WO2022250055A1 - ナノ粒子体、その製造方法およびナノ粒子体を含むナノ粒子体組成物 - Google Patents
ナノ粒子体、その製造方法およびナノ粒子体を含むナノ粒子体組成物 Download PDFInfo
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/54346—Nanoparticles
-
- 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
Definitions
- the present invention relates to nanoparticles (especially nanoparticles used for plasmon-excited fluorescence analysis), methods for producing the same, and nanoparticle compositions containing the nanoparticles (especially nanoparticles used for plasmon-excited fluorescence analysis).
- a biosensor specifically reacts a specific test substance to be detected with a specific specific binding substance to form a complex, and detects the test substance by a signal derived from the specific binding in the complex.
- the complex further includes, for example, a fluorescent substance and a metal particle in addition to the test substance and the specific binding substance (the complex includes, for example, a metal particle, a specific binding substance, a fluorescent substance, and a test substance including).
- the complex is irradiated with excitation light, surface plasmon resonance occurs in the metal particles in the complex, forming a near-field near the surface of the metal particles. This near field increases the fluorescence intensity of the fluorescent substance.
- the composite particles for immunochromatography described in Patent Document 1 have a structure in which the outside of fine particles made of metal is covered with at least one layer of silica containing at least one fluorescent substance, and specifically recognizes a target substance. It consists of fine particles surface-modified with a labeling substance.
- the surface of the metal particle is covered with a silica layer, and the fluorescent substance is fixed to the silica layer, so that the fluorescent substance excited by the near field formed by surface plasmon resonance comes into contact with the metal particle. prevent this from happening. This suppresses deactivation (quenching) of the excited fluorescent substance.
- the main object of the present invention is to provide a nanoparticle body with superior detection stability by forming a stably immobilized thin film on the surface of a metal particle. More specifically, the main object of the present invention is to provide nanoparticles that reliably capture a test substance, sufficiently suppress the quenching of a fluorescent substance, and reduce the distance between metal particles in a complex, resulting in superior detection sensitivity. It is to provide the body.
- Nanoparticles according to one embodiment of the present invention, It contains metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a nano-sized specific binding substance bound to the surface of the polymer film that specifically binds to the test substance in the specimen.
- the polymer film contains at least one selected from the group consisting of a bonding site via a sulfur atom, a positively charged group, and a hydrophobic group with the surface of the metal nanoparticles.
- FIG. 1 is a cross-sectional view schematically showing a nanoparticle body according to the first embodiment.
- FIG. 2 is an enlarged cross-sectional view of part A in FIG.
- FIG. 3 is a cross-sectional view schematically showing the nanoparticle composition according to the second embodiment.
- FIG. 4 is a cross-sectional view schematically showing a composite according to the third embodiment.
- FIG. 5 is a cross-sectional view schematically showing a composite according to a modification of the third embodiment.
- FIG. 6 is a diagram schematically showing a measuring device according to the fourth embodiment.
- FIG. 7 is a schematic diagram illustrating a method for producing a polymer film.
- FIG. 8 is a schematic diagram illustrating a method for producing a polymer film.
- FIG. 9 shows a scanning electron microscope (SEM) image of metal nanoparticles coated with a polymer film.
- the metal nanoparticles are silver nanoparticles (particle diameter: 80 nm) in FIG. 9(a) and gold nanoparticles (particle diameter: 20 nm) in FIG. 9(b).
- FIG. 10 is a diagram showing a zeta potential graph of metal nanoparticles coated with a polymer film.
- the metal nanoparticles are silver nanoparticles (particle diameter: 80 nm) in FIG. 10(a) and gold nanoparticles (particle diameter: 20 nm) in FIG. 10(b).
- FIG. 11 is a diagram showing a zeta potential graph of metal nanoparticles not coated with a polymer film.
- the metal nanoparticles are silver nanoparticles (particle diameter: 80 nm) in FIG. 11(a) and gold nanoparticles (particle diameter: 20 nm) in FIG. 11(b).
- FIG. 12 is a diagram showing fluorescence spectra of a fluorescent substance-gold nanoparticle mixed system.
- the gold nanoparticles are coated with a polymer film in FIG. 12(a) and are not coated with a polymer film in FIG. 12(b).
- 13 is a cross-sectional view schematically showing the nanoparticle body of Example 1.
- FIG. FIG. 14 shows the fluorescence spectrum of the test substance-nanoparticle system.
- FIG. 15 shows an SEM image of a composite prepared using the nanoparticles of Example 2.
- FIG. 16 is a schematic cross-sectional view showing nanoparticles in the nanoparticle composition of Example 3.
- FIG. 17 is a graph showing the absorption spectrum of the nanoparticulate composition of Example 3.
- FIG. 18 is a graph showing the fluorescence spectrum of the nanoparticulate composition of Example 3.
- FIG. 19A and 19B are schematic diagrams for explaining a method for producing a nanoparticle body of Example 4.
- FIG. 20 is a graph showing the absorption spectrum and fluorescence spectrum of the nanoparticulate composition of Example 4.
- FIG. 21 is a diagram showing the frequency distribution of sizes in Example 5.
- FIG. 22 is a diagram showing the frequency distribution of the zeta potential of Example 5.
- FIG. 23 is a schematic diagram showing the relationship between the number of washing treatments and the form of aggregates in Example 5.
- FIG. 24 is a cross-sectional view schematically showing a nanoparticle body according to the fourth embodiment.
- FIG. 25 is an enlarged schematic diagram of part A in FIG.
- FIG. 26 is a reaction scheme showing an example of a method for producing nanoparticles according to the fifth embodiment.
- FIG. 27 is a cross-sectional view schematically showing a composite according to the sixth embodiment.
- FIG. 28 is a diagram schematically showing a measuring device according to the seventh embodiment.
- FIG. 29 is a schematic diagram for explaining the method for producing nanoparticles of Example 6.
- FIG. 30 is a diagram showing the fluorescence spectrum of the nanoparticles of Example 6.
- FIG. 31 shows an SEM image of a composite prepared using the nanoparticles of Example 7.
- the nanoparticle body, the composite, and the measuring device which are embodiments of the present invention, will be described in detail below with reference to the illustrated embodiments.
- the drawings are schematic and may not reflect actual dimensions or proportions.
- the nanoparticles according to the first embodiment are It comprises metal nanoparticles, a polymer film that coats the surface of the metal nanoparticles, and a nano-sized specific binding substance bound to the surface of the polymer film that specifically binds to the test substance in the specimen.
- the polymer film contains at least one selected from the group consisting of a bonding site via a sulfur atom, a positively charged group, and a hydrophobic group with the surface of the metal nanoparticles.
- the nanoparticle body according to this embodiment comprises metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a specific binding substance bound to the polymer film.
- the nanoparticles according to this embodiment are dissolved or dispersed in a sample, and the test substance contained in the sample is captured to form a complex (second embodiment). More specifically, the complex is formed by specific binding between the specific binding substance of the nanoparticles and the test substance.
- the complex has a structure in which two nanoparticles are bound via the test substance. Thus, two metal nanoparticles are spaced apart by a certain distance by binding to the same test substance with their respective specific binding substances in a complex. Additionally, the conjugate includes a fluorescent substance.
- SPFS surface plasmon excitation enhanced fluorescence spectroscopy
- LSPR localized surface plasmon resonance
- a test substance in a specimen can be detected by measuring fluorescence intensity.
- the nanoparticles according to this embodiment are superior in detection stability. Although not bound by a specific theory, the reason is presumed as follows.
- the nanoparticle body according to the present embodiment includes metal nanoparticles and a polymer film that coats the surface of the metal nanoparticles, and the polymer film and the surface of the metal nanoparticles have sulfur atoms interposed therebetween. binding sites, positively charged, and hydrophobic groups. Therefore, the polymer film forms a relatively strong bond with the surface of the metal nanoparticles, and is stably fixed on the surfaces of the metal nanoparticles.
- FIG. 1 is a cross-sectional view schematically showing a nanoparticle body.
- a nanoparticle body 1 according to the present embodiment includes metal nanoparticles 2, a polymer film 3 covering the surface of the metal nanoparticles 2, and a specific binding substance 4 bound to the surface of the polymer film 3. Become.
- the nanoparticle body 1 can be used for plasmon-excited fluorescence analysis.
- the nanoparticulate bodies 1 can be used for surface plasmon excitation enhanced fluorescence spectroimmunoassay.
- the nanoparticles 1 can capture the test substance in the subject and form a complex containing two nanoparticles 1 and the test substance. When the complex is irradiated with excitation light, it causes localized surface plasmon resonance and forms a near-field. This near field increases the fluorescence intensity.
- the nanoparticle body 1 may also block non-specific binding sites with a blocking agent.
- the blocked nanoparticle body 1 suppresses the formation of non-specific binding of the specific binding substance 4 to substances other than the detection target (i.e., substances other than the test substance), thereby reducing background and false positive signals. , the signal-to-noise ratio (SNR) can be improved.
- Blocking agents include, for example, proteins such as bovine serum albumin (BSA), skimmed milk, and casein, and chemically synthesized polymers.
- the dispersion liquid of the nanoparticles 1 may further contain a dispersant for the purpose of improving the dispersibility of the nanoparticles 1.
- a dispersant include, for example, sodium heparin. This point will be described in detail in the nanoparticle composition according to the second embodiment.
- the metal nanoparticles 2, the polymer film 3, the specific binding substance 4, and the fluorescent substance that constitute the nanoparticle body 1 will be described below.
- Metal nanoparticles The surface of the metal nanoparticles 2 is covered with a polymer film 3 .
- the metal nanoparticles 2 interact with light having a specific wavelength, depending on the type of metal, and cause localized surface plasmon resonance.
- the plasmon resonance peaks are from 400 nm to 530 nm for silver nanoparticles and from 510 nm to 580 nm for gold nanoparticles. This depends on the particle size. For example, silver nanoparticles with a particle size of 20 nm resonate with light of a wavelength of 405 nm, and gold nanoparticles with a particle size of 20 nm resonate with light with a wavelength of 524 nm.
- the polymer film 3 covers the surfaces of the metal nanoparticles 2 .
- the polymer film 3 functions as a metal quenching molecular film.
- the polymer film 3 can arrange the fluorescent substance away from the surface of the metal nanoparticles 2 by at least the thickness of the polymer film 3 . Therefore, it is possible to suppress the quenching of the excited fluorescent substance by contacting the surface of the metal nanoparticles 2, thereby suppressing the decrease in detection sensitivity.
- the presence of the polymer film 3 can be confirmed by taking an image of the nanoparticle 1 using SEM or TEM and observing the nanoparticle 1 in the image.
- the polymer membrane 3 will be described with reference to FIG.
- FIG. 2 is an enlarged view of part A in FIG. 1, and is an enlarged cross-sectional view of the vicinity of the interface between the polymer film 3 of the nanoparticle body 1 and the surface of the metal nanoparticles 2.
- FIG. The polymer film 3 contains at least one selected from the group consisting of bonding sites 3a with the surfaces of the metal nanoparticles 2 via sulfur atoms, positively charged groups 3b, and hydrophobic groups 3c. More specifically, the polymer film 3 has bonding sites 3a via sulfur atoms with the surfaces of the metal nanoparticles 2, and primary ammonium groups (—NH 3+ ) as positively chargeable groups 3b. , hydrophobic group 3c.
- the binding site 3a binds between the surface of the metal nanoparticle 2 and the polymer film 3 via sulfur atoms.
- the positively chargeable group 3 b forms an electrostatic bond (ionic bond) b with the surface of the negatively chargeable metal nanoparticle 2 .
- Hydrophobic group 3c forms hydrophobic bond c with the surface of metal nanoparticle 2 .
- the polymer film 3 Since all of the above three bonds are relatively strong bonds with the surfaces of the metal nanoparticles 2, the polymer film 3 is stably attached to the surfaces of the metal nanoparticles 2 by at least one of the above three bonds. Fixed. As a result, peeling of the polymer film 3 from the surface of the metal nanoparticles 2 is prevented. As a result, detachment of the specific binding substance 4 due to detachment of the polymer film 3 or the like is suppressed, and a decrease in detection sensitivity is suppressed. In addition, exposure of the surface of the metal nanoparticles 2 due to peeling of the polymer film 3 is suppressed, quenching due to contact with an excited fluorescent substance is suppressed, and a decrease in detection sensitivity is suppressed.
- the polymer film 3 contains the polymer 3A, it is easier to chemically modify than the silica layer, and the need for surface modification and the like is low. As a result, the film thickness can be made smaller than that of the silica layer, and the distance between the metal nanoparticles 2 in the composite can be reduced. Therefore, a near-field is formed more efficiently, and detection sensitivity can be improved. As described above, the nanoparticles according to the present embodiment are superior in detection stability.
- the polymer 3A that constitutes the polymer film 3 is bound to the surface of the metal nanoparticle 2 by a bonding site 3a via a sulfur atom, a positively charged group 3b, and a hydrophobic group 3c. at least one selected from the group consisting of The presence of the binding site 3a via the sulfur atom, the positively charged group 3b, and the hydrophobic group 3c is confirmed by measuring the signals derived therefrom using infrared spectroscopy and nuclear magnetic resonance spectroscopy. can do.
- the polymer 3A constituting the polymer film 3 can have a site containing a disulfide bond (--S--S--) as a side chain.
- the site containing the disulfide bond can have a positively charged group 3b. Also, the site containing the disulfide bond can have a hydrophobic group 3c.
- the binding site 3a via the sulfur atom, the positively charged group 3b and the hydrophobic group 3c are described below.
- the binding sites 3a via sulfur atoms are formed, for example, by mixing metal nanoparticles 2 with a polymer having a site containing a disulfide bond as a side chain.
- a polymer having a site containing a disulfide bond as a side chain For example, when the polymer as a raw material has a hydrophobic group 3c via a disulfide bond in a side chain as shown in FIG. A site is formed (see right side in FIGS. 2 and 8).
- a positively charged group forms a strong electrostatic bond with the surface of the metal nanoparticles 2 .
- a positively charged group as used herein, is a group that has a valence of one or more and is completely positively ionized.
- the positively chargeable groups 3b are represented by the following formula (1):
- pKa is an electrically neutral group contained in the polymer 3A constituting the polymer film 3
- a positively charged group more specifically, primary ammonium group (—NH 3+ ) etc.
- an electrically neutral group more specifically, a primary amino group (—NH 2 ) etc.
- pH indicates the pH of the environment (more specifically, the specimen, etc.) in which the test substance is detected
- B indicates the electrically neutral group contained in the polymer 3A
- BH + indicates the positively charged property contained in the polymer 3A.
- group 3b A group represented by and having a pKa of 7 or more. That is, the positively charged group 3b is an environment in which the electrically neutral group of the polymer 3A constituting the polymer membrane 3 and the positively charged group 3b detect the test substance (for example, a sample with a pH of approximately 6 to 8). in which the following chemical equilibrium equation (2): A group in which the concentration of positively charged groups ([BH + ]) is at least 10 times greater than the concentration of electrically neutral groups ([B]) when an equilibrium state represented by .
- hydrophobic group 3c is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups and aliphatic chain groups.
- aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups.
- Aromatic carbocyclic groups are groups that do not contain aromatic heterocycles but contain aromatic rings in which the ring members are all carbon atoms.
- aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups and the like) and arylalkyl groups (more specifically, benzyl groups and the like).
- An aromatic heterocyclic group is a group containing an aromatic ring in which at least one of the ring member atoms is a heteroatom (more specifically, an oxygen atom, a sulfur atom, a nitrogen atom, etc.).
- aromatic heterocyclic groups include nitrogen-containing aromatic heterocyclic groups (more specifically, imidazoyl groups and pyridyl groups (pyridinyl groups), etc.), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups. groups.
- An aliphatic cyclic group is a group containing a cyclic group consisting of a non-aromatic ring without containing an aromatic ring.
- Aliphatic cyclic groups include, for example, aliphatic carbocyclic groups and aliphatic heterocyclic groups.
- Aliphatic carbocyclic groups are groups containing a non-aromatic ring in which the ring members are all carbon atoms, and include, for example, cycloalkyl groups.
- Aliphatic heterocyclic groups are groups containing a non-aromatic ring in which at least one of the ring member atoms is a heteroatom.
- An aliphatic chain group is a chain (more specifically, linear and branched) group that does not contain an aromatic ring or a non-aromatic ring.
- Aliphatic chain groups include, for example, aliphatic carbon chain groups (more specifically, alkyl groups, alkylene groups, etc.) and aliphatic heterochain groups.
- the polymer 3A that constitutes the polymer film 3 can form a hydrophobic bond between the hydrophobic group 3c that the polymer 3A can have and the surface of the metal nanoparticle 2.
- the polymer 3A constituting the polymer membrane 3 can also form other hydrophobic bonds.
- a hydrophobic group bonded to the surface of the metal nanoparticles 2 via a sulfur atom more specifically, a pyridyl group (pyridinyl group) bonded to the surface of the metal nanoparticles 2 in FIG. 8 via a sulfur atom, etc.
- a hydrophobic group more specifically, an alkylene group possessed by the polymer 3A in FIG. 8) 3c that the polymer 3A constituting the polymer membrane 3 may have.
- Hydrophobic groups bonded to the surfaces of the metal nanoparticles 2 via sulfur atoms are formed as follows.
- the binding site through the sulfur atom can be formed, for example, by mixing the metal nanoparticles 2 with a polymer having a hydrophobic group 3c in the side chain through a disulfide bond, as described above.
- the hydrophobic group 3c bound to the sulfur atom also binds to the surface of the metal nanoparticle 2. In this way, hydrophobic groups are formed that bond to the surfaces of the metal nanoparticles 2 via sulfur atoms.
- the polymer 3A that constitutes the polymer film 3 may form a bond through a sulfur atom via a site (linker portion) derived from a cross-linking agent.
- a cross-linking agent includes, for example, an amino group-sulfhydryl group cross-linking agent (more specifically, an NHS-maleimide group cross-linking agent, etc.).
- the film thickness of the polymer film 3 is preferably 1 nm to 50 nm, more preferably 1 nm to 10 nm.
- the separation distance (separation distance) is such that the near field is efficiently formed in the space between the two metal nanoparticles, so the detection sensitivity is further improved.
- the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance are arranged with a predetermined distance therebetween, so that the quenching of the fluorescent substance excited in the measurement is suppressed, and the detection sensitivity is further improved. improves.
- the separation distance (separation distance) is the minimum value (shortest distance) of the distance between the metal nanoparticle surfaces contained in the two nanoparticles that bind through the test substance in the complex.
- the specific binding substance 4 is a nano-sized (maximum size of 3 to 15 nm) substance that specifically binds to the test substance (described in the second embodiment) in the sample.
- Specific binding substances 4 include, for example, antibodies (hereinafter referred to as nanobodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA chains and RNA chains).
- nanobodies antibodies
- ligands ligands
- enzymes ligands
- nucleic acid chains more specifically, DNA chains and RNA chains.
- a nano-antibody as the specific binding substance 4 specifically binds to an antigen as a test substance at its tip (Antigen Binding Site) by an antigen-antibody reaction to form a complex.
- the ligand as the specific binding substance 4 forms a complex with the protein as the test substance by specific protein-ligand binding through the ligand-receptor reaction.
- the nucleic acid strand as the specific binding substance 4 forms a complementary nucleic acid strand pair (double strand) based on base pair complementarity.
- the enzyme as the specific binding substance 4 forms an enzyme-substrate complex with the substrate as the test substance at its active site (active center) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as hydrogen bonds and bonds resulting from intermolecular forces, hydrophobic interactions and charge interactions.
- Nanobodies are, for example, VHH (variable domain of heavy chain antibody) antibodies, Fab (Fragment Antigen Binding) antibodies, and variants thereof.
- VHH antibodies are single domain antibodies.
- a variant is an antibody in which a part of the amino acid sequence has been recombined or a substituent has been introduced within the range of specific binding to an antigen.
- Nanobodies are preferably VHH antibodies.
- the nanoantibody is a VHH antibody
- the VHH antibody has a relatively small volume, so the distance (separation distance) between the two metal nanoparticles 2 in the complex is narrowed, the near field is formed more efficiently, and the fluorescence intensity is increased. can be further increased.
- the molecular mass of the nanobody is preferably 60,000 Da or less, more preferably 30,000 Da or less, and even more preferably 20,000 Da or less.
- the relatively small volume of the Nanobodies reduces the separation distance in the complex and makes the near-field more efficient. can be formed to further increase fluorescence intensity.
- Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.
- the specific binding substance 4 may be directly bound to the polymer membrane 3, and a linker portion (more specifically, SM (PEG ) 6, etc.).
- Nanoparticle bodies 1 may further comprise a fluorescent substance.
- a fluorescent substance In this case, at least one of the surface of the polymer membrane 3 and the specific binding substance is labeled with the fluorescent substance.
- a fluorescent substance is excited by a near field formed by localized surface plasmon resonance and emits fluorescence.
- Fluorescent materials include, for example, complexes of metals such as europium and ruthenium (metal complexes), and the dyes of the Alexsa Fluor series® (Molecular Probes®).
- the fluorescent substance preferably has a large Stokes shift.
- the Stokes shift is the difference between the absorption peak wavelength (maximum excitation wavelength) in the absorption spectrum of the fluorescent substance and the fluorescence peak wavelength (maximum fluorescence wavelength) in the fluorescence spectrum.
- the Stokes shift of the fluorescent substance is large, the absorption spectrum and the fluorescence spectrum are less likely to overlap, the excitation light (scattered light) is less likely to enter the detected fluorescence, and more accurate fluorescence intensity can be measured.
- the fluorescence spectrum of the fluorescent substance be sharp.
- the fluorescence spectrum is sharp, it is difficult for the fluorescence spectrum to overlap with the absorption spectrum, so that excitation light (scattered light) is less likely to enter the fluorescence to be detected, and fluorescence intensity can be measured more accurately.
- Nanoparticle Composition is comprising a nanoparticulate body and a solvent containing the nanoparticulate body;
- the nanoparticle body includes a metal nanoparticle, a polymer film that coats the surface of the metal nanoparticle, and a specific substance that binds to the surface of the polymer film or the surface of the metal nanoparticle and specifically binds to the test substance in the specimen.
- Solvents include polyanionic polymers in addition to nanoparticles.
- the composite particles (nanoparticles) for immunochromatography described in Patent Document 1 have a structure in which the outside of fine particles (metal nanoparticles) made of metal is covered with at least one layer of silica containing at least one fluorescent substance. It consists of fine particles surface-modified with a labeling substance (specific binding substance) that specifically recognizes the target substance (test substance).
- the coating layer (coating film) covering the outside of the metal nanoparticles is surface-modified with a specific binding substance.
- the design of the nanoparticulate body may be modified as needed.
- the number of specific binding substances bound to the coated membrane is increased to sufficiently capture the desired test substance.
- the number of fluorescent substances bound to the coating film is increased. In those cases, as the number of specific binding substances and fluorescent substances increased, the dispersibility of the nanoparticles decreased, and in the worst case, the nanoparticles aggregated.
- the main object of the present embodiment is to provide a nanoparticle composition comprising nanoparticles with excellent dispersibility while maintaining design flexibility in addition to the above-described excellent detection sensitivity.
- the nanoparticulate composition according to this embodiment comprises a nanoparticulate and a solvent containing the nanoparticulate.
- the nanoparticle body has metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a specific binding substance that binds to the surface of the polymer film or the surface of the metal nanoparticles.
- a nanoparticle composition according to the present embodiment is added to a sample, and the test substance contained in the sample is captured to form a complex.
- the complex is formed by specific binding between the specific binding substance of the nanoparticles and the test substance.
- the complex has a structure in which two nanoparticles are bound via the test substance.
- two metal nanoparticles are spaced apart by a certain distance by binding to the same test substance with their respective specific binding substances in a complex.
- the complex includes a fluorescent substance.
- SPFS surface plasmon excitation enhanced fluorescence spectroscopy
- LSPR localized surface plasmon resonance
- a test substance in a specimen can be detected by measuring fluorescence intensity.
- the nanoparticle composition according to the present embodiment contains nanoparticles having excellent dispersibility while maintaining a degree of design freedom. Although not bound by a specific theory, the reason is presumed as follows.
- the nanoparticulate composition according to this embodiment comprises a nanoparticulate and a solvent containing the nanoparticulate.
- the nanoparticle body includes a metal nanoparticle, a polymer film covering the surface of the metal nanoparticle, and a specific substance that binds to the surface of the polymer film or the surface of the metal nanoparticle and specifically binds to the test substance in the sample. and a binding substance. Solvents include polyanionic polymers in addition to nanoparticles.
- the nanoparticles can form associations (aggregates) in which the individual particles are surrounded by the polyanionic polymer.
- the polyanionic polymer acts as a spacer between a plurality of nanoparticles and prevents aggregation due to contact between the nanoparticles.
- electrostatic repulsion acts between the aggregates to keep the nanoparticles away from each other.
- the nanoparticles can be dispersed singly in the nanoparticle composition according to the present embodiment. In other words, the nanoparticles are excellent in dispersibility in the nanoparticle composition according to this embodiment.
- the nanoparticles are not surface-modified to improve dispersibility. Therefore, there is room for modifying the surface of the nanoparticle body, and the degree of freedom in design can be maintained. From the above, it is considered that the nanoparticle composition according to the present embodiment contains nanoparticles having both a high degree of design freedom and excellent dispersibility.
- the structure of the nanoparticle composition will be described below.
- the nanoparticulate body composition comprises a nanoparticulate body and a solvent containing the nanoparticulate body.
- Solvents include polyanionic polymers in addition to nanoparticles. Solvents include, for example, aqueous solvents.
- the aqueous solvent contains at least water (more specifically, pure water). Examples of the aqueous solvent include water (pure water), a mixed solvent containing water and an organic solvent, and a solvent in which a salt component is dissolved (more specifically, a buffer solution or the like).
