideration of convenience for analyzing a large number of samples, one of the transparent flat substrates (such as glass) that can be attached and detached on the horizontal plane of the prism. It is desirable that it be formed on the surface.

  For convenience, the direction from the dielectric member toward the metal thin film is referred to as “up”, and the opposite direction is referred to as “down”, and the surface on the side where the metal thin film of the laminate including the dielectric member and the metal thin film is located is “Front”, the surface on which the dielectric member is located is called “back”.

  Such a member (referred to as a “sensor substrate”) composed of at least a transparent flat substrate and a metal thin film formed in an upper layer of the transparent flat substrate usually further supplies and stores various fluids. Used in combination with a member (a sheet or plate having a thickness that forms a side wall of the flow path, a top plate, etc.). These are often integrated and take the form of a chip-like structure (referred to as a “flow cell”). The flow cell is provided with an opening for introducing or discharging the fluid, and the fluid is transferred back and forth using, for example, a pump and a tube. The conditions for feeding the liquid (flow rate, time, temperature, concentration of specimen and labeled ligand, etc.) can be adjusted as appropriate.

  The metal thin film of the flow cell (sensor substrate) is made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, which is stable against oxidation and has a large electric field enhancement effect by surface plasmons. (May be in the form of an alloy), particularly preferably made of gold. 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.

  The thickness of the metal thin film made of gold, silver, aluminum, copper, platinum, or an alloy thereof is preferably 5 to 500 nm, and the thickness of the chromium thin film is preferably 1 to 20 nm so that surface plasmons are easily generated. From the viewpoint of the electric field enhancement 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, The thickness of the chromium thin film is more preferably 1 to 3 nm.

-Analyte The analyte in the present invention is not particularly limited as long as the ligand can bind to the distance at which FRET occurs. For example, a protein such as a tumor marker, a signal transmitter, a hormone (polypeptide, oligopeptide, etc.) And pathogenic microorganisms such as viruses and bacteria. In addition, nucleic acids (including single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc.), carbohydrates (including oligosaccharides, polysaccharides, sugar chains, etc.), lipids Other molecules such as can also be converted into analytes after modification treatment such as biotinylation as necessary.

  A substance that contains or may contain an analyte and that is subjected to fluorescence analysis using the detection probe of the present invention is referred to as “specimen”. For example, humans, non-human mammals (model animals, pets, etc.), blood (serum / plasma) collected from other animals, urine, nostril, saliva, feces, body cavity fluids (spinal fluid, ascites, pleural effusion, etc.) Etc. are mentioned as specimens. At the time of analysis, the sample may be mixed with various solvents (pure water, physiological saline, buffer solution, reagent solution, etc.) as necessary. Such a mixture or specimen itself, or a solution containing an analyte prepared for a predetermined purpose, which is mixed with the analyte detection reagent of the present invention, is collectively referred to as “sample” or “sample”.

[Example 1] (First embodiment of the present invention)
Production Example 1: Production of analyte detection probe
(Step 1) Preparation of metal particles (1) Preparation of gold nanoparticles: particle size 50 nm (gold colloid) To 200 ml of ultrapure water, 10 ml of a 1% aqueous solution of tetrachlorogold (III) acid tetrahydrate was added, Heated to 80 ° C. While stirring the solution, 20 ml of a 3% aqueous solution of citric acid was added thereto, and the mixture was further stirred vigorously for 20 minutes while being heated to 80 ° C. The particle size was 50 nm. The particle size was determined by measuring the volume average particle size. The volume average particle size was measured with a dynamic light scattering particle size / particle size distribution measuring device “Nanotrac UPA-EX150” (Nikkiso Co., Ltd.). Hereinafter, the particle size of the metal nanoparticles was measured by the same method.

(2) Gold nanoparticles: particle size 1.4 nm (gold colloid)
Gold nanoparticles with a particle size of 1.4 nm were purchased and obtained from Funakoshi Corporation. The product name is “Nanogold Particles, φ1.4 nm”.

