Nanoparticles and methods for producing same

Abstract

A nanoparticle body comprising: metal nanoparticles; a polymer membrane covering surfaces of the metal nanoparticles; and a nano-size specific binding substance that is bound to the surface of the polymer membrane and that specifically binds with a substance to be tested in a sample.

Description

[Technical Field]

[0001] The present invention relates to a nanoparticle (particularly a nanoparticle used in plasmon-excited fluorescence analysis), a method for producing the same, and a nanoparticle composition containing the nanoparticle (particularly a nanoparticle composition used in plasmon-excited fluorescence analysis). [Background technology]

[0002] A biosensor specifically reacts a specific test substance, which is the detection target, with a specific specific binding substance to form a complex, and detects the test substance based on a signal derived from the specific binding in the complex. In plasmon excitation fluorescence analysis, the complex contains, for example, a fluorescent substance and metal particles in addition to the test substance and specific binding substance (the complex contains, for example, metal particles, a specific binding substance, a fluorescent substance, and the test substance). When excitation light is irradiated onto the complex, surface plasmon resonance occurs in the metal particles within the complex, and a near-field is formed near the surface of the metal particles. This near-field increases the fluorescence intensity of the fluorescent substance.

[0003] The composite particles for immunochromatography described in Patent Document 1 have a structure in which the exterior of metal microparticles is covered with at least one layer of silica containing at least one type of fluorescent substance, and are composed of microparticles whose surfaces are modified with a labeling substance that specifically recognizes a target substance. The composite particles of Patent Document 1 cover the surfaces of the metal particles with a silica layer and fix the fluorescent substance to the silica layer, thereby preventing the fluorescent substance excited by the near field formed by surface plasmon resonance from coming into contact with the metal particles. This suppresses deactivation (quenching) of the excited fluorescent substance. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-220705 Summary of the Invention [Problem to be solved by the invention]

[0005] However, as a result of extensive research by the present inventors, it was found that there is room for further improvement in the detection stability of the above-mentioned sensor. Specifically, since a specific binding substance usually does not directly bind to a silica layer, it is necessary to perform surface modification of the silica layer to improve the binding to the silica layer. This increases the film thickness of the silica layer, making it impossible to sufficiently reduce the distance between the metal particles in the composite. As a result, the detection sensitivity decreases. On the other hand, when preparing a silica film, tetraethoxysilane is hydrolyzed under basic conditions to form a polymeric film. However, the formation rate is significantly affected by factors such as the reaction concentration, reaction temperature, and reaction time, making it difficult to produce a stable film.

[0006] The present invention has been made in view of such problems. Ta That is, a main object of the present invention is to provide a nanoparticle having excellent detection stability by forming a thin film stably fixed on the surface of a metal particle. More specifically, a main object of the present invention is to provide a nanoparticle having excellent detection sensitivity by reliably capturing an analyte, sufficiently suppressing quenching of a fluorescent substance, and reducing the distance between metal particles in a composite. [Means for solving the problem]

[0007] The nanoparticle according to one embodiment of the present invention comprises: The method comprises: a metal nanoparticle; a polymer film covering the surface of the metal nanoparticle; and a nano-sized specific binding substance bound to the surface of the polymer film, the specific binding substance binding specifically to a test substance in a specimen; The polymer film includes at least one selected from the group consisting of a bonding site between the polymer film and the surface of the metal nanoparticle via a sulfur atom, a positively charged group, and a hydrophobic group. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a cross-sectional view schematically showing a nanoparticle according to the first embodiment. [Figure 2] FIG. 2 is an enlarged cross-sectional view of part A in FIG. [Figure 3] FIG. 3 is a cross-sectional view schematically illustrating a nanoparticle composition according to the second embodiment. [Figure 4] FIG. 4 is a cross-sectional view schematically showing a composite according to a third embodiment. [Figure 5] FIG. 5 is a cross-sectional view schematically showing a composite according to a modified example of the third embodiment. [Figure 6] FIG. 6 is a diagram schematically showing a measurement device according to the fourth embodiment. [Figure 7] FIG. 7 is a schematic diagram illustrating a method for producing a polymer film. [Figure 8] FIG. 8 is a schematic diagram illustrating a method for producing a polymer film. [Figure 9] Figure 9 shows scanning electron microscope (SEM) images of metal nanoparticles coated with a polymer film. The metal nanoparticles are silver nanoparticles (particle diameter: 80 nm) in Figure 9(a) and gold nanoparticles (particle diameter: 20 nm) in Figure 9(b). [Figure 10] Figure 10 shows graphs of the zeta potential of metal nanoparticles coated with a polymer film. The metal nanoparticles are silver nanoparticles (particle diameter: 80 nm) in Figure 10(a) and gold nanoparticles (particle diameter: 20 nm) in Figure 10(b). [Figure 11] Figure 11 shows graphs of the zeta potential of metal nanoparticles not coated with a polymer film. The metal nanoparticles are silver nanoparticles (particle diameter: 80 nm) in Figure 11(a) and gold nanoparticles (particle diameter: 20 nm) in Figure 11(b). [Figure 12] Figure 12 shows the fluorescence spectra of a fluorescent substance-gold nanoparticle mixture. In Figure 12(a), the gold nanoparticles are coated with a polymer film, while in Figure 12(b), they are not coated with a polymer film. [Figure 13] FIG. 13 is a cross-sectional view schematically showing the nanoparticles of Example 1. As shown in FIG. [Figure 14]14 shows the fluorescence spectrum of the test substance-nanoparticle system, where the solid line shows the fluorescence spectrum when reacted with CRP antigen, and the dashed line shows the fluorescence spectrum of the blank sample. [Figure 15] FIG. 15 shows an SEM image of a composite prepared using the nanoparticles of Example 2. [Figure 16] FIG. 16 is a schematic cross-sectional view showing nanoparticles in the nanoparticle composition of Example 3. [Figure 17] FIG. 17 is a graph showing the absorption spectrum of the nanoparticulate composition of Example 3. [Figure 18] FIG. 18 is a graph showing the fluorescence spectrum of the nanoparticulate composition of Example 3. [Figure 19] FIG. 19 is a schematic diagram illustrating the method for producing the nanoparticles of Example 4. [Figure 20] FIG. 20 is a graph showing the absorption spectrum and fluorescence spectrum of the nanoparticulate composition of Example 4. [Figure 21] FIG. 21 is a diagram showing the frequency distribution of sizes in Example 5. [Figure 22] FIG. 22 is a diagram showing the frequency distribution of the zeta potential in Example 5. [Figure 23] FIG. 23 is a schematic diagram showing the relationship between the number of washing treatments and the state of the aggregate in Example 5. [Figure 24] FIG. 24 is a cross-sectional view schematically showing a nanoparticle according to the fourth embodiment. [Figure 25] FIG. 25 is an enlarged schematic view of part A in FIG. [Figure 26] FIG. 26 is a reaction scheme showing an example of a method for producing nanoparticles according to the fifth embodiment. [Figure 27] FIG. 27 is a cross-sectional view schematically showing a composite according to the sixth embodiment. [Figure 28] FIG. 28 is a diagram schematically showing a measurement device according to the seventh embodiment. [Figure 29] FIG. 29 is a schematic diagram illustrating the method for producing the nanoparticles of Example 6. [Figure 30] FIG. 30 shows the fluorescence spectrum of the nanoparticles of Example 6. [Figure 31] FIG. 31 shows an SEM image of a composite prepared using the nanoparticles of Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, the nanoparticles, composites, and measuring devices according to the embodiments of the present invention will be described in detail with reference to the drawings. Note that the drawings include schematic views and may not reflect actual dimensions or proportions.

[0010] Numerical ranges mentioned in this specification are intended to include both the lower and upper limits. For example, if a numerical range is 1 nm to 10 nm, the numerical range is interpreted as including both the lower limit "1 nm" and the upper limit "1 nm."

[0011] <First embodiment: nanoparticles> The nanoparticle according to the first embodiment is The method comprises: a metal nanoparticle; a polymer film covering the surface of the metal nanoparticle; and a nano-sized specific binding substance bound to the surface of the polymer film, the specific binding substance binding specifically to a test substance in a specimen; The polymer film contains at least one selected from the group consisting of a bonding site between the surface of the metal nanoparticle and the polymer film via a sulfur atom, a positively charged group, and a hydrophobic group.

[0012] [Test substance detection method] First, for the convenience of explaining the nanoparticles according to this embodiment and to aid in understanding thereof, a method for detecting a test substance using the nanoparticles according to this embodiment will be described. The nanoparticles according to this embodiment comprise metal nanoparticles, a polymer membrane covering the surface of the metal nanoparticles, and a specific binding substance bound to the polymer membrane. The nanoparticles according to this embodiment are dissolved or dispersed in a specimen, and a test substance contained in the specimen 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. In this way, the two metal nanoparticles are arranged at a certain distance apart in the complex by binding to their respective specific binding substances for the same test substance. Furthermore, the complex contains a fluorescent substance.

[0013] In Surface Plasmon Fluorescence Spectroscopy (SPFS), when excitation light is irradiated onto the complex, Localized Surface Plasmon Resonance (LSPR) occurs, and a near-field is efficiently formed near the surface of the metal nanoparticle (especially near the surface between two metal nanoparticles). This near-field efficiently excites the fluorescent substance in the complex, increasing its fluorescence intensity. By measuring the fluorescence intensity, the test substance in the sample can be detected.

[0014] [Mechanism of action] The nanoparticles according to this embodiment have superior detection stability. Without being bound by any particular theory, the reason for this is presumed to be as follows: The nanoparticles according to this embodiment comprise metal nanoparticles and a polymer film covering the surface of the metal nanoparticles. The polymer film has a bonding site between the surface of the metal nanoparticles and the surface of the metal nanoparticles via a sulfur atom, a positively charged basisand at least one selected from the group consisting of hydrophobic groups. Therefore, the polymer film forms a relatively strong bond with the surface of the metal nanoparticle, thereby stably immobilizing it on the surface of the metal nanoparticle. This prevents the polymer film from peeling off from the surface of the metal nanoparticle, thereby suppressing "exposure of the metal nanoparticle surface" and "detachment of specific binding substances" associated with peeling of the polymer film, thereby suppressing a decrease in detection sensitivity. Furthermore, since the polymer film is composed of a polymer, it is easier to chemically modify than a silica layer. This allows the film thickness to be smaller than that of a silica layer, thereby reducing the distance between metal particles in the composite. Therefore, detection sensitivity can be improved. From the above, it is believed that the nanoparticles according to this embodiment have superior detection stability.

[0015] [Nanoparticle structure] The structure of the nanoparticle will be described below. The nanoparticle will be described with reference to Fig. 1. Fig. 1 is a cross-sectional view showing a schematic diagram of a nanoparticle. The nanoparticle 1 according to this embodiment comprises a metal nanoparticle 2, a polymer film 3 covering the surface of the metal nanoparticle 2, and a specific binding substance 4 bound to the surface of the polymer film 3.

[0016] The nanoparticle 1 can be used in plasmon excitation fluorescence analysis. In other words, the nanoparticle 1 can be used in surface plasmon excitation enhanced fluorescence spectroscopic immunoassay. The nanoparticle 1 can capture a test substance in a test specimen and form a complex containing two nanoparticles 1 and the test substance. When excitation light is irradiated onto the complex, localized surface plasmon resonance occurs, forming a near-field. This near-field increases the fluorescence intensity.

[0017] The nanoparticle 1 may also have non-specific binding sites blocked by a blocking agent. The blocked nanoparticle 1 suppresses non-specific binding of the specific binding substance 4 to substances other than the target of detection (i.e., substances other than the test substance), reducing background and false positive signals and improving the signal-to-noise ratio (SN ratio). Examples of blocking agents include proteins such as bovine serum albumin (BSA), skim milk, and casein, as well as chemically synthesized polymers.

[0018] When the nanoparticles 1 are present in a solvent, the dispersion of the nanoparticles 1 may further contain a dispersant to improve the dispersibility of the nanoparticles 1. An example of such a dispersant is heparin sodium. This will be described in detail in the nanoparticle composition according to the second embodiment.

[0019] The metal nanoparticles 2, polymer film 3, specific binding substance 4 and fluorescent substance that constitute the nanoparticle body 1 will be described below.

