WO2010123073A1 - Procédé d'essai utilisant un détecteur à excitation de plasmons comprenant un polymère sensible au stimulus - Google Patents

Procédé d'essai utilisant un détecteur à excitation de plasmons comprenant un polymère sensible au stimulus Download PDF

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WO2010123073A1
WO2010123073A1 PCT/JP2010/057163 JP2010057163W WO2010123073A1 WO 2010123073 A1 WO2010123073 A1 WO 2010123073A1 JP 2010057163 W JP2010057163 W JP 2010057163W WO 2010123073 A1 WO2010123073 A1 WO 2010123073A1
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thin film
plasmon excitation
assay method
stimulus
substrate
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PCT/JP2010/057163
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English (en)
Japanese (ja)
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義一 栗原
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コニカミノルタホールディングス株式会社
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Publication of WO2010123073A1 publication Critical patent/WO2010123073A1/fr

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    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention relates to an assay method using a plasmon excitation sensor having a stimulus-responsive polymer. More specifically, the present invention is based on the principle of a sensor using surface plasmon excitation enhanced fluorescence spectroscopy [SPFS; Surface Plasmon-field enhanced Fluorescence Spectroscopy] and surface plasmon resonance [SPR; Surface Plasmon Resonance].
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • SPR Surface Plasmon-field enhanced Fluorescence Spectroscopy
  • SPR Surface Plasmon Resonance
  • SPR Surface Plasmon Resonance
  • ATR total reflection attenuation
  • Or refractive index is a phenomenon in which when the wave number of the evanescent wave, which is easily affected by the difference in refractive index, coincides, the reflected light attenuates, and the interaction between the ligand and the analyte on the sensor surface A difference occurs in the dielectric constant (or refractive index) of the dielectric, and as a result, the surface plasmon resonance is changed, whereby the interaction between the ligand and the analyte can be quantified.
  • a measurement chip for a biosensor having a biological substance recognition molecule (ligand) 14 bonded to is disclosed in Patent Document 1, and in addition to a change in refractive index caused by binding of an analyte to the polymer, It is described that the signal is enhanced by a change in refractive index due to a phase transition change of the polymer.
  • Patent Document 2 a dielectric sample that exhibits stimulus responsiveness due to heating, application of an electric field, or the like is brought into contact with one surface of a metal thin film and surface plasmon resonance is induced, and the state of the dielectric sample (crystal -Measurement devices capable of measuring liquid crystal phase transition, phase separation, orientation change, etc.) have been proposed.
  • Patent Document 2 does not have a technical idea of effectively using the phase transition of the dielectric sample.
  • An object of the present invention is to provide a high-sensitivity and high-accuracy assay method, the assay device, and the assay kit that can improve not only the fluorescence signal by SPFS but also the SPR signal. .
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • a dense wave surface plasmon
  • ATR total reflection
  • the present inventors have effectively reduced the distance between the fluorescent dye to be measured and the metal thin film by efficiently applying an external stimulus to the stimulus-responsive polymer, The inventors have found that the fluorescence signals of SPFS and SPR are improved, and have completed the present invention.
  • the assay method of the present invention is characterized by including at least the following steps (a) to (e).
  • the stimuli-responsive polymer includes poly N-isopropylacrylamide, polymethacrylate, polyvinyl methyl ether, polyacrylic acid, polymethacrylic acid, poly (2-ethylacrylic acid), poly (2-propylacrylic acid), poly (2- It is preferably at least one polymer selected from the group consisting of (dimethylamino) methyl methacrylate, poly (2-diethylamino) methyl methacrylate and poly (2-methoxyaniline-5-sulfonate).
  • the external stimulus is preferably a temperature change and / or a pH change.
  • the metal thin film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum, and particularly preferably formed of gold.
  • SAM self-assembled monolayer
  • the stimuli-responsive polymer is preferably immobilized on the SAM.
  • the specimen is preferably at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.
  • the apparatus of the present invention includes at least the plasmon excitation sensor and is used in the assay method.
  • the kit of the present invention also comprises a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate. And a layer for containing a plasmon excitation sensor, and is used in the assay method.
  • the present invention not only improves the fluorescence signal in SPFS but also contributes to the improvement of the SPR signal by shortening the distance between the fluorescent dye to be measured and the metal thin film by applying an external stimulus to the stimulus-responsive polymer.
  • An assay method, the assay device and the assay kit can be provided.
  • FIG. 1A schematically shows a cross-sectional view of the plasmon excitation sensor obtained in the step (b) of the assay method of the present invention
  • FIG. 1B shows the step of the assay method of the present invention
  • FIG. 3 schematically shows a cross-sectional view of the plasmon excitation sensor obtained in c
  • FIG. 2 schematically shows a cross-sectional view of the biosensor measurement chip described in Patent Document 1. As shown in FIG.
  • the assay method of the present invention comprises at least the following steps (a) to (e), and preferably further includes a washing step.