- the organic solvent is a water-miscible organic solvent, for example, alcohol (more specifically, methanol, ethanol, propanol, etc.), and at least selected from the group consisting of tetrahydrofuran, dimethylformamide, dimethylsulfoxide, etc. It is one type.
- a polyanionic polymer is a polymer having a plurality of anionic groups.
- anionic groups include at least one anionic group selected from the group consisting of carboxylate groups, sulfate groups, sulfonate groups, nitrate groups, phosphate groups, and borate groups. That is, the polyanionic polymer has at least one anionic group selected from the group consisting of carboxylate groups, sulfate groups, sulfonate groups, nitrate groups, phosphate groups, and borate groups.
- Polyanionic polymers include, for example, polyglutamic acid, heparin: , polyaspartic acid, polyacrylic acid and salts thereof, and DNA.
- the counter ions (counter cations) of the above salts include, for example, alkali metal cations (more specifically, Li + , Na + , K + , etc.) and alkaline earth metal cations (more specifically, , Mg 2+ and Ca 2+ ).
- Heparin has a sulfate group (-OSO 3 -X + ), a carboxylate group (-COO - X + ), and a sulfonate group (-SO 3 -X + ), where X + is a monovalent counter cation).
- polyglutamates examples include sodium salts: are mentioned.
- Polyglutamic acid sodium salt has carboxylic acid groups (COO ⁇ Na + ).
- the polyanionic polymer acts as a dispersing agent for the nanoparticles in the nanoparticle composition according to this embodiment.
- the function of the polyanionic polymer as a dispersant will be described with reference to FIG.
- FIG. 3 is a cross-sectional view schematically showing nanoparticles in the nanoparticle composition according to this embodiment.
- the polyanionic polymer 7 exists so as to surround individual particles of the nanoparticles 1 . That is, the nanoparticle composition contains nanoparticles 1 and associations (aggregates) 9 of polyanionic polymers 7 .
- the polyanionic polymer 7 binds to the polymer film 3 of the nanoparticle 1 by electrostatic interaction to form an aggregate 9 .
- the aggregate 9 behaves like one in the nanoparticle composition according to this embodiment.
- the polyanionic polymer 7 since the polyanionic polymer 7 exists so as to surround the nanoparticles 1, the polyanionic polymer 7 acts like a spacer between the nanoparticles 1, and the nanoparticles 1 are separated from each other. Prevents agglomeration on contact. Furthermore, since a plurality of anionic groups of the polyanionic polymer 7 are present on the outer surface of the aggregate 9, it is considered that an electric double layer is formed in the vicinity of the interface between the aggregate and the solvent. As a result, an electrostatic repulsive force (more specifically, an electric double-layer repulsive force) acts between the aggregates 9 to keep the nanoparticles 1 away from each other. As a result, the nanoparticles 1 can be dispersed singly in the nanoparticle composition according to the present embodiment. In other words, it is considered that the nanoparticles 1 are superior in dispersibility in the nanoparticle composition according to the present embodiment.
- the existence of the aggregate 9 can be confirmed by measuring the zeta potential. Specifically, a process of washing the aggregate 9 in the nanoparticle composition with a solvent is performed multiple times, and the zeta potential is measured for each washing process.
- the existence of aggregate 9 is strongly suggested when the obtained frequency distribution of a plurality of zeta potentials exhibits behavior such that it increases from a negative value as the number of times of washing increases. Such behavior of the zeta potential is not bound by any particular theory, but is presumed to be due to the following reasons.
- the zeta potential shows a negative value due to the polyanionic polymer 7 forming the aggregate 9 before the washing treatment.
- the existence of aggregate 9 can also be confirmed by measuring its size. Specifically, the treatment of washing the aggregate 9 in the nanoparticle composition with a solvent is performed multiple times, and the size is measured for each washing treatment.
- the existence of the aggregate 9 is strongly suggested when the obtained frequency distribution of multiple sizes exhibits behavior such that it increases with an increase in the number of washings. Such size behavior is not bound by any particular theory, but is presumed to be due to the following reasons.
- the washing process is performed, the plurality of polyanionic polymers 7 forming the aggregates 9 are gradually dissociated from the aggregates 9 and dissolved in the washing solvent. Specifically, in the frequency distribution of sizes subjected to a large amount of washing treatment, the peak shifts to the larger size side.
- Such size behavior is not bound by any particular theory, but is presumed to be due to the following reasons. If the washing treatment is frequently performed, the dispersibility of the nanoparticles 1 is lowered. As a result, the nanoparticles 1 aggregate to form aggregates and increase in size.
- the content of the polyanionic polymer 7 is much larger than the content of the nanoparticles 1 in the nanoparticle composition according to the present embodiment (that is, excessive). This is because, in such a case, the nanoparticles 1 are likely to be surrounded by the polyanionic polymer 7 in the nanoparticle composition according to the present embodiment. Thereby, the dispersibility of the nanoparticles 1 is further improved.
- nanoparticles (nanoparticles) -Linker-
- the nanoparticle 1 can have a linker that binds to the polymer membrane 3 as long as the degree of freedom in designing the nanoparticle 1 is not significantly reduced.
- the specific binding substance 4 is not bound to the end of this linker.
- the linker acts like a spacer between the nanoparticles 1 (acts as a steric hindrance), causing the nanoparticles 1 to aggregate due to contact with each other. to prevent In such a case, the nanoparticles 1 are further improved in dispersibility in the nanoparticle composition according to the present embodiment.
- the linker contains, for example, at least one selected from the group consisting of polyalkylene ether chains and alkyl chains.
- the polyalkylene ether chain and alkyl chain may be part or all of the linker portion.
- Examples of polyalkylene ether chains include polyalkyleneoxy groups (more specifically, polyethyleneoxy groups and the like).
- the alkyl chain is, for example, an alkylene group (more specifically, -propylene group, n-butylene group, etc.).
- FIG. 4 is a cross-sectional view schematically showing the composite.
- a complex 40 comprises a test substance 30 to be detected and two nanoparticles 10 and 20 .
- Two nanoparticulate bodies 10 and 20 are bound via a test substance 30 in a composite 40 . That is, the nanoparticles 10 and 20 according to the first embodiment form the complex according to the second embodiment, which is bound via the test substance 30 .
- One of the two nanoparticle bodies 10 and 20 is called the first nanoparticle body 10 and the other nanoparticle body is called the second nanoparticle body 20 .
- composite 40 includes first nanoparticle 10 and second nanoparticle 20 as nanoparticle 1 .
- the first nanoparticle body 10 is composed of the first metal nanoparticles 12, the first polymer film 13 covering the surface of the first metal nanoparticles 12, and the first polymer film 13. and a first fluorescent substance 16 labeled on the first polymer film 13 . That is, the first nanoparticle body 10 includes a first metal nanoparticle 12 as a metal nanoparticle, a first polymer film 13 as a polymer film, and a first specific binding substance 14 as a specific binding substance. , the first fluorescent substance 16 is labeled on the first polymer film 13 .
- the second nanoparticle body 20 includes a second metal nanoparticle 22, a second polymer film 23 covering the surface of the second metal nanoparticle 22, and a second specific binding substance bound to the second polymer film 23. 24 and a second fluorescent substance 26 labeled on the second polymer film 23 . That is, the second nanoparticle body 20 includes a second metal nanoparticle 22 as a metal nanoparticle, a second polymer film 23 as a polymer film, and a second specific binding substance 24 as a specific binding substance. , the second fluorescent substance 26 is labeled on the second polymer film 23 .
- the separation distance L is small within a range in which the excited fluorescent substances 16 and 26 are less likely to be quenched.
- the two nanoparticulate bodies 10, 20 in the composite 40 are in close proximity to each other.
- the two nanoparticle bodies 10 and 23 are in contact with each other such that the first polymer film 13 of the first nanoparticle body 10 and the second polymer film 23 of the second nanoparticle body 20 in the composite 40 are in contact with each other. 20 are close to each other.
- at least one of the first polymer film 13 of the first nanoparticle body 10 and the second polymer film 23 of the second nanoparticle body 20 in the composite 40 shrinks. The two nanoparticulate bodies 10, 20 are brought close to each other so that they are in contact with each other.
- the test substance 30 and At least one of the specific binding substances 14 and 24 that bind to the test substance 30 and the fluorescent substances 16 and 26 can be incorporated into the polymer membranes 13 and 23.
- the polymer membranes 13 and 23 are in contact with each other, for example, in the complex 40 shown in FIGS. It is believed that at least one of the binding specific binding substances 14 , 24 and the fluorescent substances 16 , 26 can be incorporated into the polymer membranes 13 , 23 .
- the films covering the surfaces of the metal nanoparticles 12 and 22 are the polymer films 13 and 23, the fluorescence intensity can be increased.
- the reasons is presumed as follows.
- the films that cover the surfaces of the metal nanoparticles 12 and 22 are the polymer films 13 and 23, and the polymer films 13 and 23 have relatively high flexibility compared to inorganic films containing inorganic oxides. Therefore, in the composite 40, the polymer films 13 and 23 can contract, and as a result, the two metal nanoparticles 12 and 22 are reduced to two film thicknesses of the polymer film (thickness of the polymer film 13 + (thickness of the polymer film 23).
- the separation distance L can be less than two film thicknesses of the polymer films.
- the plasmon enhancement effect is easily obtained, and the fluorescence intensity is further increased.
- the film thickness of the polymer film in "two film thicknesses of the polymer film” does not refer to the film thickness of the polymer films 13 and 23 at the contracted portion, which is the object of the separation distance, It is the film thickness of the polymer films 13 and 23 of the non-shrinking portion that is not to be separated (for example, T1 in FIG. 15 to be described later).
- the polymer membranes 13 and 23 include at least one selected from the group consisting of binding sites 3a via sulfur atoms, positively charged groups 3b, and hydrophobic groups 3c (for example, the polymer membrane
- the polymer 3A constituting 13 and 23 contains at least one selected from the group consisting of a binding site 3a via a sulfur atom, a positively charged group 3b, and a hydrophobic group 3c), so that the fluorescence intensity is further increased. can be made The reason is presumed as follows. In such a case, at least one of the binding site 3a, the positively charged group 3b, and the hydrophobic group 3c forms a bond with the surfaces of the metal nanoparticles 12,22.
- the polymer 3A has a network structure and coats the surfaces of the metal nanoparticles 12 and 22 in a network. Since polymer 3A has such a network structure, it has relatively high flexibility. Therefore, in the composite 40, the polymer membranes 13, 23 can be further shrunk, thereby bringing the two metal nanoparticles 12, 22 closer together than the distance corresponding to two polymer membrane thicknesses. It becomes possible to Therefore, in the present embodiment, the separation distance L can be less than two film thicknesses of the polymer film, so that the plasmon enhancement effect can be further obtained and the fluorescence intensity can be further increased.
- the polymer 3A constituting the polymer films 13 and 23 has a side chain (more specifically, a side chain end) having a binding site 3a via a sulfur atom, a positively chargeable group 3b, and At least one selected from the group consisting of hydrophobic groups 3c is included.
- fluorescence intensity can be further increased. The reason is presumed as follows. In such a case, at least one of the binding site 3a, the positively charged group 3b, and the hydrophobic group 3c forms a bond with the surfaces of the metal nanoparticles 12,22. Therefore, it is considered that the polymer 3A has a network structure and coats the surfaces of the metal nanoparticles 12 and 22 in a network with the side chains as binding sites.
- polymer 3A Since polymer 3A has such a network structure, it has relatively high flexibility. Therefore, in the composite 40, the polymer membranes 13, 23 can be further shrunk, thereby bringing the two metal nanoparticles 12, 22 closer together than the distance corresponding to two polymer membrane thicknesses. It becomes possible to Therefore, in the present embodiment, the separation distance L can be less than two film thicknesses of the polymer film, so that the plasmon enhancement effect can be further obtained and the fluorescence intensity can be further increased.
- the separation distance L between the first nanoparticle bodies 10 and the second nanoparticle bodies 20 is, for example, 12 nm to 52 nm, preferably 12 nm to 27 nm.
- the separation distance L is the distance between the first metal nanoparticles 12 and the second metal nanoparticles 22, and is the distance between the first point P1 on the surface of the first nanoparticle body 10 and the second nanoparticle body 20. It is the minimum distance of the line segment connected with the second point P2 on the surface.
- the separation distance L is 52 nm or less, when the composite 40 is irradiated with excitation light, a near-field is generated more efficiently in the space near the surface between the first and second metal nanoparticles 12 and 22. , the fluorescence intensity can be further increased.
- the polymer 3A constituting the polymer films 13 and 23 (for example, in the side chain (more specifically, at the end of the side chain)) has a binding site 3a via a sulfur atom, a positively chargeable It has at least one selected from the group consisting of group 3b and hydrophobic group 3c. Therefore, as described above, the separation distance L can be closer than the distance corresponding to two film thicknesses of the polymer film covering the surfaces of the two metal nanoparticles 12 and 22 in the composite 40. . For example, if the thickness of the polymer films 13, 23 is 5 nm, the separation distance L can be less than 10 nm (more specifically, 2-9 nm, 3-8 nm, 4-7 nm, etc.).
- the phosphors 16, 26 are preferably positioned between the first metal nanoparticles 12 and the second metal nanoparticles 22, as shown in FIG. This is because the fluorescent substances 16 and 26 are positioned in the space between the metal nanoparticles 12 and 22 because the space between the metal nanoparticles 12 and 22 is a space in which a near-field is efficiently generated, so that the fluorescence intensity is likely to be increased.
- Test substance 30 is a substance to be detected contained in the specimen.
- Test substances 30 include, for example, antigens, proteins, substrates, and nucleic acid chains.
- the test substance 30 specifically binds to the specific binding substances 14,24.
- an antigen has at least two antigenic determinants (epitopes), and forms specific binding with the first and second specific binding substances 14, 24 at the antigenic determinants.
- Antigens are, for example, proteins such as c-reactive protein, myoglobin, troponin T, troponin I, and BNP, and antigenic proteins of viruses such as influenza virus, respiratory syncytial virus, and the like.
- Test substance 30 is, for example, a test substance derived from a specimen such as blood, plasma, urine, or saliva. That is, samples containing the test substance 30 are, for example, blood, plasma, serum, urine, and saliva.
- the specimen further includes solvents and buffers (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, MES buffer, etc.). It's okay.
- PBS phosphate-buffered saline
- Tris buffer Tris buffer
- HEPES buffer Tris buffer
- MOPS buffer MOPS buffer
- MES buffer etc.
- FIG. 6 is a diagram showing a measuring device.
- the measurement apparatus 100 includes an excitation light source 110 , an excitation light irradiation optical system 120 , a reagent container 130 , a light receiving optical system 140 and a light receiving element 150 .
- the excitation light source 110 emits excitation light 112 .
- the excitation light source 110 is, for example, a laser.
- the excitation light irradiation optical system 120 adjusts the cross-sectional diameter and the like like the excitation light 112 and outputs incident excitation light 122 .
- the excitation light irradiation optical system 120 is a lens 124 and a polarizing element ( ⁇ /2 plate) 126 .
- the incident excitation light 122 output from the excitation light irradiation optical system 120 enters the reagent container 130 and irradiates the measurement sample in the reagent container 130 .
- the reagent container 130 is, for example, a detachable container (more specifically, a cell, a preparation, etc.) and a microchannel chip.
- a microchannel chip is a chip having minute channels.
- the measurement sample irradiated with the incident excitation light 122 emits fluorescence (detection light 132).
- the light-receiving optical system 140 is arranged in a direction perpendicular to the traveling direction of the incident excitation light 122 to the reagent container 130 .
- the light receiving optical system 140 can adjust the cross-sectional diameter and the like of the detection light 132 emitted from the measurement sample, remove the scattered light of the incident excitation light 122, or adjust the light intensity.
- the light receiving optical system 140 is a lens 144 and an optical filter 146 .
- Optical filters 146 are, for example, bandpass filters and dichroic mirrors.
- Fluorescence 142 that has passed through the light receiving optical system 140 is detected by the light receiving element 150 .
- the light receiving element 150 is, for example, PD, APD, PMT, CCD camera, and spectroscope.
- the light-receiving element 150 is capable of measuring the fluorescence amount of a single wavelength, measuring the fluorescence spectrum, and creating two-dimensional planar fluorescence imaging.
- FIG. 5 is a cross-sectional view schematically showing a composite according to a modification of the second embodiment.
- first and second fluorescent substances 16 and 26 may be labeled with first and second specific binding substances 14 and 24, respectively.
- the first and second fluorescent substances 16 and 26 are easily positioned between the first metal nanoparticles 12 and the second metal nanoparticles 22, and the detection intensity is improved, which is more preferable.
- one of the first and second fluorescent substances 16 and 26 may be labeled with the polymer membranes 13 and 23 and the other may be labeled with the specific binding substances 14 and 24 .
- the complex 40 is labeled with two fluorescent substances 16 and 26, but it is not limited to this.
- the number of fluorescent substances labeled on the complex 40 may be one or three or more.
- the light receiving optical system 140 in the measuring device 100 is arranged in a direction perpendicular to the traveling direction of the incident excitation light 122 to the reagent container 130, but it is not limited to this.
- the light-receiving optical system 140 may be arranged, for example, in a direction parallel to the traveling direction of the incident excitation light 122, or may be arranged in a direction forming an acute or obtuse angle with respect to the traveling direction of the incident excitation light 122. .
- the concentration of the metal nanoparticles in the dispersion liquid may be indicated by the absorbance.
- the absorbance was measured using an ultraviolet-visible spectrophotometer (“infinite M200 PRO” manufactured by TECAN Japan Co., Ltd.). Since the absorption wavelength differs depending on each sample, it is described for each sample.
- FIGS. 7 and 8 show schematic diagrams for explaining a method for producing a polymer membrane.
- poly-L-lysine (“3075” manufactured by Peptide Institute Inc.) and 3-(2-pyridyldithio)propionamide-PEG4-NHS (manufactured by Thermo Fisher Scientific, production number “26128”) ", "NHS-PEG4-SPDP”) were stirred and mixed using a small rotary incubator ("RT-30mini” manufactured by Taitec Co., Ltd.) at room temperature for 4 hours.As a result, the polymer This synthetic reaction is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamide-PEG4-NHS.
- the polymer had a hydrophobic group (pyridyl group (pyridinyl group)) 3c and a positively charged group (primary ammonium group) 3b.
- the obtained polymer was used as a silver nanoparticle as a metal nanoparticle.
- RT-30 mini manufactured by Taitec Co., Ltd.
- the polymer film 3 includes a binding site 3a via a sulfur atom on the surface of the silver nanoparticles 2 and a hydrophobic group (pyridyl group) that forms a hydrophobic bond with the surface of the silver nanoparticles 2. group)) 3c and a positively charged group (primary ammonium group) 3b that forms an electrostatic bond with the surface of the silver nanoparticles 2.
- the polymer 3A constituting the polymer film 3 has a bonding site 3a via a sulfur atom on the surface of the silver nanoparticles 2 and a hydrophobic group (pyridyl group (pyridinyl group)) 3c and a positively charged group (primary ammonium group) 3b that forms an electrostatic bond with the surface of the silver nanoparticles 2.
- An SEM image magnification: 500,000
- the films thickness of the polymer film 3 covering the silver nanoparticles 2 was measured from the SEM image (see FIG. 9(a)).
- the polymer film 3 has a bonding site 3a via a sulfur atom on the surface of the gold nanoparticles 2, a positively charged group (primary ammonium group) 3b, and a hydrophobic group (pyridyl group (pyridinyl group)) 3c. .
- An SEM image (magnification: 500,000) of the obtained gold nanoparticles 2 was created, and it was confirmed that the surfaces of the gold nanoparticles 2 were continuously coated with the polymer film 3 .
- FIG. 10 is a graph of the zeta potential of the metal nanoparticles 2 coated with the polymer film 3 (horizontal axis: zeta potential (unit: mV) and vertical axis: relative intensity (unit: arbitrary unit)).
- the metal nanoparticles 2 are silver nanoparticles (particle diameter: 80 nm) in FIG.
- FIG. 11 is a graph of the zeta potential of the metal nanoparticles 2 not covered with the polymer film 3 (horizontal axis: zeta potential (unit: mV) and vertical axis: relative intensity (unit: arbitrary unit)).
- the metal nanoparticles 2 are silver nanoparticles (particle diameter: 80 nm) in FIG. 11(a) and gold nanoparticles (particle diameter: 20 nm) in FIG. 11(b).
- the zeta potentials of silver nanoparticles (particle diameter 80 nm) and gold nanoparticles (particle diameter 20 nm) coated with the polymer film 3 are both It had a peak at a positive potential and had a shape in which the entire frequency distribution of zeta electrons fell substantially within the positive potential range. Therefore, it was confirmed that the surface of the metal nanoparticles 2 coated with the polymer film 3 (that is, the surface of the polymer film 3) was positively charged. From the results of FIGS. 10 and 11, it was confirmed that the surfaces of the metal nanoparticles 2 and the polymer film 3 have paired chargeability. Therefore, it was strongly suggested that, in the metal nanoparticles 2 coated with the polymer film 3, the surface of the metal nanoparticles 2 and the polymer film 3 form an electrostatic bond.
- a dispersion liquid of metal nanoparticles (silver nanoparticles and gold nanoparticles) was separately prepared according to Production Examples 1 and 2, and left to stand for three months after preparation. After that, the zeta potential of the metal nanoparticles 2 coated with the polymer film 3 was measured. It was confirmed that the metal nanoparticles 2 coated with the polymer film 3 left still for 3 months were positively charged like the metal nanoparticles 2 coated with the polymer film 3 immediately after preparation. Therefore, it was shown that the polymer membrane 3 is stably maintained for a long period of time (for example, 3 months).
- the metal nanoparticles 2 coated with the polymer film 3 were added to the fluorescent material solution, and the fluorescence intensity was measured with respect to the added amount, confirming that the polymer film 3 contributes to the suppression of fluorescence quenching. .
- the measurement conditions were an excitation light wavelength of 430 nm and a detection wavelength of 470 to 700 nm. Fluorescence spectra were measured by changing the amount of coated gold nanoparticle dispersion added (60 ⁇ L, 120 ⁇ L, 180 ⁇ L, and 240 ⁇ L). The results are shown in FIG. 12(a).
- FIG. 12(a) shows the fluorescence spectrum (horizontal axis: fluorescence wavelength (unit: nm ) and vertical axis: fluorescence intensity (unit: arbitrary unit)).
- the fluorescence spectrum without the addition of the coated gold nanoparticle dispersion (addition amount of the coated gold nanoparticle dispersion: 0 ⁇ L) showed a spectral shape with peaks near 614 nm and 530 nm.
- the peak at 614 nm is assigned to the ruthenium complex and the peak around 530 nm is background fluorescence.
- Fluorescent substance-gold nanoparticle mixed system not coated with polymer film hereinafter also referred to as fluorescent substance-uncoated metal nanoparticle mixed system was measured in the same manner except that it was changed to . The measurement results are shown in FIG. 12(b).
- FIG. 12(b) shows the fluorescence spectrum of the fluorescent substance-uncoated metal nanoparticles mixed system (horizontal axis: fluorescence wavelength (unit: nm) and vertical axis: fluorescence intensity (unit: arbitrary unit)).
- the fluorescence spectrum without the addition of the uncoated gold nanoparticle dispersion (added amount of uncoated gold nanoparticle dispersion: 0 ⁇ L) showed a spectral shape with peaks near 614 nm and 530 nm.
- FIG. 13 is a schematic diagram showing the structure of nanoparticles to which fluorescence-labeled antibodies are bound.
- the nanoparticle body shown in FIG. 13 is obtained by first binding a cross-linking agent to the surface of the polymer-coated metal nanoparticles, separately binding a fluorescent substance and a cross-linking agent to the nanoantibody, and then binding to the polymer-coated metal nanoparticles. and the cross-linking agent bound to the nano-antibody.
- n indicates the number of repeating units of ethylene oxide repeating units, which is 6.
- the polymer film 3 includes a bonding site 3a via a sulfur atom on the surface of the silver nanoparticle 2, a hydrophobic group (pyridyl group (pyridinyl group)) 3c that makes a hydrophobic bond with the surface of the silver nanoparticle 2, and a silver nanoparticle. It contains a positively charged group (primary ammonium group) 3b that forms an electrostatic bond with the surface of the particle 2 .
- the polymer 3A constituting the polymer film 3 has a binding site 3a via a sulfur atom on the surface of the silver nanoparticles 2 and a hydrophobic group (pyridyl group (pyridinyl group)) 3c and a positively charged group (primary ammonium group) 3b that forms an electrostatic bond with the surface of the silver nanoparticles 2 (see FIG. 8).
- a dispersion of silver nanoparticles in which the cross-linking agent SM(PEG)6 was bound to the polymer film 3 (hereinafter also referred to as polymer-coated silver nanoparticles to which the SM(PEG)6 linker is bound) was obtained.
- the SM(PEG)6 linker that attached the polymer-coated silver nanoparticles had a maleimide group.
- VHH antibody Alexa Fluor 430 carboxylic acid, succinimidyl ester (“A10169” manufactured by Invitrogen) was added to 100 ⁇ g of VHH antibody (manufactured by RePHAGEN, molecular mass 18,000 Da), and the mixture was incubated at room temperature for 1 hour in a small rotary culture machine ( It was stirred and mixed using “RT-30mini” manufactured by Taitec Co., Ltd.). As a result, a fluorescent substance-bound VHH antibody (hereinafter also referred to as a fluorescent-labeled VHH antibody) was obtained.