[0020] (metal nanoparticles) The surfaces of the metal nanoparticles 2 are coated with a polymer film 3. The metal nanoparticles 2 interact with light having a specific wavelength, which varies depending on the type of metal, and cause localized surface plasmon resonance. The plasmon resonance peak for silver nanoparticles is between 400 nm and 530 nm, while for gold nanoparticles it is between 510 nm and 580 nm. This range varies depending on the particle size. For example, silver nanoparticles with a particle diameter of 20 nm resonate with light with a wavelength of 405 nm, while gold nanoparticles with a particle diameter of 20 nm resonate with light with a wavelength of 524 nm. The particle diameter (average primary particle diameter) of the metal nanoparticles 2 is, for example, 5 nm to 100 nm. The particle diameter of the metal nanoparticles 2 can be obtained by capturing an image of the metal nanoparticles 2 using a scanning electron microscope (SEM) or a transmission electron microscope (TEM), measuring the particle diameter of the metal nanoparticles 2 in the image, and calculating the average value of multiple particle diameters (number of measurements: for example, at least 10 or more). The metal nanoparticles 2 preferably comprise gold or silver, more preferably silver.

[0021] (polymer membrane) The polymer film 3 covers the surface of the metal nanoparticle 2. The polymer film 3 functions as a metal quenching molecular film. In the composite, the polymer film 3 can dispose the fluorescent substance at a distance from the surface of the metal nanoparticle 2 by at least the thickness of the polymer film 3. This prevents the excited fluorescent substance from coming into contact with the surface of the metal nanoparticle 2 and being quenched, thereby preventing a decrease in detection sensitivity. The presence of the polymer film 3 can be confirmed by capturing an image of the nanoparticle 1 using an SEM or TEM and observing the nanoparticle 1 in the image.

[0022] The polymer film 3 will be described with reference to FIG. 2. 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 nanoparticle 2. The polymer film 3 includes at least one selected from the group consisting of a binding site 3a between the surface of the metal nanoparticle 2 and the polymer film 3 via a sulfur atom, a positively charged group 3b, and a hydrophobic group 3c. More specifically, the polymer film 3 includes a binding site 3a between the surface of the metal nanoparticle 2 and the polymer film 3 via a sulfur atom, and a primary ammonium group (-NH3 + ) and hydrophobic groups 3c. The binding sites 3a bond the surface of the metal nanoparticles 2 to the polymer film 3 via sulfur atoms. The positively charged groups 3b form electrostatic bonds (ionic bonds) b with the surface of the negatively charged metal nanoparticles 2. The hydrophobic groups 3c form hydrophobic bonds c with the surface of the metal nanoparticles 2.

[0023] Because all three of these bonds are relatively strong bonds with the surface of the metal nanoparticle 2, the polymer film 3 is stably fixed to the surface of the metal nanoparticle 2 by at least one of these three bonds. This prevents the polymer film 3 from peeling off from the surface of the metal nanoparticle 2. As a result, the detachment of the specific binding substance 4 due to peeling off of the polymer film 3 is suppressed, and a decrease in detection sensitivity is suppressed. In addition, exposure of the surface of the metal nanoparticle 2 due to peeling off of the polymer film 3 is suppressed, which suppresses quenching due to contact with excited fluorescent substances and a decrease in detection sensitivity. Furthermore, because the polymer film 3 contains polymer 3A, it is easier to chemically modify than a silica layer and requires less surface modification. This allows the film thickness to be smaller than a silica layer, thereby reducing the distance between the metal nanoparticles 2 in the composite. Therefore, a near-field is formed more efficiently, and detection sensitivity is improved. As a result, the nanoparticle body according to this embodiment has superior detection stability.

[0024] As shown in FIG. 2, the polymer 3A constituting the polymer film 3 can include at least one selected from the group consisting of a sulfur atom-mediated binding site 3a, a positively charged group 3b, and a hydrophobic group 3c between the surface of the metal nanoparticle 2 and the polymer 3A. The presence of the sulfur atom-mediated binding site 3a, the positively charged group 3b, and the hydrophobic group 3c can be confirmed by measuring signals derived therefrom using infrared spectroscopy and nuclear magnetic resonance spectroscopy. Furthermore, the polymer 3A constituting the polymer film 3 can have a site containing a disulfide bond (-SS-) as a side chain. The site containing the disulfide bond can have a positively charged group 3b. 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 will be described below.

[0025] -Binding site via sulfur atom- The binding site 3a via a sulfur atom is formed, for example, by mixing a polymer having a site containing a disulfide bond in a side chain with the metal nanoparticle 2. For example, as shown in Figure 8 described later, if the raw material polymer has a hydrophobic group 3c in the side chain via a disulfide bond, a binding site via a sulfur atom is formed between the polymer and the surface of the metal nanoparticle 2 (see the right side in Figures 2 and 8).

[0026] -Positively charged group- The positively charged group forms a strong electrostatic bond with the surface of the metal nanoparticle 2. In this specification, the positively charged group is a group that has a valence of one or more and is completely positively ionized. When considering the multiple positively charged groups 3b contained in the polymer that constitutes the polymer film 3, the positively charged group 3b can be expressed by the following mathematical formula (1):

number

[0027] The positively charged group 3b is preferably a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, or a guanidyl group (-NHC(=NH2 + )NH2).

[0028] -Hydrophobic group- The hydrophobic group 3c is at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group.

[0029] Examples of aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups. Aromatic carbocyclic groups are groups that do not include aromatic heterocyclic rings and that contain aromatic rings in which all of the ring atoms are carbon atoms. Examples of aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups, etc.) and arylalkyl groups (more specifically, benzyl groups, etc.). Aromatic heterocyclic groups are aromatic rings in which at least one of the ring atoms is a heteroatom (more specifically, oxygen atom, sulfur atom, nitrogen atom, etc.). tribe Examples of the aromatic heterocyclic group include nitrogen-containing aromatic heterocyclic groups (more specifically, imidazoyl groups and pyridyl (pyridinyl) groups), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups.

[0030] An aliphatic cyclic group is a group that does not contain an aromatic ring and contains a cyclic group consisting of a non-aromatic ring. Examples of the aliphatic cyclic group include an aliphatic carbocyclic group and an aliphatic heterocyclic group. An aliphatic carbocyclic group is a group that contains a non-aromatic ring in which all ring atoms are carbon atoms, such as a cycloalkyl group. An aliphatic heterocyclic group is a non-aromatic ring in which at least one ring atom is a heteroatom. tribe It is a group containing a ring.

[0031] The aliphatic chain group is tribe Ring and non-aromatic tribeIt is a chain-like group (more specifically, a linear or branched chain) that does not contain a ring. Examples of the aliphatic chain-like group include aliphatic carbon chain groups (more specifically, alkyl groups, alkylene groups, etc.) and aliphatic hetero chain groups.

[0032] The polymer 3A constituting the polymer film 3 can form a hydrophobic bond between the hydrophobic group 3c that the polymer 3A may have and the surface of the metal nanoparticle 2. The polymer 3A constituting the polymer film 3 can also form other hydrophobic bonds. For example, a hydrophobic bond can be formed between a hydrophobic group bonded to the surface of the metal nanoparticle 2 via a sulfur atom (more specifically, a pyridyl group (pyridinyl group) bonded to the surface of the metal nanoparticle 2 via a sulfur atom in FIG. 8 ) and a hydrophobic group 3c that the polymer 3A constituting the polymer film 3 may have (more specifically, an alkylene group 3c that the polymer 3A in FIG. 8 has). When such a hydrophobic bond is formed, the polymer film 3 is more stably fixed to the surface of the metal nanoparticle 2. The hydrophobic group bonded to the surface of the metal nanoparticle 2 via a sulfur atom can be formed as follows. As described above, the bond site via a sulfur atom can be formed, for example, by mixing a polymer having a hydrophobic group 3c in its side chain via a disulfide bond with the metal nanoparticle 2. Here, the hydrophobic groups 3c bonded to the sulfur atoms also bond to the surfaces of the metal nanoparticles 2. In this way, hydrophobic groups bonded to the surfaces of the metal nanoparticles 2 via sulfur atoms are formed.

[0033] The polymer 3A constituting the polymer membrane 3 may form a bond via a sulfur atom via a moiety (linker moiety) derived from a crosslinking agent. Examples of such a crosslinking agent include an amino group-sulfhydryl group crosslinking agent (more specifically, an NHS-maleimide group crosslinking agent, etc.).

[0034] The film thickness of the polymer film 3 is preferably 1 nm to 50 nm, and more preferably 1 nm to 10 nm. When the film thickness of the polymer film 3 is 50 nm or less, the separation distance (separation distance) is such that a near-field is efficiently formed in the space between the two metal nanoparticles, further improving detection sensitivity. Furthermore, when the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance are arranged at a predetermined distance, thereby suppressing quenching of the excited fluorescent substance during measurement and further improving detection sensitivity. In this specification, the term "separation distance" refers to the minimum distance (shortest distance) between the surfaces of metal nanoparticles contained in two nanoparticles that are bound to each other via the test substance in a complex.

[0035] (specific binding substance) The specific binding substance 4 is a nano-sized substance (having a maximum size of 3 to 15 nm) that specifically binds to a test substance (described in the second embodiment) in a sample. Examples of the specific binding substance 4 include antibodies (hereinafter referred to as nanoantibodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA and RNA chains). For example, a nanoantibody serving as the specific binding substance 4 specifically binds to an antigen serving as a test substance at its tip (antigen binding site) through an antigen-antibody reaction to form a complex. A ligand serving as the specific binding substance 4 forms a complex with a protein serving as a test substance through a specific protein-ligand bond through a ligand-receptor reaction. A nucleic acid chain serving as the specific binding substance 4 forms a pair (double strand) with a complementary nucleic acid chain based on base pair complementarity. An enzyme serving as the specific binding substance 4 forms an enzyme-substrate complex with a substrate serving as a test substance at its active site (active center) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as those resulting from hydrogen bonds, as well as intermolecular forces, hydrophobic interactions and charge interactions.

[0036] Nanobodies are, for example, variable domain heavy chain (VHH) antibodies, fragment antigen binding (Fab) antibodies, and variants thereof. VHH antibodies are single domain antibodies. Variants are antibodies that bind to antigens. specific binding The nanoantibody is an antibody in which a part of the amino acid sequence has been modified or a substituent has been introduced within the range of (I) above. The nanoantibody is preferably a VHH antibody. When the nanoantibody is a VHH antibody, the VHH antibody has a relatively small volume, which narrows the distance (separation) between the two metal nanoparticles 2 in the complex, allowing for more efficient formation of a near field and further increasing the fluorescence intensity.

[0037] 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. When the molecular mass is 60,000 Da or less (particularly 30,000 Da or less or 20,000 Da or less), the nanobody has a relatively small volume, which narrows the separation distance in the complex, more efficiently forms a near-field, and further increases the fluorescence intensity. Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.

[0038] The specific binding substance 4 may be directly bound to the polymer membrane 3, or may be indirectly bound to the polymer membrane 3 via a linker moiety (more specifically, SM(PEG)6, etc.) derived from a crosslinking agent (more specifically, an NHS-maleimide group crosslinking agent, etc.).

[0039] (fluorescent material) The nanoparticle 1 may further comprise a fluorescent substance. In this case, the fluorescent substance is labeled on at least one of the surface of the polymer film 3 and the specific binding substance. The fluorescent substance is excited by the near field formed by localized surface plasmon resonance and emits fluorescence. Examples of fluorescent substances include complexes of metals such as europium and ruthenium (metal complexes), and dyes in the Alexsa Fluor series (registered trademark) (Molecular Probes (registered trademark)).

[0040] The fluorescent substance preferably has a large Stokes shift. Here, 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. When the Stokes shift of a fluorescent substance 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, allowing for more accurate measurement of fluorescence intensity.

[0041] The fluorescent substance preferably has a sharp fluorescence spectrum. A sharp fluorescence spectrum is less likely to overlap with the absorption spectrum, making it less likely for excitation light (scattered light) to enter the fluorescence to be detected, allowing for more accurate measurement of fluorescence intensity.

[0042] <Second embodiment: nanoparticulate composition> The nanoparticle composition according to the second embodiment comprises: a nanoparticle body and a solvent containing the nanoparticle body; 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 and specifically binds to a test substance in a specimen; The solvent contains the polyanionic polymer in addition to the nanoparticles.

[0043] [Background to the development of the nanoparticle composition according to the second embodiment] The composite particles (nanoparticles) for immunochromatography described in Patent Document 1 have a structure in which the exterior of fine metal particles (metal nanoparticles) is covered with at least one layer of silica containing at least one type of fluorescent substance, and the fine particles are surface-modified with a labeling substance (specific binding substance) that specifically recognizes a target substance (analyte). In this way, the nanoparticles described in Patent Document 1 are surface-modified with a specific binding substance on a coating layer (coating film) that covers the exterior of the metal nanoparticles.