  • Step (b) A step of causing the plasmon excitation sensor obtained through the step (a) to react with a conjugate of a second ligand and a fluorescent dye.
  • Step (e) A step of calculating the amount of the analyte contained in the sample from the measurement result obtained in the step (d).
  • Cleaning step a step of cleaning at least one of the surface of the plasmon excitation sensor obtained through the step (a) and the surface of the plasmon excitation sensor obtained through the step (b).
  • Step (a) includes: a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimuli-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate.
  • the plasmon excitation sensor used in the present invention includes a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus responsiveness formed on the other surface of the thin film that is not in contact with the substrate
  • a SAM Self-Assembled Monolayer; a self-assembled monolayer formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate;
  • the stimuli-responsive polymer can be immobilized on the SAM.
  • a spacer layer made of a dielectric may be appropriately formed for the purpose of preventing the metal quenching of the fluorescent dye by the metal thin film.
  • the spacer layer is preferably formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate.
  • a transparent flat substrate is used as a flat substrate that supports the structure of the plasmon excitation sensor.
  • the transparent flat substrate is used as the flat substrate because light irradiation to a metal thin film described later is performed through the flat substrate.
  • the transparent flat substrate used in the present invention is not particularly limited as long as the object of the present invention is achieved.
  • the transparent flat substrate may be made of glass, or may be made of plastic such as polycarbonate [PC] or cycloolefin polymer [COP].
  • the refractive index [n d ] at the d line (589.3 nm) is preferably 1.40 to 2.20, and the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.
  • the size (vertical ⁇ horizontal) is not particularly limited.
  • the transparent transparent substrate made of glass is “BK7” (refractive index [n d ] 1.52) and “LaSFN9” (refractive index [n d ] 1.85) manufactured by Shot Japan Co., Ltd. as commercially available products.
  • K-PSFn3 reffractive index [n d ] 1.84
  • K-LaSFn17 reffractive index [n d ] 1.88
  • K-LaSFn22 reffractive index
  • Ratio [n d ] 1.90) and “S-LAL10” (refractive index [n d ] 1.72) manufactured by OHARA INC. Are preferable from the viewpoint of optical properties and detergency.
  • the transparent flat substrate is preferably cleaned with acid and / or plasma before forming a metal thin film on the surface.
  • As the cleaning treatment with an acid it is preferable to immerse in 0.001 to 1N hydrochloric acid for 1 to 3 hours.
  • Examples of the plasma cleaning treatment include a method of immersing in a plasma dry cleaner (“PDC200” manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.
  • PDC200 plasma dry cleaner
  • a metal thin film is formed on one surface of the transparent flat substrate. This metal thin film has a role of generating surface plasmon excitation by light irradiated from a light source, generating an electric field, and causing emission of a fluorescent dye.
  • the metal thin film formed on one surface of the transparent flat substrate is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and more preferably made of gold. preferable. These metals may be in the form of their alloys. Such metal species are suitable because they are stable against oxidation and increase in electric field due to surface plasmons is large.
  • a glass flat substrate When a glass flat substrate is used as the transparent flat substrate, it is preferable to form a chromium, nickel chromium alloy or titanium thin film in advance in order to bond the glass and the metal thin film more firmly.
  • Examples of a method for forming a metal thin film on a transparent flat substrate include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and / or a metal thin film by sputtering or vapor deposition.
  • the thickness of the metal thin film is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm.
  • the thickness of the thin film is preferably 1 to 20 nm.
  • gold 20 to 70 nm
  • silver 20 to 70 nm
  • aluminum 10 to 50 nm
  • copper 20 to 70 nm
  • platinum 20 to 70 nm
  • alloys thereof 10 to 70 nm
  • chromium The thickness of the thin film is more preferably 1 to 3 nm.
  • the thickness of the metal thin film is within the above range because surface plasmons are easily generated. Moreover, if it is a metal thin film which has such thickness, a magnitude
  • Spacer layer made of dielectric As the dielectric used for forming the spacer layer made of a dielectric, various optically transparent inorganic substances, natural or synthetic polymers can be used. Among them, it is preferable to contain silicon dioxide [SiO 2 ] or titanium dioxide [TiO 2 ] because it is excellent in chemical stability, production stability and optical transparency.
  • the thickness of the spacer layer made of a dielectric is usually 10 nm to 1 mm, and is preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. On the other hand, it is preferably 200 nm to 1 mm from the viewpoint of electric field enhancement, and more preferably 400 nm to 1,600 nm from the stability of the effect of electric field enhancement.
  • Examples of the method for forming the spacer layer made of a dielectric material include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or an application with a spin coater.
  • SAM Self-Assembled Monolayer
  • the fluorescence measurement is performed in a state where the specimen and the fluorescent dye are captured on the metal thin film via the ligand.