- a reducing agent TCEP (77720" manufactured by ThermoFisher SCIENTIFIC) was added in a molar ratio of 2 equivalents to the fluorescence-labeled VHH antibody to which the SPDP linker was bound. -100") and stirred to mix.
- a VHH antibody bound with a fluorescent substance and a reduced SPDP linker (hereinafter also called a reduced SPDP linker) was obtained (hereinafter also called a fluorescence-labeled VHH antibody bound with a reduced SPDP linker).
- the reduced SPDP linkers had thiol groups (-SH groups) generated by reduction of disulfide bonds.
- the maleimide group of the SM(PEG)6 linker reacted with the thiol group of the reduced SPDP linker to obtain nanoparticles to which the fluorescence-labeled VHH antibody was bound via the linker portion (see FIG. 13).
- the film thickness of the polymer film 3 was 8.31 nm (expected value M) ⁇ 1.89 nm (standard deviation ⁇ ), and the film thickness variation ( ⁇ /M) was 13.7%.
- the resulting nanoparticulate composition consisted of the nanoparticles shown in FIG. 13 and a solvent containing sodium heparin as the polyanionic polymer.
- a measurement sample was placed in a measurement container, and the measurement container was installed in a fluorescence spectrophotometer (TECAN Japan Co., Ltd. “infinite M200PRO”).
- the measurement container was irradiated with a wavelength of 430 nm, and the fluorescence spectrum was measured. Fluorescence spectra were measured under the measurement conditions of a detection wavelength of 470 nm to 700 nm and an optical path length of 3.5 mm.
- the fluorescent substance contained in the nanoparticles shows an absorption spectrum with a peak at 430 nm and a fluorescence spectrum with a peak at 520 nm.
- Silver nanoparticles (50 nm in diameter) contained in the nanoparticle body exhibit an absorption spectrum with a peak at 430 nm. Fluorescence spectra of blank samples were also measured in the same manner.
- the results of the obtained fluorescence spectra are shown in FIG.
- the horizontal axis indicates fluorescence wavelength (unit: nm), and the vertical axis indicates fluorescence intensity.
- the fluorescence spectrum of the “sample to which CRP antigen was added” indicated by the solid line had a spectral shape with a peak with a maximum fluorescence intensity of about 50,000 near 520 nm.
- the fluorescence spectrum of the "blank sample” obtained had a spectral shape with a peak with a maximum fluorescence intensity of about 43,000.
- the fluorescence intensity of the sample to which CRP antigen was added was generally greater than that of the blank sample. Thus, a significant difference was observed between the fluorescence intensity of the CRP antigen-added sample and the fluorescence intensity of the blank sample.
- the fluorescence intensity of the blank sample is due to the fluorescence emitted by the fluorescent substance directly excited by absorbing the excitation light (light with a wavelength of 430 nm).
- the fluorescence intensity of the sample to which the CRP antigen was added was the fluorescence emitted by the fluorescent substance directly excited by absorbing the excitation light, as well as the proximity generated by surface plasmon resonance on the surface of the metal nanoparticles. It is due to the fluorescence emitted by the fluorescent substance indirectly excited by the field. Therefore, the significant difference in the observed fluorescence intensity is attributed to the fluorescence emitted by the fluorescent substance indirectly excited by the near-field formed by the surface plasmon resonance on the metal nanoparticle surface. From the above, it is considered that the fluorescence enhancement effect was obtained in the sample to which the CRP antigen of Example 1 was added.
- Example 2 Shrinkage of polymer membrane
- nanoparticles were prepared in the same manner as in Example 1 described above, except that the following three conditions were changed.
- the nanoparticle of Example 2 was a nanoparticle to which a fluorescence-labeled VHH antibody was bound via a linker portion.
- Fluorescent substance is Alexa Fluor 430 carboxylic acid, succinimidyl ester ("A10169” manufactured by Invitrogen) to the chemical formula of [Chemical 3]: (“Ruthenium(II)tris(Bipyridyl)-C5-NHS ester” manufactured by Tokyo Chemical Industry Co., Ltd.).
- -Crosslinking agent- A cross-linking agent (NHS-bipyridyl disulfide cross-linking agent) to the fluorescently labeled VHH antibody is converted from 3-(2-pyridyldithio)propionamide-PEG4-NHS ("NHS-PEG4-SPDP" manufactured by Tokyo Kasei Co., Ltd.) to 3-(2 -pyridyldithio)propionamide-PEG4-NHS (manufactured by Thermo Fisher Scientific, product number “26128”, “NHS-PEG4-SPDP”).
- C Reactive Protein (“00-AGN-AP-CRP-00” manufactured by ADVY CHEMICAL) (hereinafter also referred to as CRP antigen) as a test substance was added to the phosphate buffer solution of the nanoparticles of Example 2, and the mixture was added at room temperature. and 5 minutes using a small rotary culture machine ("RT-30mini” manufactured by Taitec Co., Ltd.) to prepare a measurement sample containing a sandwich-type complex.
- the nanoparticle body of Example 2 had a polymer film coating the surface of the metal nanoparticles, as described above.
- the polymer constituting this polymer film has a primary ammonium group as a positively charged group at the end of its side chain, a pyridyl group as a hydrophobic group, and a sulfur atom between the surface of the metal nanoparticle. It had a binding site via
- FIG. 15 shows an SEM image of a composite prepared using the nanoparticles of Example 2.
- the separation distance L1 between the metal nanoparticles in the nanoparticles in the composite and the thickness T1 of the polymer film other than between the metal nanoparticles in the nanoparticles are measured and compared.
- the thickness T1 of the polymer film was the thickness of the polymer film in the non-shrinking portion, which was not subject to the separation distance.
- the separation distance L1 was smaller than the thickness (T1 ⁇ 2) corresponding to two thicknesses of the polymer film. Therefore, in the composite prepared with the nanoparticle composition of Example 2, the polymer film shrinks, and the two metal nanoparticles in the composite correspond to two thicknesses of the polymer film (T1 ⁇ 2). It was found that they were closer than the separation distance between them. This strongly suggested that the plasmon enhancement effect was further obtained and the fluorescence intensity was further increased.
- silica-coated silver nanoparticles manufactured by nanoComposix, silver nanoparticle diameter (core particle diameter) 50 nm, silica film thickness 20 nm
- aqueous dispersion prepared by diluted with water to prepare an aqueous dispersion of silica-coated silver nanoparticles. .
- the obtained aqueous dispersion was used as a measurement sample.
- aggregates in which two particles were aggregated were also present in the measurement sample. This aggregate was located and evaluated.
- Example 2 In the same manner as in Example 2, an SEM image (500K magnification) was taken, and from the SEM image, the thickness T2 of the inorganic film in the aggregate and the distance L2 between the two metal nanoparticles were measured and compared. As a result, the separation distance L2 was about twice the thickness T2 of the inorganic film. Note that the thickness T2 of the inorganic film was the thickness of the inorganic film at a portion not subject to the separation distance.
- Example 3 Preparation of various nanoparticulate body compositions
- a nanoparticulate composition as shown in FIG. 16 was prepared.
- a dispersion of poly-L-lysine (“3075” manufactured by Peptide Institute) as a polymer and silver nanoparticles (“AGCB50-1M” manufactured by nanocomposite, diameter 50 nm, OD 430 0.1) as metal nanoparticles. It was added to 1 mL, and stirred and mixed at room temperature (25° C.) for 4 hours using a small rotary incubator (“RT-30mini” manufactured by Taitec Co., Ltd.).
- the polymer film has a binding site via a sulfur atom on the surface of the silver nanoparticles, a hydrophobic group that forms a hydrophobic bond with the surface of the silver nanoparticles, and a positive charge that forms an electrostatic bond with the surface of the silver nanoparticles. It contains a neutral group (primary ammonium group) and an electrically neutral group (primary amino group).
- the polymer composing the polymer film has a binding site via a sulfur atom on the surface of the silver nanoparticles, a hydrophobic group that forms a hydrophobic bond with the surface of the silver nanoparticles, and a static bond with the surface of the silver nanoparticles. It has a positively charged group (primary ammonium group) that forms an electrical bond and an electrically neutral group (primary amino group).
- fluorescent-labeled polymer-coated silver nanoparticles A succinimidyl ester (“Alexa Fluor (registered trademark) 430 NHS ester” manufactured by Thermo Fisher Scientific) as a fluorescent substance was added to 1 mL of the polymer-coated silver nanoparticle dispersion, and the microrotation culture was performed at room temperature for 1 hour. The mixture was stirred and mixed using a machine (“RT-30mini” manufactured by Taitec Co., Ltd.). As a result, a dispersion of polymer-coated silver nanoparticles in which the fluorescent substance was bound to the polymer film (hereinafter also referred to as fluorescent-labeled polymer-coated silver nanoparticles) was obtained.
- the nanoparticle composition of Example 3 was put into the measurement container.
- the measurement container was placed in an ultraviolet-visible spectrophotometer (“infinite M200 PRO” manufactured by TECAN Japan Co., Ltd.) to measure the absorption spectrum. Measurement conditions were a measurement wavelength range of 400 to 600 nm.
- FIG. 17 shows the absorption spectrum of the nanoparticle composition of Example 3. The absorption spectrum obtained had a peak at 455 nm.
- the nanoparticulate composition of Example 3 was placed in a measurement container with an optical path length of 1 cm.
- the measurement container was placed in a fluorophotometer (“infinite M200 PRO” manufactured by TECAN Japan Co., Ltd.) to measure the fluorescence spectrum.
- the measurement conditions were an excitation light wavelength of 430 nm and a detection wavelength of 470-700 nm.
- FIG. 18 shows the fluorescence spectrum of the nanoparticle composition of Example 3.
- FIG. The fluorescence spectrum obtained had a peak at 535 nm.
- Example 4 Nanoparticulate composition comprising nanoparticles bound to a polymer membrane by a linker
- a method of making a nanoparticulate body composition comprising nanoparticulate bodies will now be described with reference to FIG.
- FIG. 19 is a schematic diagram for explaining a method for producing a nanoparticulate body composition comprising the nanoparticulate bodies. A dispersion of polymer-coated silver nanoparticles was prepared in the same manner as in Example 1.
- succinimidyl ester (“Alexa Fluor (registered trademark) 430 NHS ester” manufactured by Thermo Fisher Scientific) as a fluorescent substance and SM (PEG) 6 (PEGylated, long-chain SMCC crosslinker) as a crosslinker )
- SM (PEG) 6 PEGylated, long-chain SMCC crosslinker
- 22105 manufactured by ThermoFisher SCIENTIFIC
- heparin sodium (“081-00136” manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a polyanionic polymer, and the mixture was incubated at room temperature for 4 hours in a small rotary culture machine (“RT-30mini” manufactured by Taitec Co., Ltd.). was used to stir and mix.
- a nanoparticle composition comprising polymer-coated silver nanoparticles (nanoparticles) bound with a fluorescent substance and a linker and a polyanionic polymer was prepared.
- the absorption spectrum of the resulting nanoparticles was measured in the same manner as in Example 1.
- the absorption spectrum of the nanoparticle composition of Example 4 is shown in FIG. 20(a).
- the obtained absorption spectrum had a peak at 450 nm.
- the fluorescence spectrum of the resulting nanoparticle composition was measured in the same manner as in Example 1.
- the fluorescence spectrum of the nanoparticle composition of Example 4 is shown in FIG. 20(b).
- the fluorescence spectrum obtained had a peak at 540 nm. It was confirmed that the nanoparticles were not aggregated by the fact that the maximum wavelength of the absorption spectrum did not shift to the long wavelength side, and that the fluorescent substance was bound to the nanoparticles by the fact that fluorescence could be detected. did.
- Example 5 Dispersibility evaluation of nanoparticles by polyanionic polymer
- APCB80-1M dispersion liquid of silver nanoparticles
- OD 455 0.1
- poly-L-lysine 3075
- RT-30mini small rotary culture machine
- the polymer film consists of a hydrophobic group (alkylene group) that forms a hydrophobic bond with the surface of the silver nanoparticles and an electrically neutral group (primary amino group) that can form an electrostatic bond with the anionic group of the polyanionic polymer.
- the polymer that constitutes the polymer film is composed of a hydrophobic group (alkylene group) that forms a hydrophobic bond with the surface of the silver nanoparticles and an electrical medium capable of forming an electrostatic bond with the anionic group of the polyanionic polymer. and a functional group (primary amino group).
- Polyglutamic acid sodium salt (manufactured by Sigma-Aldrich Co., Ltd. "P4886" molecular weight 50,000 to 100,000) as a polyanionic polymer was added to 1 mL of the polymer-coated silver nanoparticle dispersion. The mixture was stirred and mixed using a rotary culture machine (“RT-30mini” manufactured by Taitec Co., Ltd.). As a result, a nanoparticle composition comprising polymer-coated silver nanoparticles and a polyanionic polymer was obtained.
- RT-30mini manufactured by Taitec Co., Ltd.
- Example 4 composition not subjected to cleaning treatment (hereinafter also referred to as 0-time cleaning composition)) and a composition subjected to two cleaning treatments (hereinafter referred to as 2 (also referred to as a cleaning treatment composition) was prepared.
- the washing treatment is performed by adding pure water as a solvent so that the concentration of the nanoparticles in the composition is 1/2, vigorously stirring, and after stirring, at least a centrifuge (centrifuge) (manufactured by Co., Ltd.)
- a centrifuge centrifuge
- a series of treatments are shown in which the nanoparticles are sedimented under the conditions of 12,000 G and 30 minutes using Tomy Seiko's "MX307", the supernatant is removed, and pure water is newly added. This operation was defined as one washing treatment. When the washing treatment was performed three times, agglomeration occurred and the recovery rate decreased significantly.
- FIG. 21 is a diagram showing the frequency distribution of sizes.
- FIG. 21(a) shows the size frequency distribution when the number of washings is 0, and
- FIG. 21(b) shows the size frequency distribution when the number of washings is two.
- the horizontal axis indicates the size (unit: nm, logarithmic scale), and the vertical axis indicates the scattering intensity ratio (SID) (unit: none).
- the size frequency distribution has a shape with one peak near 100 nm when the number of washing treatments is 0, and the number of washing treatments is 2 (two-time washing treatment composition ) had a peak in a region larger than 100 nm and had a broadened shape.
- the peak of the frequency distribution is shifted to the larger side of the size, and the frequency distribution is broadened. It is thought that things have arisen. That is, as shown in FIGS. 23(a) and 23(b), the number of polyanionic polymers surrounding the nanoparticles gradually decreases due to the washing treatment with a solvent (more specifically, pure water).
- FIG. 22 is a diagram showing the frequency distribution of zeta potential.
- FIG. 22(a) shows the frequency distribution of the zeta potential when the number of washing treatments is 0, and
- FIG. 22(b) shows the frequency distribution of the zeta potential when the number of washing treatments is two.
- the horizontal axis indicates zeta potential (unit: mV)
- the vertical axis indicates frequency (unit: arbitrary unit).
- the frequency distribution of the zeta potential has a peak in the negative potential region when the number of washings is 0, and the frequency distribution when the number of washings is two. peak existed in the region of positive potential.
- negative potential ⁇ positive potential As a result of the change in the potential of the peak of the frequency distribution before and after the washing treatment (negative potential ⁇ positive potential), as the number of washing treatments increases from 0 to 2, the negatively charged The number of polyanionic polymers gradually decreased, strongly suggesting that the positively charged polymer film was exposed (see: change from FIG. 23(a) to 23(b)).
- nanoparticles and nanoparticle compositions according to this embodiment can be used to detect specific test substances in specimens using plasmon-excited fluorescence spectrometry.
- the present invention relates to nanoparticles, particularly nanoparticles used in plasmon-excited fluorescence analysis, and methods for producing the same. (Background technology)
- a biosensor specifically reacts a specific test substance to be detected with a specific specific binding substance to form a complex, and detects the test substance by a signal derived from the specific binding in the complex.
- complexes include, for example, metal particles, specific binding substances, fluorescent substances and test substances.
- surface plasmon resonance occurs in the metal particles in the complex, forming a near-field near the surface of the metal particles. This near field increases the fluorescence intensity of the fluorescent substance.
- the composite particles for immunochromatography described in Patent Document 1 have a structure in which the outside of fine particles made of metal is covered with at least one layer of silica containing at least one fluorescent substance, and specifically recognizes a target substance. It consists of fine particles surface-modified with a labeling substance. That is, in the composite particle of Patent Document 1, the surface of the metal particle is covered with a silica layer, and the fluorescent substance and labeling substance are immobilized on the silica layer. (Prior art document) (Patent document)
- Patent Document 1 JP 2011-220705 A (outline of the invention) (Problems to be solved by the invention)
- the surfaces of metal particles are covered with a silica layer, and the silica layer is further modified with a fluorescent substance and a labeling substance. Therefore, in the production of nanoparticles, the site of the fluorescent substance to be labeled (labeling site) on the silica layer competes with the site of the labeling substance to be bound (binding site). This makes it difficult to achieve the desired increase in fluorescent material in the nanoparticulate body.
- the main object of the present invention is to provide a nanoparticle having a novel labeling mode of a fluorescent substance capable of labeling a larger number of fluorescent substances, and a method for producing the same. (means to solve the problem)
- Nanoparticles Comprising metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a fluorescent substance,
- the fluorescent substance is labeled on the surface of the metal nanoparticles
- the polymer film includes binding sites via sulfur atoms between the surfaces of the metal nanoparticles.
- a method for producing nanoparticles according to one embodiment of the present invention comprises: A polymer to which a fluorescent substance binds via a disulfide bond is mixed with metal nanoparticles to label the surface of the metal nanoparticles with the fluorescent substance and to form a polymer film on the surface of the metal nanoparticles. forming. (Effect of the invention)
- a nanoparticle according to an embodiment of the present invention has a novel labeling mode for fluorescent substances, and can label a larger amount of fluorescent substances. (Brief description of the drawing)
- FIG. 24 is a cross-sectional view schematically showing a nanoparticle body according to a fourth embodiment.
- FIG. 25 is an enlarged schematic view of part A in FIG.
- FIG. 26 is a reaction scheme showing an example of a method for producing nanoparticles according to the fifth embodiment.
- FIG. 27 is a sectional view schematically showing a composite according to the sixth embodiment.
- FIG. 28 is a diagram schematically showing a measuring device according to the seventh embodiment.
- FIG. 29 is a schematic diagram illustrating a method for producing a nanoparticle body of Example 6.
- FIG. 30 FIG. 30 is a diagram showing the fluorescence spectrum of the nanoparticles of Example 6.
- FIG. 31 is a SEM image of a composite prepared using the nanoparticles of Example 7.
- nanoparticle body its manufacturing method, composite body, and measuring device, which are embodiments of the present invention, will be described in detail with reference to the illustrated embodiments.
- the drawings are schematic and may not reflect actual dimensions or proportions.
- Nanoparticles are Comprising metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a fluorescent substance, The fluorescent substance is labeled on the surface of the metal nanoparticles,
- the polymer film contains binding sites via sulfur atoms between the surface of the metal nanoparticles.
- the nanoparticles according to this embodiment are useful for detecting test substances.
- the nanoparticle body according to this embodiment includes metal nanoparticles, a polymer film covering the surfaces of the metal nanoparticles, and a fluorescent substance labeled on the surfaces of the metal nanoparticles.
- the nanoparticle bodies according to this embodiment may further comprise a specific binding substance bound to the polymer membrane.
- a specific binding substance can specifically bind to a test substance in a sample.
- a nanoparticle to which a specific binding substance is bound is also referred to as a "specifically binding nanoparticle".
- the test substance contained in the specimen is captured to form a complex (described in detail in the sixth embodiment). More specifically, the complex is formed by specific binding between the specific binding substance of the specific binding nanoparticle and the test substance.
- the complex has a structure (sandwich type structure) in which two specific binding nanoparticles bind via one test substance. In this way, the two metal nanoparticles are spaced apart at a certain distance by binding the specific binding substances possessed by the two nanoparticles in the complex to the same test substance.
- SPFS surface plasmon excitation enhanced fluorescence spectroscopy
- LSPR localized surface plasmon resonance
- a test substance in a specimen can be detected by measuring fluorescence intensity.
- the nanoparticles according to the present embodiment have a novel labeling mode for fluorescent substances that can label more fluorescent substances. That is, in the nanoparticles according to the present embodiment, the novel labeling mode of the fluorescent substance can label more fluorescent substances.
- the nanoparticle body according to this embodiment includes metal nanoparticles, a polymer film covering the surfaces of the metal nanoparticles, and a fluorescent substance labeled on the surfaces of the metal nanoparticles.
- the fluorescent substance is labeled on the surface of the metal nanoparticles
- a substance that specifically binds to a site different from the surface of the metal nanoparticles in the nanoparticle body for example, a specific functional group of the polymer that constitutes the polymer film.
- the labeling site where the fluorescent substance is labeled in the nanoparticle is different from the binding site where the specific binding substance binds, it is possible to further increase the number of fluorescent substances in the nanoparticle. From the above, it is considered that the nanoparticles according to the present embodiment can label more fluorescent substances. Furthermore, the nanoparticles according to the present embodiment are capable of labeling a larger amount of fluorescent substances, and are therefore excellent in detection sensitivity.
- FIG. 24 is a cross-sectional view schematically showing a nanoparticle body.
- the nanoparticle body 1 includes metal nanoparticles 2, a polymer film 3 covering the surface of the metal nanoparticles 2, and a label on the surface of the metal nanoparticles 2. and a fluorescent material 6 that As shown in FIG. 24(b), the nanoparticle body 1 may further contain a specific binding substance 4 that is bound to the surface of the polymer membrane 3 and specifically binds to the test substance in the specimen.
- the nanoparticle body 1 can be used for plasmon-excited fluorescence analysis.
- the nanoparticulate bodies 1 can be used for surface plasmon excitation enhanced fluorescence spectroimmunoassay.
- the nanoparticles 1 can capture the test substance in the subject and form a complex containing two nanoparticles 1 and one test substance. When the complex is irradiated with excitation light, it causes localized surface plasmon resonance and forms a near-field. This near field increases the fluorescence intensity.
- the nanoparticle body 1 may also block non-specific binding sites with a blocking agent.
- the blocked nanoparticle body 1 suppresses the formation of non-specific binding of the specific binding substance 4 to substances other than the detection target (i.e., substances other than the test substance), thereby reducing background and false positive signals. , the signal-to-noise ratio (SNR) can be improved. In such a case, detection sensitivity can be further improved.
- Blocking agents include, for example, bovine serum albumin (BSA), proteins such as skimmed milk and casein, and chemically synthesized polymers.
- the dispersion liquid of the nanoparticles 1 may further contain a dispersant for the purpose of improving the dispersibility of the nanoparticles 1.
- a dispersant include, for example, sodium heparin.
- Metal nanoparticles The surface of the metal nanoparticles 2 is covered with a polymer film 3 .
- the metal nanoparticles 2 interact with light having a specific wavelength, depending on the type of metal, and cause localized surface plasmon resonance. There is a plasmon resonance peak from 400 nm to 530 nm for silver nanoparticles and from 510 nm to 580 nm for gold nanoparticles. This depends on the particle size. For example, silver nanoparticles with a particle size of 20 nm resonate with light with a wavelength of 405 nm.
- Gold nanoparticles with a particle size of 20 nm resonate with light with a wavelength of 524 nm.
- the particle size (average primary particle size) of the metal nanoparticles 2 is, for example, 5 nm to 100 nm.
- the particle size of the metal nanoparticles 2 is obtained by capturing an image of the metal nanoparticles 2 using a scanning electron microscope (SEM) or a transmission electron microscope (TEM) and measuring the particle size of the metal nanoparticles 2 in the image. It can be obtained by calculating the average value of a plurality of particle sizes (the number of measurements: for example, at least 10 or more).
- the metal nanoparticles 2 preferably comprise gold or silver, more preferably silver.
- the polymer film 3 covers the surfaces of the metal nanoparticles 2 .
- the polymer film 3 functions as a metal quenching film.
- the polymer film 3 can be arranged so that the fluorescent substance 6 is separated from the surface of the metal nanoparticles 2 by at least the thickness of the polymer film 3 . Therefore, it is possible to prevent the excited fluorescent substance 6 from coming into contact with the surface of the metal nanoparticle 2 and quenching, thereby suppressing a decrease in detection sensitivity.
- the presence of the polymer film 3 can be confirmed by taking an image of the nanoparticle 1 using SEM or TEM and observing the nanoparticle 1 in the image.
- the polymer membrane 3 will be described with reference to FIG.
- FIG. 25 is an enlarged view of part A in FIG. 24, and is an enlarged schematic view of the vicinity of the interface between the polymer film 3 of the nanoparticle body 1 and the surface of the metal nanoparticles 2.
- FIG. The polymer film 3 includes bonding sites 3a with the surfaces of the metal nanoparticles 2 via sulfur atoms.
- Polymer membrane 3 may further include at least one selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c. More specifically, the polymer film 3 has a binding site 3a via a sulfur atom between the surface of the metal nanoparticle 2 and a primary ammonium group (—NH 3 + ) and a hydrophobic group 3c.
- the binding site 3a binds between the surface of the metal nanoparticle 2 and the polymer film 3 via sulfur atoms.
- the positively chargeable group 3 b forms an electrostatic bond (ionic bond) b with the surface of the negatively chargeable metal nanoparticle 2 .
- Hydrophobic group 3c forms hydrophobic bond c with the surface of metal nanoparticle 2 .
- the polymer film 3 is stably fixed on the surface of the metal nanoparticles 2 by binding sites 3a. Furthermore, the polymer film 3 can be more stably fixed to the surface of the metal nanoparticles 2 by the hydrophobic bond c and the electrostatic bond b by the positively charged group 3b. Since the polymer film 3 is thus stably fixed on the surfaces of the metal nanoparticles 2, the phosphor 6 and the surfaces of the metal nanoparticles 2 can be stably separated by a predetermined distance. Therefore, in this embodiment, the quenching of the excited fluorescent substance is suppressed, and the decrease in detection sensitivity can be suppressed.