(3) Preparation of alloy nanoparticles: particle diameter 10 nm (alloy colloid) Au-Ag alloy colloid (Au: Ag = 50: 50 weight ratio) is used as an Au source Na ₃ Au (SO ₃ ) ₂ aqueous solution, Ag source A predetermined amount of AgNO ₃ aqueous solution is mixed to prepare an aqueous solution with a total metal solid content of 0.03 wt%, and trisodium citrate dihydrate is added and dissolved in an equimolar amount with respect to the metal component to obtain a raw material aqueous solution. It was. After this raw material aqueous solution was heated to 40 ° C., an aqueous solution in which 0.5 mole amount of NaBH ₄ was dissolved with respect to the metal component of the raw material aqueous solution was dropped in 5 minutes to carry out a reduction reaction. The obtained colloid was confirmed to have an average particle size of about 10 nm and an almost uniform particle size distribution.

(Step 2) Preparation of SAM-coated metal particles The metal particles prepared in (1) to (3) of Step 1 are immersed in a 2 μM ethanol solution of 10-carboxy-1-decanethiol, and the surface is made of 10-carboxy-1-decanethiol. The following SAM-coated metal particles were prepared.

(Step 3) Preparation of Silica-Coated Metal Particles 100 g of the metal particle dispersion prepared in step 1 (1) or (3) was sampled, and 1% by weight NaOH aqueous solution was added to the dispersion. The pH was adjusted to 10.5, the temperature was raised to 95 ° C. and heated for 30 minutes. Thereafter, 1.3 g of an acidic silicic acid solution having a SiO ₂ concentration of 3% by weight was added over 15 minutes to prepare a silica-coated gold nanoparticle dispersion. After separating the particles from this dispersion, it was dried at 120 ° C. for 1 hour to prepare silica-coated gold nanoparticles. The thickness of the silica layer was about 3 nm.

A silane coupling agent (Me) ₂ SiCl— (CH ₂ ) _m —CO—NHS (where NHS represents N-Hydroxysulfosuccinimide) (Altech Co., Ltd.) is added to the suspension of the silica-coated gold nanoparticles. Was added to prepare silica-coated gold nanoparticles having NHS ester on the surface.

(Step 4) Preparation of fluorescently labeled antibody The fluorescently labeled antibodies shown in Table 1 were prepared using a fluorescent substance labeling kit. For example, labeled antibody 1 is labeled “Cy5” for anti-αfetoprotein 1D5 IgG1-κ antibody, labeled antibody 4 is labeled “Alexa Fluor 647” for anti-αfetoprotein 6D2 IgG2a-κ antibody, according to the protocol included in the kit. (CyDye Antibody Labeling Kits). Similarly, other labeled antibodies were labeled on 1D5 or 6D2 according to the protocol attached to each kit. Labeled antibody 4/1 (Alexa Fluor647 / Cy5), labeled antibody 5/2 (FITC / TRITC), and labeled antibody 6/3 (R-PE / APC) each form a FRET pair (donor / acceptor). .

(Step 5) Immobilization of fluorescently labeled antibodies on SAM-coated metal particles
N-Hydroxysulfosuccinimide (NHS: manufactured by Dojindo Laboratories) and 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride (EDC: manufactured by Dojindo Laboratories) each at a concentration of 50 mg / mL in a 25 mM MES buffer Dissolved in. To the resulting NHS solution and EDC solution mixed solution, the metal particles coated with SAM prepared in Step 2 were added, and the particle surfaces were modified with succinimide groups. Then, the fluorescently labeled antibody prepared in Step 4 is added, incubated at room temperature for 30 minutes, and the succinimide group on the surface of the metal particle is reacted with the amino group of the anti-αfetoprotein antibody to immobilize the fluorescently labeled antibody on the surface of the metal particle did. Unreacted succinimide groups on the surface of the metal particles were eliminated by adding tris (hydroxymethyl) aminomethane. In this way, analyte detection probes 1 to 8, 11 and 12 shown in Table 2 were produced.

(Step 6) Immobilization of fluorescently labeled antibody on silica-coated metal particles For silica-coated metal particles whose surface has already been modified with succinimide groups prepared in Step 3, the fluorescent label prepared in Step 4 was added to the dispersion. The antibody was added, incubated at room temperature for 30 minutes, and the succinimide group (NHS ester) on the surface of the metal particle was reacted with the amino group of the anti-αfetoprotein antibody to immobilize the fluorescently labeled antibody on the surface of the metal particle. Unreacted succinimide groups on the surface of the metal particles were eliminated by adding tris (hydroxymethyl) aminomethane. In this way, the analyte detection probes 9, 10, 13, and 14 shown in Table 2 were produced.