  • the stimulus-responsive polymer is fixed to the metal thin film via the SAM. Is desirable. That is, the SAM has a role as a foundation when the stimulus-responsive polymer is fixed to the metal thin film.
  • the SAM contains a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.), in particular.
  • a carboxyalkanethiol having about 4 to 20 carbon atoms for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.
  • 10-carboxy-1-decanethiol is used.
  • Carboxyalkanethiol having 4 to 20 carbon atoms has properties such as little optical influence of SAM formed using it, that is, high transparency, low refractive index, and thin film thickness. Therefore, it is preferable.
  • the method for forming the SAM is not particularly limited, and a conventionally known method can be used.
  • a transparent glass substrate having a metal thin film formed thereon is immersed in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories). .
  • the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the gold thin film to form a SAM.
  • a general stimulus-responsive polymer that is, a polymer that changes a higher-order structure in response to external stimuli such as heat, light, and pH is used.
  • a temperature-responsive polymer or a pH-responsive polymer that can reversibly change the higher order structure by changing heat or pH.
  • the layer containing the stimulus-responsive polymer preferably contains the stimulus-responsive polymer and a polymer such as carboxydextran for immobilizing the ligand.
  • a polymer such as carboxydextran
  • examples of such a polymer include carboxymethyldextran.
  • polysaccharides obtained by polymerizing glucose such as dextran, glycogen, starch (amylose, amylopectin), cellulose, glucan ( ⁇ 1,3-glucan), and the like. As long as it has a reactive functional group for immobilizing, there is no particular limitation.
  • the layer 3 containing the stimulus-responsive polymer causes a volume change due to a phase transition from (A) to (B) at the lower critical solution temperature [LCST] or a specific pH. Therefore, the fluorescent dye included in the conjugate 4 of the captured fluorescent dye and the second ligand is efficiently excited by being closer to the metal thin film.
  • the “volume change” of the polymer in the present invention means shrinkage.
  • poly (N-substituted acrylamide), poly (N-substituted methacrylamide), poly (N, N-disubstituted acrylamide), polyvinyl ethers and the like can be used.
  • poly N-isopropylacrylamide is preferred because the phase transition temperature is approximately 30 ° C. and relatively close to body temperature.
  • pH-responsive polymer examples include polyacrylic acid, polymethacrylic acid, poly (2-ethylacrylic acid), poly (2-propylacrylic acid), poly (2-dimethylamino) methyl methacrylate, poly (2-diethylamino). ) Methyl methacrylate, poly (2-methoxyaniline-5-sulfonate) and the like.
  • polyacrylic acid is preferred because the phase transition pH is relatively close to the pH of a living body, around pH 7.4.
  • the weight-average molecular weight of the stimulus-responsive polymer is preferably 1,000 to 1,000,000.
  • the distance between the fluorescent dye captured by the polymer and the metal thin film is preferably within about 200 nm.
  • the stimulus-responsive polymer in the present invention can be obtained by a known polymerization method by dissolving the monomer constituting the stimulus-responsive polymer in a solvent.
  • N-isopropylacrylamide is copolymerized using an initiator.
  • the initiator in radical polymerization, azobisisobutyronitrile [AIBN], 2,2-azobis (2-amidinopropane) dihydrochloride [ABAH], 2- [N- (2-hydroxyethyl) carbamoyl] propyl are used. It is preferable to use -2-dithiobenzoate.
  • crosslinking agent when producing a gel using the obtained poly-N-isopropylacrylamide.
  • the crosslinking agent include N, N′-methylenebisacrylamide, ethylene glycol dimethacrylate and the like.
  • Such stimuli-responsive polymers are bonded to the metal thin film (preferably SAM) at a density of preferably 1 to 100 ng / mm 2 , more preferably 1 to 10 ng / mm 2 .
  • the density of the stimulus-responsive polymer is within the above range, the balance between the density and the density is good, i.e., the analyte in the channel and the ligand immobilized on the stimulus-responsive polymer are sufficiently dense to contact each other. It is preferable because it is so coarse that substances other than the analyte in the flow path are not retained.
  • Examples of the method for forming the layer containing the stimuli-responsive polymer include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or a coating with a spin coater.
  • the first ligand is immobilized on the layer containing the stimulus-responsive polymer. This ligand is used for the purpose of immobilizing (capturing) the analyte in the specimen on the plasmon excitation sensor.
  • the ligand used in step (a) is referred to as “first ligand” in order to distinguish it from the ligand (“second ligand”) used in step (b) described later. .
  • ligand refers to a molecule or molecular fragment that can specifically recognize (or be recognized) and bind to an analyte contained in a specimen.
  • molecules or “molecular fragments” include nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), which may be single-stranded or double-stranded, etc., Alternatively, nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modifications thereof Examples include, but are not limited to, molecules and complexes.