[0044] However, the inventors of the present invention have conducted extensive research into the above-described sensors and found that there is room for further improvement in dispersibility while maintaining the design flexibility of the nanoparticles. Specifically, the design of the nanoparticles may be modified as needed. For example, the number of specific binding substances bound to the coating film may be increased to adequately capture the desired analyte. Furthermore, the number of fluorescent substances bound to the coating film may be increased to increase the detected fluorescence intensity. In these cases, as the number of specific binding substances and fluorescent substances increases, the dispersibility of the nanoparticles decreases, and in the worst case, the nanoparticles may aggregate. On the other hand, when the dispersing agent is bonded to the coating film to improve the dispersibility of the nanoparticles, the number of bonding sites on the coating film decreases, which reduces the degree of freedom in designing the nanoparticles. Thus, it has not been possible to fully achieve both high degree of freedom in designing and excellent dispersibility.

[0045] The main object of this embodiment is to provide a nanoparticle composition containing nanoparticles that not only have the excellent detection sensitivity described above but also have excellent dispersibility while maintaining design freedom.

[0046] [Test substance detection method] First, for the convenience of explaining the nanoparticle composition according to this embodiment and to aid in its understanding, an example of a method for detecting a test substance using the nanoparticle composition according to this embodiment will be described. The nanoparticle composition according to this embodiment comprises nanoparticles and a solvent containing nanoparticles. The nanoparticles have metal nanoparticles, a polymer membrane covering the surface of the metal nanoparticles, and a specific binding substance that binds to the surface of the polymer membrane or the surface of the metal nanoparticles. The nanoparticle composition according to this embodiment is added to a specimen to capture an analyte contained in the specimen and form a complex. More specifically, the complex is formed by specific binding between the specific binding substance of the nanoparticles and the analyte. The complex has a structure in which two nanoparticles are bound via the analyte. In this way, the two metal nanoparticles are spaced apart at a certain distance in the complex by binding to their respective specific binding substances for the same analyte. Furthermore, the complex contains a fluorescent substance.

[0047] In Surface Plasmon Fluorescence Spectroscopy (SPFS), when excitation light is irradiated onto the complex, Localized Surface Plasmon Resonance (LSPR) occurs, and a near-field is efficiently formed near the surface of the metal nanoparticle (especially near the surface between two metal nanoparticles). This near-field efficiently excites the fluorescent substance in the complex, increasing its fluorescence intensity. By measuring the fluorescence intensity, the test substance in the sample can be detected.

[0048] [Mechanism of action] The nanoparticle composition according to this embodiment contains nanoparticles with excellent dispersibility while maintaining design flexibility. Without being bound by any particular theory, the reason for this is presumed to be as follows. The nanoparticle composition according to this embodiment contains nanoparticles and a solvent containing nanoparticles. The nanoparticles include metal nanoparticles, a polymer membrane covering the surfaces of the metal nanoparticles, and a specific binding substance that binds to the surface of the polymer membrane or the surface of the metal nanoparticles and specifically binds to an analyte in a specimen. The solvent contains a polyanionic polymer in addition to the nanoparticles. Therefore, in the nanoparticle composition according to this embodiment, the nanoparticles can form aggregates (aggregates) in which individual particles are surrounded by the polyanionic polymer. The polyanionic polymer acts as a spacer between multiple nanoparticles, preventing aggregation due to contact between the nanoparticles. Furthermore, because multiple anionic groups of the polyanionic polymer are present on the outer surface of the aggregates, electrostatic repulsion acts between the aggregates, keeping the nanoparticles apart. This allows the nanoparticles to be dispersed as single particles in the nanoparticle composition according to this embodiment. That is, the nanoparticles have excellent dispersibility in the nanoparticle composition according to this embodiment. As described above, the nanoparticles are not surface-modified to improve dispersibility. This allows for surface modification of the nanoparticles, maintaining design flexibility. Therefore, the nanoparticle composition according to this embodiment is believed to contain nanoparticles that offer both high design flexibility and excellent dispersibility.

[0049] [Configuration of nanoparticle composition] The composition of the nanoparticle composition will be described below: The nanoparticle composition comprises nanoparticles and a solvent containing the nanoparticles.

[0050] (solvent) The solvent contains polyanionic polymers in addition to nanoparticles. The solvent includes, for example, an aqueous solvent. The aqueous solvent contains at least water (more specifically, pure water). Examples of aqueous solvents 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, etc.). The organic solvent is an organic solvent that is miscible with water, such as alcohol (more specifically, methanol, ethanol, propanol, etc.), as well as tetrahydrofuran, dimethylformamide, and dimethyl sulfoxide. Doka At least one selected from the group consisting of:

[0051] [Polyanionic polymers] A polyanionic polymer is a polymer having a plurality of anionic groups. Examples of the anionic groups include 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. In other words, a 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.

[0052] Polyanionic polymers include, for example, polyglutamic acid, heparin: [ka] The counter ion (counter cation) of the salt is, for example, an alkali metal cation (more specifically, Li + , Na + , and K. + etc.), as well as cations of alkaline earth metals (more specifically, Mg 2+ and Ca 2+ Heparin is a sulfate group (-OSO3 - X + ), carboxylate group (-COO - X +), and sulfonate groups (-SO3 - X + ) (where X + is a monovalent countercation).

[0053] Polyglutamates include, for example, sodium salts: [ka] Polyglutamic acid sodium salt is a carboxylate salt (COO - Na + )

[0054] The polyanionic polymer acts as a dispersant 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. 3. FIG. 3 is a cross-sectional view schematically illustrating nanoparticles in the nanoparticle composition according to this embodiment. In the nanoparticle composition according to this embodiment, the polyanionic polymer 7 exists so as to surround each individual particle of the nanoparticles 1. In other words, the nanoparticle composition contains an association (aggregate) 9 between the nanoparticles 1 and the polyanionic polymer 7. The polyanionic polymer 7 binds to the polymer film 3 of the nanoparticles 1 through electrostatic interaction to form the association 9. The association 9 behaves as a single entity in the nanoparticle composition according to this embodiment.

[0055] In this way, the polyanionic polymer 7 surrounds the nanoparticles 1, and the polyanionic polymer 7 acts as a spacer between the nanoparticles 1, preventing aggregation due to contact between the nanoparticles 1. Furthermore, since multiple anionic groups of the polyanionic polymer 7 are present on the outer surface of the aggregate 9, it is believed that an electric double layer is formed near the interface between the aggregate and the solvent. This causes electrostatic repulsion (more specifically, electric double layer repulsion) between the aggregates 9, keeping the nanoparticles 1 away from each other. As a result, the nanoparticles 1 can be dispersed alone in the nanoparticle composition of this embodiment. In other words, it is believed that the nanoparticles 1 have superior dispersibility in the nanoparticle composition of this embodiment.

[0056] The presence of the aggregate 9 can be confirmed by measuring the zeta potential. Specifically, the aggregate 9 in the nanoparticle composition is washed with a solvent multiple times, and the zeta potential is measured after each washing. If the frequency distribution of the obtained zeta potentials shows a behavior in which the number of washings increases from a negative value, the presence of the aggregate 9 is strongly suggested. While not bound by any particular theory, this behavior of the zeta potential is presumed to be due to the following reason. Before the washing treatment, the zeta potential exhibits a negative value due to the polyanionic polymer 7 that forms the aggregate 9. As the washing treatment is performed, the multiple polyanionic polymers 7 that form the aggregate 9 gradually dissociate from the aggregate 9 and dissolve in the washing solvent. Therefore, as the number of polyanionic polymers 7 decreases from the aggregate 9 as the washing treatment is performed, the zeta potential increases and approaches the zeta potential of the nanoparticle 1.

[0057] The presence of aggregates 9 can also be confirmed by size measurement. Specifically, aggregates 9 in the nanoparticle composition are washed with a solvent multiple times, and the size is measured after each washing. If the resulting size frequency distribution shows an increase with the number of washings, the presence of aggregates 9 is strongly suggested. While not bound by any particular theory, this size behavior is presumed to be due to the following reason. As washing is performed, the multiple polyanionic polymers 7 that form aggregates 9 gradually dissociate from the aggregates 9 and dissolve in the washing solvent. Specifically, in the size frequency distribution for which more washings have been performed, the peak shifts to the larger size side. While not bound by any particular theory, this size behavior is presumed to be due to the following reason. As washing is performed more frequently, the dispersibility of nanoparticles 1 decreases. As a result, nanoparticles 1 aggregate to form aggregates, resulting in an increase in size.

[0058] The content of polyanionic polymer 7 is preferably much greater (i.e., in excess) than the content of nanoparticles 1 in the nanoparticle composition of this embodiment. This is because, in this case, the nanoparticles 1 are more likely to be surrounded by polyanionic polymer 7 in the nanoparticle composition of this embodiment. This further improves the dispersibility of the nanoparticles 1.

[0059] (nanoparticles) -Linker- The nanoparticles 1 can have a linker that binds to the polymer membrane 3, provided that this does not significantly reduce the degree of freedom in designing the nanoparticles 1. This linker does not have a specific binding substance 4 bound to its end. When the nanoparticles 1 have a linker that binds to the polymer membrane 3, the linker acts as a spacer between the nanoparticles 1 (acts as a steric hindrance), preventing aggregation due to contact between the nanoparticles 1. In this case, the dispersibility of the nanoparticles 1 in the nanoparticle composition according to this embodiment is further improved.

[0060] The linker includes, for example, at least one selected from the group consisting of a polyalkylene ether chain and an alkyl chain. The polyalkylene ether chain and the alkyl chain may be a part of the linker portion, or may be the entirety of the linker portion. The polyalkylene ether chain is, for example, a polyalkyleneoxy group (more specifically, a polyethyleneoxy group, etc.). The alkyl chain is, for example, an alkylene group (more specifically, n -propylene group, n-butylene group, etc.).

[0061] <Third embodiment: composite> The complex will be described with reference to FIG. 4. FIG. 4 is a cross-sectional view showing a schematic diagram of the complex. The complex 40 comprises an analyte 30, which is the detection target, and two nanoparticles 10, 20. In the complex 40, the two nanoparticles 10, 20 are bound via the analyte 30. That is, the nanoparticles 10, 20 according to the first embodiment are bound via the analyte 30 to form the complex according to the second embodiment. One of the two nanoparticles 10, 20 will be referred to as the first nanoparticle 10, and the other nanoparticle will be referred to as the second nanoparticle 20. In this way, the complex 40 comprises the first nanoparticle 10 and the second nanoparticle 20 as the nanoparticles 1.

[0062] In the composite 40, the first nanoparticles 10 comprise first metal nanoparticles 12, a first polymer film 13 covering the surfaces of the first metal nanoparticles 12, a first specific binding substance 14 bound to the surface of the first polymer film 13, and a first fluorescent substance 16 labeled on the first polymer film 13. That is, the first nanoparticles 10 comprise first metal nanoparticles 12 as metal nanoparticles, the first polymer film 13 as a polymer film, and the first specific binding substance 14 as a specific binding substance, and the first fluorescent substance 16 is labeled on the first polymer film 13. The second nanoparticles 20 comprise second metal nanoparticles 22, a second polymer film 23 covering the surfaces of the second metal nanoparticles 22, a second specific binding substance 24 bound to the second polymer film 23, and a second fluorescent substance 26 labeled on the second polymer film 23. That is, the second nanoparticle body 20 includes second metal nanoparticles 22 as metal nanoparticles, a second polymer film 23 as a polymer film, and a second specific binding substance 24 as a specific binding substance, and the second fluorescent substance 26 is labeled on the second polymer film 23.

[0063] From the viewpoint of further increasing the fluorescence intensity, it is preferable that the separation distance L is small, within a range in which the excited fluorescent substances 16, 26 are not easily quenched. More specifically, in a preferred embodiment, the two nanoparticles 10, 20 in the composite 40 are close to each other. In a more preferred embodiment, the two nanoparticles 10, 20 are close to each other so that the first polymer film 13 of the first nanoparticle 10 and the second polymer film 23 of the second nanoparticle 20 in the composite 40 come into contact. In an even more preferred embodiment, the two nanoparticles 10, 20 are close to each other so that at least one of the polymer films 13 of the first nanoparticle 10 and the second polymer film 23 of the second nanoparticle 20 in the composite 40 contracts and comes into contact.

[0064] In a further preferred embodiment, when at least one of polymer films 13, 23 shrinks and comes into contact with each other, for example, in complex 40 shown in Figures 4-5, it is believed that at least one of analyte 30, specific binding substances 14, 24 that bind to analyte 30, and fluorescent substances 16, 26 can be inserted into polymer films 13, 23. Furthermore, in a more preferred embodiment, when polymer films 13, 23 come into contact with each other, for example, in complex 40 shown in Figures 4-5, it is believed that at least one of analyte 30, specific binding substances 14, 24 that bind to analyte 30, and fluorescent substances 16, 26 can be inserted into polymer films 13, 23, as in the further preferred embodiment.