Measurement example 1
First, a detection probe (two types: 6D2, 1D5 immobilized, one type depending on the experiment) immobilized with the anti-αfetoprotein antibody prepared in Preparation Example 1 was dispersed in the solution. A solution containing αfetoprotein (AFP), which is an antigen of anti-αfetoprotein antibody, was added thereto (see FIG. 3).

  The state before the addition of the antigen (0 min) was taken as the initial value, and the fluorescence amount was measured 10 minutes (10 min) and 20 minutes (20 min) after the addition of the antigen. A value obtained by dividing the signals of 10 min and 20 min by the signal of 0 min was calculated as the performance S / N value of the detection probe, and the performance of the detection probe was evaluated. The detection device (fluorescence plate reader) used in this measurement example is Fluoroscan Ascent (Thermo Fisher Scientific, Inc. (USA)). Further, the filters attached to this detection apparatus were an excitation filter: 584 nm and a measurement filter: 612 nm. The results are shown in Table 3.

  From the results of Experiment Nos. 1-1 and 1-2 in Table 3, it was confirmed that almost no signal was obtained only by using one type of detection probe. From the results of Experiment No. 1-3, it was confirmed that when gold nanoparticles with a diameter of 1.4 nm were used, the plasmon was not generated on the surface of the gold fine particles, so the electric field enhancement effect could not be obtained and only a very small signal could be obtained. did.

On the other hand, from the results of Experiment Nos. 1-4, 1-5, and 1-6, when two types of detection probes that are FRET pairs were used, the signal gradually increased with the addition of the antigen, and no antigen was added. It was confirmed that a significantly higher signal value was obtained. In addition, using three types of FRET pairs, it was confirmed that a sufficient signal-to-noise ratio was obtained, although the signal values were different. From the results of Experiment No. 1-7, it was confirmed that the fine S / N ratio was obtained with the fine particles coated with the dielectric layer (SiO ₂ ) on the surface of the gold nanoparticle because the electric field enhancement effect was high. From the results of Experiment No. 1-8, it was confirmed that a sufficient S / N ratio was obtained even when an alloy of gold and silver was used. In Experiment No. 1-9, in which the alloys of both the probes (A) and (D) are covered with a dielectric layer (SiO ₂ ), an S / N ratio higher than that in Experiment No. 1-8 was obtained. In Experiment Nos. 1-10 and 1-11, in which only one of the probes (A) or (D) is covered with a dielectric layer (SiO ₂ ), both probes are dielectric layers (SiO ₂ ). Although it is not equivalent to the experiment No. 1-7 coated, it was confirmed that a sufficient S / N ratio was obtained.

Measurement example 2
First, a detection probe (two types: 6D2, 1D5 immobilized, one type depending on the experiment) immobilized with the anti-αfetoprotein antibody prepared in Preparation Example 1 was dispersed in the solution. A solution containing αfetoprotein (AFP), which is an antigen of anti-αfetoprotein antibody, was added thereto.

  Immediately after the addition, the solution was fed to the flow cell of the SPFS measuring apparatus (detection system) shown in FIG. 4 and stopped when the flow cell was sufficiently filled with the solution. Thereafter, excitation light was irradiated from the bottom of the gold film to generate a strong electric field due to plasmons on the gold film surface, and the amount of excited fluorescence was measured over time.

  The results are shown in Table 4. The same tendency as in Measurement Example 1 using a fluorescent plate reader (Fluoroscan Ascent) is observed, but the S / N ratio is improved as compared with Measurement Example 1 due to the effect of surface plasmon by the gold thin film of the detection device. It was confirmed. Furthermore, it was confirmed that the detection probe having a dielectric layer on the gold nanoparticle surface has the highest performance.

[Example 2] (Second embodiment of the present invention)
Preparation Example 2: Preparation of analyte detection probe (Step 1) Preparation of metal particles (1) Preparation of gold nanoparticles: particle size of 50 nm (gold colloid) Tetrachlorogold (III) acid tetrahydrate in 200 ml of ultrapure water 10 ml of a 1% aqueous solution of the product was added and heated to 80 ° C. While stirring the solution, 20 ml of a 3% aqueous solution of citric acid was added thereto, and the mixture was further stirred vigorously for 20 minutes while being heated to 80 ° C. The particle size was 50 nm.