  • proteins examples include antibodies and the like, specifically, anti- ⁇ -fetoprotein [AFP] monoclonal antibody (available from Nippon Medical Laboratory, Inc.), anti-carcinoembryonic antigen [CEA Monoclonal antibodies, anti-CA19-9 monoclonal antibodies, anti-PSA monoclonal antibodies, and the like.
  • AFP anti- ⁇ -fetoprotein
  • CEA anti-carcinoembryonic antigen
  • the term “antibody” includes polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments.
  • a carboxyl group possessed by a polymer having a reactive functional group such as carboxymethyldextran preferably contained in a layer containing a stimulus-responsive polymer, is converted into a water-soluble carbodiimide [WSC] ( For example, active esterification with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC] and the like and N-hydroxysuccinimide [NHS], and the carboxyl group thus active esterified
  • WSC water-soluble carbodiimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • the surface of the plasmon excitation sensor is treated with a blocking agent such as bovine serum albumin [BSA]. It is preferable to do.
  • a blocking agent such as bovine serum albumin [BSA]. It is preferable to do.
  • first ligand the ligand used in step (a) is referred to as “first ligand” in order to distinguish it from the ligand (“second ligand”) used in the following step (b).
  • the first ligand is the same as the “ligand” described above.
  • “specimen” refers to various samples to be measured by the assay method of the present invention.
  • samples of the “specimen” include blood (serum / plasma), urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc., and appropriately diluted in a desired solvent, buffer, etc. May be used.
  • blood serum / plasma
  • urine nasal fluid
  • saliva saliva
  • stool body cavity fluid
  • body cavity fluid spinal fluid, ascites, pleural effusion, etc.
  • contact means that a surface on which a ligand or the like of a plasmon excitation sensor is immobilized is immersed in the liquid supply, and an object contained in the liquid supply is referred to as the plasmon excitation sensor.
  • the “contact” between the specimen and the plasmon excitation sensor means that the specimen is included in the liquid feed circulating in the flow path, and only the one surface on which the ligand of the plasmon excitation sensor is immobilized is the liquid feed.
  • a mode in which the plasmon excitation sensor and the specimen are brought into contact with each other in a state of being immersed therein is preferable.
  • the above-mentioned “flow path” is a rectangular tube or cylinder (tube) that can efficiently deliver a small amount of drug solution and can change or circulate the solution feeding speed in order to promote the reaction. ). Further, as the shape of this flow path, the vicinity of the place where the plasmon excitation sensor is installed preferably has a rectangular tube structure, and the vicinity of the place where the drug solution is delivered preferably has a cylindrical (tube) shape.
  • the material is a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material in the plasmon excitation sensor part; a light-transmitting material such as polyolefin such as polyethylene, and a silicone rubber, Teflon ( (Registered trademark), polyethylene, polypropylene, and other polymers are used.
  • a light-transmitting material such as polyolefin such as polyethylene, and a silicone rubber, Teflon ( (Registered trademark), polyethylene, polypropylene, and other polymers are used.
  • the channel structure is not necessarily made of a light-transmitting material unless the channel is kept in a fixed shape during fluorescence measurement and the detection of fluorescence generated by plasmon excitation is not hindered.
  • other parts may be partially or entirely made of a chemically stable material other than the light transmissive material.
  • the surface of the plasmon excitation sensor on which the metal thin film (or preferably SAM) exists is defined as the bottom surface, for example, the surface is opposed to the bottom surface.
  • the ceiling surface may be made of a light transmissive material, and the side surface may be made of a chemically stable material other than the light transmissive material.
  • the other parts for example, the side surfaces are not necessarily rigid bodies as long as a certain shape is maintained at the time of fluorescence measurement, and may have an appropriate elasticity to ensure a sealing property.
  • the ceiling surface may be composed of polymethyl methacrylate [PMMA] and the side surface may be composed of silicone rubber.
  • the vertical and horizontal sections of the channel of the plasmon excitation sensor unit are independently about 100 nm to 1 mm.
  • the position of the liquid feeding inlet for introducing the liquid feeding from the drug delivery section to the plasmon excitation sensor section and the position of the liquid feeding outlet for discharging the liquid feeding from the plasmon excitation sensor section are both obstructing the fluorescence measurement. Unless it becomes, it will not specifically limit.
  • the flow path of the plasmon excitation sensor section has a rectangular parallelepiped structure, it is convenient to prepare the flow path for the liquid feed inlet and the liquid feed outlet on the ceiling surface. Either one or both of the liquid discharge ports may be provided on the side surface.
  • the method for fixing the plasmon excitation sensor to the flow path is not particularly limited as long as the flow path is maintained in a certain shape and the fluorescence measurement is not hindered.
  • a silicone rubber sheet or O-ring having a certain thickness is formed on the surface on which the metal thin film of the plasmon excitation sensor is formed. Is formed by placing a light-transmitting top plate (for example, a PMMA substrate) provided with a liquid feeding inlet and a liquid feeding outlet thereon. Examples include a method of forming a ceiling surface of a road, and then crimping and fixing them with an appropriate fastener.