[0065] In this embodiment, the films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, which can increase the fluorescence intensity. The reason for this is presumed to be as follows: The films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, which have relatively high flexibility compared to inorganic films containing inorganic oxides. Therefore, in the composite 40, the polymer films 13 and 23 can contract, which allows the two metal nanoparticles 12 and 22 to be closer than a distance equivalent to two polymer film thicknesses (the film thickness of the polymer film 13 plus the film thickness of the polymer film 23). In other words, because the films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, the separation distance L can be less than two polymer film thicknesses. This makes it easier to obtain the plasmon enhancement effect, further increasing the fluorescence intensity. In this specification, the thickness of the polymer film in "two thicknesses of the polymer film" does not refer to the thickness of the polymer film 13, 23 in the contracted portion that is the target of the separation distance, but to the thickness of the polymer film 13, 23 in the non-contracted portion that is not the target of separation (for example, T1 in Figure 15 described later).

[0066] In this embodiment, the polymer films 13 and 23 contain at least one selected from the group consisting of sulfur-atom-mediated binding sites 3a, positively charged groups 3b, and hydrophobic groups 3c (for example, the polymer 3A constituting the polymer films 13 and 23 contains at least one selected from the group consisting of sulfur-atom-mediated binding sites 3a, positively charged groups 3b, and hydrophobic groups 3c), thereby further increasing the fluorescence intensity. The reason for this is presumed to be as follows: In this case, at least one of the binding sites 3a, positively charged groups 3b, and hydrophobic groups 3c forms a bond with the surface of the metal nanoparticles 12 and 22. Therefore, the polymer 3A has a mesh-like structure, which is thought to coat the surfaces of the metal nanoparticles 12 and 22 in a mesh-like manner. Because the polymer 3A has this mesh-like structure, it also has relatively high flexibility. Therefore, in the composite 40, the polymer films 13 and 23 can further contract, allowing the two metal nanoparticles 12 and 22 to be closer to each other than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two times the thickness of the polymer film, and the plasmon enhancement effect is further obtained, further increasing the fluorescence intensity.

[0067] In a preferred embodiment, the polymer 3A constituting the polymer film 13, 23 includes at least one selected from the group consisting of a binding site 3a, a positively charged group 3b, and a hydrophobic group 3c via a sulfur atom in its side chain (more specifically, at the side chain terminal). In a preferred embodiment, the fluorescence intensity can be further increased. The reason for this is presumed to be 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 surface of the metal nanoparticle 12, 22. Therefore, the polymer 3A has a mesh-like structure, and it is thought that the side chain serves as a binding site to coat the surface of the metal nanoparticle 12, 22 in a mesh-like manner. Because of this mesh-like structure, the polymer 3A has relatively high flexibility. Therefore, in the composite 40, the polymer film 13, 23 can further contract, allowing the two metal nanoparticles 12, 22 to be closer than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two times the thickness of the polymer film, and the plasmon enhancement effect is further obtained, further increasing the fluorescence intensity.

[0068] The separation distance L between the first nanoparticles 10 and the second nanoparticles 20 is, for example, 12 nm to 52 nm, and 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 at which the line segment connecting a first point P1 on the surface of the first nanoparticles 10 and a second point P2 on the surface of the second nanoparticles 20 is the shortest. If the separation distance L is 52 nm or less, when excitation light is irradiated onto the complex 40, a near-field is generated more efficiently in the space near the surfaces between the first and second metal nanoparticles 12, 22, thereby further increasing the fluorescence intensity.

[0069] In a preferred embodiment, the polymer 3A constituting the polymer film 13, 23 has at least one selected from the group consisting of a sulfur-atom-mediated bonding site 3a, a positively charged group 3b, and a hydrophobic group 3c (for example, in a side chain (more specifically, at the end of the side chain)). Therefore, as described above, the separation distance L can be closer than the distance equivalent to twice the thickness of the polymer film covering the surfaces of the two metal nanoparticles 12, 22 in the composite 40. For example, when the thickness of the polymer film 13, 23 is 5 nm, the separation distance L can be less than 10 nm (more specifically, 2 to 9 nm, 3 to 8 nm, 4 to 7 nm, etc.).

[0070] (fluorescent material) 4, the fluorescent substances 16, 26 are preferably positioned between the first metal nanoparticle 12 and the second metal nanoparticle 22. This is because the space between the metal nanoparticles 12, 22 is one in which a near-field is efficiently generated, and therefore, by positioning the fluorescent substances 16, 26 in the space between the metal nanoparticles 12, 22, the fluorescence intensity is likely to be increased.

[0071] (Test substance) The test substance 30 is a substance to be detected that is contained in a sample. Examples of the test substance 30 include antigens, proteins, substrates, and nucleic acid chains. The test substance 30 specifically binds to the specific binding substances 14, 24. For example, an antigen has at least two antigenic determinants (epitopes) that form specific bonds with the first and second specific binding substances 14, 24 at the antigenic determinants. Examples of antigens include proteins such as c-reactive protein, myoglobin, troponin T, troponin I, and BNP, as well as antigenic proteins of viruses such as influenza virus and respiratory syncytial virus. The test substance 30 is derived from a sample such as blood, plasma, urine, or saliva. In other words, examples of samples containing the test substance 30 include blood, plasma, serum, urine, and saliva. The sample may further contain a solvent and a buffer solution (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, MES buffer, etc.).

[0072] <Fourth embodiment: Measuring device> The measurement device will be described with reference to Fig. 6. Fig. 6 is a diagram showing the measurement device. As shown in Fig. 6, the measurement device 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 of the excitation light 112, such as by focusing it, and outputs incident excitation light 122. The excitation light irradiation optical system 120 includes a lens 124 and a polarizing element (λ / 2 plate) 126. The incident excitation light 122 output from the excitation light irradiation optical system 120 enters a reagent container 130 and is irradiated onto the measurement sample in the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell, a slide, etc.) or a microchannel chip. The microchannel chip is a chip having a minute channel. When the reagent container 130 is a microchannel chip, for example, the nanoparticles (reagent) according to the first embodiment and a sample can be mixed and continuously supplied. This eliminates the need to prepare a measurement sample by mixing it in advance, enabling continuous measurement.

[0073] The measurement sample irradiated with the incident excitation light 122 emits fluorescence (detection light 132). The light-receiving optical system 140 is disposed perpendicular to the direction of travel of the incident excitation light 122 into the reagent container 130. The light-receiving optical system 140 adjusts the cross-sectional diameter of the detection light 132 emitted from the measurement sample, and can remove scattered light from the incident excitation light 122 or adjust the light intensity. The light-receiving optical system 140 includes a lens 144 and an optical filter 146. The optical filter 146 is, for example, a band-pass filter or a dichroic mirror.

[0074] The fluorescence 142 that passes through the light-receiving optical system 140 is detected by the light-receiving element 150. The light-receiving element 150 is, for example, a PD, an APD, a PMT, a CCD camera, or a spectrometer. The light-receiving element 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring a fluorescence spectrum, and creating a two-dimensional fluorescence image.

[0075] The present invention is not limited to the above-described embodiment, and design modifications are possible within the scope of the gist of the present invention.

[0076] In the third embodiment, the first and second fluorescent substances 16 and 26 are labeled on the first and second polymer films 13 and 23, respectively. However, this is not limiting. For example, FIG. 5 is a cross-sectional view schematically illustrating a complex according to a modified example of the second embodiment. As shown in FIG. 5, the first and second fluorescent substances 16 and 26 may be labeled on the first and second specific binding substances 14 and 24, respectively. This is more preferable because it makes it easier to position the first and second fluorescent substances 16 and 26 between the first metal nanoparticle 12 and the second metal nanoparticle 22, improving detection intensity. Alternatively, one of the first and second fluorescent substances 16 and 26 may be labeled on the polymer films 13 and 23, and the other may be labeled on the specific binding substances 14 and 24.

[0077] In the third embodiment, the complex 40 is labeled with two fluorescent substances 16 and 26, but this is not limiting. For example, the number of fluorescent substances labeled on the complex 40 may be one or three or more.

[0078] In the fourth embodiment, the light-receiving optical system 140 in the measuring device 100 is disposed in a direction perpendicular to the traveling direction of the incident excitation light 122 into the reagent container 130, but is not limited to this. The light-receiving optical system 140 may be disposed, for example, in a direction parallel to the traveling direction of the incident excitation light 122, or in a direction forming an acute angle or an obtuse angle with respect to the traveling direction of the incident excitation light 122. [Example]

[0079] The present invention will be described in more detail below using examples. However, the present invention is not limited to the following examples. Furthermore, unless otherwise specified, parts and percentages in the examples are by mass.

[0080] In the examples and comparative examples, the concentration of metal nanoparticles in a dispersion liquid is sometimes expressed as absorbance. The absorbance was measured using an ultraviolet-visible spectrophotometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.). The absorption wavelength differs depending on the sample, so it is listed for each sample. The number subscripted to the absorbance notation OD indicates the absorption wavelength. For example, OD 455 =0.1 indicates that the absorbance at a wavelength of 455 nm is 0.1.

[0081] [Preparation of polymer membranes] (Production Example 1) A method for producing a polymer film will be described with reference to Figures 7 and 8. Figures 7 and 8 show schematic diagrams for explaining the method for producing a polymer film. As shown in Figure 7, poly-L-lysine (Peptide Institute, Inc., "3075") and 3-(2-pyridyldithio)propionamido-PEG4-NHS (Thermo Fisher Scientific, serial number "26128" or "NHS-PEG4-SPDP") were mixed at room temperature for 4 hours using a small rotating incubator (Tatec Corporation, "RT-30mini"). This resulted in a polymer. This synthesis reaction was a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacked the NHS ester group of 3-(2-pyridyldithio)propionamido-PEG4-NHS. The synthesized polymer had a hydrophobic group (pyridyl group) 3c and a positively charged group (primary ammonium group) 3b. The resulting polymer was then mixed with silver nanoparticles (nanocomposix, "AGCB80-1M," 80 nm in diameter, OD 1000) as metal nanoparticles. 455 The mixture was added to 1 mL of a dispersion of silver nanoparticles coated with polymer film 3 (pH 7.0; pH ...

[0082] As shown in Figure 8, the polymer film 3 contains sulfur-atom-mediated binding sites 3a on the surface of the silver nanoparticles 2, hydrophobic groups (pyridyl groups (pyridinyl groups)) 3c that form hydrophobic bonds with the surface of the silver nanoparticles 2, and positively charged groups (primary ammonium groups) 3b that form electrostatic bonds with the surface of the silver nanoparticles 2. Specifically, the polymer 3A constituting the polymer film 3 contains sulfur-atom-mediated binding sites 3a on the surface of the silver nanoparticles 2, hydrophobic groups (pyridyl groups (pyridinyl groups)) 3c that form hydrophobic bonds with the surface of the silver nanoparticles 2, and positively charged groups (primary ammonium groups) 3b that form electrostatic bonds with the surface of the silver nanoparticles 2. SEM images (magnification: 500,000x) of the resulting silver nanoparticles 2 were created, confirming that the surfaces of the silver nanoparticles 2 were continuously coated with the polymer film 3. The thickness of the polymer film 3 covering the silver nanoparticles 2 was also measured from the SEM image (see Figure 9(a)). The thickness of the polymer film was 4.98 nm (expected value (average) M) ±2.7 nm (standard deviation σ, number of measurements n=7), and the variation in film thickness (σ / M) was 27.4%.

[0083] (Production Example 2) Silver nanoparticles of Production Example 1 ("AGCB80-1M" manufactured by nanocomposix, diameter 80 nm, OD 455 1 mL of the dispersion of gold nanoparticles (SigmaAl dr Made by ich "753610-25ml", diameter 20nm, OD 520A dispersion of gold nanoparticles 2 coated with a polymer film 3 (hereinafter also referred to as a coated gold nanoparticle dispersion) was prepared in the same manner as in Production Example 1, except that the amount of dispersion was changed to 1 mL of a polymer film (M = 0.1). The polymer film 3 has sulfur-atom-mediated binding sites 3a on the surface of the gold nanoparticles 2, positively charged groups (primary ammonium groups) 3b, and hydrophobic groups (pyridyl groups (pyridinyl groups)) 3c. An SEM image (magnification 500,000x) 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. Furthermore, from the SEM image, the thickness of the polymer film of the gold nanoparticles 2 was 8.64 nm (expected value M) ± 0.58 nm (standard deviation σ, number of measurements n = 6), and the film thickness variation (σ / M) was 4.36% (see Figure 9(b)).