- the polymer film 3 can be configured to contain the polymer 3A, it is easier to chemically modify than the silica layer, and the need for surface modification and the like is low. Thereby, the film thickness can be made smaller than that of the silica layer, and the distance between the metal nanoparticles 2 in the composite can be appropriately reduced. Therefore, the near field is formed more efficiently, and the detection sensitivity can be further improved.
- the polymer 3A that can constitute the polymer film 3 has a bonding site 3a with the surface of the metal nanoparticle 2 via a sulfur atom, a positively charged group 3b and a hydrophobic group. at least one selected from the group consisting of 3c.
- the presence of the binding site 3a via the sulfur atom, the positively charged group 3b and the hydrophobic group 3c is confirmed by measuring the signals derived therefrom using infrared spectroscopy and nuclear magnetic resonance spectroscopy. be able to.
- the binding sites 3a via sulfur atoms are formed, for example, by mixing metal nanoparticles 2 with a polymer having a site containing a disulfide bond as a side chain.
- the polymer film 3 may be directly bonded to the surface of the metal nanoparticles 2, and a linker portion derived from a cross-linking agent (more specifically, SM(PEG) n (where n is 4, 6 and 8 etc.)) to the surface of the metal nanoparticles 2 indirectly.
- a cross-linking agent more specifically, SM(PEG) n (where n is 4, 6 and 8 etc.)
- cross-linking agents include, for example, amino group-sulfhydryl group cross-linking agents (more specifically, NHS (N-hydroxysuccinimide)-maleimide group cross-linking agents, etc.).
- the positively chargeable group 3b can form a relatively strong electrostatic bond b with the surface of the metal nanoparticle 2.
- the positively chargeable group 3b in this specification is a completely positively ionized group having a valence of 1 or more.
- the positively-chargeable groups 3b are represented by the following formula (1): (Number 1) [In the formula (1), pKa is an electrically neutral group contained in the polymer 3A constituting the polymer film 3, and if positively charged, a positively charged group (more specifically, Primary ammonium group (—NH 3 + ) etc.) 3b (hereinafter also referred to as an electrically neutral group) (more specifically, primary amino group (—NH 2 ) etc.).
- pH indicates the pH of the environment (more specifically, the specimen, etc.) in which the test substance is detected
- B indicates an electrically neutral group contained in polymer 3A
- BH + indicates a positive electrode contained in polymer 3A. showing the chargeable group 3b] A group represented by and having a pKa of 7 or more. That is, the positively charged group 3b is an environment in which the electrically neutral group of the polymer 3A constituting the polymer membrane 3 and the positively charged group 3b detect the test substance (for example, a sample with a pH of about 6 to 8). in which the following chemical equilibrium equation (2): (Chem. 1) When the equilibrium state represented by Say.
- Hydrophobic group - Hydrophobic group 3 c can form hydrophobic bond c with the surface of metal nanoparticle 2 .
- the hydrophobic group is, for example, at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups and aliphatic chain groups.
- the aromatic cyclic group includes, for example, an aromatic carbocyclic group and an aromatic heterocyclic group.
- Aromatic carbocyclic groups are groups that do not contain aromatic heterocycles but contain aromatic rings in which the ring members are all carbon atoms. Examples of aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups and the like) and arylalkyl groups (more specifically, benzyl groups and the like).
- An aromatic heterocyclic group is a group containing an aromatic ring in which at least one of the ring member atoms is a heteroatom (more specifically, an oxygen atom, a sulfur atom, a nitrogen atom, etc.). Examples of aromatic heterocyclic groups include nitrogen-containing aromatic heterocyclic groups (more specifically, pyridyl groups (pyridinyl groups), etc.), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups. be done.
- An aliphatic cyclic group is a group containing a cyclic group consisting of a non-aromatic ring without containing an aromatic ring.
- Aliphatic cyclic groups include, for example, aliphatic carbocyclic groups and aliphatic heterocyclic groups.
- Aliphatic carbocyclic groups are groups containing a non-aromatic ring in which the ring members are all carbon atoms, and include, for example, cycloalkyl groups.
- Aliphatic heterocyclic groups are groups containing a non-aromatic ring in which at least one of the ring member atoms is a heteroatom.
- An aliphatic chain group is a chain (more specifically, linear and branched) group that does not contain aromatic rings and non-aromatic rings.
- Aliphatic chain groups include, for example, aliphatic carbon chain groups (more specifically, alkyl groups, alkylene groups, etc.) and aliphatic heterochain groups.
- An example of an alkyl group is a butyl group.
- the alkylene group is, for example, an n-butylene group.
- the polymer 3A constituting the polymer film 3 may be directly bonded to the surface of the metal nanoparticles 2, and a linker portion derived from a cross-linking agent (more specifically, SM(PEG) n (here , n may be indirectly attached to the surface of the metal nanoparticles 2 via 4, 6 and 8, etc.)).
- a cross-linking agent includes, for example, an amino group-sulfhydryl group cross-linking agent (more specifically, an NHS-maleimide group cross-linking agent, etc.).
- the film thickness of the polymer film 3 is preferably 1 nm to 50 nm, more preferably 1 nm to 10 nm.
- the separation distance (separation distance) is such that the near field is efficiently formed in the space between the two metal nanoparticles 2, so the detection sensitivity is further improved.
- the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance are arranged with a predetermined distance therebetween, so that the quenching of the fluorescent substance excited in the measurement is suppressed, and the detection sensitivity is further improved. improves.
- the separation distance (separation distance) is the minimum value (shortest distance) of the distance between the metal nanoparticle surfaces contained in the two nanoparticles that bind through the test substance in the complex.
- a fluorescent substance 6 is labeled on the surface of the metal nanoparticles 2 .
- the labeling site of the fluorescent substance 6 is the surface of the metal nanoparticles 2 .
- the binding site of the specific binding substance 4 is the polymer membrane 3 as described later.
- the labeling site of the fluorescent substance 6 is different from the binding site of the specific binding substance 4 . Therefore, the number of fluorescent substances 6 can be sufficiently increased compared to nanoparticles having the same labeling site and binding site. Therefore, in this embodiment, the detection sensitivity is superior.
- the fluorescent material 6 includes binding sites via sulfur atoms with the surface of the metal nanoparticles 2 .
- the fluorescent substance 6 is bound to the surface of the metal nanoparticles 2 via sulfur atoms.
- Such a bond can be formed, for example, by mixing metal nanoparticles 2 as starting materials with a fluorescent substance 6 having a disulfide bond.
- the fluorescent substance 6 is preferably labeled on the surface of the metal nanoparticles 2 via a linker (for example, an alkylene group containing an amide group, etc.).
- a linker for example, an alkylene group containing an amide group, etc.
- the linker portion is positioned between the fluorescent substance 6 and the surface of the metal nanoparticle 2, and the excited fluorescent substance 6 becomes the metal nanoparticle. It is easy to prevent contact with the surface of the particles 2 . This is thought to further suppress fluorescence quenching.
- the fluorescent substance 6 is excited by the near field formed by localized surface plasmon resonance and emits fluorescence.
- fluorescent substances include metal complexes (metal complexes) such as europium and ruthenium.
- Ruthenium complexes include, for example, tris(bipyridine)ruthenium (II), which may have a counter anion.
- the fluorescent substance 6 preferably has a large Stokes shift.
- the Stokes shift is the difference between the absorption peak wavelength (maximum excitation wavelength) in the absorption spectrum of the fluorescent substance 6 and the fluorescence peak wavelength (maximum fluorescence wavelength) in the fluorescence spectrum of the fluorescent substance 6 .
- the Stokes shift of the fluorescent material 6 is large, the absorption spectrum and the fluorescence spectrum are less likely to overlap, and excitation light (scattered light) is less likely to enter the fluorescence to be detected, making it possible to measure fluorescence intensity more accurately.
- the fluorescence spectrum of the fluorescent substance 6 is preferably sharp. When the fluorescence spectrum is sharp, it is difficult to overlap with the absorption spectrum of the fluorescent substance 6, so that excitation light (scattered light) is less likely to enter the fluorescence to be detected, and more accurate fluorescence intensity can be measured.
- Nanoparticle body 1 may further comprise a specific binding substance 4 .
- the specific binding substance 4 is a nano-sized (maximum size of 3 to 15 nm) substance that specifically binds to the test substance (described in the sixth embodiment) in the sample.
- the detection sensitivity is superior.
- This description focuses on the fluorescent substance 6, but from a different point of view, let us focus on the specific binding substance 4.
- FIG. In this embodiment the binding site of the specific binding substance 4 in the nanoparticle body 1 is different from the labeling site of the fluorescent substance 6 . Therefore, for the same reason as the fluorescent substance 6, the number of specific binding substances 4 in the nanoparticle body 1 can be increased. Therefore, it is considered that the nanoparticles 1 according to the present embodiment can bind more specific binding substances 4 .
- the detection sensitivity can be further improved.
- the reason is presumed as follows.
- the specific binding nanoparticles collide (encounter) with the test substance in the sample. Due to the collision, the specific binding nanoparticle and the test substance form a bond (specific bond) at their respective specific sites, resulting in the formation of a complex.
- specific binding is formed at a constant rate with respect to the number of collisions between the specific binding nanoparticles and the test substance.
- the specific binding nanoparticles collide with the test substance they do not necessarily have a configurational relationship that is advantageous for forming a specific bond. That is, in order to form specific binding, the specific sites of the specific binding nanoparticles and the test substance must be in contact or at least close to each other during the collision.
- the specific site is, for example, when the specific binding substance is an antibody and the test substance is an antigen, the antibody's antigen binding site (Antigen Binding Site) and the antigen's antigenic determinant (Antigenic Determinant or Epitope).
- the nanoparticles according to the present embodiment can bind a larger amount of the specific binding substance 4, so that the complex can be easily formed, and the detection sensitivity can be further improved.
- the specific binding substance 4 is, for example, at least one selected from the group consisting of antibodies (hereinafter referred to as nanoantibodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA chains and RNA chains).
- the nanoparticle 1 to which such a specific binding substance 4 is bound is superior in detection sensitivity.
- a nano-antibody as the specific binding substance 4 specifically binds to an antigen as a test substance at its tip (antigen-binding site) by an antigen-antibody reaction to form a complex.
- the ligand as the specific binding substance 4 forms a complex with the protein as the test substance by specific protein-ligand binding through the ligand-receptor reaction.
- the nucleic acid strand as the specific binding substance 4 forms a complementary nucleic acid strand pair (double strand) based on base pair complementarity.
- the enzyme as the specific binding substance 4 forms an enzyme-substrate complex with the substrate as the test substance at its active site (active center) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as hydrogen bonds and bonds resulting from intermolecular forces, hydrophobic interactions and charge interactions.
- Nanobodies are, for example, VHH (variable domain of heavy chain antibody) antibodies, fragmented antibodies (more specifically, Fab (Fragment Antigen Binding) antibodies, etc.) and at least one selected from the group consisting of variants thereof is one.
- VHH antibodies are single domain antibodies.
- a variant is an antibody in which a part of the amino acid sequence has been recombined or a substituent has been introduced within the range of specific binding to an antigen.
- these nanobodies are at least one selected from the group consisting of VHH antibodies, fragmented antibodies, and variants thereof, these nanobodies have a relatively small volume, so that the two metal nanoparticles 2 in the complex By narrowing the distance between them (separation distance), the near field can be formed more efficiently and the fluorescence intensity can be further increased.
- the molecular mass of the nanobody is preferably 60,000 Da or less, more preferably 30,000 Da or less, and even more preferably 20,000 Da or less.
- the relatively small volume of the Nanobodies reduces the separation distance in the complex and makes the near-field more efficient. can be formed to further increase fluorescence intensity.
- Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.
- the specific binding substance 4 may be directly bound to the polymer membrane 3, and a linker portion derived from a cross-linking agent (more specifically, SM(PEG) n (where n is 4, 6 and 8, etc.))).
- a cross-linking agent includes, for example, an amino group-sulfhydryl group cross-linking agent (more specifically, an NHS-maleimide group cross-linking agent, etc.).
- the metal nanoparticles 2 are mixed with the polymer to which the fluorescent substance 6 bonds via disulfide bonds, and the fluorescent substance 6 is added to the surface of the metal nanoparticles 2. while labeling the metal nanoparticles 2 and forming a polymer film 3 on the surface of the metal nanoparticles 2 (hereinafter also referred to as a “fluorescence labeling film forming step”).
- the method for producing the nanoparticle body 1 according to the fifth embodiment includes a step of forming a fluorescent labeling film in which the fluorescent labeling on the surface of the metal nanoparticles and the formation of the polymer film can be performed in parallel.
- the steps of fluorescent labeling can be simplified and the cost can be reduced as compared with the conventional technology.
- the fluorescent substance 6 can be labeled at a site different from that of the specific binding substance 4, so that the fluorescent labeling density in the nanoparticle body 1 can be increased.
- the method for producing the nanoparticle 1 may further include a step of thiolating a fluorescent substance, a step of fluorescently labeling a polymer, and a step of binding a specific binding substance.
- FIG. 26 is a reaction scheme showing an example of a method for producing the nanoparticle body 1.
- fluorescent substance thiolation step In the fluorescent substance thiolation step, the fluorescent substance 6 is thiolated. More specifically, in the reaction scheme, as represented by reaction formula (R-1), (A) a fluorescent substance having an ester bond (hereinafter also referred to as a fluorescent substance) and (B) having an amino group A thiol (hereinafter also referred to as aminoalkanethiol) is reacted to form (C) an amide bond to synthesize a thiolated fluorescent substance (hereinafter also referred to as a thiolated fluorescent substance).
- reaction formula (R-1) A fluorescent substance having an ester bond (hereinafter also referred to as a fluorescent substance) and (B) having an amino group A thiol (hereinafter also referred to as aminoalkanethiol) is reacted to form (C) an amide bond to synthesize a thiolated fluorescent substance (hereinafter also referred to as a thiolated fluorescent substance).
- reaction (R-1) The reaction represented by reaction formula (R-1) (hereinafter also referred to as reaction (R-1)) can proceed, for example, in a buffer solution.
- the reaction ratio (molar ratio) between (A) the fluorescent substance and (B) the aminoalkanethiol is, for example, 2:1 to 1:2.
- reaction (R-1) the reaction temperature is, for example, 20-30°C.
- the reaction time is, for example, 0.5 to 2 hours.
- Reaction (R-1) can also be carried out under stirring conditions.
- R 1 in the fluorescent substance represents an alkylene group.
- Such an alkylene group includes, for example, an alkylene group having 2 to 5 carbon atoms (more specifically, an ethylene group, a propylene group, a butylene group and a pentylene group).
- T 1 in the fluorescent substance indicates a terminal group.
- Such a terminal group T1 includes, for example, an NHS group.
- Aminoalkanethiol includes, for example, aminoethanethiol (cysteamine).
- R2 in aminoalkanethiol represents an alkylene group.
- the alkylene group represented by R 2 is the same as the alkylene group represented by R 1 described above.
- Aminoalkanethiol may be in the form of a salt. Examples of such salts include hydrochlorides of aminoalkanethiols (more specifically, cysteamine hydrochloride and the like).
- R 1 and R 2 in (C) thiolated fluorescent substance are the same as R 1 in (A) fluorescent substance and R 2 in (B) aminoalkanethiol, respectively.
- polymer fluorescent labeling step In the polymer fluorescent labeling step, the polymer is fluorescently labeled. More specifically, in the reaction scheme, as represented by reaction formula (R-2), (C) a thiolated fluorescent substance and (D) a polymer having a disulfide bond (hereinafter also referred to as a polymer) to synthesize (E) a polymer labeled with a fluorescent substance (hereinafter also referred to as a fluorescently labeled polymer).
- reaction formula (R-2) C
- D a polymer having a disulfide bond
- E a polymer labeled with a fluorescent substance
- reaction (R-2) the molar ratio of (C) the thiolated fluorescent substance and (D) the disulfide bond of the polymer is For example, 3:1 to 1:1.
- the reaction temperature is, for example, 30-40°C.
- the reaction time is, for example, 0.5 to 2 hours.
- Reaction (R-2) can also be carried out under stirring conditions.
- fluorescent-labeled film forming step In the fluorescent-labeled film forming step, (E) a fluorescent-labeled polymer (a polymer to which the fluorescent substance 6 binds via a disulfide bond) and (F) the metal nanoparticles 2 are mixed to convert the fluorescent substance 6 into metal nanoparticles. A polymer film 3 is formed on the surface of the metal nanoparticles 2 while labeling the surfaces of the particles 2 . That is, in the fluorescent labeling film forming step, (F) labeling of the surface of the metal nanoparticles 2 with the fluorescent substance 6 and (F) formation of the polymer film 3 on the surface of the metal nanoparticles 2 are performed in parallel. can.
- the formed polymer film 3 includes (F) bonding sites 3a with the surface of the metal nanoparticles 2 via sulfur atoms. Note that in the reaction represented by the reaction formula (R-3) in FIG. 4 shows an enlarged schematic diagram of the vicinity of the interface with the surface of the .
- reaction (R-3) the reaction time is, for example, 12-36 hours.
- the reaction temperature is, for example, 20-30°C.
- Reaction (R-3) can also be carried out under stirring conditions.
- reaction (R-3) with the aim of labeling the surface of the (F) metal nanoparticles 2 with the fluorescent substance 6 as much as possible, until the reaction (R-3) does not substantially proceed, (F)
- (E) Fluorescent labeling polymer is excessively applied to the surface of the metal nanoparticles 2 .
- a nanoparticulate body 1 can be produced comprising a fluorescent material 6 containing a
- the binding site of the polymer film 3 that binds to the specific binding substance 4 is, for example, (E) the functional group of the fluorescently labeled polymer.
- Such functional groups are, for example, at least one functional group selected from the group consisting of amino groups, carboxyl groups, thiol groups, N-hydroxysuccinimide groups (NHS groups) and maleimide groups.
- the metal nanoparticles 2, the polymer film 3 covering the surface of the metal nanoparticles 2, and the metal nanoparticles Nanoparticulate bodies 1 can be produced comprising fluorescent substances 6 labeled on the surface of particles 2 and specific binding substances 4 bound to polymer membranes 3 .
- optional steps may further comprise:
- FIG. 27 is a cross-sectional view schematically showing the composite.
- a complex 40 comprises a test substance 30 to be detected and two nanoparticles 10 and 20 .
- Two nanoparticulate bodies 10 and 20 are bound via a test substance 30 in a composite 40 . That is, the nanoparticles 10 and 20 according to the fifth embodiment form the complex according to the sixth embodiment, which is bound via the test substance 30 .
- One of the two nanoparticle bodies 10 and 20 is called the first nanoparticle body 10 and the other nanoparticle body is called the second nanoparticle body 20 .
- composite 40 includes first nanoparticle 10 and second nanoparticle 20 as nanoparticle 1 .
- the first nanoparticle body 10 includes the first metal nanoparticles 12 as metal nanoparticles, the first polymer film 13 as the polymer film, and the first fluorescent substance 16 as the fluorescent substance.
- the second nanoparticle body 20 includes a second metal nanoparticle 22 as a metal nanoparticle, a second polymer film 23 as a polymer film, and a second fluorescent substance 26 as a fluorescent substance. At least one of the surface of the first metal nanoparticles 12 and the surface of the second metal nanoparticles 22 is labeled with the fluorescent substances 16 and 26 .
- the first nanoparticle body 10 includes the first metal nanoparticles 12, the first polymer film 13 covering the surface of the first metal nanoparticles 12, and the first metal nanoparticles 12. and a first fluorescent material 16 labeled on the surface.
- the second nanoparticle body 20 includes a second metal nanoparticle 22, a second polymer film 23 covering the surface of the second metal nanoparticle 22, and a second fluorescence labeled on the surface of the second metal nanoparticle 22. material 26;
- the first nanoparticle body 10 further contains a first specific binding substance 14 as a specific binding substance
- the second nanoparticle body 20 further contains a second specific binding substance 24 as a specific binding substance.
- the first nanoparticle body 10 further comprises a first specific binding substance 14 bound to the first polymer membrane 13, and the second nanoparticle body 20 is bound to the second polymer membrane 23. further comprising a second specific binding substance 24.
- the separation distance L is small within a range in which the excited fluorescent substances 16 and 26 are less likely to be quenched.
- the two nanoparticulate bodies 10, 20 in the composite 40 are in close proximity to each other.
- the two nanoparticle bodies 10 and 23 are in contact with each other such that the first polymer film 13 of the first nanoparticle body 10 and the second polymer film 23 of the second nanoparticle body 20 in the composite 40 are in contact with each other. 20 are close to each other.
- at least one of the first polymer film 13 of the first nanoparticle body 10 and the second polymer film 23 of the second nanoparticle body 20 in the composite 40 shrinks. The two nanoparticulate bodies 10, 20 are brought close to each other so that they are in contact with each other.
- the test substance 30 and the test substance it is believed that at least one of the specific binding substances 14 , 24 that bind to 30 and the fluorescent substances 16 , 26 can be incorporated into the polymer membranes 13 , 23 .
- the polymer membranes 13 and 23 are in contact with each other, for example, the complex 40 shown in FIG. It is believed that at least one of the specific binding substances 14,24 and the fluorescent substances 16,26 can be incorporated into the polymer membranes 13,23.
- the films covering the surfaces of the metal nanoparticles 12 and 22 are the polymer films 13 and 23, the fluorescence intensity can be increased.
- the reasons is presumed as follows.
- the films that cover the surfaces of the metal nanoparticles 12 and 22 are the polymer films 13 and 23, and the polymer films 13 and 23 have relatively high flexibility compared to inorganic films containing inorganic oxides. For this reason, in the composite 40, the polymer films 13 and 23 can contract, and as a result, the two metal nanoparticles 12 and 22 are reduced to two film thicknesses of the polymer film (thickness of the polymer film 13 + (thickness of the polymer film 23).
- the separation distance L can be less than two film thicknesses of the polymer films.
- the plasmon enhancement effect is easily obtained, and the fluorescence intensity is further increased.
- the film thickness of the polymer film in "two film thicknesses of the polymer film” does not mean the film thickness of the polymer films 13 and 23 at the contracted portion, which is the object of the separation distance, It is the film thickness of the polymer films 13 and 23 of the non-shrinking portion that is not to be separated (for example, T 1 in FIG. 31 to be described later).
- the polymer films 13 and 23 contain binding sites 3a via sulfur atoms between the surfaces of the metal nanoparticles.
- the polymer 3A that constitutes the polymer films 13 and 23 includes a binding site 3a via a sulfur atom. This can further increase the fluorescence intensity. The reason is presumed as follows. In such a case, since the binding site 3a forms a bond with the surfaces of the metal nanoparticles 12 and 22, the polymer 3A has a network structure and is considered to cover the surfaces of the metal nanoparticles 12 and 22 in a network. Since polymer 3A has such a network structure, it has relatively high flexibility.
- the polymer membranes 13, 23 can be further shrunk, thereby bringing the two metal nanoparticles 12, 22 closer together than the distance corresponding to two polymer membrane thicknesses. It becomes possible to Therefore, in the present embodiment, the separation distance L can be less than two film thicknesses of the polymer film, so that the plasmon enhancement effect can be further obtained and the fluorescence intensity can be further increased.
- polymer films 13 and 23 have, in addition to binding sites 3a via sulfur atoms, at least selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c between the surfaces of the metal nanoparticles. Further includes one. This further increases the fluorescence intensity.
- at least one selected from the group consisting of the positively charged group 3b and the hydrophobic group 3c forms a bond with the surfaces of the metal nanoparticles 12 and 22, so that the polymer 3A has a network structure, It is thought that the surfaces of the metal nanoparticles 12 and 22 are coated in a mesh pattern. Since polymer 3A has such a network structure, it has relatively high flexibility.
- the polymer membranes 13, 23 can be further shrunk, thereby bringing the two metal nanoparticles 12, 22 closer together than the distance corresponding to two polymer membrane thicknesses. It becomes possible to Therefore, in the present embodiment, the separation distance L can be less than two film thicknesses of the polymer film, so that the plasmon enhancement effect can be further obtained and the fluorescence intensity can be further increased.
- the polymer 3A that constitutes the polymer films 13 and 23 includes a binding site 3a via a sulfur atom in its side chain (more specifically, the end of the side chain).
- fluorescence intensity can be further increased. The reason is presumed as follows. In such a case, the binding sites 3a form bonds with the surfaces of the metal nanoparticles 12,22. Therefore, it is considered that the polymer 3A has a network structure and coats the surfaces of the metal nanoparticles 12 and 22 in a network with the side chains as binding sites. Since polymer 3A has such a network structure, it has relatively high flexibility.
- the polymer membranes 13, 23 can be further shrunk, thereby bringing the two metal nanoparticles 12, 22 closer together than the distance corresponding to two polymer membrane thicknesses. It becomes possible to Therefore, in the present embodiment, the separation distance L can be less than two film thicknesses of the polymer film, so that the plasmon enhancement effect can be further obtained and the fluorescence intensity can be further increased.
- the polymer 3A constituting the polymer membranes 13 and 23 is selected from the group consisting of the positively charged group 3b and the hydrophobic group 3c on its side chain (more specifically, the side chain end). including at least one
- fluorescence intensity can be further increased. The reason is presumed as follows. In such cases, the positively charged groups 3b and/or the hydrophobic groups 3c form bonds with the surfaces of the metal nanoparticles 12,22. Therefore, it is considered that the polymer 3A has a network structure and coats the surfaces of the metal nanoparticles 12 and 22 in a network with the side chains as binding sites. Since polymer 3A has such a network structure, it has relatively high flexibility.
