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WO2011096394A1 - Sonde de detection d'analyte et procede de detection d'analyte mettant ladite sonde en oeuvre - Google Patents

Sonde de detection d'analyte et procede de detection d'analyte mettant ladite sonde en oeuvre Download PDF

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Publication number
WO2011096394A1
WO2011096394A1 PCT/JP2011/052029 JP2011052029W WO2011096394A1 WO 2011096394 A1 WO2011096394 A1 WO 2011096394A1 JP 2011052029 W JP2011052029 W JP 2011052029W WO 2011096394 A1 WO2011096394 A1 WO 2011096394A1
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Prior art keywords
analyte detection
detection probe
metal
metal particles
phosphor
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PCT/JP2011/052029
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English (en)
Japanese (ja)
Inventor
智典 金子
英隆 二宮
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Konica Minolta Inc
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Konica Minolta Inc
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Priority to JP2011552784A priority Critical patent/JP5853703B2/ja
Publication of WO2011096394A1 publication Critical patent/WO2011096394A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the present invention relates to a probe for detecting an analyte contained in a specimen and an assay using the same.
  • 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.
  • 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 antibodies, nucleic acids, and receptor proteins.
  • 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 (oligonucleotide or the like) and at least ten molecules having a light emitting activity (fluorescent organic dye or the like) 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).
  • a measurement system using FRET excitation energy transfer due to resonance of a phosphor
  • FRET excitation energy transfer due to resonance of a phosphor
  • 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.
  • FRET excitation energy transfer due to resonance of a phosphor
  • An object of the present invention is to provide an analysis method having improved sensitivity as compared with the conventional method and a detection probe used for the same while taking advantage of a liquid-liquid system that can analyze an analyte quickly and easily.
  • the inventor of the present invention has described the metal particles so that the phosphor serving as a donor and acceptor of FRET is disposed in a region sandwiched between two or more metal particles that cause localized plasmon resonance, that is, a region in which a significant electric field enhancement effect is obtained. And a measurement system that binds two or more detection probes having a phosphor to one target substance, for example, the phosphor is supported on a ligand that binds to an analyte, or the phosphor is a polysaccharide.
  • the inventors have found that the above-described problems can be solved by supporting a large number of such linear polymers on the present invention, and the present invention has been completed.
  • 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.
  • 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.
  • An analyte detection probe is provided on the metal particle.
  • the first aspect of the analyte detection probe includes at least a metal particle that causes plasmon resonance, one or more ligands bound to the metal particle, and a donor or acceptor in one or more FRETs bound to the ligand. And phosphors that can be used.
  • At least metal particles that cause localized plasmon resonance when irradiated with excitation light one or more ligands bonded to the metal particles, and the metal particles And one or more linear polymers bonded to each other and a phosphor that can be a donor or an acceptor in one or more FRET bonded to the linear polymers.
  • analyte a substance (mainly a biological substance) to be subjected to fluorescence analysis using a detection probe
  • ligand a substance that specifically binds to the analyte
  • 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 volume average particle diameter of the metal particles is preferably 10 to 100 nm.
  • the metal particles are preferably those whose surfaces are coated with a layer made of a dielectric.
  • the dielectric layer preferably includes, for example, a SiO 2 layer, and the thickness of the dielectric layer is preferably 1 to 20 nm.
  • 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.
  • the present invention provides an analyte detection reagent characterized by containing the probe (D) and the probe (A).
  • the step (1) of bringing the probe (D) and the probe (A) contained in the analyte detection reagent into contact with the analyte to form a complex thereof And a step (2) of measuring a fluorescent signal generated when the complex is irradiated with excitation light.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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”.
  • the detection probe 1 and the detection probe 2 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.
  • 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.
  • 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.
  • 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 that cause 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.
  • the metal particles of the detection probe of the present invention may be partially or entirely covered with a layer made of a dielectric.
  • 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.
  • a layer made of SiO 2 as the dielectric layer.
  • 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.
  • 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.
  • 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 the reaction conditions when forming the dielectric layer according to the material of the dielectric layer. Can be within the above range.
  • 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.
  • the analyte is a protein
  • an antibody immunoglobulin
  • epitope a specific structural unit
  • a polyclonal antibody or a monoclonal antibody that recognizes different epitopes is used as the antibody of the probe (D) having a donor fluorophore and the antibody of the probe (A) having an acceptor fluorophore.
  • the probes (D) and (A) compete with each other, and both probes cannot bind to one molecule of protein, and FRET does not occur.
  • 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.
  • an antibody not including the Fab region and Fc region
  • 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.