[0084] [Measurement method and results] (Charging properties of metal nanoparticle surfaces and polymer films) The zeta potential of metal nanoparticles 2 and metal nanoparticles 2 coated with a polymer film 3 was measured using a zeta potential measurement device (MALVERN "ZETA SIZER Nanoseries nano-ZS"). Figure 10 is a graph of the zeta potential of metal nanoparticles 2 coated with a 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 Figure 10(a) and gold nanoparticles (particle diameter: 20 nm) in Figure 10(b). Figure 11 is a graph of the zeta potential of metal nanoparticles 2 not coated with a 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 Figure 11(a) and gold nanoparticles (particle diameter: 20 nm) in Figure 11(b).

[0085] 11(a)-(b), the zeta potentials of silver nanoparticles (particle diameter 80 nm) and gold nanoparticles (particle diameter 20 nm) both had peaks at negative potentials, and the frequency distribution of the zeta potentials was almost entirely within the negative potential range. Therefore, it was confirmed that the surfaces of the metal nanoparticles 2 that were not coated with the polymer film 3 were negatively charged. As shown in FIGS. 10(a) and 10(b), the zeta potentials of silver nanoparticles (particle diameter: 80 nm) and gold nanoparticles (particle diameter: 20 nm) whose surfaces are covered with the polymer film 3 both have a peak at a positive potential. rank The entire frequency distribution of had a shape that was almost entirely within the range of positive potential. Therefore, it was confirmed that the surface of the metal nanoparticles 2 coated with the polymer film 3 (i.e., the surface of the polymer film 3) was positively charged. 10 and 11, it was confirmed that the surfaces of the metal nanoparticles 2 and the polymer film 3 have opposing charges. This strongly suggests that in the metal nanoparticles 2 coated with the polymer film 3, the surfaces of the metal nanoparticles 2 and the polymer film 3 form electrostatic bonds.

[0086] Furthermore, a dispersion of metal nanoparticles (silver nanoparticles and gold nanoparticles) was prepared separately according to Production Examples 1 and 2 and allowed to stand for three months after preparation. Thereafter, 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 that had been allowed to stand for three months were positively charged, similar to the metal nanoparticles 2 coated with the polymer film 3 immediately after preparation. This demonstrated that the polymer film 3 could be stably maintained for a long period of time (for example, three months).

[0087] (Inhibition of fluorescence quenching) Metal nanoparticles 2 (dispersion liquid) coated with a polymer film 3 were added to a fluorescent substance solution, and the fluorescence intensity relative to the amount added was measured, confirming that the polymer film 3 contributes to suppressing fluorescence quenching.

[0088] (Fluorescent material-coated metal nanoparticle mixture system) Tris(2,2'-bipyridyl)ruthenium(II) Chloride Hexahydrate (Tokyo Chemical Industry Co., Ltd.) as a Fluorescent Material 16, 26 industryA mixed solution was prepared by adding the coated gold nanoparticle dispersion of Production Example 2 to 5 μL of a dimethyl sulfoxide solution (concentration: 10 mg / ml) of TECAN T1655 (T1655 Co., Ltd.). The mixed solution was placed in a microplate. The measurement container was placed in a fluorometer (TECAN Japan "infinite M200 PRO"), and the fluorescence spectrum was measured. The measurement conditions were an excitation wavelength of 430 nm and a detection wavelength of 470 to 700 nm. The fluorescence spectrum was measured by varying the amount of coated gold nanoparticle dispersion added (60 μL, 120 μL, 180 μL, and 240 μL). The results are shown in Figure 12(a).

[0089] Figure 12(a) shows the fluorescence spectrum (horizontal axis: fluorescence wavelength (unit: nm) and vertical axis: fluorescence intensity (unit: arbitrary unit)) of a fluorescent substance-polymer film-coated gold nanoparticle mixed system (hereinafter also referred to as a fluorescent substance-coated metal nanoparticle mixed system). The fluorescence spectrum without the addition of coated gold nanoparticle dispersion (addition amount of coated gold nanoparticle dispersion: 0 μL) exhibited a spectral shape with peaks near 614 nm and 530 nm. The peak at 614 nm is attributed to the ruthenium complex, and the peak near 530 nm is background fluorescence. Using the fluorescence spectrum at 0 μL of added coated gold nanoparticle dispersion as a reference, it was confirmed that as the amount of added coated gold nanoparticle dispersion was increased from 60 μL (60 μL → 240 μL), the intensity of the fluorescence spectrum (fluorescence intensity) decreased continuously overall.

[0090] (Fluorescent material-uncoated metal nanoparticle mixture system) The coated gold nanoparticle dispersion of Production Example 2 was mixed with gold nanoparticles (SigmaAl dr ich "753610-25ml", diameter 20nm, OD 520 The fluorescence spectrum of a fluorescent substance-gold nanoparticle mixture not coated with a polymer film (hereinafter also referred to as a fluorescent substance-uncoated metal nanoparticle mixture) was measured in the same manner, except that the dispersion was changed to a dispersion of fluorescein-coated gold nanoparticles with a fluorescein-coated gold nanoparticle film (K = 0.1) (hereinafter also referred to as an uncoated gold nanoparticle dispersion). The measurement results are shown in Figure 12(b).

[0091] Figure 12(b) shows the fluorescence spectrum of the fluorescent substance-uncoated metal nanoparticle mixture (horizontal axis: fluorescence wavelength (unit: nm) and vertical axis: fluorescence intensity (unit: arbitrary unit)). The fluorescence spectrum without the addition of uncoated gold nanoparticle dispersion (amount of uncoated gold nanoparticle dispersion added: 0 μL) exhibited a spectral shape with peaks near 614 nm and 530 nm. Using the fluorescence spectrum when 0 μL of uncoated gold nanoparticle dispersion was added as the reference, it was confirmed that the intensity of the entire fluorescence spectrum continuously decreased as the amount of uncoated gold nanoparticle dispersion added increased (60 μL → 240 μL).

[0092] (Fluorescence intensity comparison) As shown in Figures 12(a) and 12(b), the intensity of the fluorescence spectra continuously decreased as the amount of dispersion added (coated gold nanoparticle dispersion in Figure 12(a) and uncoated gold nanoparticle dispersion in Figure 12(b)) increased. Comparing the intensities of these fluorescence spectra, the intensity of the fluorescence spectrum shown in Figure 12(a) was greater than the intensity of the fluorescence spectrum shown in Figure 12(b) as the amount of dispersion added increased, confirming that the decrease in fluorescence intensity was suppressed. In other words, in Figure 12(a), the gold nanoparticles are coated with a polymer film, which is thought to prevent direct contact of the fluorescent substance, ruthenium complex, with the gold nanoparticles, thereby suppressing the decrease in fluorescence intensity. On the other hand, in Figure 12(b), the gold nanoparticles are not coated with a polymer film, which is thought to result in direct contact of the ruthenium complex with the gold nanoparticles, resulting in a significant decrease in fluorescence intensity. In this way, it was demonstrated that the polymer film placed on the surface of the gold nanoparticles suppresses fluorescence quenching.

[0093] [Example 1] [Preparation of nanoparticles bound to fluorescently labeled antibodies (preparation of nanoparticle compositions)] Figure 13 is a schematic diagram showing the structure of a nanoparticle bound to a fluorescently labeled antibody. The nanoparticle shown in Figure 13 was prepared by first binding a crosslinker to the surface of a polymer-coated metal nanoparticle, then separately binding a fluorescent substance and a crosslinker to a nanoantibody, and then binding the crosslinker bound to the polymer-coated metal nanoparticle with the crosslinker bound to the nanoantibody. Details of the preparation of the nanoparticle bound to a fluorescently labeled antibody are described below. In Figure 13, n represents the number of repeating ethylene oxide units, which is 6.

[0094] (polymer-coated silver nanoparticles) First, metal nanoparticles were prepared using nanocomposix's AGCB80-1M, diameter 80 nm, OD 455 =0.1" to "nanocomposix's "AGCB50-1M", diameter 50nm, OD 455 = 0.1”, a dispersion of silver nanoparticles 2 coated with a polymer film 3 (hereinafter also referred to as polymer-coated silver nanoparticles) was obtained in the same manner as in Production Example 1 above. The obtained polymer film 3 covered the entire surface of the silver nanoparticles. The polymer film 3 contained binding sites 3a via sulfur atoms on the surface of the silver nanoparticles 2, hydrophobic groups (pyridyl groups (pyridinyl groups)) 3c that form hydrophobic bonds with the surface of the silver nanoparticles 2, and positively charged groups (primary ammonium groups) 3b that form electrostatic bonds with the surface of the silver nanoparticles 2. In other words, the polymer 3A that constitutes the polymer film 3 has binding sites 3a via sulfur atoms on the surface of the silver nanoparticles 2, hydrophobic groups (pyridyl groups (pyridinyl groups)) 3c that form hydrophobic bonds with the surface of the silver nanoparticles 2, and positively charged groups (primary ammonium groups) 3b that form electrostatic bonds with the surface of the silver nanoparticles 2 (see Figure 8).

[0095] (Binding of crosslinkers to polymer-coated silver nanoparticles) Next, to 1 mL of the prepared dispersion of polymer-coated silver nanoparticles, the crosslinker SM(PEG)6 (PEGylated, long-chain SMCC crosslinker) (ThermoFisher Scientific, "22105") and heparin sodium (Fujifilm Wako Pure Chemical Industries, "081-00136") were added, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator (Titec Corporation, "RT-30mini"). As a result, a dispersion of silver nanoparticles with the crosslinker SM(PEG)6 bound to the polymer membrane 3 (hereinafter referred to as polymer-coated silver nanoparticles bound with SM(PEG)6 linkers) was obtained. The SM(PEG)6 linkers bound to the polymer-coated silver nanoparticles contained maleimide groups.

[0096] (Fluorescent labeling of VHH antibodies) Alexa Fluor 430 carboxylic acid, succinimidyl ester (Invitrogen, "A10169") was added to 100 μg of VHH antibody (RePHAGEN, molecular mass 18,000 Da) and mixed at room temperature for 1 hour using a small rotary incubator (Tatec Corporation, "RT-30mini") to obtain fluorescently conjugated VHH antibody (hereinafter referred to as fluorescently labeled VHH antibody).

[0097] (Binding of cross-linking agent to fluorescently labeled VHH antibody) Next, the fluorescently labeled VHH antibody was crosslinked with 3-(2-pyridyldithio)propionamido-PEG4-NHS (Tokyo Chemical Industry Co., Ltd.) as an NHS-bipyridyl disulfide crosslinker. industry An 8-fold molar equivalent of "NHS-PEG4-SPDP" (manufactured by Taitec Co., Ltd.) was added, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator ("RT-30mini" manufactured by Taitec Co., Ltd.). As a result, a VHH antibody bound to a fluorescent substance and an SPDP linker (hereinafter referred to as SPDP The resulting antibody was a fluorescently labeled VHH antibody with a linker attached thereto.

[0098] (Thiolation of fluorescently labeled VHH antibodies bound to crosslinkers) Next, a 2-fold molar equivalent of the reducing agent TCEP (ThermoFisher Scientific, "77720") was added to the SPDP linker-conjugated fluorescently labeled VHH antibody, and the mixture was stirred and mixed using a mixer (BioSan, "TS-100") at 37°C for 1 hour. As a result, a VHH antibody (hereinafter also referred to as a fluorescently labeled VHH antibody conjugated with a reduced SPDP linker) was obtained, in which a fluorescent substance and a reduced SPDP linker (hereinafter also referred to as a reduced SPDP linker) were conjugated. The reduced SPDP linker contained a thiol group (-SH group) generated by reduction of the disulfide bond.

[0099] (Binding of fluorescently labeled VHH antibodies to silver nanoparticles) Next, a dispersion of polymer-coated silver nanoparticles (OD 430 A fluorescently labeled VHH antibody bound to a reduced SPDP linker was added to a 1000-well plate (M = 0.1) and stirred overnight at room temperature using a small rotary incubator (RT-30mini, manufactured by Taitec Corporation). As a result, the maleimide group of the SM(PEG)6 linker reacted with the thiol group of the reduced SPDP linker, yielding nanoparticles to which the fluorescently labeled VHH antibody was bound via the linker (see Figure 13). The thickness of the polymer film 3 was 8.31 nm (expected value M) ± 1.89 nm (standard deviation σ), with a film thickness variation (σ / M) of 13.7%. The resulting nanoparticle composition contained the nanoparticles shown in Figure 13 and a solvent containing sodium heparin as a polyanionic polymer.