- the polymer membranes 13, 23 can be further shrunk, thereby bringing the two metal nanoparticles 12, 22 closer together than the distance corresponding to two polymer membrane thicknesses. It becomes possible to Therefore, in the present embodiment, the separation distance L can be less than two film thicknesses of the polymer film, so that the plasmon enhancement effect can be further obtained and the fluorescence intensity can be further increased.
- the separation distance L between the first nanoparticle bodies 10 and the second nanoparticle bodies 20 is, for example, 12 nm to 52 nm, preferably 12 nm to 27 nm.
- the separation distance L is the distance between the first metal nanoparticles 12 and the second metal nanoparticles 22, and is the distance between the first point P1 on the surface of the first nanoparticle body 10 and the second nanoparticle body 20. It is the minimum distance of the line segment connected with the second point P2 on the surface.
- the separation distance L is 52 nm or less, when the composite 40 is irradiated with excitation light, a near-field is generated more efficiently in the space near the surface between the first and second metal nanoparticles 12 and 22. , the fluorescence intensity can be further increased.
- the films that cover the surfaces of the metal nanoparticles 12 and 22 are the polymer films 13 and 23, and the polymer films 13 and 23 contain binding sites 3a via sulfur atoms between the surfaces of the metal nanoparticles. Therefore, as described above, the separation distance L can be closer than the distance corresponding to two film thicknesses of the polymer film covering the surfaces of the two metal nanoparticles 12 and 22 in the composite 40. . For example, if the thickness of the polymer films 13, 23 is 5 nm, the separation distance L can be less than 10 nm (more specifically, 2-9 nm, 3-8 nm, 4-7 nm, etc.).
- the fluorescent substances 16, 26 are positioned at least between the first metal nanoparticles 12 and the second metal nanoparticles 22 in the composite 40, as shown in FIG. This is because the fluorescent substances 16 and 26 are positioned in the space between the metal nanoparticles 12 and 22 because the space between the metal nanoparticles 12 and 22 is a space in which a near-field is efficiently generated, so that the fluorescence intensity is likely to be increased.
- Test substance 30 is a substance to be detected contained in the specimen.
- Test substances 30 include, for example, antigens, proteins, substrates, and nucleic acid chains.
- the test substance 30 specifically binds to the specific binding substances 14,24.
- an antigen has at least two antigenic determinants and forms specific binding with the first and second specific binding substances 14, 24 at the antigenic determinants.
- Antigens are, for example, proteins such as c-reactive protein, myoglobin, troponin T, troponin I, and BNP, and antigenic proteins of viruses such as influenza virus, respiratory syncytial virus, and the like.
- Test substance 30 is, for example, a test substance derived from a specimen such as blood, plasma, urine, or saliva.
- samples containing the test substance 30 are, for example, blood, plasma, serum, urine, and saliva.
- the specimen further includes solvents and buffers (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, MES buffer, etc.). It's okay.
- PBS phosphate-buffered saline
- Tris buffer Tris buffer
- HEPES buffer Tris buffer
- MOPS buffer MOPS buffer
- MES buffer etc.
- FIG. 28 is a diagram schematically showing a measuring device according to the seventh embodiment.
- the measurement apparatus 100 includes an excitation light source 110 , an excitation light irradiation optical system 120 , a reagent container 130 , a light receiving optical system 140 and a light receiving element 150 .
- the excitation light source 110 emits excitation light 112 .
- the excitation light source 110 is, for example, a laser.
- the excitation light irradiation optical system 120 adjusts the cross-sectional diameter and the like like the excitation light 112 and outputs incident excitation light 122 .
- the excitation light irradiation optical system 120 is a lens 124 and a polarizing element ( ⁇ /2 plate) 126 .
- the incident excitation light 122 output from the excitation light irradiation optical system 120 enters the reagent container 130 and irradiates the measurement sample in the reagent container 130 .
- the reagent container 130 is, for example, a removable container (more specifically, a cell, a preparation, etc.) and a microchannel chip.
- a microchannel chip is a chip having minute channels.
- the measurement sample irradiated with the incident excitation light 122 emits fluorescence (detection light 132).
- the light-receiving optical system 140 is arranged in a direction perpendicular to the traveling direction of the incident excitation light 122 to the reagent container 130 .
- the light receiving optical system 140 can adjust the cross-sectional diameter and the like of the detection light 132 emitted from the measurement sample, remove the scattered light of the incident excitation light 122, or adjust the light intensity.
- the light receiving optical system 140 is a lens 144 and an optical filter 146 .
- Optical filters 146 are, for example, bandpass filters and dichroic mirrors.
- Fluorescence 142 that has passed through the light receiving optical system 140 is detected by the light receiving element 150 .
- the light receiving element 150 is, for example, PD, APD, PMT, CCD camera, and spectroscope.
- the light-receiving element 150 is capable of measuring the fluorescence amount of a single wavelength, measuring the fluorescence spectrum, and creating two-dimensional planar fluorescence imaging.
- each of the nanoparticles 1, 10, and 20 is labeled with four fluorescent substances 6, 16, and 26, but the present invention is not limited to this.
- the number of fluorescent substances labeled on the nanoparticles 1, 10, 20 may be 5 or more.
- the present invention is not limited to this.
- the number of specific binding substances 4, 14, 24 labeled on the nanoparticles 1, 10, 20 may be 5 or more, respectively.
- the light-receiving optical system 140 in the measurement device 100 is arranged in a direction perpendicular to the traveling direction of the incident excitation light 122 to the reagent container 130, but it is not limited to this.
- the light-receiving optical system 140 may be arranged, for example, in a direction parallel to the traveling direction of the incident excitation light 122, or may be arranged in a direction forming an acute or obtuse angle with respect to the traveling direction of the incident excitation light 122. . (Example)
- the concentration of the metal nanoparticles in the dispersion liquid may be indicated by the absorbance.
- the absorbance was measured using an ultraviolet-visible spectrophotometer (“infinite M200 PRO” manufactured by TECAN Japan Co., Ltd.).
- Example 6 Preparation of nanoparticles
- the nanoparticles of Example 6 were produced as follows. (Introduction of thiol group to ruthenium complex derivative) (b) cysteamine hydrochloride (“A0296” manufactured by Tokyo Chemical Industry Co., Ltd.) and (a) NHS-labeled Ru complex derivative (“Ruthenium (II) tris(Bipyridyl)-C5-NHS ester” manufactured by Tokyo Chemical Industry Co., Ltd.) and were stirred and mixed using a small rotary incubator ("RT-30 mini” manufactured by Taitec Co., Ltd.) at room temperature (for example, 25 ° C.) for 1 hour.As a result, at the end of the ligand of the Ru complex A thiol group was introduced, and this synthetic reaction is represented by the reaction formula (r-1): (Chemical 2) is a nucleophilic substitution reaction in which (b) the primary amino group of cysteamine hydrochloride attacks the NHS ester group of (a) the
- reaction formula (r-4) (Chemical 3) is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamide-PEG4-NHS, as shown in .
- Ru complex-labeled polymer (hereinafter also referred to as "Ru complex-labeled polymer") in which a Ru complex is bound to the side chain via an SPDP linker and a disulfide bond was obtained.
- the synthesized (e) Ru complex-labeled polymer had a hydrophobic group (n-butylene group) and a positively charged group (primary ammonium group).
- This synthesis reaction is represented by reaction formula (r-2): (Chem. 4)
- the thiol group of the Ru complex-SH attacks the disulfide group of the (d) disulfide group-containing polymer in a thiol nucleophilic reaction.
- FIG. 29 shows a schematic diagram for explaining the method for producing a nanoparticle body of Example 6. As shown in FIG. 29
- FIG. 30 shows the fluorescence spectrum of the nanoparticles of Example 6 (horizontal axis: fluorescence wavelength (unit: nm) and vertical axis: fluorescence intensity (unit: arbitrary unit)).
- the fluorescence spectrum obtained had a peak at about 620 nm.
- the shape of this fluorescence spectrum almost matched the shape of the fluorescence spectrum of the Ru complex. From this result, it was confirmed that the nanoparticles of Example 6 were labeled with the Ru complex.
- Example 7 Preparation of nanoparticles
- the nanoparticles were prepared by labeling the polymer film of the polymer-coated silver nanoparticles with a fluorescent substance.
- a nanoparticle composition containing the nanoparticles of Example 7 was prepared by adding a polyanionic polymer to the nanoparticles.
- This synthetic reaction is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamide-PEG4-NHS.
- RT-30mini manufactured by Taitec Co., Ltd.
- a dispersion of silver nanoparticles in which the cross-linking agent SM(PEG)6 was bound to the polymer film (hereinafter also referred to as polymer-coated silver nanoparticles bound with SM(PEG)6 linker) was obtained.
- the SM(PEG)6 linker that attached the polymer-coated silver nanoparticles had a maleimide group.
- a reducing agent TCEP (77720" manufactured by ThermoFisher SCIENTIFIC) was added in a molar ratio of 2 equivalents to the fluorescence-labeled VHH antibody to which the SPDP linker was bound. -100") and stirred to mix.
- a VHH antibody bound with a fluorescent substance and a reduced SPDP linker (hereinafter also called a reduced SPDP linker) was obtained (hereinafter also called a fluorescence-labeled VHH antibody bound with a reduced SPDP linker).
- the reduced SPDP linkers had thiol groups (-SH groups) generated by reduction of disulfide bonds.
- nanoparticulate composition consisted of the nanoparticles of Example 7 and a solvent containing sodium heparin as the polyanionic polymer.
- FIG. 31 shows an SEM image of a composite prepared using the nanoparticles of Example 7.
- the separation distance L 1 between the metal nanoparticles of the nanoparticles in the composite and the thickness T 1 of the polymer film other than between the metal nanoparticles of the nanoparticles was measured and compared.
- the thickness T1 of the polymer film was the thickness of the polymer film in the non-shrinking portion that was not subject to the separation distance.
- the separation distance L 1 was smaller than the thickness (T 1 ⁇ 2) corresponding to two thicknesses of the polymer film. Therefore, in the composite prepared with the nanoparticles of Example 7, the polymer film shrinks, and the two metal nanoparticles in the composite correspond to two thicknesses of the polymer film (T 1 ⁇ 2). It turns out that they are closer than they are apart. This strongly suggested that the plasmon enhancement effect was further obtained and the fluorescence intensity was further increased.
- silica-coated silver nanoparticles manufactured by nanoComposix, silver nanoparticle diameter (core particle diameter) 50 nm, silica film thickness 20 nm
- aqueous dispersion prepared by diluted with water to prepare an aqueous dispersion of silica-coated silver nanoparticles. .
- the obtained aqueous dispersion was used as a measurement sample.
- aggregates in which two particles were aggregated were also present in the measurement sample. This aggregate was located and evaluated.
- Example 7 In the same manner as in Example 7, an SEM image (500K magnification) was taken, and from the SEM image, the thickness T2 of the inorganic film in the aggregate and the distance L2 between the two metal nanoparticles were measured and compared. As a result, the separation distance L2 was about twice the thickness T2 of the inorganic film. Note that the thickness T2 of the inorganic film was the thickness of the inorganic film at a portion not subject to the separation distance. (Description of symbols)
- Embodiments according to the present invention also include the following aspects.
- It contains metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a nano-sized specific binding substance bound to the surface of the polymer film that specifically binds to the test substance in the specimen.
- the hydrophobic group is at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group.
- the nanoparticle body according to any one of [1] to [14], wherein the metal nanoparticles comprise gold or silver.
- the nanoparticle bodies include a first nanoparticle body and a second nanoparticle body,
- the first nanoparticle body includes a first metal nanoparticle as the metal nanoparticle, a first polymer film as the polymer film, and a first specific binding substance as the specific binding substance
- the second nanoparticle body includes a second metal nanoparticle as the metal nanoparticle, a second polymer film as the polymer film, and a second specific binding substance as the specific binding substance, At least one of the first polymer membrane and the second polymer membrane, and the first specific binding substance and the second specific binding substance is labeled with a fluorescent substance, [1] to [16] ]
- the nanoparticle body according to any one of .
- Embodiments according to the present invention also include the following aspects.
- [1] Comprising metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a fluorescent substance, The fluorescent substance is labeled on the surface of the metal nanoparticles, A nanoparticle body, wherein the polymer film includes a binding site via a sulfur atom between the surface of the metal nanoparticle.