(2) Gold nanoparticles: particle size 1.4 nm (gold colloid)
Gold nanoparticles with a particle size of 1.4 nm were purchased and obtained from Funakoshi Corporation. The product name is “Nanogold Particles, φ1.4 nm”.

(3) Preparation of alloy nanoparticles: particle diameter 10 nm (alloy colloid) Au-Ag alloy colloid (Au: Ag = 50: 50 weight ratio) is used as an Au source Na ₃ Au (SO ₃ ) ₂ aqueous solution, Ag source A predetermined amount of AgNO ₃ aqueous solution is mixed to prepare an aqueous solution with a total metal solid content of 0.03 wt%, and trisodium citrate dihydrate is added and dissolved in an equimolar amount with respect to the metal component to obtain a raw material aqueous solution. It was. After this raw material aqueous solution was heated to 40 ° C., an aqueous solution in which 0.5 mole amount of NaBH ₄ was dissolved with respect to the metal component of the raw material aqueous solution was dropped in 5 minutes to carry out a reduction reaction. The obtained colloid was confirmed to have an average particle size of about 10 nm and an almost uniform particle size distribution.

(Step 2) Preparation of fluorescently labeled dextran
N-Hydroxysulfosuccinimide (NHS: manufactured by Dojindo Laboratories) and 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride (EDC: manufactured by Dojindo Laboratories) are 11.5 mg / mL and 19.2 mg / mL, respectively. Was dissolved in MilliQ water. The obtained NHS solution and EDC solution were mixed with 5 μL each of carboxymethyl dextran (molecular weight 500,000 Da) and mixed. After further dilution with 300 μL of PBS, a phosphor (Cy5 or Alexa Fluor 647) was added. The labeling reaction was carried out by incubating at room temperature for 30 minutes, followed by purification of fluorescently labeled dextran by ultrafiltration. Alexa Fluor647 / Cy5 forms a FRET pair (donor / acceptor).

(Step 3) Introduction of organic sulfur molecule into antibody
N-Hydroxysulfosuccinimide (NHS: manufactured by Dojindo Laboratories) and 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride (EDC: manufactured by Dojindo Laboratories) each at a concentration of 50 mg / mL in a 25 mM MES buffer Dissolved in.

  10-Carboxy-1-decanethiol is added to the resulting NHS solution and EDC solution mixed solution, followed by anti-αfetoprotein antibody (1D5 IgG1-κ antibody or 6D2 IgG2a-κ antibody), and at room temperature. Incubated. Thereafter, unreacted carboxydecanethiol was removed by ultrafiltration to purify the antibody-carboxydecanethiol complex.

(Step 4) Introduction of organic sulfur molecule into fluorescently labeled dextran For introduction of organic sulfur molecule into fluorescent dextran prepared in Step 2, a reductive amination reaction was applied. When an aldehyde or ketone is reacted with a primary amine / secondary amine in the presence of a hydride reducing agent, reductive amination occurs and the corresponding amine is obtained. The reaction was carried out under conditions using sodium cyanoborohydride (NaBH ₃ CN) as a reducing agent, and an amine was formed between the reducing end of fluorescent dextran and the amino group of 11-amino-1-undecanthiol. After purification of the product, a white powder obtained by lyophilization was obtained.

(Step 5) Immobilization of antibody and fluorescently labeled dextran on metal particles The antibody-organic sulfur molecule prepared in Step 3 and the fluorescent dextran-organic sulfur molecule prepared in Step 4 are mixed at a final concentration of 1 μM, respectively. By adding the gold nanoparticles (1) to (3) prepared in (1), the nanoparticles in which the antibody and the fluorescent dextran were immobilized at an appropriate density were formed. In this way, analyte detection probes 15 to 20 shown in Table 5 were produced.

Measurement example 3
First, a detection probe (two types: 6D2, 1D5 immobilized, one type depending on the experiment) in which the anti-αfetoprotein antibody prepared in Preparation Example 2 was immobilized was dispersed in the solution. A solution containing αfetoprotein (AFP), which is an antigen of anti-αfetoprotein antibody, was added thereto (see FIG. 3).