[0100] [Detection of test substances by surface plasmon excitation-enhanced fluorescence spectroscopic immunoassay] The nanoparticles thus obtained were added to a phosphate buffer solution containing C Reactive Protein (ADVY CHEMICA) as the test substance. Company L The sample containing the sandwich complex (OD 200) was added to the plate and stirred at room temperature for 5 minutes using a small rotary incubator (TAITEC Corporation's RT-30mini).455 A blank sample was prepared in the same manner as the measurement sample, except that the test substance was not added.

[0101] (Surface plasmon excitation enhanced fluorescence spectroscopic immunoassay) The measurement sample was placed in a measurement container, and the measurement container was placed in a fluorescence spectrophotometer (TECAN Japan, Inc., "infiniteM200PRO"). Light with a wavelength of 430 nm was irradiated onto the measurement container, and the fluorescence spectrum was measured. The fluorescence spectrum was measured under 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 exhibits an absorption spectrum with a peak at 430 nm and a fluorescence spectrum with a peak at 520 nm. The silver nanoparticles (diameter 50 nm) contained in the nanoparticles exhibit an absorption spectrum with a peak at 430 nm. The fluorescence spectrum of a blank sample was also measured in the same manner.

[0102] The obtained fluorescence spectrum results are shown in Figure 14. In the figure, the horizontal axis represents the fluorescence wavelength (unit: nm), and the vertical axis represents the fluorescence intensity. In Figure 14, the fluorescence spectrum of the "sample with added CRP antigen," shown by the solid line, exhibited a spectral shape with a peak at approximately 520 nm with a maximum fluorescence intensity of approximately 50,000, whereas the fluorescence spectrum of the "blank sample," shown by the dashed line, exhibited a spectral shape with a peak with a maximum fluorescence intensity of approximately 43,000. The fluorescence intensity of the sample with added CRP antigen was generally greater than that of the blank sample. Thus, a significant difference was observed between the fluorescence intensity of the sample with added CRP antigen and that of the blank sample.

[0103] The fluorescence intensity of the blank sample is due to the fluorescence emitted by the fluorescent substance that is directly excited by absorbing the excitation light (light with a wavelength of 430 nm).In contrast, the fluorescence intensity of the sample to which the CRP antigen was added is due to the fluorescence emitted by the fluorescent substance that is directly excited by absorbing the excitation light, as well as the fluorescence emitted by the fluorescent substance that is indirectly excited by the near-field formed by surface plasmon resonance on the surface of the metal nanoparticles. Therefore, the significant difference in the observed fluorescence intensity is considered to be due to the fluorescence emitted by the fluorescent substance indirectly excited by the near field formed by the surface plasmon resonance on the surface of the metal nanoparticle. 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.

[0104] [Example 2: Contraction of polymer membrane] (Preparation of nanoparticles) The nanoparticles of Example 2 were prepared in the same manner as in Example 1, except for the following three changes: The nanoparticles of Example 2 were nanoparticles in which fluorescently labeled VHH antibodies were bound via a linker moiety.

[0105] -Fluorescent material- The fluorescent substance was prepared from Alexa Fluor 430 carboxylic acid, succinimidyl ester (Invitrogen "A10169") [chemical 4 ] Chemical formula: [ka] The compound was changed to an NHS-labeled Ru complex derivative represented by the formula ("Ruthenium(II)tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd.).

[0106] -Crosslinking agent- The crosslinker (NHS-bipyridyl disulfide crosslinker) for fluorescently labeled VHH antibodies was 3-(2-pyridyldithio)propionamido-PEG4-NHS (Tokyo Chemical Industry Co., Ltd.). industry The PEG-4-NHS copolymer was changed from "NHS-PEG4-SPDP" manufactured by Thermo Fisher Scientific Co., Ltd. to 3-(2-pyridyldithio)propionamido-PEG4-NHS (product number "26128" and "NHS-PEG4-SPDP" manufactured by Thermo Fisher Scientific Co., Ltd.).

[0107] -Metal nanoparticles- Metal nanoparticles were "NANOCOMPOSIX AGCB50-1M" with a diameter of 50 nm and an OD455 =0.1" to "nanocomposix's "AGCB80-1M", diameter 80nm, OD 455 =0.1".

[0108] (Preparation of the complex) The nanoparticles of Example 2 were dissolved in phosphate buffer solution, and C Reactive Protein (manufactured by ADVY CHEMICAL, "00-AGN-AP-CRP-00") was added as a test substance. )of The cells were then incubated at room temperature for 5 minutes in a small rotating incubator (Taitec Co., Ltd.). Company The nanoparticles of Example 2 were stirred using a centrifuge (RT-30mini) to prepare a measurement sample containing a sandwich-type complex. As described above, the nanoparticles of Example 2 had a polymer film covering the surface of the metal nanoparticles. The polymer constituting this polymer film had 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 binding site between the polymer and the surface of the metal nanoparticle via a sulfur atom.

[0109] (SEM image capture) Using a scanning electron microscope (Regulus 8220 manufactured by Hitachi High-Technologies Corporation), an SEM image (magnification 100K) of the composite in the measurement sample obtained in Example 2 was taken. FIG. 15 shows an SEM image of the composite prepared using the nanoparticles of Example 2. In the SEM image obtained as shown in FIG. 15, the separation distance L1 between the metal nanoparticles of the nanoparticles in the composite and the thickness T1 of the polymer film other than between the metal nanoparticles of the nanoparticles were measured and compared. Note that the thickness T1 of the polymer film was the thickness of the unshrunk portion of the polymer film that was not subject to the separation distance. As a result, the separation distance L1 was smaller than the thickness (T1 × 2) equivalent to twice the thickness of the polymer film. Therefore, in the composite prepared with the nanoparticle composition of Example 2, the polymer film contracted, and the two metal nanoparticles in the composite were closer than the separation distance equivalent to twice the thickness of the polymer film (T1 × 2). This strongly suggests that the plasmon enhancement effect was further enhanced, further increasing the fluorescence intensity.

[0110] [Reference Example 1: Shrinkage of inorganic film] In order to clarify the technical significance of the shrinkage of the polymer film shown in Example 2, an aggregate of metal nanoparticles coated with an inorganic film (Reference Example 1) was examined as a comparative example.

[0111] (Preparation of measurement sample) Silica-coated silver nanoparticles (manufactured by nanoComposix, silver nanoparticle particle diameter (core particle diameter) 50 nm, silica film thickness 20 nm) were diluted with water to prepare an aqueous dispersion of silica-coated silver nanoparticles. The resulting aqueous dispersion was used as the measurement sample. Specifically, in addition to the primary particles of silica-coated silver nanoparticles, the measurement sample also contained aggregates of two particles. These aggregates were identified and evaluated. As in Example 2, SEM images (500K magnification) were taken, and the thickness T2 of the inorganic film in the aggregate and the separation distance L2 between two metal nanoparticles were measured from the SEM images and compared. As a result, the separation distance L2 was approximately twice the thickness T2 of the inorganic film. Note that the thickness T2 of the inorganic film was the thickness of the inorganic film in the portion not subject to the separation distance.

[0112] Example 3: Preparation of various nanoparticulate compositions (polymer-coated silver nanoparticles) The nanoparticle composition shown in Figure 16 was prepared. Poly-L-lysine (Peptide Institute, "3075") was used as the polymer, and silver nanoparticles (nanocomposix, "AGCB50-1M", diameter 50 nm, OD 100) were used as the metal nanoparticles. 430The solution was added to 1 mL of a dispersion of silver nanoparticles (a pH 7.0 or higher) containing 100% ammonium hydroxide (pH 7.0) and incubated at room temperature (25°C) for 4 hours under stirring using a small rotary incubator (RT-30mini, manufactured by Taitec Corporation). This resulted in a dispersion of silver nanoparticles coated with a polymer membrane (polymer-coated silver nanoparticles). The polymer membrane contains sulfur-atom-mediated bonding sites on the surface of the silver nanoparticles, hydrophobic groups that form hydrophobic bonds with the surface of the silver nanoparticles, positively charged groups (primary ammonium groups) that form electrostatic bonds with the surface of the silver nanoparticles, and electrically neutral groups (primary amino groups). In other words, the polymer that makes up the polymer membrane contains sulfur-atom-mediated bonding sites on the surface of the silver nanoparticles, hydrophobic groups that form hydrophobic bonds with the surface of the silver nanoparticles, positively charged groups (primary ammonium groups) that form electrostatic bonds with the surface of the silver nanoparticles, and electrically neutral groups (primary amino groups).

[0113] (Fluorescently labeled polymer-coated silver nanoparticles) A fluorescent succinimidyl ester (Alexa Fluor® 430 NHS ester, manufactured by Thermo Fisher Scientific) was added to 1 mL of the polymer-coated silver nanoparticle dispersion, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator (RT-30mini, manufactured by Taitec Corporation). As a result, a dispersion of polymer-coated silver nanoparticles with the fluorescent substance bound to the polymer membrane (hereinafter also referred to as fluorescently labeled polymer-coated silver nanoparticles) was obtained.

[0114] To 1 mL of the dispersion of fluorescently labeled polymer-coated silver nanoparticles, 0.1% of polyglutamic acid sodium salt (Sigma-Aldrich "P4886" molecular weight 50,000-100,000) was added as a polyanionic polymer, and the mixture was stirred overnight at room temperature using a small rotary incubator (Tatec Corporation "RT-30mini") to obtain a nanoparticle composition (solvent: pure water) containing fluorescently labeled polymer-coated silver nanoparticles (i.e., nanoparticles) and polyglutamic acid sodium salt.

[0115] (SEM image capture) Using a scanning electron microscope (Hitachi High-Tech Corporation, "Regulus 8220"), SEM images (magnification 200,000x) of the nanoparticles in the resulting nanoparticle composition were taken. Observation of the SEM image confirmed that the nanoparticles were dispersed singly in the nanoparticle composition. Furthermore, the SEM image confirmed that the surfaces of the silver nanoparticles in the nanoparticles were continuously coated with a polymer film. The film thickness of the polymer film coating the silver nanoparticles was also measured from the SEM image. The film thickness of the polymer film was approximately 11 nm.

[0116] (Absorption spectrum measurement) The nanoparticle composition of Example 3 was placed in a measurement container. The measurement container was placed in an ultraviolet-visible spectrophotometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.) and the absorption spectrum was measured. The measurement conditions were a measurement wavelength range of 400 to 600 nm. Figure 17 shows the absorption spectrum of the nanoparticle composition of Example 3. The obtained absorption spectrum had a peak at 455 nm.

[0117] (Fluorescence spectrum measurement) The nanoparticle composition of Example 3 was placed in a measurement vessel with an optical path length of 1 cm. The measurement vessel was placed in a fluorometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.) and the fluorescence spectrum was measured. The measurement conditions were an excitation wavelength of 430 nm and a detection wavelength of 470 to 700 nm. Figure 18 shows the fluorescence spectrum of the nanoparticle composition of Example 3. The obtained fluorescence spectrum had a peak at 535 nm.

[0118] Example 4: Nanoparticulate composition comprising nanoparticulate bodies with linkers attached to a polymer membrane A method for preparing a nanoparticle composition containing nanoparticles will be described with reference to FIG. 19. FIG. 19 is a schematic diagram illustrating a method for preparing a nanoparticle composition containing nanoparticles. A dispersion of polymer-coated silver nanoparticles was prepared in the same manner as in Example 1. To 1 mL of this dispersion, succinimidyl ester (Alexa Fluor® 430 NHS ester, manufactured by Thermo Fisher Scientific) as a fluorescent substance and SM(PEG)6 (PEGylated, long-chain SMCC crosslinker) (22105, manufactured by Thermo Fisher Scientific) as a crosslinker were simultaneously added, and the mixture was stirred and mixed at room temperature for 4 hours using a small rotary incubator (RT-30mini, manufactured by Taitec Corporation). As a result, a dispersion of polymer-coated silver nanoparticles to which the fluorescent substance and linker were bound was obtained. Furthermore, heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd., product number 081-00136) was added as a polyanionic polymer, and the mixture was stirred and mixed at room temperature for 4 hours using a small rotary incubator (Tatec Corporation, product number RT-30mini). As a result, a nanoparticle composition was prepared comprising polymer-coated silver nanoparticles (nanoparticles) bound to a fluorescent substance and a linker, and a polyanionic polymer.