- [2] The nanoparticle according to [1], wherein the polymer constituting the polymer film contains the binding site in its side chain.
- [3] The nanoparticle body according to [1] or [2], wherein the fluorescent substance includes a binding site via a sulfur atom to the surface of the metal nanoparticle.
- the polymer constituting the polymer film further includes at least one selected from the group consisting of the positively charged group and the hydrophobic group in its side chain.
- the polymer film contains at least the positively charged group,
- the nanoparticle according to [5] which is one type.
- the polymer membrane contains at least the hydrophobic group, The nanoparticle according to [5], wherein the hydrophobic group is at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group and an aliphatic chain group.
- the nanoparticle bodies include a first nanoparticle body and a second nanoparticle body,
- the first nanoparticle body includes first metal nanoparticles as the metal nanoparticles and a first polymer film as the polymer film
- the second nanoparticle body includes second metal nanoparticles as the metal nanoparticles and a second polymer film as the polymer film
- the nanoparticle body according to any one of [1] to [13], wherein at least one of the surface of the first metal nanoparticles and the surface of the second metal nanoparticles is labeled with the fluorescent substance. .
- the nanoparticle according to [14] wherein the first nanoparticulate and the second nanoparticulate form a complex bound via a test substance.
- Embodiments according to the present invention also include the following aspects.
- a nanoparticulate composition comprising a nanoparticulate and a solvent containing the nanoparticulate,
- the nanoparticle body binds to the metal nanoparticles, the coating film covering the surface of the metal nanoparticles, the surface of the coating film or the surface of the metal nanoparticles, and is specific to the test substance in the specimen.
- a specific binding substance that binds to A nanoparticle composition, wherein the solvent contains a polyanionic polymer in addition to the nanoparticles.
- the polyanionic polymer has at least one anionic group selected from the group consisting of a carboxylate group, a sulfate group, a sulfonate group, a nitrate group, a phosphate group, and a borate group; A nanoparticulate body composition as described.
- the polyanionic polymer is at least one selected from the group consisting of polyglutamic acid, heparin, polyaspartic acid, polyacrylic acid and salts thereof, and DNA. Particulate composition.
- the coating film is An inorganic film containing an inorganic oxide, or a polymer containing at least one selected from the group consisting of a bonding site via a sulfur atom between the surface of the metal nanoparticles, a positively charged group, and a hydrophobic group
- the coating film is the polymer film, [ 4].
- nanoparticle composition according to any one of [1] to [6], wherein the specific binding substance is crosslinked with at least one selected from the group consisting of polyalkylene ether chains and alkyl chains. .
- the solvent comprises an aqueous solvent.
- the nanoparticle bodies include a first nanoparticle body and a second nanoparticle body,
- the first nanoparticle body includes a first metal nanoparticle as the metal nanoparticle, a first coating film as the coating film, and a first specific binding substance as the specific binding substance
- the second nanoparticle body includes a second metal nanoparticle as the metal nanoparticle, a second coating film as the coating film, and a second specific binding substance as the specific binding substance, [1] to [13], wherein at least one of the first coating film and the second coating film, and the first specific binding substance and the second specific binding substance is labeled with a fluorescent substance.
- a nanoparticulate composition according to any one of the preceding.
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Abstract
Description
プラズモン励起蛍光分析では、複合体は、例えば、被験物質、特異結合物質の他に、蛍光物質と金属粒子をさらに含む(複合体は、例えば、金属粒子、特異結合物質、蛍光物質、および被験物質を含む)。励起光が複合体に照射されると、複合体内の金属粒子で表面プラズモン共鳴が発生し、金属粒子の表面近傍で近接場が形成される。この近接場によって蛍光物質の蛍光強度が増大される。
一方、シリカ膜を調製する場合、テトラエトキシシランを塩基性条件下で加水分解して重合的に形成させるが、その形成速度が反応濃度や反応温度、反応時間などの影響を大きく受け、安定して作製することが困難である。
金属ナノ粒子と、該金属ナノ粒子の表面を被覆する高分子膜と、該高分子膜の表面に結合された、検体中の被験物質と特異的に結合するナノサイズの特異結合物質とを含んで成り、
前記高分子膜が、前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性基、および疎水性基からなる群より選択される少なくとも1つを含む。
第1実施形態に係るナノ粒子体は、
金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、高分子膜の表面に結合された、検体中の被験物質と特異的に結合するナノサイズの特異結合物質とを含んで成り、
高分子膜が、金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性基、および疎水性基からなる群より選択される少なくとも1つを含む。
はじめに、本実施形態に係るナノ粒子体の説明の便宜上、その理解を助ける目的で、本実施形態に係るナノ粒子体を用いた被験物質の検出方法を説明する。
本実施形態に係るナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、高分子膜に結合した特異結合物質とを含んで成る。本実施形態に係るナノ粒子体を検体に溶解または分散させて、検体中に含まれる被験物質を捕捉して複合体(第2実施形態)を形成する。より具体的には、複合体は、ナノ粒子体の特異結合物質と、被験物質とが特異的に結合することによって形成される。複合体は、被験物質を介して2つのナノ粒子体が結合する構造を有する。このように、2つの金属ナノ粒子は、複合体において同一の被験物質に対してそれぞれの特異結合物質と結合することによって、一定の距離で離間して配置されている。さらに、複合体は、蛍光物質を含む。
本実施形態に係るナノ粒子体は、検出安定性により優れる。特定の理論に拘束されるわけではないが、その理由は以下のように推測される。本実施形態に係るナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜とを含んで成り、高分子膜は、金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性、および疎水性基からなる群より選択された少なくとも1つを含む。よって、高分子膜は、金属ナノ粒子の表面と比較的強い結合を形成するため、金属ナノ粒子の表面に安定的に固定される。これにより、高分子膜の金属ナノ粒子表面からの剥離等が防止され、ひいては「金属ナノ粒子表面の露出」、および高分子膜の剥離等に伴う「特異結合物質の脱離」が抑制され、検出感度の低下が抑制される。さらに、高分子膜は、高分子を含んで構成されているため、シリカ層に比べ、化学修飾しやすい。これにより、シリカ層に比べ膜厚を小さくして、複合体における金属粒子間距離を減少させることができる。よって、検出感度を向上させることができる。以上から、本実施形態に係るナノ粒子体は、検出安定性により優れると考えられる。
以下、ナノ粒子体の構成を説明する。図1を参照して、ナノ粒子体を説明する。図1は、ナノ粒子体を模式的に示す断面図である。本実施形態に係るナノ粒子体1は、金属ナノ粒子2と、金属ナノ粒子2の表面を被覆する高分子膜3と、高分子膜3の表面に結合された特異結合物質4とを含んで成る。
金属ナノ粒子2は、その表面を高分子膜3で被覆されている。金属ナノ粒子2は、金属の種類によって異なるが、特定の波長を有する光と相互作用し、局在表面プラズモン共鳴を起こす。銀ナノ粒子では400nmから530nm、金ナノ粒子では510nmから580nmにプラズモンの共鳴ピークがある。これは粒子径により異なる。例えば、粒子径が20nmの銀からなるナノ粒子は、波長405nmの光と共鳴し、粒子径が20nmの金からなるナノ粒子は、波長524nmの光と共鳴する。金属ナノ粒子2の粒子径(平均一次粒子径)は、例えば、5nm~100nmである。金属ナノ粒子2の粒子径は、走査型電子顕微鏡(SEM)または透過型電子顕微鏡(TEM)を用いて、金属ナノ粒子2の画像を撮像し、画像での金属ナノ粒子2の粒子径を測長し、複数の粒子径の平均値(測定数:例えば、少なくとも10以上)を算出することにより得ることができる。
金属ナノ粒子2は、好ましくは金または銀を含んで成り、より好ましくは銀を含んで成る。
高分子膜3は金属ナノ粒子2の表面を被覆する。高分子膜3は、金属消光分子膜として機能する。複合体においては、高分子膜3は、少なくとも高分子膜3の厚み分、金属ナノ粒子2の表面から蛍光物質を離間して配置させることができる。このため、励起した蛍光物質が金属ナノ粒子2の表面に接触して消光することを抑制し、検出感度の低下を抑制できる。高分子膜3の存在は、SEMまたはTEMを用いて、ナノ粒子体1の画像を撮像し、画像でのナノ粒子体1を観察し、確認することができる。
以下、硫黄原子を介した結合部位3a、正帯電性基3bおよび疎水性基3cを説明する。
硫黄原子を介した結合部位3aは、例えば、側鎖としてジスルフィド結合を含む部位を有する高分子と、金属ナノ粒子2とを混合することで形成される。原料としての高分子が、例えば、後述の図8に示すように、側鎖にジスルフィド結合を介して疎水性基3cを有する場合、金属ナノ粒子2の表面との間に硫黄原子を介した結合部位を形成する(図2および図8中の右側参照)。
正帯電性基は、金属ナノ粒子2の表面と強い静電結合を形成する。正帯電性基は、本明細書において、1価以上の価数を有し、完全に正に電離した基である。高分子膜3を構成する高分子に含まれる複数の正帯電性基3bを考慮した場合、正帯電性基3bは、下記の数式(1):
で表されるpKaが7以上である基をいう。つまり、正帯電性基3bは、高分子膜3を構成する高分子3Aの電気的中性基および正帯電性基3bとが被験物質を検出する環境(例えば、pHおおよそ6~8の検体)中で、下記の化学平衡式(2):
疎水性基3cは、芳香族環状基、脂肪族環状基、および脂肪族鎖状基からなる群より選択される少なくとも1種である。
なお、本明細書において、離間距離(離隔距離)とは、複合体において被験物質を介して結合する2つのナノ粒子体にそれぞれ含まれる金属ナノ粒子表面間の距離の最小値(最短距離)をいう。
特異結合物質4は、検体中の被験物質(第2実施形態にて説明する)と特異的に結合するナノサイズ(最長が3~15nmであるサイズ)の物質である。特異結合物質4としては、例えば、抗体(以下、ナノ抗体と称する)、リガンド、酵素、ならびに核酸鎖(より具体的には、DNA鎖およびRNA鎖)が挙げられる。例えば、特異結合物質4としてのナノ抗体は、抗原抗体反応により、その先端部(抗原結合部位:Antigen Binding Site)で、被験物質としての抗原と特異的に結合して複合体を形成する。特異結合物質4としてのリガンドは、被験物質としてのタンパク質と、リガンド・レセプター反応によって特異的なタンパク質-リガンド結合して複合体を形成する。特異結合物質4としての核酸鎖は、塩基対の相補性に基づいて、相補的な関係にある核酸鎖と核酸鎖の対(二本鎖)を形成する。特異結合物質4としての酵素は、その活性部位(活性中心)で基質特異性(立体特異性)に基づいて被験物質としての基質と酵素-基質複合体を形成する。これらの特異的結合は、非共有結合であり、例えば、水素結合、ならびに分子間力、疎水的相互作用および電荷的相互作用に起因する結合である。
ナノ粒子体1は、蛍光物質をさらに含んで成ってもよい。この場合、蛍光物質は高分子膜3の表面および特異結合物質の少なくとも一方に標識される。蛍光物質は、局在表面プラズモン共鳴で形成される近接場により励起され、蛍光を発する。蛍光物質は、例えば、ユーロピウムおよびルテニウムのような金属の錯体(金属錯体)、ならびにAlexsa Fluorシリーズ(登録商標)(Molecular Probes(登録商標))の色素が挙げられる。
第2実施形態に係るナノ粒子体組成物は、
ナノ粒子体と、ナノ粒子体を含む溶媒とを含んで成り、
ナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、高分子膜の表面または金属ナノ粒子の表面に結合し、検体中の被験物質と特異的に結合する特異結合物質とを有し、
溶媒には、ナノ粒子体に加えてポリアニオン系高分子が含まれる。
特許文献1に記載のイムノクロマトグラム用複合粒子(ナノ粒子体)は、金属からなる微粒子(金属ナノ粒子)の外側が、少なくとも一種の蛍光物質を含有するシリカの少なくとも1層で覆われた構造を持ち、標的物質(被験物質)を特異的に認識する標識物質(特異結合物質)で表面修飾された微粒子からなる。このように、特許文献1に記載のナノ粒子体は、金属ナノ粒子の外側を覆う被覆層(被覆膜)に特異結合物質を表面修飾している。
一方、被覆膜に分散剤を結合させてナノ粒子体の分散性を向上させると、被覆膜上の結合サイトが減少し、ナノ粒子体の設計の自由度が損なわれるという問題が生じる。このように、高い設計自由度と、優れた分散性とを十分に両立させることができなかった。
はじめに、本実施形態に係るナノ粒子体組成物の説明の便宜上、その理解を助ける目的で、本実施形態に係るナノ粒子体組成物を用いた被験物質の検出方法の一例を説明する。
本実施形態に係るナノ粒子体組成物は、ナノ粒子体と、ナノ粒子体を含む溶媒とを含んで成る。ナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、高分子膜の表面または金属ナノ粒子の表面に結合する特異結合物質とを有する。本実施形態に係るナノ粒子体組成物を検体に添加して、検体中に含まれる被験物質を捕捉して複合体を形成する。より具体的には、複合体は、ナノ粒子体の特異結合物質と、被験物質とが特異的に結合することによって形成される。複合体は、被験物質を介して2つのナノ粒子体が結合する構造を有する。このように、2つの金属ナノ粒子は、複合体において同一の被験物質に対してそれぞれの特異結合物質と結合することによって、一定の距離で離間して配置されている。さらに、複合体は、蛍光物質を含む。
本実施形態に係るナノ粒子体組成物は、設計自由度を維持しつつ、分散性により優れるナノ粒子体を含んで成る。特定の理論に拘束されるわけではないが、その理由は以下のように推測される。本実施形態に係るナノ粒子体組成物は、ナノ粒子体と、ナノ粒子体を含む溶媒とを含んで成る。ナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、高分子膜の表面または金属ナノ粒子の表面に結合し、検体中の被験物質と特異的に結合する特異結合物質とを有する。溶媒には、ナノ粒子体に加えてポリアニオン系高分子が含まれる。このため、本実施形態に係るナノ粒子体組成物において、ナノ粒子体は、その個々の粒子体がポリアニオン系高分子により取り囲まれた会合体(集合体)を形成し得る。ポリアニオン系高分子が複数のナノ粒子体間のスペーサとして作用し、ナノ粒子体同士の接触による凝集を防止する。さらに、会合体の外表面には、ポリアニオン系高分子のアニオン性基が複数存在するため、会合体間で静電的斥力が作用し、ナノ粒子体同士を互いに遠ざける。これにより、本実施形態に係るナノ粒子体組成物中でナノ粒子体は単体で分散することができる。つまり、ナノ粒子体は本実施形態に係るナノ粒子体組成物において分散性に優れる。
上述のように、ナノ粒子体は、分散性を向上させるために表面修飾を施していない。このため、ナノ粒子体の表面を修飾する余地があり、設計の自由度を維持することができる。以上から、本実施形態に係るナノ粒子体組成物は、高い設計自由度と、優れた分散性とを兼ね備えるナノ粒子体を含んで成ると考えられる。
以下、ナノ粒子体組成物の構成を説明する。ナノ粒子体組成物は、ナノ粒子体と、ナノ粒子体を含む溶媒とを含んで成る。
溶媒には、ナノ粒子体に加えてポリアニオン系高分子が含まれる。溶媒は、例えば、水性溶媒を含む。水性溶媒は、少なくとも水(より具体的には、純水)を含んで成る。水性溶媒としては、例えば、水(純水)、水と有機溶媒とを含む混合溶媒、および塩成分が溶解している溶媒(より具体的には、緩衝液等)である。前記有機溶媒は、水と混和する有機溶媒であり、例えば、アルコール(より具体的には、メタノール、エタノールおよびプロパノール等)、ならびにテトラヒドロフラン、ジメチルホルムアミド、およびジメチルスルホキシド等からなる群より選択される少なくとも1種である。
ポリアニオン系高分子は、複数のアニオン性基を有する高分子である。アニオン性基としては、例えば、カルボン酸塩基、硫酸塩基、スルホン酸塩基、硝酸塩基、リン酸塩基、およびホウ酸塩基からなる群より選択される少なくとも1種のアニオン性基が挙げられる。つまり、ポリアニオン系高分子は、カルボン酸塩基、硫酸塩基、スルホン酸塩基、硝酸塩基、リン酸塩基、およびホウ酸塩基からなる群より選択される少なくとも1種のアニオン性基を有する。
-リンカー-
ナノ粒子体1は、ナノ粒子体1の設計自由度を著しく低下させない範囲で、高分子膜3に結合するリンカーを有することができる。このリンカーは、その末端に特異結合物質4が結合されていない。ナノ粒子体1が高分子膜3に結合するリンカーを有すると、リンカーがナノ粒子体1間のスペーサのように作用して(立体障害として作用して)、ナノ粒子体1同士の接触による凝集を防止する。かかる場合、ナノ粒子体1は本実施形態に係るナノ粒子体組成物における分散性がさらに向上する。
図4を参照して、複合体を説明する。図4は、複合体を模式的に示す断面図である。複合体40は、検出対象である被験物質30と、2つのナノ粒子体10,20とを含んで成る。2つのナノ粒子体10,20は、複合体40において、被験物質30を介して結合されている。つまり、第1実施形態に係るナノ粒子体10,20は、被験物質30を介して結合された、第2実施形態に係る複合体を形成する。2つのナノ粒子体10,20のうち、一方を第1ナノ粒子体10と称し、もう一方のナノ粒子体を第2ナノ粒子体20と称する。このように複合体40は、ナノ粒子体1として第1ナノ粒子体10と第2ナノ粒子体20とを含む。
蛍光物質16,26は、図4に示すように、第1金属ナノ粒子12と第2金属ナノ粒子22との間に位置づけられていることが好ましい。金属ナノ粒子12,22間では近接場が効率的に生じる空間であるため、金属ナノ粒子12,22間の空間に蛍光物質16,26が位置づけられることで蛍光強度が増大されやすいからである。
被験物質30は、検体に含まれる検出対象となる物質である。被験物質30としては、例えば、抗原、タンパク質、基質、および核酸鎖が挙げられる。被験物質30は、特異結合物質14,24と特異的に結合する。例えば、抗原は、少なくとも2つの抗原決定基(epitope)を有し、抗原決定基で第1,第2特異結合物質14,24と特異的結合を形成する。抗原は、例えば、c反応性タンパク質、ミオグロビン、トロポニンT、トロポニンI、およびBNP等のようなタンパク質、ならびにインフルエンザウイルス、およびRSウイルス等のようなウイルスの抗原タンパク質である。被験物質30は、例えば、血液、血漿、尿、または唾液のような検体に由来する被験物質である。つまり、被験物質30を含む検体としては、例えば、血液、血漿、血清、尿、および唾液である。検体は、溶媒および緩衝液(より具体的には、リン酸緩衝生理食塩水(phosphate-buffered saline:PBS)、Tris緩衝液、HEPES緩衝液、MOPS緩衝液、およびMES緩衝液など)をさらに含んでもよい。
図6を参照して、測定装置を説明する。図6は、測定装置を示す図である。図6に示すように、測定装置100は、励起用光源110と、励起光照射光学系120と、試薬容器130と、受光光学系140と、受光素子150とを備える。
励起用光源110は、励起光112を照射する。励起用光源110は、例えば、レーザーである。励起光照射光学系120は、励起光112の集光のように断面径の調整等を行い、入射励起光122を出力する。励起光照射光学系120は、レンズ124および偏光素子(λ/2板)126である。励起光照射光学系120から出力した入射励起光122は、試薬容器130に入射し、試薬容器130内の測定試料に照射される。試薬容器130は、例えば、着脱可能な容器(より具体的には、セル、およびプレパラート等)、およびマイクロ流路チップである。マイクロ流路チップは、微小な流路を有するチップである。試薬容器130がマイクロ流路チップである場合、例えば、第1実施形態に係るナノ粒子体(試薬)と検体とを混合して連続的に供給することができる。このため、あらかじめ測定試料を混合して調製する必要がなく、連続的に測定することが可能となる。
(製造例1)
図7~8を参照して、高分子膜の作製方法を説明する。図7~8は、高分子膜の作製方法を説明するための模式図を示す。図7に示すように、ポリ-L-リシン(株式会社ペプチド研究所製「3075」)と、3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(Thermo Fisher Scientific社製、製造番号「26128」、「NHS-PEG4-SPDP」)とを、室温および4時間の条件で、小型回転培養器(タイテック株式会社製「RT-30mini)を用いて攪拌し、混合した。その結果、高分子を得た。この合成反応は、ポリ-L-リシンの第1級アミノ基が3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHSのNHSエステル基に攻撃する求核置換反応である。合成した高分子は、疎水性基(ピリジル基(ピリジニル基))3cと、正帯電性基(第1アンモニウム基)3bとを有していた。得られた高分子を金属ナノ粒子としての銀ナノ粒子(nanocomposix製「AGCB80-1M」,直径80nm,OD455=0.1)の分散液1mLに、添加し、室温およびオーバーナイトの条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜3で被覆された銀ナノ粒子の分散液を得た。
製造例1の銀ナノ粒子(nanocomposix製「AGCB80-1M」,直径80nm,OD455=0.1)の分散液1mLを金ナノ粒子(SigmaAldirich製「753610-25ml」,直径20nm,OD520=0.1)の分散液1mLに変更した以外は製造例1と同様にして高分子膜3で被覆された金ナノ粒子2の分散液(以下、被覆金ナノ粒子分散液とも称する)を調製した。高分子膜3は、金ナノ粒子2の表面に硫黄原子を介した結合部位3a、正帯電性基(第1級アンモニウム基)3b、および疎水性基(ピリジル基(ピリジニル基))3cを有する。得られた金ナノ粒子2のSEM画像(倍率500,000倍)を作成し、金ナノ粒子2の表面が高分子膜3によって連続的に被覆されていることが確認された。また、SEM画像から金ナノ粒子2の高分子膜の膜厚は、8.64nm(期待値M)±0.58nm(標準偏差σ,測定数n=6)であり、膜厚のばらつき(σ/M)は4.36%であった(図9(b)参照)。
(金属ナノ粒子表面および高分子膜の帯電性)
ゼータ電位測定装置(MALVERN社製「ZETA SIZER Nanoseries nano-ZS」)を用いて、金属ナノ粒子2、および高分子膜3で被覆された金属ナノ粒子2のゼータ電位を測定した。図10は、高分子膜3で被覆された金属ナノ粒子2のゼータ電位のグラフ(横軸:ゼータ電位(単位:mV)および縦軸:相対強度(単位:任意単位))である。金属ナノ粒子2は、図10(a)では銀ナノ粒子(粒子径:80nm)であり、図10(b)では金ナノ粒子(粒子径:20nm)である。図11は、高分子膜3で被覆されていない金属ナノ粒子2のゼータ電位のグラフ(横軸:ゼータ電位(単位:mV)および縦軸:相対強度(単位:任意単位))である。金属ナノ粒子2は、図11(a)では銀ナノ粒子(粒子径:80nm)であり、図11(b)では金ナノ粒子(粒子径:20nm)である。
図10(a)~(b)に示すように、高分子膜3でその表面が被覆された、銀ナノ粒子(粒子径80nm)および金ナノ粒子(粒子径20nm)のゼータ電位は、いずれもプラス電位にピークを有し、そのゼータ電子の度数分布全体がプラス電位の範囲にほぼおさまる形状を有していた。このため、高分子膜3で被覆された金属ナノ粒子2の表面(すなわち、高分子膜3の表面)は、正帯電していることが確認された。
図10~図11の結果から、金属ナノ粒子2の表面と、高分子膜3とは対となる帯電性を有することが確認された。よって、高分子膜3で被覆された金属ナノ粒子2では、金属ナノ粒子2の表面と高分子膜3とが静電結合を形成することが強く示唆された。
蛍光物質溶液に高分子膜3で被覆された金属ナノ粒子2(の分散液)を添加し、添加量に対する蛍光強度を測定し、高分子膜3が蛍光消光の抑制に寄与することを確認した。
蛍光物質16,26としての塩化トリス(2,2’-ビピリジル)ルテニウム(II)六水和物(Tris(2,2'-bipyridyl)ruthenium(II)Chloride Hexsahydrate)(東京化成株式会社 T1655))のジメチルスルホキシド溶液(濃度10mg/ml)5μLに、製造例2の被覆金ナノ粒子分散液を添加して混合し混合液を調製した。混合液をマイクロプレートに入れた。測定容器を蛍光光度計(TECANジャパン株式会社製「infinite M200 PRO」)に配置して、蛍光スペクトルを測定した。測定条件は、励起光の波長430nm、検出波長470~700nmであった。被覆金ナノ粒子分散液の添加量(60μL,120μL,180μL,および240μL)を変化させて、蛍光スペクトルを測定した。その結果を図12(a)に示す。
製造例2の被覆金ナノ粒子分散液を金ナノ粒子(SigmaAldirich社製「753610-25ml」,直径20nm,OD520=0.1)の分散液(以下、非被覆金ナノ粒子分散液とも称する)に変更した以外は同様にして、蛍光物質-高分子膜で被覆されていない金ナノ粒子混合系(以下、蛍光物質-非被覆金属ナノ粒子混合系とも称する)の蛍光スペクトルを測定した。測定結果を図12(b)に示す。
蛍光スペクトルの強度は、図12(a)および図12(b)に示すように、分散液(図12(a)では被覆金ナノ粒子分散液、図12(b)では非被覆金ナノ粒子分散液)の添加量が増加するにつれて、いずれも連続的に減少することが確認された。それらの蛍光スペクトルの強度を比較すると、分散液の添加量が増加するにつれて、図12(a)に示す蛍光スペクトルの強度は、図12(b)に示す蛍光スペクトルの強度に比べ大きく、蛍光強度の減少が抑制されていることが確認された。