  • 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.
  • SAM silane coupling agent, etc.
  • SAM reagents examples 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.
  • silane coupling agent examples 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.
  • 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.
  • 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.
  • linear polymers 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 linear polymers include polysaccharides such as dextran, glycogen, starch (amylose, amylopectin), cellulose, glucan ( ⁇ 1,3-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.
  • polysaccharides such as dextran, glycogen, starch (amylose, amylopectin), cellulose, glucan (
  • the molecular weight of the linear polymer may be adjusted within an appropriate range in consideration of the number of phosphors to be bonded.
  • carboxymethyl dextran preferably has an average molecular weight in the range of about 3000 to 100,000, 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.
  • 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).
  • silane coupling agent a molecule having a group or the like
  • the above molecule can be reacted with a linear polymer first, and the resulting linear polymer derivative can be reacted with a metal particle.
  • 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
  • the detection probe of the present invention includes a fluorescent substance for fluorescently labeling an analyte as one of the constituent elements.
  • 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.
  • donor FRET donor
  • acceptor a phosphor serving as an acceptor
  • the combination of the FRET pair 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, ⁇ -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 /
  • 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).
  • 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 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 phosphor can be bound to various ligands according to a known method.
  • an antibody protein
  • 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.
  • the ligand 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.
  • linker molecule such as an alkylene group having about 3 to 20 carbon atoms or a polyethylene glycol chain.
  • 1 to 4 phosphors can be bound per molecule of avidin by biotinylating the ligand, reacting this with avidin (including streptavidin, etc.), and then reacting with 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.
  • the phosphor can be bound to a linear polymer (polymer polysaccharide) according to a known method.
  • a linear polymer polymer polysaccharide
  • hydroxyl groups in linear polymers aldehyde groups formed by oxidative cleavage of a portion of 1,2-diol moieties in constituent monosaccharides with metaperiodic acid, etc.
  • An introduced reactive functional group for example, a carboxyl group possessed by carboxymethyldextran
  • 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.
  • 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.
  • linker molecule such as an alkylene group having about 3 to 20 carbon atoms or a polyethylene glycol chain.
  • biotinylated phosphors per molecule of avidin 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.
  • FRET fluorescence resonance spectroscopy
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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”).
  • 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.
  • 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.
  • FRET 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.
  • 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.
  • 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.
  • a measuring device such as a fluorescence plate reader
  • SPFS Surface Plasmon- You may perform using the measuring apparatus for field (enhanced (fluorescence) Spectroscopy: surface plasmon excitation enhanced fluorescence spectroscopy).
  • 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.
  • 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.
  • the effect of irradiating the complex of the analyte and the detection probe with excitation light (evanescent wave) stronger than usual is obtained.
  • LPFS Localized surface Plasmon-field enhanced Fluorescence Spectroscopy
  • plasmon localized plasmon
  • 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 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.
  • 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.
  • 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.
  • 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 can be adjusted as appropriate.
  • the metal thin film of the flow cell 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.
  • 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.
  • the analyte in the present invention is not particularly limited as long as the ligand can bind to the distance at which FRET occurs.
  • a protein such as a tumor marker, a signal transmitter, a hormone (polypeptide, oligopeptide, etc.) And pathogenic microorganisms such as viruses and bacteria.
  • 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.
  • specimen a substance that contains or possibly contains an analyte and that is subjected to fluorescence analysis using the detection probe of the present invention.
  • human 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.
  • the sample may be mixed with various solvents (pure water, physiological saline, buffer solution, reagent solution, etc.) as necessary.
  • sample 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 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. 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.).
  • the particle size of the metal nanoparticles was measured by the same method.
  • 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, ⁇ 1.4 nm”.
  • Step 2 Preparation of SAM-coated metal particles
  • the metal particles prepared in (1) to (3) of Step 1 are immersed in a 2 ⁇ M 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.
  • NHS represents N-Hydroxysulfosuccinimide
  • Step 4 Preparation of fluorescently labeled antibody
  • the fluorescently labeled antibodies shown in Table 1 were prepared using a fluorescent substance labeling kit.
  • labeled antibody 1 is labeled “Cy5” for anti- ⁇ fetoprotein 1D5 IgG1- ⁇ antibody
  • labeled antibody 4 is labeled “Alexa Fluor 647” for anti- ⁇ fetoprotein 6D2 IgG2a- ⁇ antibody, according to the protocol included in the kit.