  The state before the addition of the antigen (0 min) was taken as the initial value, and the fluorescence amount was measured 10 minutes (10 min) and 20 minutes (20 min) after the addition of the antigen. A value obtained by dividing the signals of 10 min and 20 min by the signal of 0 min was calculated as the performance S / N value of the detection probe, and the performance of the detection probe was evaluated. The detection device (fluorescence plate reader) used in this measurement example is Fluoroscan Ascent (Thermo Fisher Scientific, Inc. (USA)). Further, the filters attached to this detection apparatus were an excitation filter: 584 nm and a measurement filter: 612 nm.

  The results are shown in Table 6. From the results of Experiment Nos. 3-1 and 3-2, it was confirmed that almost no signal was obtained only by using one type of detection probe. From the results of Experiment No.3-3, when gold nanoparticles with a diameter of 1.4 nm were used, it was confirmed that the plasmon was not generated on the surface of the gold fine particles, so the electric field enhancement effect could not be obtained, and only a very small signal could be obtained. did.

  On the other hand, from the results of Experiment No. 3-4, when two types of detection probes that are FRET pairs were used, the signal gradually increased with the addition of the antigen, and a significantly higher signal value was obtained compared to the state without the addition of the antigen. It was confirmed that From the results of Experiment No. 3-5, it was confirmed that a sufficient S / N ratio was obtained even when an alloy of gold and silver was used.

  In addition, the value of Experiment No. 3-4 signal and S / N ratio are significantly higher than Experiment No. 1-4 of the said Example 1 using the same fluorescent substance (donor and acceptor). That is, in this comparison, the probe of the second embodiment using the fluorescence-labeled dextran is preferable because the higher signal value and S / N ratio can be obtained than the probe of the first embodiment using the fluorescence-labeled antibody. It was shown that.

Measurement example 4
First, a detection probe (two types: 6D2, 1D5 immobilized, one type depending on the experiment) in which the anti-αfetoprotein antibody prepared in Preparation Example 2 was immobilized was dispersed in the solution. A solution containing αfetoprotein (AFP), which is an antigen of anti-αfetoprotein antibody, was added thereto.

  Immediately after the addition, the solution was fed to the flow cell of the SPFS measuring apparatus (detection system) shown in FIG. 4 and stopped when the flow cell was sufficiently filled with the solution. Thereafter, excitation light was irradiated from the bottom of the gold film to generate a strong electric field due to plasmons on the gold film surface, and the amount of excited fluorescence was measured over time.

  The results are shown in Table 7. The same tendency as in Measurement Example 3 using a fluorescent plate reader (Fluoroscan Ascent) is observed, but the S / N ratio is improved as compared with Measurement Example 3 due to the effect of surface plasmon by the gold thin film of the detection device. It was confirmed.

1: Probe (D)
2: Probe (A)
10: Metal particle 11: Coating layer 20: Ligand (for example, antibody)
30: Linear polymer 31: Phosphor (donor)
32: Phosphor (acceptor)
40: Analyte (eg antigen)
50: Composite 100: Measuring device (system) for SPFS
101: Glass substrate (S-LAL100, Au: Cr = 49: 1 nm)
102: Flow cell 103: Triangular prism (S-LAL100, n = 1.72)
104: Laser diode (635nm, 0.95mW)
105: Lens 106: Polarizing prism 107: Photo diode 108: Rotating stage 109: Objective lens (× 20)
110: Interference filter (670nm)
111: CCD camera 120: PC
121: Stage controller 150: SPFS unit 151: SPR unit

Claims (21)