つまり、図12(a)では、金ナノ粒子が高分子膜で被覆されているため、金ナノ粒子への蛍光物質であるルテニウム錯体の直接的な接触が回避され、蛍光強度の減少が抑制されていると考えられる。一方、図12(b)では、金ナノ粒子が高分子膜で被覆されていないため、金ナノ粒子へのルテニウム錯体が直接的に接触して、蛍光強度が大きく減少したものと考えられる。このように、金ナノ粒子表面に配置された高分子膜が蛍光消光を抑制することが示された。
[蛍光標識抗体を結合したナノ粒子体の作製(ナノ粒子体組成物の調製)]
図13は、蛍光標識した抗体を結合したナノ粒子体の構造を示す模式図である。図13に示すナノ粒子体は、はじめに、高分子被膜金属ナノ粒子の表面に架橋剤を結合させ、別途、ナノ抗体に蛍光物質および架橋剤を結合させ、次いで、高分子被膜金属ナノ粒子に結合した架橋剤と、ナノ抗体に結合した架橋剤とを結合させて作製した。以下、蛍光標識抗体を結合したナノ粒子体の作製の詳細について説明する。なお、図13中、nは、エチレンオキシド繰り返し単位の繰り返し単位数を示し、6を示す。
まず、金属ナノ粒子を「nanocomposix製「AGCB80-1M」,直径80nm,OD455=0.1」から「nanocomposix製「AGCB50-1M」,直径50nm,OD455=0.1」に変更した以外は上述の製造例1と同様の方法で、高分子膜3で被覆された銀ナノ粒子2(以下、高分子被膜銀ナノ粒子とも称する)の分散液を得た。
得られた高分子膜3は、銀ナノ粒子の全表面を被覆していた。高分子膜3は、銀ナノ粒子2の表面に硫黄原子を介した結合部位3aと、銀ナノ粒子2の表面と疎水結合をする疎水性基(ピリジル基(ピリジニル基))3cと、銀ナノ粒子2の表面と静電結合を形成する正帯電性基(第1級アンモニウム基)3bとを含んでいる。つまり、高分子膜3を構成する高分子3Aは、銀ナノ粒子2の表面に硫黄原子を介した結合部位3aと、銀ナノ粒子2の表面と疎水結合をする疎水性基(ピリジル基(ピリジニル基))3cと、銀ナノ粒子2の表面と静電結合を形成する正帯電性基(第1級アンモニウム基)3bとを有する(図8参照)。
次いで、作製した高分子被膜銀ナノ粒子の分散液1mLに、さらに架橋剤SM(PEG)6 (PEGylated, long-chain SMCC crosslinker)(ThermoFisherSCIENTIFIC社製「22105」)およびヘパリンナトリウム(富士フイルム和光純薬株式会社製「081-00136」)を添加し、室温および1時間の条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜3に架橋剤SM(PEG)6が結合した銀ナノ粒子(以下、SM(PEG)6リンカーが結合した高分子被膜銀ナノ粒子とも称する)の分散液を得た。高分子被膜銀ナノ粒子に結合するSM(PEG)6リンカーは、マレイミド基を有していた。
VHH抗体(RePHAGEN社製,分子質量18,000Da)100μgに対してAlexa Fluor 430 carboxylic acid, succinimidyl ester(invitrogen社製「A10169」)を添加し、室温および1時間の条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質が結合したVHH抗体(以下、蛍光標識VHH抗体とも称する)を得た。
次いで、蛍光物質標識VHH抗体にNHS-ビピリジルジスルフィド架橋剤としての3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(東京化成株式会社製「NHS-PEG4-SPDP」)をモル比で8倍当量加え、室温および1時間の条件で小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質およびSPDPリンカーが結合したVHH抗体(以下、SDPDリンカーが結合した蛍光標識VHH抗体とも称する)を得た。
次いで、SPDPリンカーが結合した蛍光標識VHH抗体に対して、還元剤TCEP(ThermoFisherSCIENTIFIC社製「77720」)をモル比で2倍当量加え、37℃および1時間の条件で攪拌機(BioSan社製「TS-100」)を用いて攪拌し、混合した。その結果、蛍光物質と、還元されたSPDPリンカー(以下、還元SPDPリンカーとも称する)とが結合したVHH抗体(以下、還元SPDPリンカーが結合した蛍光標識VHH抗体とも称する)を得た。還元SPDPリンカーは、ジスルフィド結合の還元により生成されたチオール基(-SH基)を有していた。
次いで、マレイミド基を有するSM(PEG)6リンカーが結合した高分子被膜銀ナノ粒子の分散液(OD430=0.1)に対して還元SPDPリンカーが結合した蛍光標識VHH抗体を加え、室温およびオーバーナイトの条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、SM(PEG)6リンカーのマレイミド基と還元SPDPリンカーのチオール基とが反応して、蛍光標識VHH抗体がリンカー部を介して結合したナノ粒子体を得た(図13参照)。高分子膜3の膜厚は、8.31nm(期待値M)±1.89nm(標準偏差σ)であり、膜厚のばらつき(σ/M)は13.7%であった。得られたナノ粒子体組成物は、図13に示すナノ粒子体と、ポリアニオン系高分子としてヘパリンナトリウムを含む溶媒とを含んで成っていた。
得られたナノ粒子体のリン酸塩緩衝溶液に被験物質としてのC Reactive Protein(ADVY CHEMICALシグマアルドリッチ社製「00-AGN-AP-CRP-00」)(以下、CRP抗原とも称する)を添加し、室温および5分の条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌して、サンドイッチ型の複合体を含む測定試料(OD455=0.4)を調製した。また、被験物質を添加しなかったこと以外は測定試料と同様にしてブランク試料を調製した。
測定試料を測定容器に入れ、蛍光分光光度計(TECANジャパン株式会社「infiniteM200PRO」)に測定容器を設置した。波長430nmを測定容器に照射し、蛍光スペクトルを測定した。蛍光スペクトルは、検出波長470nm~700nmおよび光路長3.5mmの測定条件で測定された。なお、ナノ粒子体に含まれる蛍光物質は、430nmにピークを有する吸収スペクトルを示し、520nmにピークを有する蛍光スペクトルを示す。ナノ粒子体に含まれる銀ナノ粒子(直径50nm)は、430nmにピークを有する吸収スペクトルを示す。
ブランク試料の蛍光スペクトルも同様に測定した。
よって、観測された蛍光強度の有意差は、金属ナノ粒子表面での表面プラズモン共鳴によって形成された近接場によって間接的に励起した蛍光物質が発した蛍光に起因すると考えられる。
以上から、実施例1のCRP抗原を加えた試料において、蛍光増強効果が得られたものと考えられる。
(ナノ粒子体の調製)
実施例2のナノ粒子体の調製では、以下の3つの条件を変更した以外は、上述した実施例1と同様にしてナノ粒子体を調製した。実施例2のナノ粒子体は、蛍光標識VHH抗体がリンカー部を介して結合したナノ粒子体であった。
蛍光物質をAlexa Fluor 430 carboxylic acid, succinimidyl ester(invitrogen社製「A10169」)から[化3]の化学式:
蛍光標識VHH抗体への架橋剤(NHS-ビピリジルジスルフィド架橋剤)を3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(東京化成株式会社製「NHS-PEG4-SPDP」)から3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(Thermo Fisher SCIENTIFIC社製 製品番号「26128」、「NHS-PEG4-SPDP」)に変更した。
金属ナノ粒子を「nanocomposix製「AGCB50-1M」,直径50nm,OD455=0.1」から「nanocomposix製「AGCB80-1M」,直径80nm,OD455=0.1」に変更した。
実施例2のナノ粒子体のリン酸緩衝液に被験物質としてのC Reactive Protein(ADVY CHEMICAL社製「00-AGN-AP-CRP-00」)(以下、CRP抗原とも称する)を添加し、室温および5分の条件で小型回転培養機(タイテック株式会社社製「RT-30mini」)を用いて攪拌して、サンドイッチ型の複合体を含む測定試料を調製した。実施例2のナノ粒子体は、上述のように、金属ナノ粒子の表面を被覆する高分子膜を有していた。この高分子膜を構成する高分子は、その側鎖の末端に正帯電性基としての第1級アンモニウム基と、疎水性基としてのピリジル基と、金属ナノ粒子の表面との間に硫黄原子を介した結合部位とを有していた。
走査型電子顕微鏡(株式会社日立ハイテク製「Regulus8220」)を用いて、得られた実施例2の測定試料中の複合体のSEM画像(倍率100K倍)を撮像した。図15に、実施例2のナノ粒子体を用いて調製した複合体のSEM画像を示す。図15に示すように得られたSEM画像において、複合体におけるナノ粒子体の金属ナノ粒子間の離隔距離L1と、前記ナノ粒子体の金属ナノ粒子体間以外の高分子膜の厚みT1とを測定し、比較した。なお、高分子膜の厚みT1は、離隔距離の対象ではない収縮していない部分の高分子膜の厚みであった。
その結果、離隔距離L1は、高分子膜の厚み2つ分に相当する厚み(T1×2)よりも小さかった。このため、実施例2のナノ粒子体組成物で調製した複合体では、高分子膜が収縮し、複合体の2つの金属ナノ粒子が高分子膜の厚み2つ分(T1×2)に相当する離隔距離よりも近接していることが分かった。これにより、プラズモン増強効果がさらに得られ、蛍光強度がさらに増大していることが強く示唆された。
実施例2で示した高分子膜の収縮性の技術的意義を明確にすべく、比較対象として無機膜で被覆された金属ナノ粒子の凝集物(参考例1)を検討した。
シリカ被覆された銀ナノ粒子(nanoComposix製、銀ナノ粒子の粒子径(コア粒子径)50nm、シリカ膜の厚み20nm)を水で希釈して、シリカ被覆した銀ナノ粒子の水分散液を調製した。得られた水分散液を測定サンプルとした。詳しくは、測定サンプル中には、シリカ被覆された銀ナノ粒子の一次粒子状態の他に、2粒子が凝集した凝集体も存在していた。この凝集体を探し出し、評価した。
実施例2と同様に、SEM画像(500K倍)を撮像し、SEM画像から凝集体における無機膜の厚みT2と、2つの金属ナノ粒子間の離隔距離L2とを測定し、比較した。その結果、離隔距離L2は、無機膜の厚みT2の約2倍であった。なお、無機膜の厚みT2は、離隔距離の対象ではない部分の無機膜の厚みであった。
(高分子被膜銀ナノ粒子)
図16に示すナノ粒子体組成物を調製した。高分子としてのポリ-L-リジン(ペプチド研究所製「3075」)を金属ナノ粒子としての銀ナノ粒子(nanocomposix社製「AGCB50―1M」,直径50nm,OD430=0.1)の分散液1mLに、添加し、室温(25℃)および4時間の条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜で被覆された銀ナノ粒子(高分子被膜銀ナノ粒子)の分散液を得た。高分子膜は、銀ナノ粒子の表面に硫黄原子を介した結合部位と、銀ナノ粒子の表面と疎水結合を形成する疎水性基と、銀ナノ粒子の表面と静電結合を形成する正帯電性基(第1級アンモニウム基)と、電気的中性基(第1級アミノ基)とを含む。つまり、高分子膜を構成する高分子は、銀ナノ粒子の表面に硫黄原子を介した結合部位と、銀ナノ粒子の表面と疎水結合を形成する疎水性基と、銀ナノ粒子の表面と静電結合を形成する正帯電性基(第1級アンモニウム基)と、電気的中性基(第1級アミノ基)とを有する。
高分子被膜銀ナノ粒子の分散液1mLに蛍光物質としてのサクシンイミジルエステル(Thermo Fisher Scientific製「Alexa Fluor(登録商標)430 NHS エステル」)を添加し、室温および1時間の条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質が高分子膜に結合した高分子被膜銀ナノ粒子(以下、蛍光標識高分子被膜銀ナノ粒子とも称する)の分散液を得た。
走査型電子顕微鏡(株式会社日立ハイテク製「Regulus8220」)を用いて、得られたナノ粒子体組成物中のナノ粒子体のSEM画像(倍率200,000倍)を撮像した。SEM画像を観察し、ナノ粒子体がナノ粒子体組成物において単体で分散していることが確認された。さらに、SEM画像から、ナノ粒子体において銀ナノ粒子の表面が高分子膜によって連続的に被覆されていることが確認された。また、SEM画像から銀ナノ粒子を被覆する高分子膜の膜厚を測定した。高分子膜の膜厚は、約11nmであった。
実施例3のナノ粒子体組成物を測定容器に投入した。測定容器を紫外可視分光光度計(TECANジャパン株式会社製「infinite M200 PRO」)に配置して、吸収スペクトルを測定した。測定条件は、測定波長範囲400~600nmであった。図17に実施例3のナノ粒子体組成物の吸収スペクトルを示す。得られた吸収スペクトルは、455nmにピークを有していた。
実施例3のナノ粒子体組成物を光路長1cmの測定容器に入れた。測定容器を蛍光光度計(TECANジャパン株式会社製「infinite M200 PRO」)に配置して、蛍光スペクトルを測定した。測定条件は、励起光の波長430nmおよび検出波長470~700nmであった。図18に実施例3のナノ粒子体組成物の蛍光スペクトルを示す。得られた蛍光スペクトルは、535nmにピークを有していた。
図19を参照してナノ粒子体を含んで成るナノ粒子体組成物の作製方法を説明する。図19は、ナノ粒子体を含んで成るナノ粒子体組成物の作製方法を説明するための模式図である。実施例1と同様にして高分子被膜銀ナノ粒子の分散液を調製した。この分散液1mLに、蛍光物質としてのサクシンイミジルエステル(Thermo Fisher Scientific社製「Alexa Fluor(登録商標)430 NHS エステル」)と、架橋剤としてのSM(PEG)6(PEGylated, long-chain SMCC crosslinker)(ThermoFisherSCIENTIFIC社製「22105」)を同時に添加し、室温および4時間の条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質およびリンカーが結合した高分子被膜銀ナノ粒子の分散液を得た。さらに、ポリアニオン系高分子としてヘパリンナトリウム(富士フイルム和光純薬株式会社製「081-00136」)を加え、室温および4時間の条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質およびリンカーが結合した高分子被膜銀ナノ粒子(ナノ粒子体)と、ポリアニオン系高分子とを含んで成るナノ粒子体組成物を調製した。
得られたナノ粒子体について実施例1と同様にして吸収スペクトルを測定した。図20(a)に実施例4のナノ粒子体組成物の吸収スペクトルを示す。得られた吸収スペクトルは、450nmにピークを有していた。
また、得られたナノ粒子体組成物について実施例1と同様にして蛍光スペクトルを測定した。図20(b)に実施例4のナノ粒子体組成物の蛍光スペクトルを示す。得られた蛍光スペクトルは、540nmにピークを有していた。吸収スペクトルの極大波長が長波長側にシフトしていないことよりナノ粒子体が凝集していないことを確認し、また、蛍光検出できることより、蛍光物質がナノ粒子体に結合されていることを確認した。
(ナノ粒子体組成物の調製)
金属ナノ粒子としての銀ナノ粒子(nanocomposix社製「AGCB80-1M」,直径80nm,OD455=0.1)の分散液1mLに、ポリ-L-リシン(株式会社ぺプチド研究所製「3075」)を添加し、室温およびオーバーナイトの条件で、小型回転培養機(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜で被覆された銀ナノ粒子(高分子被膜銀ナノ粒子)の分散液を得た。
測定試料として、実施例4のナノ粒子体組成物(洗浄処理を施していない組成物(以下、0回洗浄処理組成物とも称する))と、2回洗浄処理を施した組成物(以下、2回洗浄処理組成物とも称する)とを準備した。洗浄処理とは、組成物中のナノ粒子体の濃度が1/2となるように溶媒として純水を添加して激しく攪拌し、攪拌後測定前に少なくとも遠心機(遠心分離機)(株式会社トミー精工製「MX307」)を用いて12,000Gおよび30分間の条件でナノ粒子体を沈降させた後、上清を取り除き、あらたに純水を添加する一連の処理を示す。この操作を洗浄処理回数1回とした。なお、洗浄処理を3回施すと、凝集が生じ、回収率が著しく減少した。
会合体のサイズ測定と同様に、2つの測定試料を準備した。ゼータ電位測定装置(MALVERN PANALYTICAL製「ZETA SIZER Nanoseries nano-ZS」)を用いて、上記調製した2つの測定試料における会合体のゼータ電位を測定した。図22は、ゼータ電位の度数分布を示す図である。図22(a)は、洗浄処理回数0回でのゼータ電位の度数分布を示し、図22(b)は、洗浄処理回数2回でのゼータ電位の度数分布を示す。図22中、横軸はゼータ電位(単位:mV)を示し、縦軸は度数(単位:任意単位)を示す。
2 ・・・金属ナノ粒子
3 ・・・高分子膜
3A ・・・高分子膜を構成する高分子
3a ・・・硫黄原子を介した結合部位
3b ・・・正帯電性基
3c ・・・疎水性基
4 ・・・特異結合物質
7 ・・・ポリアニオン系高分子
9 ・・・会合体
10 ・・・第1ナノ粒子体
12 ・・・第1金属ナノ粒子
13 ・・・第1高分子膜
14 ・・・第1特異結合物質
16 ・・・第1蛍光物質
20 ・・・第2ナノ粒子体
22 ・・・第2金属ナノ粒子
23 ・・・第2高分子膜
24 ・・・第2特異結合物質
26 ・・・第2蛍光物質
30 ・・・被験物質
40 ・・・複合体
L ・・・離隔距離(離間距離)
(発明の名称)ナノ粒子体およびその製造方法
(技術分野)
(背景技術)
プラズモン励起蛍光分析では、複合体は、例えば、金属粒子、特異結合物質、蛍光物質および被験物質を含む。励起光が複合体に照射されると、複合体内の金属粒子で表面プラズモン共鳴が発生し、金属粒子の表面近傍で近接場が形成される。この近接場によって蛍光物質の蛍光強度が増大される。
(先行技術文献)
(特許文献)
(発明の概要)
(発明が解決しようとする課題)
(課題を解決するための手段)
金属ナノ粒子と、該金属ナノ粒子の表面を被覆する高分子膜と、蛍光物質とを含んで成り、
前記蛍光物質は、前記金属ナノ粒子の表面に標識されており、
前記高分子膜が前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位を含む。
蛍光物質がジスルフィド結合を介して結合する高分子と、金属ナノ粒子とを混合して、前記蛍光物質を前記金属ナノ粒子の表面に標識しつつ、かつ前記金属ナノ粒子の表面に高分子膜を形成する工程を含んで成る。
(発明の効果)
(図面の簡単な説明)
(図25)図25は、図24のA部拡大模式図である。
(図26)図26は、第5実施形態に係るナノ粒子体の製造方法の一例を示す反応スキームである。
(図27)図27は、第6実施形態に係る複合体を模式的に示す断面図である。
(図28)図28は、第7実施形態に係る測定装置を模式的に示す図である。
(図29)図29は、実施例6のナノ粒子体の製造方法を説明する模式図である。
(図30)図30は、実施例6のナノ粒子体の蛍光スペクトルを示す図である。
(図31)図31は、実施例7のナノ粒子体を用いて調製した複合体のSEM画像を示す図である。
(発明を実施するための形態)
第5実施形態に係るナノ粒子体は、
金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、蛍光物質とを含んで成り、
蛍光物質は、金属ナノ粒子の表面に標識されており、
高分子膜が金属ナノ粒子の表面との間に硫黄原子を介した結合部位を含む。
はじめに、本実施形態に係るナノ粒子体の説明の便宜上、その理解を助ける目的で、本実施形態に係るナノ粒子体を用いた被験物質の検出方法を説明する。
本実施形態に係るナノ粒子体は、被験物質の検出に有用である。本実施形態に係るナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、金属ナノ粒子の表面に標識されている蛍光物質とを含んで成る。本実施形態に係るナノ粒子体は、高分子膜に結合した特異結合物質をさらに含み得る。特異結合物質は、検体中の被験物質と特異的に結合することができる。以下、このように特異結合物質が結合したナノ粒子体を「特異結合性ナノ粒子体」とも称する。
本実施形態に係るナノ粒子体は、より多くの蛍光物質を標識し得る、蛍光物質の新規の標識態様を有する。つまり、本実施形態に係るナノ粒子体では、蛍光物質の新規の標識態様は、より多くの蛍光物質を標識し得る。
特定の理論に拘束されるわけではないが、その理由は以下のように推測される。本実施形態に係るナノ粒子体は、金属ナノ粒子と、金属ナノ粒子の表面を被覆する高分子膜と、金属ナノ粒子の表面に標識されている蛍光物質とを含んで成る。蛍光物質が金属ナノ粒子の表面に標識されているため、ナノ粒子体の金属ナノ粒子の表面とは異なるサイト(例えば、高分子膜を構成する高分子が有する特定の官能基)に特異結合物質を結合させることが可能となる。このようにナノ粒子体における蛍光物質が標識される標識サイトが、特異結合物質が結合する結合サイトと相違するため、ナノ粒子体における蛍光物質の数をより増加させることが可能となる。以上から、本実施形態に係るナノ粒子体は、より多くの蛍光物質を標識し得ると考えられる。さらに、本実施形態に係るナノ粒子体は、より多くの蛍光物質を標識し得るため、検出感度に優れる。
以下、ナノ粒子体の構成を説明する。図24を参照して、ナノ粒子体を説明する。図24は、ナノ粒子体を模式的に示す断面図である。本実施形態に係るナノ粒子体1は、図24(a)に示すように、金属ナノ粒子2と、金属ナノ粒子2の表面を被覆する高分子膜3と、金属ナノ粒子2の表面に標識する蛍光物質6とを含んで成る。ナノ粒子体1は、図24(b)に示すように、高分子膜3の表面に結合された、検体中の被験物質と特異的に結合する特異結合物質4をさらに含み得る。
金属ナノ粒子2は、その表面を高分子膜3で被覆されている。金属ナノ粒子2は、金属の種類によって異なるが、特定の波長を有する光と相互作用し、局在表面プラズモン共鳴を起こす。銀ナノ粒子では400nmから530nmに、金ナノ粒子では510nmから580nmにプラズモンの共鳴ピークがある。これは粒子径により異なる。例えば、粒子径が20nmの銀からなるナノ粒子は、波長405nmの光と共鳴する。粒子径が20nmの金からなるナノ粒子は、波長524nmの光と共鳴する。金属ナノ粒子2の粒子径(平均一次粒子径)は、例えば、5nm~100nmである。金属ナノ粒子2の粒子径は、走査型電子顕微鏡(SEM)または透過型電子顕微鏡(TEM)を用いて、金属ナノ粒子2の画像を撮像し、画像での金属ナノ粒子2の粒子径を測長し、複数の粒子径の平均値(測定数:例えば、少なくとも10以上)を算出することにより得ることができる。
金属ナノ粒子2は、好ましくは金または銀を含んで成り、より好ましくは銀を含んで成る。
高分子膜3は金属ナノ粒子2の表面を被覆する。高分子膜3は、金属消光膜として機能する。複合体においては、高分子膜3は、少なくとも高分子膜3の厚み分、金属ナノ粒子2の表面から蛍光物質6を離間して配置させることができる。このため、励起した蛍光物質6が金属ナノ粒子2の表面に接触して消光することを抑制し、検出感度の低下を抑制できる。高分子膜3の存在は、SEMまたはTEMを用いて、ナノ粒子体1の画像を撮像し、画像でのナノ粒子体1を観察し、確認することができる。
硫黄原子を介した結合部位3aは、例えば、側鎖としてジスルフィド結合を含む部位を有する高分子と、金属ナノ粒子2とを混合することで形成される。
正帯電性基3bは、金属ナノ粒子2の表面と比較的強い静電結合bを形成することができる。正帯電性基3bは、本明細書において、1価以上の価数を有し、完全に正に電離した基である。高分子膜3を構成する高分子に含まれる複数の正帯電性基3bを考慮した場合、正帯電性基3bは、下記の数式(1):
(数1)
[数式(1)中、pKaは高分子膜3を構成する高分子3Aに含まれる電気的に中性な基であって、正に帯電すれば、正帯電性基(より具体的には、第1級アンモニウム基(-NH3 +)等)3bとなり得る基(以下、電気的中性基とも称する)(より具体的には、第1アミノ基(-NH2)等)のpKaを示し、pHは被験物質を検出する環境(より具体的には、検体等)のpHを示し、Bは高分子3Aに含まれる電気的中性基を示し、BH+は高分子3Aに含まれる正帯電性基3bを示す]
で表されるpKaが7以上である基をいう。つまり、正帯電性基3bは、高分子膜3を構成する高分子3Aの電気的中性基および正帯電性基3bとが被験物質を検出する環境(例えば、pHおおよそ6~8の検体)中で、下記の化学平衡式(2):
(化1)
で表される平衡状態を形成している場合であって、正帯電性基3bの濃度([BH+])が電気的中性基の濃度([B])に比べ10倍以上大きい基をいう。
疎水性基3cは、金属ナノ粒子2の表面との間で疎水結合cを形成することができる。
疎水性基は、例えば、芳香族環状基、脂肪族環状基および脂肪族鎖状基からなる群より選択される少なくとも1種である。
なお、本明細書において、離間距離(離隔距離)とは、複合体において被験物質を介して結合する2つのナノ粒子体にそれぞれ含まれる金属ナノ粒子表面間の距離の最小値(最短距離)をいう。
蛍光物質6は、金属ナノ粒子2の表面に標識されている。蛍光物質6の標識サイトは金属ナノ粒子2の表面である。これに対して、特異結合物質4の結合サイトは後述するように高分子膜3である。このように、本実施形態に係るナノ粒子体1では、蛍光物質6の標識サイトは特異結合物質4の結合サイトと異なる。よって、標識サイトと結合サイトとが同一のナノ粒子体に比べ、蛍光物質6の数を十分に増加させることができる。よって、本実施形態では、検出感度により優れる。
ナノ粒子体1は、特異結合物質4をさらに含んで成ってもよい。特異結合物質4は、検体中の被験物質(第6実施形態にて説明する)と特異的に結合するナノサイズ(最長が3~15nmであるサイズ)の物質である。
このような事項を考慮すると、特異結合性ナノ粒子体により多くの特異結合物質4が結合されていれば、前記衝突における特定の部位間の接触および近接の確率が増加する。よって、本実施形態に係るナノ粒子体は、より多くの特異結合物質4を結合し得るため、複合体を形成しやすく、検出感度をさらに向上させることができると考えられる。
第5実施形態に係るナノ粒子体1の製造方法は、蛍光物質6がジスルフィド結合を介して結合する高分子と、金属ナノ粒子2とを混合して、蛍光物質6を金属ナノ粒子2の表面に標識しつつ、かつ金属ナノ粒子2の表面に高分子膜3を形成する工程(以下、「蛍光標識膜形成工程」とも称する)を含んで成る。
第5実施形態に係るナノ粒子体1の製造方法では、金属ナノ粒子表面への蛍光標識と高分子膜形成とを並行して実施できる蛍光標識膜形成工程を含んで成る。このため、従来技術に対して蛍光標識の工程を簡素化でき、コストを低減することができる。また、本製造方法では、蛍光物質6を特異結合物質4とは異なるサイトに標識できるため、ナノ粒子体1における蛍光標識密度を高めることができる。
ナノ粒子体1の製造方法は、蛍光標識膜形成工程に加え、蛍光物質チオール化工程、高分子蛍光標識工程および特異結合物質結合工程をさらに含んで成ってもよい。
蛍光物質チオール化工程では、蛍光物質6をチオール化する。より具体的には、反応スキーム中、反応式(R-1)で表されるように、(A)エステル結合を有する蛍光物質(以下、蛍光物質とも称する)と、(B)アミノ基を有するチオール(以下、アミノアルカンチオールとも称する)とを反応させて、(C)アミド結合を形成してチオール化した蛍光物質(以下、チオール化蛍光物質とも称する)を合成する。
高分子蛍光標識工程では、高分子を蛍光標識する。より具体的には、反応スキーム中、反応式(R-2)で表されるように、(C)チオール化蛍光物質と、(D)ジスルフィド結合を有する高分子(以下、高分子とも称する)とを反応させて、(E)蛍光物質が標識された高分子(以下、蛍光標識高分子とも称する)を合成する。
蛍光標識膜形成工程では、(E)蛍光標識高分子(蛍光物質6がジスルフィド結合を介して結合する高分子)と、(F)金属ナノ粒子2とを混合して、蛍光物質6を金属ナノ粒子2の表面に標識しつつ、かつ金属ナノ粒子2の表面に高分子膜3を形成する。つまり、蛍光標識膜形成工程では、(F)金属ナノ粒子2の表面への蛍光物質6の標識と、(F)金属ナノ粒子2の表面での高分子膜3の形成とを並行して実施できる。このように蛍光標識と膜形成とを2つの工程ではなく1つの工程で実施できるため、コスト低減の観点から好ましい。形成された高分子膜3は、(F)金属ナノ粒子2の表面との間に硫黄原子を介した結合部位3aを含む。なお、図26中の反応式(R-3)で表される反応(以下、反応(R-3)とも称する)では、その生成物としてナノ粒子体1の高分子膜3と金属ナノ粒子2の表面との界面付近の拡大模式図を示している。
特異結合物質結合工程では、高分子膜3に特異結合物質4を結合させる。特異結合物質4と結合する高分子膜3の結合サイトとしては、例えば、(E)蛍光標識高分子の官能基である。このような官能基は、例えば、アミノ基、カルボキシル基、チオール基、N-ヒドロキシスクシンイミド基(NHS基)およびマレイミド基からなる群より選択される少なくとも1種の官能基である。
図27を参照して、複合体を説明する。図27は、複合体を模式的に示す断面図である。複合体40は、検出対象である被験物質30と、2つのナノ粒子体10,20とを含んで成る。2つのナノ粒子体10,20は、複合体40において、被験物質30を介して結合されている。つまり、第5実施形態に係るナノ粒子体10,20は、被験物質30を介して結合された、第6実施形態に係る複合体を形成する。2つのナノ粒子体10,20のうち、一方を第1ナノ粒子体10と称し、もう一方のナノ粒子体を第2ナノ粒子体20と称する。このように複合体40は、ナノ粒子体1として第1ナノ粒子体10と第2ナノ粒子体20とを含む。
詳しくは、複合体40において、第1ナノ粒子体10は、第1金属ナノ粒子12と、第1金属ナノ粒子12の表面を被覆する第1高分子膜13と、第1金属ナノ粒子12の表面に標識された第1蛍光物質16とを含んで成る。第2ナノ粒子体20は、第2金属ナノ粒子22と、第2金属ナノ粒子22の表面を被覆する第2高分子膜23と、第2金属ナノ粒子22の表面に標識された第2蛍光物質26とを含んで成る。
詳しくは、第1ナノ粒子体10は、第1高分子膜13に結合された第1特異結合物質14をさらに含んで成り、第2ナノ粒子体20は、第2高分子膜23に結合された第2特異結合物質24をさらに含んで成る。
蛍光物質16,26は、図27に示すように、複合体40において第1金属ナノ粒子12と第2金属ナノ粒子22との間に少なくとも位置づけられていることが好ましい。金属ナノ粒子12,22間では近接場が効率的に生じる空間であるため、金属ナノ粒子12,22間の空間に蛍光物質16,26が位置づけられることで蛍光強度が増大されやすいからである。
被験物質30は、検体に含まれる検出対象となる物質である。被験物質30としては、例えば、抗原、タンパク質、基質、および核酸鎖が挙げられる。被験物質30は、特異結合物質14,24と特異的に結合する。例えば、抗原は、少なくとも2つの抗原決定基を有し、抗原決定基で第1,第2特異結合物質14,24と特異的結合を形成する。抗原は、例えば、c反応性タンパク質、ミオグロビン、トロポニンT、トロポニンI、およびBNP等のようなタンパク質、ならびにインフルエンザウイルス、およびRSウイルス等のようなウイルスの抗原タンパク質である。被験物質30は、例えば、血液、血漿、尿、または唾液のような検体に由来する被験物質である。つまり、被験物質30を含む検体としては、例えば、血液、血漿、血清、尿、および唾液である。検体は、溶媒および緩衝液(より具体的には、リン酸緩衝生理食塩水(phosphate-buffered saline:PBS)、Tris緩衝液、HEPES緩衝液、MOPS緩衝液、およびMES緩衝液など)をさらに含んでもよい。