  • a silicone rubber sheet having an appropriate thickness and having a hole having an arbitrary shape and size is used as a material constituting the side surface structure, the inner periphery of the hole has a plasmon. Since it becomes the side structure of the flow path of an excitation sensor part, since the flow path which has a required shape and a size can be formed easily, it is preferable.
  • a perforated polydimethylsiloxane (PDMS) sheet having a flow path height of 0.5 mm is first formed on the surface on which the metal thin film of the plasmon excitation sensor is formed.
  • a PMMA substrate on which a liquid feeding inlet and a liquid feeding outlet are provided in advance is placed on the polydimethylsiloxane [PDMS] sheet, and then the PMMA substrate and the PMMA substrate are placed on the polydimethylsiloxane [PDMS] sheet.
  • a method in which a polydimethylsiloxane [PDMS] sheet and the plasmon excitation sensor are pressure-bonded and fixed with a fastener such as a screw is preferred.
  • a silicone rubber sheet or O-ring and a light-transmitting top plate are pressure-bonded and fixed to the plasmon excitation sensor, an appropriate spacer made of a material such as silicone rubber or stainless steel is attached as necessary. You may use together.
  • a gold substrate is directly formed on a plastic integrally molded product or a separately prepared gold substrate is fixed. Then, after forming a layer made of a polymer based on gold colloid (preferably SAM) and immobilizing the ligand on the gold surface, the lid is covered with an integrally molded plastic product corresponding to the top plate of the flow path. Can be manufactured. If necessary, the prism can be integrated into the flow path.
  • SAM gold colloid
  • Such “liquid feeding” is preferably the same as the solvent or buffer in which the specimen is diluted, and examples thereof include phosphate buffered saline [PBS] and Tris buffered saline [TBS]. It is not particularly limited.
  • the temperature and time for circulating the liquid supply vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. ⁇ 1 to 60 minutes, preferably 37 ° C. ⁇ 5 to 15 minutes.
  • the initial concentration of the analyte contained in the specimen being sent may be 100 ⁇ g / mL to 0.0001 pg / mL.
  • the total amount of liquid fed, that is, the volume of the flow path is usually 0.0001 to 20 mL, preferably 0.01 to 1 mL.
  • the flow rate of the liquid feeding is usually 1 to 2,000 ⁇ L / min, preferably 5 to 500 ⁇ L / min.
  • the cleaning step is a step of cleaning at least one of the surface of the plasmon excitation sensor obtained through the step (a) and the surface of the plasmon excitation sensor obtained through the step (b) described later. This washing step is preferably included in at least one of before and after step (b).
  • washing solution used in the washing step for example, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer solution used in the reaction of steps (a) and (b), Those containing 00001 to 1% by weight are desirable.
  • the temperature and flow rate at which the cleaning liquid is circulated are preferably the same as the “temperature and flow rate at which the liquid feed is circulated” in step (a).
  • the time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.
  • Step (b) is a step of reacting the plasmon excitation sensor obtained through the step (a) with a conjugate of the second ligand and the fluorescent dye.
  • fluorescent dye is a general term for substances that emit fluorescence by irradiating predetermined excitation light in the present invention, or excited by using an electric field effect. Including luminescence.
  • the fluorescent dye used in the present invention is not particularly limited as long as it is not quenched due to light absorption by the metal thin film, and may be any known fluorescent dye.
  • fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred.
  • fluorescent dyes examples include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes. (Applied Biosystems Japan Co., Ltd.), Coumarin family fluorescent dye (Invitrogen Corp.), Rhodamine family fluorescent dye (GE Healthcare Bioscience Co., Ltd.), Cyanine family fluorescent dye, Indocarbocyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanide dye Millie's fluorescent dye, BODIPY® family fluorescent dye (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (Registered trademark) dye series (manufactured by Invitrogen Corp.) and the like, and further, U.S. Patent
  • Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.
  • the fluorescent dye is not limited to the organic fluorescent dye.
  • rare earth complex fluorescent dyes such as Eu and Tb can also be used as fluorescent dyes in the present invention.
  • rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there.
  • An example of a commercially available rare earth complex-based fluorescent dye is ATBTA-Eu 3+ .
  • a fluorescent dye having a maximum fluorescence wavelength in a wavelength region where light absorption by the metal contained in the metal thin film is small when performing fluorescence measurement described later For example, when gold is used as the metal thin film, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 600 nm or more in order to minimize the influence of light absorption by the gold thin film. Therefore, in this case, it is particularly desirable to use a fluorescent dye having a maximum fluorescence wavelength in the near infrared region, such as Cy5, Alexa Fluor (registered trademark) 647.
  • a fluorescent dye having the maximum fluorescence wavelength in the near-infrared region can minimize the influence of light absorption by iron derived from blood cell components in the blood. Is also useful.
  • a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more it is desirable to use.