At least a metal particle that causes localized plasmon resonance when irradiated with excitation light, one or more ligands bound on the metal particle, and a donor or acceptor in one or more FRETs retained on the metal particle An analyte detection probe composed of a phosphor that can be
Within a region where FRET occurs within a region where an electric field enhancement effect by localized plasmon resonance generated from two or more metal particles is obtained when two or more of the probes form a complex with an analyte via a ligand. The phosphor is held on the metal particles so that the phosphor can be disposed;
An analyte detection probe, wherein the surface of the metal particles is coated with a layer made of a dielectric.   At least a metal particle that causes localized plasmon resonance when irradiated with excitation light, one or more ligands bound to the metal particle, and a phosphor that can be a donor or acceptor in one or more FRETs bound to the ligand The analyte detection probe according to claim 1, comprising:   At least metal particles that cause localized plasmon resonance when irradiated with excitation light, one or more ligands bound to the metal particles, one or more linear polymers bound to the metal particles, The analyte detection probe according to claim 1, wherein the analyte detection probe is composed of a phosphor that can be a donor or an acceptor in one or more FRETs bonded to a linear polymer.   The analyte detection probe according to any one of claims 1 to 3, wherein the metal particles are particles made of a metal selected from the group consisting of gold, silver, copper, aluminum, platinum, and an alloy of two or more thereof.   The analyte detection probe according to claim 1, wherein the metal particles have a volume average particle diameter of 10 to 100 nm. The analyte detection probe according to claim 1, wherein the layer made of a dielectric includes a layer made of SiO ₂ .   The analyte detection probe according to claim 1, wherein the dielectric layer has a thickness of 1 to 20 nm. The analyte detection probe according to any one of claims 1 to 7, wherein the ligand is an antibody or a Fab fragment or F (ab ') ₂ fragment thereof. An analyte detection probe (hereinafter referred to as “probe (D)”) having a phosphor serving as a donor in FRET and an analyte detection probe (hereinafter referred to as “probe (A)”) having a fluorescent molecule serving as an acceptor. An analyte detection reagent containing
The probe (D) and the probe (A) are each
At least a metal particle that causes localized plasmon resonance when irradiated with excitation light, one or more ligands bound on the metal particle, and a donor or acceptor in one or more FRETs retained on the metal particle An analyte detection probe composed of a phosphor that can be
Within a region where FRET occurs within a region where an electric field enhancement effect by localized plasmon resonance generated from two or more metal particles is obtained when two or more of the probes form a complex with an analyte via a ligand. An analyte detection reagent, wherein the phosphor is held on the metal particles so that the phosphor can be disposed. The probe (D) and the probe (A) are each
At least a metal particle that causes localized plasmon resonance when irradiated with excitation light, one or more ligands bound to the metal particle, and a phosphor that can be a donor or acceptor in one or more FRETs bound to the ligand The analyte detection reagent according to claim 9, comprising: The probe (D) and the probe (A) are each
At least metal particles that cause localized plasmon resonance when irradiated with excitation light, one or more ligands bound to the metal particles, one or more linear polymers bound to the metal particles, The analyte detection reagent according to claim 9, wherein the analyte detection reagent is composed of a phosphor that can be a donor or an acceptor in one or more FRET bonded to a linear polymer. In each of the probe (D) and the probe (A),
The analyte detection reagent according to any one of claims 9 to 11, wherein the metal particles are particles made of a metal selected from the group consisting of gold, silver, copper, aluminum, platinum, and an alloy of two or more thereof. In each of the probe (D) and the probe (A),
The analyte detection reagent according to any one of claims 9 to 12, wherein the volume average particle diameter of the metal particles is 10 to 100 nm. In each of the probe (D) and the probe (A),
The analyte detection reagent according to claim 9, wherein the surface of the metal particles is coated with a layer made of a dielectric. In each of the probe (D) and the probe (A),
The analyte detection reagent according to claim 14 , wherein the dielectric layer comprises a layer made of SiO ₂ . In each of the probe (D) and the probe (A),
The analyte detection reagent according to claim 14 or 15 , wherein the dielectric layer has a thickness of 1 to 20 nm. In each of the probe (D) and the probe (A),
The analyte detection reagent according to any one of claims 9 to 16, wherein the ligand is an antibody or a Fab fragment or F (ab ') ₂ fragment thereof.   A step (1) of bringing the probe (D) and probe (A) contained in the analyte detection reagent according to any one of claims 9 to 17 into contact with an analyte to form a complex thereof, and And (2) a step of measuring a fluorescence signal generated when the complex is irradiated with excitation light.   The step (2) irradiates the excitation light from the back surface of the metal thin film in a state where the complex is located in the upper region of the metal thin film formed on the dielectric member using the SPFS measuring device. The analyte detection method according to claim 18, wherein the method is performed. At least a metal particle that causes localized plasmon resonance when irradiated with excitation light, one or more ligands bound to the metal particle, and a phosphor that can be a donor or acceptor in one or more FRETs bound to the ligand And consists of
An analyte detection probe, wherein the surface of the metal particles is coated with a layer made of a dielectric. At least metal particles that cause localized plasmon resonance when irradiated with excitation light, one or more ligands bound to the metal particles, one or more linear polymers bound to the metal particles, A phosphor that can be a donor or acceptor in one or more FRET bonded to a linear polymer,
An analyte detection probe, wherein the surface of the metal particles is coated with a layer made of a dielectric.