図28を参照して、測定装置を説明する。図28は、第7実施形態に係る測定装置を模式的に示す図である。図28に示すように、測定装置100は、励起用光源110と、励起光照射光学系120と、試薬容器130と、受光光学系140と、受光素子150とを備える。
励起用光源110は、励起光112を照射する。励起用光源110は、例えば、レーザーである。励起光照射光学系120は、励起光112の集光のように断面径の調整等を行い、入射励起光122を出力する。励起光照射光学系120は、レンズ124および偏光素子(λ/2板)126である。励起光照射光学系120から出力した入射励起光122は、試薬容器130に入射し、試薬容器130内の測定試料に照射される。試薬容器130は、例えば、着脱可能な容器(より具体的には、セル、およびプレパラート等)、およびマイクロ流路チップである。マイクロ流路チップは、微小な流路を有するチップである。試薬容器130がマイクロ流路チップである場合、例えば、第5実施形態に係るナノ粒子体(試薬)と検体とを混合して連続的に供給することができる。このため、あらかじめ測定試料を混合して調製する必要がなく、連続的に測定することが可能となる。
(実施例)
以下のようにして実施例6のナノ粒子体を作製した。
(ルテニウム錯体誘導体へのチオール基の導入)
(b)システアミン塩酸塩(東京化成工業株式会社製「A0296」)と、(a)NHS標識Ru錯体誘導体(東京化成工業株式会社製「Ruthenium(II)tris(Bipyridyl)-C5-NHS ester」)とを室温(例えば、25℃)および1時間の条件で小型回転培養器(タイテック株式会社製「RT-30mini)を用いて攪拌し、混合した。その結果、Ru錯体の配位子の末端にチオール基を導入した。この合成反応は、反応式(r-1):
(化2)
に示すように、(b)システアミン塩酸塩の第1級アミノ基が(a)NHS標識Ru錯体誘導体のNHSエステル基に攻撃する求核置換反応である。これにより、(c)チオール基を有するRu錯体誘導体(以下、「Ru錯体-SH」とも称する)を作製した。得られた(c)Ru錯体-SHにおけるRu錯体部分は蛍光物質である。
また一方で、ポリ-L-リシン(株式会社ペプチド研究所製「3075」)と、3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(Thermo Fisher Scientific社製、製造番号「26128」、「NHS-PEG4-SPDP」)とを、室温および4時間の条件で、小型回転培養器(タイテック株式会社製「RT-30mini)を用いて攪拌し、混合した。その結果、(d)側鎖にSPDPリンカーおよびジスルフィド結合を介してピリジル基(ピリジニル基)を有する高分子(以下、「ジスルフィド基を有する高分子」とも称する)を得た。この合成反応は、反応式(r-4):
(化3)
で示すように、ポリ-L-リシンの第1級アミノ基が3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHSのNHSエステル基に攻撃する求核置換反応である。
(c)Ru錯体-SHおよび(d)ジスルフィド基を有する高分子を37℃および1時間の条件で攪拌および混合した。その結果、(e)側鎖にSPDPリンカーおよびジスルフィド結合を介してRu錯体が結合した高分子(以下、「Ru錯体標識高分子」とも称する)を得た。合成した(e)Ru錯体標識高分子は、疎水性基(n-ブチレン基)と、正帯電性基(第1アンモニウム基)とを有していた。この合成反応は、反応式(r-2):
(化4)
に示すように、(c)Ru錯体-SHのチオール基が(d)ジスルフィド基を有する高分子のジスルフィド基に攻撃するチオール求核反応である。
得られた(e)Ru錯体標識高分子を(f)金属ナノ粒子としての銀ナノ粒子(nanocomposix製「AGCB80-1M」,直径80nm,OD455=0.1)の分散液1mLに、添加し、室温およびオーバーナイトの条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜で被覆され、かつ蛍光物質で標識された銀ナノ粒子の分散液を得た。この反応では、銀の還元作用によって、(e)Ru錯体標識高分子のジスルフィド結合が開裂する。開裂によって生じたポリLリジン部分と、ルテニウム錯体部分とがそれぞれ銀ナノ粒子の表面と硫黄原子を介して結合する。その結果、図29に示すように、蛍光物質としてのRu錯体が硫黄原子を介して銀ナノ粒子表面に標識され、かつ高分子膜が硫黄原子を介して銀ナノ粒子表面に結合する。つまり、蛍光物質の標識と、高分子膜の形成とが並行して進行するものであった。なお、図29は、実施例6のナノ粒子体の製造方法を説明する模式図を示す。
以下の方法によって、実施例6のナノ粒子体に蛍光物質が標識されていることを確認した。得られたナノ粒子体を測定容器に入れた。測定容器を蛍光光度計(TECANジャパン株式会社製「infinite M200 PRO」)に配置して、蛍光スペクトルを測定した。測定条件は、励起光の波長430nm、検出波長490~700nmであった。測定された蛍光スペクトルを図30に示す。
実施例7のナノ粒子体の調製では、まず、高分子被膜銀ナノ粒子の高分子膜に蛍光物質を標識してナノ粒子体を調製した。次いで、ナノ粒子体にポリアニオン系高分子を加えて実施例7のナノ粒子体を含むナノ粒子体組成物を調製した。
(高分子被膜銀ナノ粒子)
ポリ-L-リシン(株式会社ペプチド研究所製「3075」)と、3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(Thermo Fisher SCIENTIFIC社製 製品番号「26128」、「NHS-PEG4-SPDP」)とを、室温および4時間の条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子を得た。この合成反応は、ポリ-L-リシンの第1級アミノ基が3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHSのNHSエステル基に攻撃する求核置換反応である。得られた高分子を金属ナノ粒子としての銀ナノ粒子の分散液(nanocomposix社製「AGCB80-1M」,直径80nm,OD430=0.1)1mLに、添加し、室温およびオーバーナイトの条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜で被覆された銀ナノ粒子(以下、高分子被膜銀ナノ粒子とも称する)の分散液を得た。
次いで、作製した高分子被膜銀ナノ粒子の分散液1mLに、さらに架橋剤SM(PEG)6 (PEGylated, long-chain SMCC crosslinker)(ThermoFisherSCIENTIFIC社製「22105」)およびヘパリンナトリウム(富士フイルム和光純薬株式会社製「081-00136」)を添加し、室温および1時間の条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、高分子膜に架橋剤SM(PEG)6が結合した銀ナノ粒子(以下、SM(PEG)6リンカーが結合した高分子被膜銀ナノ粒子とも称する)の分散液を得た。高分子被膜銀ナノ粒子に結合するSM(PEG)6リンカーは、マレイミド基を有していた。
VHH抗体(RePHAGEN社製,分子質量18,000Da)100μgに対してNHS標識Ru錯体誘導体(東京化成工業株式会社製「Ruthenium(II)tris(Bipyridyl)-C5-NHS ester」)を添加し、室温および1時間の条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質が結合したVHH抗体(以下、蛍光標識VHH抗体とも称する)を得た。
次いで、蛍光標識VHH抗体にNHS-ビピリジルジスルフィド架橋剤としての3-(2-ピリジルジチオ)プロピオンアミド-PEG4-NHS(Thermo Fisher SCIENTIFIC社製製品番号「26128」、「NHS-PEG4-SPDP」)をモル比で8倍当量加え、室温および1時間の条件で小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、蛍光物質およびSPDPリンカーが結合したVHH抗体(以下、SPDPリンカーが結合した蛍光標識VHH抗体とも称する)を得た。
次いで、SPDPリンカーが結合した蛍光標識VHH抗体に対して、還元剤TCEP(ThermoFisherSCIENTIFIC社製「77720」)をモル比で2倍当量加え、37℃および1時間の条件で攪拌機(BioSan社製「TS-100」)を用いて攪拌し、混合した。その結果、蛍光物質と、還元されたSPDPリンカー(以下、還元SPDPリンカーとも称する)とが結合したVHH抗体(以下、還元SPDPリンカーが結合した蛍光標識VHH抗体とも称する)を得た。還元SPDPリンカーは、ジスルフィド結合の還元により生成されたチオール基(-SH基)を有していた。
次いで、マレイミド基を有するSM(PEG)6リンカーが結合した高分子被膜銀ナノ粒子の分散液(OD430=0.1)に対して還元SPDPリンカーが結合した蛍光標識VHH抗体を加え、室温およびオーバーナイトの条件で、小型回転培養器(タイテック株式会社製「RT-30mini」)を用いて攪拌し、混合した。その結果、SM(PEG)6リンカーのマレイミド基と還元SPDPリンカーのチオール基とが反応して、蛍光標識VHH抗体がリンカー部を介して結合した銀ナノ粒子体の分散液(ナノ粒子体組成物)を得た。得られたナノ粒子体組成物は、実施例7のナノ粒子体と、ポリアニオン系高分子としてヘパリンナトリウムを含む溶媒とを含んで成っていた。
(複合体の調製)
作製したナノ粒子体組成物の分散液35μLに対してリン酸緩衝液(富士フイルム和光純薬株式会社社「PBS-T」)65μLおよび被験物質としてのC Reactive Protein(ADVY CHEMICAL社製「00-AGN-AP-CRP-00」)(以下、CRP抗原とも称する)を添加し、室温および5分の条件で小型回転培養機(タイテック株式会社社製「RT-30mini」)を用いて攪拌して、サンドイッチ型の複合体を含む測定試料を調製した。
走査型電子顕微鏡(株式会社日立ハイテク製「Regulus8220」)を用いて、得られた実施例7の測定試料中の複合体のSEM画像(倍率100K倍)を撮像した。図31に、実施例7のナノ粒子体を用いて調製した複合体のSEM画像を示す。図31に示すように得られたSEM画像において、複合体におけるナノ粒子体の金属ナノ粒子間の離隔距離L1と、前記ナノ粒子体の金属ナノ粒子体間以外の高分子膜の厚みT1とを測定し、比較した。なお、高分子膜の厚みT1は、離隔距離の対象ではない収縮していない部分の高分子膜の厚みであった。
その結果、離隔距離L1は、高分子膜の厚み2つ分に相当する厚み(T1×2)よりも小さかった。このため、実施例7のナノ粒子体で調製した複合体では、高分子膜が収縮し、複合体の2つの金属ナノ粒子が高分子膜の厚み2つ分(T1×2)に相当する離隔距離よりも近接していることが分かった。これにより、プラズモン増強効果がさらに得られ、蛍光強度がさらに増大していることが強く示唆された。
実施例7で示した高分子膜の収縮性の技術的意義を明確にすべく、比較対象として無機膜で被覆された金属ナノ粒子の凝集物(参考例1)を検討した。
シリカ被覆された銀ナノ粒子(nanoComposix製、銀ナノ粒子の粒子径(コア粒子径)50nm、シリカ膜の厚み20nm)を水で希釈して、シリカ被覆した銀ナノ粒子の水分散液を調製した。得られた水分散液を測定サンプルとした。詳しくは、測定サンプル中には、シリカ被覆された銀ナノ粒子の一次粒子状態の他に、2粒子が凝集した凝集体も存在していた。この凝集体を探し出し、評価した。
実施例7と同様に、SEM画像(500K倍)を撮像し、SEM画像から凝集体における無機膜の厚みT2と、2つの金属ナノ粒子間の離隔距離L2とを測定し、比較した。その結果、離隔距離L2は、無機膜の厚みT2の約2倍であった。なお、無機膜の厚みT2は、離隔距離の対象ではない部分の無機膜の厚みであった。
(符号の説明)
2 ・・・金属ナノ粒子
3 ・・・高分子膜
3A ・・・高分子膜を構成する高分子
3a ・・・硫黄原子を介した結合部位
3b ・・・正帯電性基
3c ・・・疎水性基
4 ・・・特異結合物質
6 ・・・蛍光物質
10 ・・・第1ナノ粒子体
12 ・・・第1金属ナノ粒子
13 ・・・第1高分子膜
14 ・・・第1特異結合物質
16 ・・・第1蛍光物質
20 ・・・第2ナノ粒子体
22 ・・・第2金属ナノ粒子
23 ・・・第2高分子膜
24 ・・・第2特異結合物質
26 ・・・第2蛍光物質
30 ・・・被験物質
40 ・・・複合体
L ・・・離隔距離(離間距離)
[1]
金属ナノ粒子と、該金属ナノ粒子の表面を被覆する高分子膜と、該高分子膜の表面に結合された、検体中の被験物質と特異的に結合するナノサイズの特異結合物質とを含んで成り、
前記高分子膜が、前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性基、および疎水性基からなる群より選択される少なくとも1つを含む、ナノ粒子体。
[2]
前記高分子膜を構成する高分子が、その側鎖に前記結合部位、前記正帯電性基、および前記疎水性基からなる群より選択される少なくとも1つを含む、[1]に記載のナノ粒子体。
[3]
前記高分子膜が、前記硫黄原子を介した前記結合部位を少なくとも含む、[1]または[2]に記載のナノ粒子体。
[4]
前記高分子膜が、前記正帯電性基を少なくとも含む、[1]~[3]のいずれか1つに記載のナノ粒子体。
[5]
前記正帯電性基は、第1級アンモニウム基、第2級アンモニウム基、第3級アンモニウム基、第4級アンモニウム基およびグアニジル基(-NHC(=NH2+)NH2)からなる群より選択される少なくとも1種である、[4]に記載のナノ粒子体。
[6]
前記高分子膜が、前記疎水性基を少なくとも含む、[1]~[5]のいずれか1つに記載のナノ粒子体。
[7]
前記疎水性基は、芳香族環状基、脂肪族環状基、および脂肪族鎖状基からなる群より選択される少なくとも1種である、[6]に記載のナノ粒子体。
[8]
前記高分子膜を構成する高分子が、側鎖としてジスルフィド結合を含む部位を有する、[1]~[7]のいずれか1つに記載のナノ粒子体。
[9]
前記ジスルフィド結合を含む部位が前記正帯電性基を有する、[3]または[4]に従属する[8]に記載のナノ粒子体。
[10]
前記ジスルフィド結合を含む部位が前記疎水性基を有する、請求項5または6に従属する請求項8に記載のナノ粒子体。
[11]
前記高分子膜の膜厚は、1nm~10nmである、[1]~[10]のいずれか1つに記載のナノ粒子体。
[12]
プラズモン励起蛍光分析に用いるナノ粒子体である、[1]~[11]のいずれか1つに記載のナノ粒子体。
[13]
前記特異結合物質がナノ抗体である、[1]~[12]のいずれか1つに記載のナノ粒子体。
[14]
前記特異結合物質がVHH抗体である、[1]~[13]のいずれか1つに記載のナノ粒子体。
[15]
前記金属ナノ粒子が金または銀を含んで成る、[1]~[14]のいずれか1つに記載のナノ粒子体。
[16]
前記高分子膜の表面および前記特異結合物質の少なくとも一方に、蛍光物質が標識されている、[1]~[15]のいずれか1つに記載のナノ粒子体。
[17]
前記ナノ粒子体として第1ナノ粒子体と第2ナノ粒子体とが含まれ、
前記第1ナノ粒子体は、前記金属ナノ粒子としての第1金属ナノ粒子と、前記高分子膜としての第1高分子膜と、前記特異結合物質としての第1特異結合物質とを含み、
前記第2ナノ粒子体は、前記金属ナノ粒子としての第2金属ナノ粒子と、前記高分子膜としての第2高分子膜と、前記特異結合物質としての第2特異結合物質とを含み、
蛍光物質は、前記第1高分子膜および前記第2高分子膜、ならびに前記第1特異結合物質および前記第2特異結合物質のうちの少なくとも1つに標識されている、[1]~[16]のいずれか1つに記載のナノ粒子体。
[18]
前記第1ナノ粒子体と前記第2ナノ粒子体とが前記被験物質を介して結合された複合体を形成する、[17]に記載のナノ粒子体。
[19]
前記被験物質が、血液、血漿、尿、または唾液である前記検体に由来する被験物質である、[18]に記載のナノ粒子体。
[20]
前記複合体において、前記第1ナノ粒子体と前記第2ナノ粒子体との間に前記蛍光物質が位置づけられている、[18]または[19]に記載のナノ粒子体。
[1]
金属ナノ粒子と、該金属ナノ粒子の表面を被覆する高分子膜と、蛍光物質とを含んで成り、
前記蛍光物質は、前記金属ナノ粒子の表面に標識されており、
前記高分子膜が前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位を含む、ナノ粒子体。
[2]
前記高分子膜を構成する高分子が、その側鎖に前記結合部位を含む、[1]に記載のナノ粒子体。
[3]
前記蛍光物質は、前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位を含む、[1]または[2]に記載のナノ粒子体。
[4]
前記高分子膜に結合された、検体中の被験物質と特異的に結合する特異結合物質をさらに含んで成る、[1]~[3]のいずれか1つに記載のナノ粒子体。
[5]
前記高分子膜が、正帯電性基および疎水性基からなる群より選択される少なくとも1つをさらに含む、[1]~[4]のいずれか1つに記載のナノ粒子体。
[6]
前記高分子膜を構成する高分子が、その側鎖に前記正帯電性基および前記疎水性基からなる群より選択される少なくとも1つをさらに含む、[5]に記載のナノ粒子体。
[7]
前記高分子膜が、前記正帯電性基を少なくとも含み、
前記正帯電性基は、第1級アンモニウム基、第2級アンモニウム基、第3級アンモニウム基、第4級アンモニウム基およびグアニジル基(-NHC(=NH2+)NH2)からなる群より選択される少なくとも1種である、[5]に記載のナノ粒子体。
[8]
前記高分子膜が、前記疎水性基を少なくとも含み、
前記疎水性基は、芳香族環状基、脂肪族環状基および脂肪族鎖状基からなる群より選択される少なくとも1種である、[5]に記載のナノ粒子体。
[9]
前記高分子膜の膜厚は、1nm~10nmである、[1]~[8]のいずれか1つに記載のナノ粒子体。
[10]
プラズモン励起蛍光分析に用いるナノ粒子体である、[1]~[9]のいずれか1つに記載のナノ粒子体。
[11]
前記特異結合物質が、ナノ抗体、リガンド、酵素および核酸鎖からなる群より選択される少なくとも1つである、[4]に従属する[5]~[10]のいずれか1つに記載のナノ粒子体。
[12]
前記特異結合物質が、VHH抗体、断片化抗体およびそれらの変異体からなる群より選択される少なくとも1つのナノ抗体である、[4]に従属する[5]~[11]のいずれか1つに記載のナノ粒子体。
[13]
前記金属ナノ粒子が金または銀を含んで成る、請求項1~12のいずれか1項に記載のナノ粒子体。
[14]
前記ナノ粒子体として第1ナノ粒子体と第2ナノ粒子体とが含まれ、
前記第1ナノ粒子体は、前記金属ナノ粒子としての第1金属ナノ粒子と、前記高分子膜としての第1高分子膜とを含み、
前記第2ナノ粒子体は、前記金属ナノ粒子としての第2金属ナノ粒子と、前記高分子膜としての第2高分子膜とを含み、
前記蛍光物質は、前記第1金属ナノ粒子の表面および前記第2金属ナノ粒子の表面の少なくとも1つに標識されている、[1]~[13]のいずれか1つに記載のナノ粒子体。
[15]
前記第1ナノ粒子体と前記第2ナノ粒子体とが被験物質を介して結合された複合体を形成する、[14]に記載のナノ粒子体。
[16]
前記被験物質が、血液、血漿、尿、または唾液である検体に由来する被験物質である、[15]に記載のナノ粒子体。
[17]
前記複合体において、前記第1ナノ粒子体と前記第2ナノ粒子体との間に少なくとも前記蛍光物質が位置づけられている、[15]または[16]に記載のナノ粒子体。
[18]
蛍光物質がジスルフィド結合を介して結合する高分子と、金属ナノ粒子とを混合して、前記蛍光物質を前記金属ナノ粒子の表面に標識しつつ、かつ前記金属ナノ粒子の表面に高分子膜を形成する工程を含んで成る、ナノ粒子体の製造方法。
[1]
ナノ粒子体と、該ナノ粒子体を含む溶媒とを含んで成るナノ粒子体組成物であって、
前記ナノ粒子体は、金属ナノ粒子と、該金属ナノ粒子の表面を被覆する被覆膜と、該被覆膜の表面または前記金属ナノ粒子の表面に結合し、検体中の被験物質と特異的に結合する特異結合物質とを有し、
前記溶媒には、前記ナノ粒子体に加えてポリアニオン系高分子が含まれる、ナノ粒子体組成物。
[2]
前記ポリアニオン系高分子は、カルボン酸塩基、硫酸塩基、スルホン酸塩基、硝酸塩基、リン酸塩基、およびホウ酸塩基からなる群より選択される少なくとも1種のアニオン性基を有する、[1]に記載のナノ粒子体組成物。
[3]
前記ポリアニオン系高分子は、ポリグルタミン酸、ヘパリン、ポリアスパラギン酸、ポリアクリル酸およびそれらの塩、ならびにDNAからなる群より選択される少なくとも1種である、[1]または[2]に記載のナノ粒子体組成物。
[4]
前記被覆膜は、
無機酸化物を含む無機膜、または
前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性基、および疎水性基からなる群より選択される少なくとも1つを含む高分子膜
である、[1]~[3]のいずれか1つに記載のナノ粒子体組成物。
[5]
前記被覆膜は前記高分子膜であり、
前記高分子膜を構成する高分子は、側鎖の末端に前記硫黄原子を介した結合部位、前記正帯電性基、および前記疎水性基からなる群より選択される少なくとも1つを有する、[4]に記載のナノ粒子体組成物。
[6]
前記特異結合物質はナノ抗体である、[1]~[5]のいずれか1つに記載のナノ粒子体組成物。
[7]
前記特異結合物質は、ポリアルキレンエーテル鎖、およびアルキル鎖からなる群より選択される少なくとも1種で架橋されている、[1]~[6]のいずれか1つに記載のナノ粒子体組成物。
[8]
前記ナノ粒子体の個々の粒子体を取り囲むように、前記ポリアニオン系高分子が存在する、[1]~[7]のいずれか1つに記載のナノ粒子体組成物。
[9]
前記溶媒は水性溶媒を含む、[1]~[8]のいずれか1つに記載のナノ粒子体組成物。
[10]
前記ポリアニオン系高分子と、前記ナノ粒子体との会合体を含む、請求項1~9のいずれか1項に記載のナノ粒子体組成物。
[11]
プラズモン励起蛍光分析に用いるナノ粒子体である、[1]~[10]のいずれか1つに記載のナノ粒子体組成物。
[12]
前記金属ナノ粒子が金または銀を含んで成る、[1]~[11]のいずれか1項に記載のナノ粒子体組成物。
[13]
前記被覆膜の表面および前記特異結合物質の少なくとも一方に、蛍光物質が標識されている、[1]~[12]のいずれか1つに記載のナノ粒子体組成物。
[14]
前記ナノ粒子体として第1ナノ粒子体と第2ナノ粒子体とが含まれ、
前記第1ナノ粒子体は、前記金属ナノ粒子としての第1金属ナノ粒子と、前記被覆膜としての第1被覆膜と、前記特異結合物質としての第1特異結合物質とを含み、
前記第2ナノ粒子体は、前記金属ナノ粒子としての第2金属ナノ粒子と、前記被覆膜としての第2被覆膜と、前記特異結合物質としての第2特異結合物質とを含み、
蛍光物質は、前記第1被覆膜および前記第2被覆膜、ならびに前記第1特異結合物質および前記第2特異結合物質の少なくとも1つに標識されている、[1]~[13]のいずれか1つに記載のナノ粒子体組成物。
[15]
前記第1ナノ粒子体と前記第2ナノ粒子体とが前記被験物質を介して結合された複合体を形成する、[14]に記載のナノ粒子体組成物。
[16]
前記複合体において、前記第1ナノ粒子体と前記第2ナノ粒子体との間に前記蛍光物質が位置づけられている、[15]に記載のナノ粒子体組成物。
[17]
前記被験物質が、血液、血漿、尿、または唾液に由来する被験物質である、[1]~[16]のいずれか1つに記載のナノ粒子体組成物。
Claims (40)
- 金属ナノ粒子と、該金属ナノ粒子の表面を被覆する高分子膜と、該高分子膜の表面に結合された、検体中の被験物質と特異的に結合するナノサイズの特異結合物質とを含んで成る、ナノ粒子体。
- 前記高分子膜が、前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性基、および疎水性基からなる群より選択される少なくとも1つを含む、請求項1に記載のナノ粒子体。
- さらに、蛍光物質を含んで成り、
前記蛍光物質は、前記金属ナノ粒子の表面、前記高分子膜の表面および前記特異結合物質からなる群より選択される少なくともいずれかに標識されている、請求項2に記載のナノ粒子体。 - 前記蛍光物質は、前記金属ナノ粒子の表面に標識されており、
前記蛍光物質は、前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位を含む、請求項3に記載のナノ粒子体。 - 前記高分子膜を構成する高分子が、その側鎖に前記結合部位、前記正帯電性基、および前記疎水性基からなる群より選択される少なくとも1つを含む、請求項2または3に記載のナノ粒子体。
- 前記高分子膜が、前記硫黄原子を介した前記結合部位を少なくとも含む、請求項2または3に記載のナノ粒子体。
- 前記高分子膜が、前記正帯電性基を少なくとも含む、請求項2または3に記載のナノ粒子体。
- 前記正帯電性基は、第1級アンモニウム基、第2級アンモニウム基、第3級アンモニウム基、第4級アンモニウム基およびグアニジル基(-NHC(=NH2 +)NH2)からなる群より選択される少なくとも1種である、請求項7に記載のナノ粒子体。
- 前記高分子膜が、前記疎水性基を少なくとも含む、請求項2または3に記載のナノ粒子体。
- 前記疎水性基は、芳香族環状基、脂肪族環状基、および脂肪族鎖状基からなる群より選択される少なくとも1種である、請求項9に記載のナノ粒子体。
- 前記高分子膜を構成する高分子が、側鎖としてジスルフィド結合を含む部位を有する、請求項2または3に記載のナノ粒子体。
- 前記高分子膜を構成する高分子が、側鎖としてジスルフィド結合を含む部位を有し、
前記ジスルフィド結合を含む部位が前記正帯電性基を有する、請求項7に記載のナノ粒子体。 - 前記高分子膜を構成する高分子が、側鎖としてジスルフィド結合を含む部位を有し、
前記ジスルフィド結合を含む部位が前記疎水性基を有する、請求項9に記載のナノ粒子体。 - 前記高分子膜の膜厚は、1nm~10nmである、請求項2または3に記載のナノ粒子体。
- プラズモン励起蛍光分析に用いるナノ粒子体である、請求項2または3に記載のナノ粒子体。
- 前記特異結合物質が、ナノ抗体、リガンド、酵素および核酸鎖からなる群より選択される少なくとも1つである、請求項2または3に記載のナノ粒子体。
- 前記特異結合物質が、VHH抗体、断片化抗体およびそれらの変異体からなる群より選択される少なくとも1つのナノ抗体である、請求項2または3に記載のナノ粒子体。
- 前記金属ナノ粒子が金または銀を含んで成る、請求項2または3に記載のナノ粒子体。
- 前記ナノ粒子体として第1ナノ粒子体と第2ナノ粒子体とが含まれ、
前記第1ナノ粒子体は、前記金属ナノ粒子としての第1金属ナノ粒子と、前記高分子膜としての第1高分子膜と、前記特異結合物質としての第1特異結合物質とを含み、
前記第2ナノ粒子体は、前記金属ナノ粒子としての第2金属ナノ粒子と、前記高分子膜としての第2高分子膜と、前記特異結合物質としての第2特異結合物質とを含み、
蛍光物質は、前記第1金属ナノ粒子の表面および前記第2金属ナノ粒子の表面、前記第1高分子膜および前記第2高分子膜、ならびに前記第1特異結合物質および前記第2特異結合物質のうちの少なくとも1つに標識されている、請求項2または3に記載のナノ粒子体。 - 前記第1ナノ粒子体と前記第2ナノ粒子体とが前記被験物質を介して結合された複合体を形成する、請求項19に記載のナノ粒子体。
- 前記被験物質が、血液、血漿、尿、または唾液である前記検体に由来する被験物質である、請求項19に記載のナノ粒子体。
- 蛍光物質をさらに含んで成り、
前記第1ナノ粒子体と前記第2ナノ粒子体とが前記被験物質を介して結合された複合体において、前記第1ナノ粒子体と前記第2ナノ粒子体との間に前記蛍光物質が位置づけられている、請求項19に記載のナノ粒子体。 - 蛍光物質がジスルフィド結合を介して結合する高分子と、金属ナノ粒子とを混合して、前記蛍光物質を前記金属ナノ粒子の表面に標識しつつ、かつ前記金属ナノ粒子の表面に高分子膜を形成する工程を含んで成る、ナノ粒子体の製造方法。
- 請求項1に記載のナノ粒子体と、該ナノ粒子体を含む溶媒とを含んで成るナノ粒子体組成物であって、
前記特異結合物質は、前記高分子膜の表面または前記金属ナノ粒子の表面に結合し、
前記溶媒には、前記ナノ粒子体に加えてポリアニオン系高分子が含まれる、ナノ粒子体組成物。 - 前記ポリアニオン系高分子は、カルボン酸塩基、硫酸塩基、スルホン酸塩基、硝酸塩基、リン酸塩基、およびホウ酸塩基からなる群より選択される少なくとも1種のアニオン性基を有する、請求項24に記載のナノ粒子体組成物。
- 前記ポリアニオン系高分子は、ポリグルタミン酸、ヘパリン、ポリアスパラギン酸、ポリアクリル酸およびそれらの塩、ならびにDNAからなる群より選択される少なくとも1種である、請求項24または25に記載のナノ粒子体組成物。
- 前記高分子膜は、
前記金属ナノ粒子の表面との間に硫黄原子を介した結合部位、正帯電性基、および疎水性基からなる群より選択される少なくとも1つを含む、請求項24または25に記載のナノ粒子体組成物。 - 前記高分子膜を構成する高分子は、側鎖の末端に前記硫黄原子を介した結合部位、前記正帯電性基、および前記疎水性基からなる群より選択される少なくとも1つを有する、請求項24または25に記載のナノ粒子体組成物。
- 前記特異結合物質はナノ抗体である、請求項24または25に記載のナノ粒子体組成物。
- 前記特異結合物質は、ポリアルキレンエーテル鎖、およびアルキル鎖からなる群より選択される少なくとも1種で架橋されている、請求項24または25に記載のナノ粒子体組成物。
- 前記ナノ粒子体の個々の粒子体を取り囲むように、前記ポリアニオン系高分子が存在する、請求項24または25に記載のナノ粒子体組成物。
- 前記溶媒は水性溶媒を含む、請求項24または25に記載のナノ粒子体組成物。
- 前記ポリアニオン系高分子と、前記ナノ粒子体との会合体を含む、請求項24または25に記載のナノ粒子体組成物。
- プラズモン励起蛍光分析に用いるナノ粒子体である、請求項24または25に記載のナノ粒子体組成物。
- 前記金属ナノ粒子が金または銀を含んで成る、請求項24または25に記載のナノ粒子体組成物。
- 前記高分子膜の表面および前記特異結合物質の少なくとも一方に、蛍光物質が標識されている、請求項24または25に記載のナノ粒子体組成物。
- 前記ナノ粒子体として第1ナノ粒子体と第2ナノ粒子体とが含まれ、
前記第1ナノ粒子体は、前記金属ナノ粒子としての第1金属ナノ粒子と、前記高分子膜としての第1高分子膜と、前記特異結合物質としての第1特異結合物質とを含み、
前記第2ナノ粒子体は、前記金属ナノ粒子としての第2金属ナノ粒子と、前記高分子膜としての第2高分子膜と、前記特異結合物質としての第2特異結合物質とを含み、
蛍光物質は、前記第1高分子膜および前記第2高分子膜、ならびに前記第1特異結合物質および前記第2特異結合物質の少なくとも1つに標識されている、請求項24または25に記載のナノ粒子体組成物。 - 前記第1ナノ粒子体と前記第2ナノ粒子体とが前記被験物質を介して結合された複合体を形成する、請求項37に記載のナノ粒子体組成物。
- 前記複合体において、前記第1ナノ粒子体と前記第2ナノ粒子体との間に前記蛍光物質が位置づけられている、請求項38に記載のナノ粒子体組成物。
- 前記被験物質が、血液、血漿、尿、または唾液に由来する被験物質である、請求項24または25に記載のナノ粒子体組成物。
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