  • CyDye Antibody Labeling Kits CyDye Antibody Labeling Kits
  • 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
  • labeled antibody 6/3 R-PE / APC
  • Step 5 Immobilization of fluorescently labeled antibody to SAM-coated metal particles
  • N-Hydroxysulfosuccinimide (NHS: manufactured by Dojindo Laboratories) and 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride (EDC: Dojindo Research) (Manufactured by Toshosha) were each dissolved in 25 mM MES buffer at a concentration of 50 mg / mL.
  • NHS 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride
  • 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- ⁇ fetoprotein 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, the 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
  • 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- ⁇ fetoprotein 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.
  • a detection probe two types: 6D2, 1D5 immobilized, one type depending on the experiment
  • the anti- ⁇ fetoprotein antibody prepared in Preparation Example 1 was dispersed in the solution.
  • AFP ⁇ fetoprotein
  • the state before 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 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 a fluoroscan ascent (Thermo Fisher Fisher Scientific, Inc. (USA)).
  • 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.
  • a detection probe two types: 6D2, 1D5 immobilized, one type depending on the experiment
  • the anti- ⁇ fetoprotein antibody prepared in Preparation Example 1 was dispersed in the solution.
  • AFP ⁇ fetoprotein
  • the solution was sent 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.
  • 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.
  • 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
  • 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, ⁇ 1.4 nm”.
  • 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) It was dissolved in MilliQ water at concentrations of 11.5 mg / mL and 19.2 mg / mL. The obtained NHS solution and EDC solution were mixed with 5 ⁇ L each of carboxymethyl dextran (molecular weight 500,000 Da) and mixed.
  • NHS fluorescently labeled dextran N-Hydroxysulfosuccinimide
  • EDC 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride
  • 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: Dojindo Laboratories) and 1-Ethyl-3- [3-dimethylamino] propyl] carbodiimide hydrochloride (EDC: Dojindo Laboratories) ) Each was dissolved in 25 mM MES buffer at a concentration of 50 mg / mL.
  • 10-carboxy-1-decanethiol is added to the resulting NHS solution and EDC solution mixed solution, followed by addition of anti- ⁇ fetoprotein antibody (1D5DIgG1- ⁇ antibody or 6D2 IgG2a- ⁇ antibody) at room temperature. Incubated. Thereafter, unreacted carboxydecanethiol was removed by ultrafiltration, and the antibody-carboxydecanethiol complex was purified.
  • Step 4 Introduction of organic sulfur molecule into fluorescently labeled dextran
  • a reductive amination reaction was applied.
  • 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.
  • NaBH 3 CN sodium cyanoborohydride
  • 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 were mixed at a final concentration of 1 ⁇ M, respectively.
  • nanoparticles having an antibody and a fluorescent dextran immobilized at an appropriate density were formed. In this way, analyte detection probes 15 to 20 shown in Table 5 were produced.
  • a detection probe two types: 6D2, 1D5 immobilized, one type depending on the experiment
  • the anti- ⁇ fetoprotein antibody prepared in Preparation Example 2 was immobilized was dispersed in the solution.
  • AFP ⁇ fetoprotein
  • the state before 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 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 a fluoroscan ascent (Thermo Fisher Fisher Scientific, Inc. (USA)).
  • the filters attached to this detection apparatus were an excitation filter: 584 nm and a measurement filter: 612 nm.
  • a detection probe two types: 6D2, 1D5 immobilized, one type depending on the experiment
  • the anti- ⁇ fetoprotein antibody prepared in Preparation Example 2 was immobilized was dispersed in the solution.
  • AFP ⁇ fetoprotein
  • the solution was sent 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.

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Abstract

L'invention concerne un procédé d'analyse qui offre une sensibilité améliorée tout en tirant avantage du fait que les systèmes liquide-liquide permettent une analyse rapide et commode. L'invention concerne également une sonde utilisée dans le procédé. La sonde de détection d'analyte comprend au moins : une particule métallique qui subit une résonance plasmonique localisée lorsqu'elle est éclairée par une lumière d'excitation ; un ou plusieurs ligands liés à la particule métallique ; et au moins un chromophore fluorescent supporté par la particule métallique qui peut servir de donneur ou d'accepteur de FRET. La sonde est caractérisée en ce que le chromophore fluorescent est supporté par la particule métallique, de telle sorte que ce dernier peut être disposé dans la plage dans laquelle un FRET se produit, dans une région où une amélioration du champ électrique se produit à cause de la résonance plasmonique localisée intervenant dans deux particules métalliques ou plus lorsque deux des sondes ou plus forment un complexe avec l'analyte via les ligands desdites sondes.
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