[0119] (Absorption and Fluorescence Spectral Measurements) The absorption spectrum of the obtained nanoparticles was measured in the same manner as in Example 1. Figure 20(a) shows the absorption spectrum of the nanoparticle composition of Example 4. The obtained absorption spectrum had a peak at 450 nm. The fluorescence spectrum of the nanoparticle composition obtained was measured in the same manner as in Example 1. Figure 20(b) shows the fluorescence spectrum of the nanoparticle composition of Example 4. The obtained fluorescence spectrum had a peak at 540 nm. The fact that the maximum wavelength of the absorption spectrum did not shift to the longer wavelength side confirmed that the nanoparticles were not aggregated, and the fact that fluorescence could be detected confirmed that the fluorescent substance was bound to the nanoparticles.

[0120] Example 5: Evaluation of dispersibility of nanoparticles made from polyanionic polymers (Preparation of nanoparticulate composition) Silver nanoparticles (AGCB80-1M manufactured by nanocomposix, diameter 80 nm, OD 455 Poly-L-lysine (Peptide Research Institute, Inc., "3075") was added to 1 mL of a dispersion of silver nanoparticles (pH = 0.1), and the mixture was stirred overnight at room temperature using a small rotary incubator (Tatec Corporation, "RT-30mini"). As a result, a dispersion of silver nanoparticles coated with a polymer membrane (polymer-coated silver nanoparticles) was obtained.

[0121] The polymer membrane has hydrophobic groups (alkylene groups) that form hydrophobic bonds with the surface of the silver nanoparticles and electrically neutral groups (primary amino groups) that can form electrostatic bonds with the anionic groups of the polyanionic polymer. That is, the polymer that constitutes the polymer membrane has hydrophobic groups (alkylene groups) that form hydrophobic bonds with the surface of the silver nanoparticles and electrically neutral groups (primary amino groups) that can form electrostatic bonds with the anionic groups of the polyanionic polymer.

[0122] To 1 mL of the dispersion of polymer-coated silver nanoparticles, polyglutamic acid sodium salt (Sigma-Aldrich "P4886" molecular weight 50,000-100,000) as a polyanionic polymer was added, and the mixture was stirred and mixed at room temperature for 4 hours using a small rotary incubator (Taitec Corporation "RT-30mini"), resulting in a nanoparticle composition comprising polymer-coated silver nanoparticles and the polyanionic polymer.

[0123] (Aggregate size measurement) As measurement samples, the nanoparticle composition of Example 4 (a composition that was not washed (hereinafter also referred to as the zero-washed composition)) and a composition that was washed twice (hereinafter also referred to as the twice-washed composition) were prepared. The washing treatment refers to a series of steps: adding pure water as a solvent to the composition so that the concentration of nanoparticles in the composition was halved, stirring vigorously, and then, before measurement, sedimenting the nanoparticles using a centrifuge (MX307, manufactured by Tomy Seiko Co., Ltd.) at 12,000 G for 30 minutes, removing the supernatant, and adding fresh pure water. This procedure was counted as one washing treatment. Note that washing three times caused aggregation and a significant decrease in recovery rate.

[0124] The aggregate size (particle diameter) of the two measurement samples prepared above was measured using a dynamic light scattering measurement device ("ZETA SIZER Nanoseries nano-ZS" manufactured by MALVERN PANALYTICAL). Figure 21 shows the size frequency distribution. Figure 21(a) shows the size frequency distribution after 0 washing treatments, and Figure 21(b) shows the size frequency distribution after 2 washing treatments. In Figure 21, the horizontal axis shows size (unit: nm, logarithmic scale), and the vertical axis shows the scattering intensity ratio (SID) (unit: no).

[0125] As shown in Figures 21(a)-(b), the size frequency distribution had a peak near 100 nm when the number of cleaning treatments was 0, and a peak in the region larger than 100 nm when the number of cleaning treatments was 2 (two-time cleaning treatment composition), resulting in a broadened shape. As the cleaning treatment shifted the peak of the frequency distribution to the larger size side and broadened the frequency distribution, it is believed that the size of the measurement object increased as the number of cleaning treatments increased from 0 to 2. In other words, Figure 23(a) 、 Figure 23(b) , Figure 23(c) and Figure 23(d)As shown in Figure 23(a), the washing process with a solvent (more specifically, pure water) gradually reduces the number of polyanionic polymers surrounding the nanoparticles, making it difficult for the electrostatic repulsion between the aggregates to act, which strongly suggests that the nanoparticles aggregate to form aggregates, the size of which gradually increases (see Figure 23(a) to Figure 23(b)). figure twenty three( d )). In FIG. 23, the specific binding substance and fluorescent substance in the nanoparticles are omitted for the sake of convenience.

[0126] From the above, the behavior of the size frequency distribution with respect to the number of washing treatments strongly suggests the presence of aggregates in the nanoparticle composition, in which polyanionic polymers are aggregated so as to surround the nanoparticles.

[0127] (Measuring the zeta potential of aggregates) Similar to the aggregate size measurement, two measurement samples were prepared. The zeta potential of the aggregates in the two measurement samples prepared above was measured using a zeta potential measurement device ("ZETA SIZER Nanoseries nano-ZS" manufactured by MALVERN PANALYTICAL). Figure 22 shows the frequency distribution of the zeta potential. Figure 22(a) shows the frequency distribution of the zeta potential when the number of washing treatments was 0, and Figure 22(b) shows the frequency distribution of the zeta potential when the number of washing treatments was 2. In Figure 22, the horizontal axis shows the zeta potential (unit: mV), and the vertical axis shows the frequency (unit: arbitrary unit).

[0128] As shown in Figures 22(a) and 22(b), the zeta potential frequency distribution peaked in the negative potential region when the number of washing treatments was 0, and in the positive potential region when the number of washing treatments was 2. This change in the peak potential of the frequency distribution before and after washing treatment (negative potential → positive potential) strongly suggests that as the number of washing treatments increased from 0 to 2, the number of negatively charged polyanionic polymers surrounding the nanoparticles gradually decreased, exposing the positively charged polymer film (see the change from Figure 23(a) to 23(b)).

[0129] From the above, the behavior of the frequency distribution of zeta potential with respect to the number of washing treatments strongly suggests the presence of aggregates in the nanoparticle composition in which polyanionic polymers are aggregated to surround the nanoparticles. [Industrial Applicability]

[0130] The nanoparticles and nanoparticle compositions according to this embodiment can be used to detect a specific analyte in a specimen using plasmon excitation fluorescence analysis. [Explanation of symbols]

[0131] 1. Nanoparticles 2. Metal nanoparticles 3...Polymer membrane 3A: Polymers that make up the polymer membrane 3a: Sulfur atom-mediated binding site 3b: Positively charged group 3c...Hydrophobic group 4...specific binding substance 7. Polyanionic polymers 9. Aggregates 10. First nanoparticle 12. First metal nanoparticles 13...first polymer membrane 14...first specific binding substance 16. First fluorescent material 20 Second nanoparticle 22 Secondary metal nanoparticles 23...Second polymer membrane 24...Second specific binding substance 26 Second fluorescent material 30 Test substance 40 Complex L...Separation distance (separation distance)

[0132] Aspect 2 (Title of invention) Nanoparticles and their manufacturing method (Technical field)

[0133] The present invention relates to a nanoparticle, particularly to a nanoparticle for use in plasmon-induced fluorescence analysis, and to a method for producing the same. (Background technology)

[0134] A biosensor specifically reacts a specific test substance, which is the detection target, with a specific specific binding substance to form a complex, and detects the test substance based on a signal derived from the specific binding in the complex. In plasmon excitation fluorescence analysis, the complex contains, for example, metal particles, a specific binding substance, a fluorescent substance, and an analyte. When excitation light is irradiated onto the complex, surface plasmon resonance occurs in the metal particles within the complex, forming a near-field near the surface of the metal particles. This near-field enhances the fluorescence intensity of the fluorescent substance.

[0135] The composite particles for immunochromatography described in Patent Document 1 have a structure in which the exterior of metal particles is covered with at least one layer of silica containing at least one type of fluorescent substance, and the particles are surface-modified with a labeling substance that specifically recognizes a target substance. That is, the composite particles in Patent Document 1 have the surfaces of metal particles covered with a silica layer, and the fluorescent substance and labeling substance are fixed to the silica layer. (Prior art document) (Patent document)

[0136] (Patent Document 1) JP 2011-220705 A (Summary of the Invention) (Problem to be solved by the invention)

[0137] However, as a result of extensive research by the present inventors, it has been found that there is room for further improvement in the detection sensitivity of nanoparticles as described below. Specifically, the composite particles described in Patent Document 1 have metal particle surfaces covered with a silica layer, which is further modified with a fluorescent substance and a labeling substance. Therefore, during the production of nanoparticles, the sites (labeling sites) of the fluorescent substance to be labeled in the silica layer compete with the sites (binding sites) of the labeling substance to be bound. This makes it difficult to increase the fluorescent substance in the nanoparticles as desired.

[0138] The present invention has been made in view of the above-mentioned problems, and a primary object of the present invention is to provide a nanoparticle having a novel labeling mode for fluorescent substances that can label a larger number of fluorescent substances, and a method for producing the same. (Means for solving the problem)

[0139] The nanoparticle according to one embodiment of the present invention comprises: The present invention relates to a method for manufacturing a fluorescent material comprising: a metal nanoparticle; a polymer film covering the surface of the metal nanoparticle; and a fluorescent substance. the fluorescent substance is labeled on the surface of the metal nanoparticle; The polymer film contains binding sites between the surface of the metal nanoparticles and the polymer film via sulfur atoms.

[0140] A method for producing a nanoparticle according to one embodiment of the present invention includes the steps of: The method comprises a step of mixing a polymer to which a fluorescent substance is bonded via a disulfide bond with metal nanoparticles, thereby labeling the surface of the metal nanoparticles with the fluorescent substance and forming a polymer film on the surface of the metal nanoparticles. (Effects of the Invention)

[0141] The nanoparticle according to one embodiment of the present invention has a novel labeling mode for fluorescent substances and can label a larger number of fluorescent substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] FIG. 24 is a cross-sectional view schematically showing a nanoparticle 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 cross-sectional view schematically showing a composite body according to a sixth embodiment. FIG. 28 is a diagram schematically showing a measurement device according to a seventh embodiment. FIG. 29 is a schematic diagram illustrating the method for producing the nanoparticles of Example 6. FIG. 30 shows 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. (Mode for Carrying Out the Invention)

[0143] Hereinafter, the nanoparticles, their manufacturing method, composite, and measuring device according to the present invention will be described in detail with reference to the illustrated embodiments. Note that the drawings include schematic views and may not reflect actual dimensions or proportions.

[0144] Numerical ranges mentioned in this specification are intended to include the lower and upper limits themselves, unless otherwise specified, such as "less than," "greater than," or "smaller." For example, in the case of a numerical range such as 1 nm to 50 nm, unless otherwise specified, the numerical range is interpreted as including the lower limit of "1 nm" and the upper limit of "50 nm."

[0145] <Fifth embodiment: nanoparticles> The nanoparticle according to the fifth embodiment is The present invention comprises metal nanoparticles, a polymer film covering the surfaces 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 between the surface of the metal nanoparticles and the polymer film via sulfur atoms.

[0146] [Test substance detection method] First, for the convenience of explaining the nanoparticles according to this embodiment and to aid in understanding thereof, a method for detecting a test substance using the nanoparticles according to this embodiment will be described. The nanoparticles according to this embodiment are useful for detecting analytes. The nanoparticles according to this embodiment comprise metal nanoparticles, a polymer membrane covering the surface of the metal nanoparticles, and a fluorescent substance labeled on the surface of the metal nanoparticles. The nanoparticles according to this embodiment may further comprise a specific binding substance bound to the polymer membrane. The specific binding substance is capable of specifically binding to the analyte in a specimen. Hereinafter, nanoparticles to which a specific binding substance is bound in this manner will also be referred to as "specifically binding nanoparticles."

[0147] The specific-binding nanoparticles are dissolved or dispersed in a specimen, and 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 nanoparticles and the test substance. The complex has a structure (sandwich structure) in which two specific-binding nanoparticles are bound via one test substance. In this way, the specific binding substances possessed by the two nanoparticles in the complex bind to the same test substance, and the two metal nanoparticles are arranged at a certain distance apart.

[0148] In Surface Plasmon Fluorescence Spectroscopy (SPFS), when excitation light is irradiated onto the complex, Localized Surface Plasmon Resonance (LSPR) occurs, and a near-field is efficiently formed near the surface of the metal nanoparticle (especially near the surface between two metal nanoparticles). This near-field efficiently excites the fluorescent substance in the complex, increasing its fluorescence intensity. By measuring the fluorescence intensity, the test substance in the sample can be detected.