  • the “conjugate comprising the second ligand and the fluorescent dye” is preferably an antibody capable of recognizing and binding to the analyte (target antigen) contained in the specimen when a secondary antibody is used as the ligand.
  • the second ligand is a ligand used for the purpose of labeling the analyte with a fluorescent dye, and may be the same as or different from the first ligand.
  • the primary antibody used as the first ligand is a polyclonal antibody
  • the secondary antibody used as the second ligand may be a monoclonal antibody or a polyclonal antibody, but the primary antibody is monoclonal.
  • the secondary antibody is preferably a monoclonal antibody that recognizes an epitope that the primary antibody does not recognize, or a polyclonal antibody.
  • a complex in which a second analyte that competes with an analyte (target antigen) contained in a specimen (competitive antigen; however, different from the target antigen) and a secondary antibody are bound in advance.
  • the embodiment to be used is also preferable. Such an embodiment is preferable because the amount of fluorescent signal (fluorescent signal) and the amount of target antigen can be proportional.
  • Carboxyimide [WSC] for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC] etc.
  • NHS N-hydroxysuccinimide
  • a method of immobilization a method of reacting and immobilizing a secondary antibody having a iodoacetamide and a thiol group and a fluorescent dye, respectively; a biotinylated fluorescent dye and a streptavidinized secondary antibody (or streptavidinized)
  • the concentration of the thus-prepared “conjugate of the second ligand and the fluorescent dye” during the feeding is preferably 0.001 to 10,000 ⁇ g / mL, and more preferably 1 to 1,000 ⁇ g / mL.
  • the temperature, time, and flow rate at which the liquid is circulated are the same as in step (a).
  • Step (c) The step (c) is a step of applying an external stimulus to which the stimulus-responsive polymer can respond to the plasmon excitation sensor obtained through the step (b).
  • the external stimulus is appropriately selected depending on the type of stimulus-responsive polymer used.
  • NIPAM poly-N-isopropylacrylamide
  • the phase transition temperature of NIPAM is around 30 ° C. Is equivalent to raising the temperature to At that time, the temperature of the liquid feeding is controlled using a plasmon excitation sensor and a temperature control device attached to the flow path.
  • polyacrylic acid which is a pH-responsive polymer
  • the phase transition pH of polyacrylic acid is around pH 4, so the pH is lowered to about 3.0 or less as an external stimulus. It corresponds to.
  • Step (d) refers to laser light from the surface opposite to the surface on which the metal thin film is formed of the transparent flat substrate of the plasmon excitation sensor obtained through the step (c) via a prism. , And the amount of fluorescence emitted from the excited fluorescent dye is measured.
  • the light source used in the assay method of the present invention is not particularly limited as long as it can cause plasmon excitation in a metal thin film, but in terms of unity of wavelength distribution and intensity of light energy, laser light is used. Is preferably used as the light source. It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through the optical filter.
  • the surface plasmon is generated on the surface of the metal thin film by the laser light irradiation under the total reflection attenuation condition [ATR]. Due to the electric field enhancement effect of surface plasmons, the fluorescent dye is excited by photons that are increased by several tens to several hundred times the amount of photons irradiated. Note that the amount of photon increase due to the electric field enhancement effect depends on the refractive index of the transparent flat substrate, the metal species and the film thickness of the metal thin film, but is usually about 10 to 20 times that of gold.
  • the fluorescent dye In the fluorescent dye, electrons in the molecule are excited by light absorption, move to the first electron excited state in a short time, and when returning from this state (level) to the ground state, the wavelength of the wavelength corresponding to the energy difference Fluoresce.
  • laser light examples include a semiconductor having a wavelength of 200 to 900 nm, an LD of 0.001 to 1,000 mW, a wavelength of 230 to 800 nm (resonance wavelength is determined by the metal species used in the metal thin film), and a semiconductor of 0.01 to 100 mW. A laser etc. are mentioned.
  • the “prism” is intended to allow laser light through various filters to efficiently enter the plasmon excitation sensor, and preferably has the same refractive index as that of the transparent flat substrate.
  • various prisms for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape.
  • a 60-degree dispersion prism may be used.
  • Examples of such commercially available prisms include those similar to the above-mentioned commercially available “glass-made transparent flat substrate”.
  • optical filter examples include a neutral density [ND] filter and a diaphragm lens.
  • the “darkening (ND) filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.
  • the “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons.
  • Cut filters are: external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattering light (excitation light originated from plasmon A filter that removes various types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor), autofluorescence of the enzyme fluorescent substrate, and examples thereof include interference filters and color filters. It is done.
  • the “condensing lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system.
  • a simple condensing system a commercially available objective lens (for example, manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be used.
  • the magnification of the objective lens is preferably 10 to 100 times.
  • the “SPFS detector” is preferably a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics) from the viewpoint of ultra-high sensitivity. Also, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed.
  • Step (e) is a step of calculating the amount of analyte contained in the sample from the measurement result obtained in step (d).
  • the analyte is a molecule or molecular fragment capable of specifically recognizing (or recognizing and binding) the first ligand immobilized on the polymer, and the “molecule” or “molecular fragment”
  • nucleic acids include, for example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., which may be single-stranded or double-stranded, or nucleosides, nucleotides and modified molecules thereof).
  • Proteins polypeptides, oligopeptides, etc.
  • amino acids including modified amino acids
  • carbohydrates oligosaccharides, polysaccharides, sugar chains, etc.
  • lipids or their modified molecules, complexes, etc.
  • it may be an oncofetal antigen such as AFP [ ⁇ -fetoprotein], a tumor marker, a signal transmitter, a hormone, etc.
  • the apparatus of the present invention includes at least the plasmon excitation sensor and is used for the assay method, and is for carrying out the assay method of the present invention.
  • the “device” includes, in addition to the plasmon excitation sensor, for example, a light source, various optical filters, a prism, a cut filter, a condensing lens, a surface plasmon excitation enhanced fluorescence detection unit, a temperature change unit (heater), and the like. It is preferable to have a liquid feeding system combined with a plasmon excitation sensor when handling sample liquid, washing liquid, labeled antibody liquid, and the like.
  • the type of liquid feeding system is not limited as long as the object of the present invention can be achieved. For example, a microchannel device connected to a liquid pump may be used.
  • the surface plasmon resonance [SPR] detector that is, the angle variable unit for adjusting the optimum angle of the photodiode, SPR and SPFS as a light receiving sensor dedicated to SPR (in order to obtain the total reflection attenuation [ATR] condition by the servomotor)
  • the angle of 45 to 85 ° can be changed by synchronizing the photodiode and the light source, and the resolution is preferably 0.01 ° or more.)
  • the information input to the surface plasmon excitation enhanced fluorescence detector is processed.
  • a computer for the purpose may also be included.
  • liquid feed pump for example, a micro pump suitable for a small amount of liquid feed, a syringe pump with high feed accuracy and low pulsation, which is preferable but cannot be circulated, a simple and excellent handleability but a small amount of liquid feed
  • a tube pump may be difficult.
  • the kit of the present invention comprises at least a transparent flat substrate; a metal thin film formed on one surface of the substrate; and a stimulus-responsive polymer formed on the other surface of the thin film that is not in contact with the substrate. And a plasmon excitation sensor substrate including the layer to be used. The substrate is used in the assay method.
  • the kit of the present invention except for the substrate for the plasmon excitation sensor, except for the first ligand, the second ligand and the sample, for example, 1 All necessary substances except ligands such as secondary antibodies and antigens (that is, the analyte contained in the sample is not limited to antigens and may be antibodies), and samples and secondary antibodies. It is preferable to include.
  • ligands such as secondary antibodies and antigens (that is, the analyte contained in the sample is not limited to antigens and may be antibodies), and samples and secondary antibodies. It is preferable to include.
  • the kit of the present invention blood or serum as a specimen, and an antibody against a specific tumor marker, the content of the specific tumor marker can be detected with high sensitivity and high accuracy. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.
  • a preclinical noninvasive cancer carcinoma in situ
  • such a “kit” includes the plasmon excitation sensor substrate; a carboxyalkanethiol having about 4 to 20 carbon atoms for forming the SAM; a fluorescent dye; a dissolution for dissolving or diluting the specimen.
  • Liquid or diluent various reaction reagents and washing reagents for reacting a plasmon excitation sensor with a specimen, and various equipment or materials necessary for carrying out the assay method of the present invention and the above-mentioned “device” It can also be included.
  • kit element a standard material for preparing a calibration curve, a manual, a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of samples may be included.
  • the unreacted antibody and the unreacted enzyme were purified using a molecular weight cut filter (manufactured by Nihon Millipore) to obtain an Alexa Fluor (registered trademark) 647-labeled anti-AFP monoclonal antibody solution.
  • the obtained gold substrate was set on a spin coater so that the surface on which the gold thin film was formed was exposed, and 100 ⁇ L of poly N-isopropylacrylamide ethanol solution (1 mg / mL) was dropped near the center of the substrate, and 1,000 rpm Rotate for 1 minute.
  • an anti- ⁇ -fetoprotein [AFP] monoclonal antibody (1D5; 2.5 mg / mL, manufactured by Japan Medical Laboratory) was diluted to 20 ⁇ g / mL with 10 mM acetate buffer (pH 5.0). The resulting solution is flowed at 10 ⁇ L / min for 10 minutes to immobilize the antibody on carboxymethyldextran.
  • a plasmon excitation sensor using a temperature-responsive polymer as a stimulus-responsive polymer was subjected to blocking treatment by flowing an aqueous solution of 1M ethanolamine hydrochloride (manufactured by SIGMA; pH 8.5) at 10 ⁇ L / min for 10 minutes. Is made.
  • the plasmon excitation sensor obtained in Production Example 3 uses a pH responsive polymer as a stimulus responsive polymer.
  • ⁇ -fetoprotein AFP; 2.0 mg / mL, manufactured by Acris Antibodies GmbH
  • AFP 2.0 mg / mL
  • PBS buffer pH 7.4
  • the diluted solution is contacted by flowing at 10 ⁇ L / min for 20 minutes.
  • a diluted solution prepared by adjusting the labeled secondary antibody obtained in Preparation Example 1 to 2.5 ⁇ g / mL with 1% BSA PBS buffer (pH 7.4) was further added at 10 ⁇ L / min. React by flowing for 20 minutes.
  • a TBS solution pH 7.4 containing 0.005% Tween 20 is flowed at 10 ⁇ L / min for 10 minutes.
  • an external stimulus is applied by changing the temperature setting from 25 ° C. to 37 ° C. by the temperature control device attached to the plasmon excitation sensor and the flow path.
  • steps (d) and (e) laser light is irradiated via a prism, and a fluorescence signal is measured from a CCD camera attached to the apparatus.
  • a fluorescence signal measures SPFS signal value when it observes from CCD 1 minute after adding external stimulus.
  • Example 2 In Example 1, the plasmon excitation sensor used in step (a) was obtained in Preparation Example 3 instead of that obtained in Preparation Example 2, and 10 mM glycine was used as an external stimulus in Step (c) instead of temperature change.
  • the assay method is carried out in the same manner as in Example 1 except that a pH change of flowing an aqueous hydrochloric acid solution (pH 3.0) at 10 ⁇ L / min for 10 minutes is performed, and the fluorescence signal is measured.
  • Example 1 the step (c) is not performed, that is, the assay method is performed in the same manner as in Example 1 except that all the steps of Example 1 are performed at 25 ° C., and the fluorescence signal is measured.
  • Example 2 the assay method is carried out in the same manner as in Example 1 except that step (c) is not carried out and all steps are carried out at 37 ° C., and the fluorescence signal is measured.
  • Example 3 The assay method is performed in the same manner as in Example 2 except that step (c) is not performed in Example 2, that is, all steps of Example 2 are performed at pH 7.4, and the fluorescence signal is measured.
  • Example 2 the assay method is carried out in the same manner as in Example 2 except that step (c) is not carried out and all steps are carried out at pH 3.0, and the fluorescence signal is measured.
  • Table 2 shows the predicted results.
  • the assay method of the present invention can be detected with high sensitivity and high accuracy, for example, even a very small amount of tumor marker contained in blood can be detected.
  • the presence of pre-clinical non-invasive cancer (carcinoma in situ) that cannot be detected by such methods can also be predicted with high accuracy.

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Abstract

L'invention porte sur un procédé d'essai hautement sensible et hautement précis, par lequel non seulement un signal SPR mais encore un signal de fluorescence en SPFS peut être amélioré ; sur un dispositif pour l'essai, et sur un coffret pour l'essai. De façon spécifique, l'invention porte sur un procédé d'essai caractérisé par le fait qu'il comprend les étapes suivantes (a) à (e) : (a) une étape de mise en contact d'un échantillon avec un détecteur à excitation de plasmons spécifique ; (b) une étape de réaction du détecteur de l'étape (a) avec un conjugué d'un second ligand avec un colorant fluorescent ; (c) une étape d'application d'un stimulus externe au détecteur de l'étape (b) ; (d) une étape d'irradiation de la surface, sur le côté inverse de la surface sur laquelle est formé un film métallique, d'un substrat plat transparent dans le détecteur de l'étape (c) avec une lumière laser à travers un prisme, et de mesure de la quantité de la fluorescence émise par le colorant fluorescent ainsi excité ; et (e) une étape de calcul de la quantité d'un analyte contenu dans ledit échantillon sur la base des résultats de mesure obtenus à l'étape (d).
PCT/JP2010/057163 2009-04-24 2010-04-22 Procédé d'essai utilisant un détecteur à excitation de plasmons comprenant un polymère sensible au stimulus WO2010123073A1 (fr)

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US10371704B2 (en) 2013-12-27 2019-08-06 Konica Minolta, Inc. Method for discriminating between prostate cancer and a benign prostate disease
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US10527615B2 (en) 2012-04-27 2020-01-07 Konica Monolta, Inc. Antigen detection method which uses lectin and comprises enzyme treatment step
US10222385B2 (en) 2013-12-27 2019-03-05 Konica Minolta, Inc. Methods for discriminating between prostate cancer and a benign prostate-disease
US10371704B2 (en) 2013-12-27 2019-08-06 Konica Minolta, Inc. Method for discriminating between prostate cancer and a benign prostate disease
CN108931647A (zh) * 2018-07-06 2018-12-04 深圳信息职业技术学院 光纤免疫传感器、检测装置及光纤免疫传感器的制作方法

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