[0149] [Mechanism of action] The nanoparticle according to the present embodiment has a novel labeling mode of the fluorescent substance that can label a larger number of fluorescent substances. That is, in the nanoparticle according to the present embodiment, the novel labeling mode of the fluorescent substance can label a larger number of fluorescent substances. Without being bound by any particular theory, the reason for this is presumed to be as follows. The nanoparticle according to this embodiment comprises metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, and a fluorescent substance labeled on the surface of the metal nanoparticles. Because the fluorescent substance is labeled on the surface of the metal nanoparticles, it becomes possible to bind a specific binding substance to a site (e.g., a specific functional group possessed by a polymer constituting the polymer film) different from the surface of the metal nanoparticles of the nanoparticles. In this way, the labeling site on the nanoparticle where the fluorescent substance is labeled differs from the binding site on which the specific binding substance binds, making it possible to further increase the number of fluorescent substances in the nanoparticle. From the above, it is believed that the nanoparticle according to this embodiment can label a larger number of fluorescent substances. Furthermore, because the nanoparticle according to this embodiment can label a larger number of fluorescent substances, it has excellent detection sensitivity.

[0150] [Nanoparticle structure] The structure of the nanoparticle will be described below. The nanoparticle will be described with reference to FIG. 24. FIG. 24 is a cross-sectional view showing a schematic diagram of a nanoparticle. As shown in FIG. 24(a), the nanoparticle 1 according to this embodiment comprises a metal nanoparticle 2, a polymer film 3 covering the surface of the metal nanoparticle 2, and a fluorescent substance 6 labeling the surface of the metal nanoparticle 2. As shown in FIG. 24(b), the nanoparticle 1 may further comprise a specific binding substance 4 bound to the surface of the polymer film 3, which specifically binds to an analyte in a specimen.

[0151] The nanoparticle 1 can be used in plasmon excitation fluorescence analysis. In other words, the nanoparticle 1 can be used in surface plasmon excitation enhanced fluorescence spectroscopic immunoassay. The nanoparticle 1 can capture a test substance in a test sample and form a complex containing two nanoparticles 1 and one test substance. When the complex is irradiated with excitation light, localized surface plasmon resonance occurs, forming a near-field. This near-field increases the fluorescence intensity.

[0152] The nanoparticle 1 may also have non-specific binding sites blocked by a blocking agent. The blocked nanoparticle 1 suppresses the formation of non-specific binding of the specific binding substance 4 to substances other than the target of detection (i.e., substances other than the test substance), reducing background and false positive signals and improving the signal-to-noise ratio (SN ratio). In such cases, the detection sensitivity can be further improved. Examples of blocking agents include proteins such as bovine serum albumin (BSA), skim milk, and casein, as well as chemically synthesized polymers.

[0153] When the nanoparticles 1 are present in a solvent, the dispersion of the nanoparticles 1 may further contain a dispersant to improve the dispersibility of the nanoparticles 1. Examples of such dispersants include heparin sodium.

[0154] (metal nanoparticles) The surfaces of the metal nanoparticles 2 are coated with a polymer film 3. The metal nanoparticles 2 interact with light having a specific wavelength, which varies depending on the type of metal, and cause localized surface plasmon resonance. Silver nanoparticles have a plasmon resonance peak between 400 nm and 530 nm, while gold nanoparticles have a plasmon resonance peak between 510 nm and 580 nm. This varies depending 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 can be obtained by capturing an image of the metal nanoparticles 2 using a scanning electron microscope (SEM) or a transmission electron microscope (TEM), measuring the particle diameters of the metal nanoparticles 2 in the image, and calculating the average value of multiple particle diameters (number of measurements: for example, at least 10 or more). The metal nanoparticles 2 preferably comprise gold or silver, more preferably silver.

[0155] (polymer membrane) The polymer film 3 covers the surface of the metal nanoparticle 2. The polymer film 3 functions as a metal quenching film. In the composite, the polymer film 3 can dispose the fluorescent substance 6 at a distance from the surface of the metal nanoparticle 2 by at least the thickness of the polymer film 3. This prevents the excited fluorescent substance 6 from coming into contact with the surface of the metal nanoparticle 2 and quenching, thereby preventing a decrease in detection sensitivity. The presence of the polymer film 3 can be confirmed by capturing an image of the nanoparticle 1 using an SEM or TEM and observing the nanoparticle 1 in the image.

[0156] The polymer film 3 will be described with reference to FIG. 25. 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 nanoparticle 2. The polymer film 3 includes binding sites 3a between the polymer film 3 and the surface of the metal nanoparticle 2 via sulfur atoms. The polymer film 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 includes primary ammonium groups (-NH3 + ) and a hydrophobic group 3c. The binding site 3a bonds the surface of the metal nanoparticle 2 to the polymer film 3 via a sulfur atom. The positively charged group 3b forms an electrostatic bond (ionic bond) b with the surface of the negatively charged metal nanoparticle 2. The hydrophobic group 3c forms a hydrophobic bond c with the surface of the metal nanoparticle 2.

[0157] All three of the above bonds are relatively strong bonds with the surface of the metal nanoparticle 2. The polymer film 3 is stably fixed to the surface of the metal nanoparticle 2 by the binding site 3a. Furthermore, the polymer film 3 can be even more stably fixed to the surface of the metal nanoparticle 2 by the hydrophobic bond c and the electrostatic bond b formed by the positively charged group 3b. Because the polymer film 3 is stably fixed to the surface of the metal nanoparticle 2 in this way, the fluorescent substance 6 and the surface of the metal nanoparticle 2 can be stably separated by a predetermined distance. Therefore, in this embodiment, quenching of the excited fluorescent substance is suppressed, and a decrease in detection sensitivity can be suppressed.

[0158] Furthermore, because the polymer film 3 can be configured to include the polymer 3A, it is easier to chemically modify than a silica layer, and the need for surface modification and the like is reduced. This allows the film thickness to be smaller than that of a silica layer, and the distance between the metal nanoparticles 2 in the composite to be appropriately reduced. This allows for more efficient formation of a near-field, further improving detection sensitivity.

[0159] 25, the polymer 3A that can constitute the polymer film 3 can include at least one selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c, in addition to a binding site 3a that is bonded to the surface of the metal nanoparticle 2 via a sulfur atom. The presence of the binding site 3a via a sulfur atom, the positively charged group 3b, and the hydrophobic group 3c can be confirmed by measuring signals derived therefrom using infrared spectroscopy and nuclear magnetic resonance spectroscopy.

[0160] -Binding site via sulfur atom- The binding site 3a via a sulfur atom is formed, for example, by mixing a polymer having a site containing a disulfide bond as a side chain with the metal nanoparticle 2.

[0161] The polymer film 3 may be directly bonded to the surface of the metal nanoparticles 2, or may be bonded to the surface of the metal nanoparticles 2 via a linker moiety derived from a crosslinking agent (more specifically, SM(PEG) n (where n is 4, 6, 8, etc.)) to the surface of the metal nanoparticle 2. Examples of such crosslinkers include amino group-sulfhydryl group crosslinkers (more specifically, NHS (N-hydroxysuccinimide)-maleimide group crosslinkers, etc.).

[0162] -Positively charged group- The positively chargeable group 3b can form a relatively strong electrostatic bond b with the surface of the metal nanoparticle 2. In this specification, the positively chargeable group 3b is a group that has a valence of one or more and is completely positively ionized. When considering the multiple positively chargeable groups 3b contained in the polymer that constitutes the polymer film 3, the positively chargeable group 3b can be expressed by the following mathematical formula (1): (Number 1) TIFF0007811942000006.tif18164 [In formula (1), pKa is an electrically neutral group contained in the polymer 3A constituting the polymer film 3. If it is positively charged, it is a positively charged group (more specifically, a primary ammonium group (-NH3 +) etc.)) 3b) (hereinafter also referred to as an electrically neutral group) (more specifically, a primary amino group (-NH2) etc.), pH indicates the pH of the environment in which the analyte is detected (more specifically, a sample etc.), B indicates an electrically neutral group contained in polymer 3A, and BH + indicates the positively charged group 3b contained in the polymer 3A. That is, the positively charged group 3b is a group having a pKa of 7 or more, which is represented by the following chemical equilibrium formula (2): In an environment for detecting an analyte (e.g., a sample having a pH of approximately 6 to 8), the positively charged group 3b and the electrically neutral group of the polymer 3A constituting the polymer film 3 are reacted with each other in accordance with the following chemical equilibrium formula (2): (chemical 1) When an equilibrium state represented by TIFF0007811942000007.tif17164 is formed, the concentration of the positively charged group 3b ([BH + ]) is more than 10 times greater than the concentration of electrically neutral groups ([B]).

[0163] The positively charged group 3b is preferably a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, or a guanidyl group (-NHC(=NH2 + )NH2).

[0164] -Hydrophobic group- The hydrophobic group 3 c can form a hydrophobic bond c with the surface of the metal nanoparticle 2 . The hydrophobic group is, for example, at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group.

[0165] Examples of aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups. Aromatic carbocyclic groups are groups containing an aromatic ring in which all ring atoms are carbon atoms, but not containing aromatic heterocycles. Examples of aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups, etc.) and arylalkyl groups (more specifically, benzyl groups, etc.). Aromatic heterocyclic groups are groups containing an aromatic ring in which at least one ring atom is a heteroatom (more specifically, oxygen atom, sulfur atom, 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.

[0166] Aliphatic cyclic groups are groups that do not contain aromatic rings and contain cyclic groups consisting of non-aromatic rings. Examples of aliphatic cyclic groups include aliphatic carbocyclic groups and aliphatic heterocyclic groups. Aliphatic carbocyclic groups are groups that contain non-aromatic rings whose ring atoms are all carbon atoms, such as cycloalkyl groups. Aliphatic heterocyclic groups are groups that contain non-aromatic rings whose ring atoms are at least one heteroatom.

[0167] The aliphatic chain group is a chain (more specifically, a linear or branched chain) group that does not contain an aromatic ring or a non-aromatic ring. Examples of the aliphatic chain group include an aliphatic carbon chain group (more specifically, an alkyl group, an alkylene group, etc.) and an aliphatic hetero chain group. Examples of the alkyl group include a butyl group. Examples of the alkylene group include an n-butylene group.

[0168] The polymer 3A constituting the polymer film 3 may be directly bound to the surface of the metal nanoparticle 2, or may be bound to the surface of the metal nanoparticle 2 via a linker moiety derived from a crosslinking agent (more specifically, SM(PEG) n (where n is 4, 6, 8, etc.)) may be indirectly bound to the surface of the metal nanoparticle 2. Examples of such crosslinkers include amino group-sulfhydryl group crosslinkers (more specifically, NHS-maleimide group crosslinkers, etc.).

[0169] The film thickness of the polymer film 3 is preferably 1 nm to 50 nm, and more preferably 1 nm to 10 nm. When the film thickness of the polymer film 3 is 50 nm or less, the separation distance (separation distance) is such that a near-field is efficiently formed in the space between the two metal nanoparticles 2, further improving detection sensitivity. Furthermore, when the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance are arranged at a predetermined distance, thereby suppressing quenching of the excited fluorescent substance during measurement and further improving detection sensitivity. In this specification, the term "separation distance" refers to the minimum distance (shortest distance) between the surfaces of metal nanoparticles contained in two nanoparticles that are bound to each other via the test substance in a complex.

[0170] (fluorescent material) The fluorescent substance 6 is labeled on the surface of the metal nanoparticle 2. The labeled site of the fluorescent substance 6 is the surface of the metal nanoparticle 2. In contrast, the binding site of the specific binding substance 4 is the polymer membrane 3, as will be described later. As described above, in the nanoparticle 1 according to this embodiment, the labeled 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 in which the labeled site and the binding site are the same. Therefore, this embodiment has superior detection sensitivity.

[0171] 25, the fluorescent substance 6 includes a bonding site between the fluorescent substance 6 and the surface of the metal nanoparticle 2 via a sulfur atom. In this case, the fluorescent substance 6 is bonded to the surface of the metal nanoparticle 2 via the sulfur atom. Such a bond can be formed, for example, by mixing the metallic nanoparticle 2 as a starting material with the fluorescent substance 6 having a disulfide bond.

[0172] From the viewpoint of suppressing fluorescence quenching, it is preferable that the fluorescent substance 6 is labeled on the surface of the metal nanoparticle 2 via a linker moiety (for example, an alkylene group containing an amide group). When the fluorescent substance 6 is labeled on the surface of the metal nanoparticle 2 via a linker moiety, the linker moiety is located between the fluorescent substance 6 and the surface of the metal nanoparticle 2, which makes it easier to prevent the excited fluorescent substance 6 from coming into contact with the surface of the metal nanoparticle 2. This is thought to further suppress fluorescence quenching.

[0173] The fluorescent substance 6 is excited by the near field formed by localized surface plasmon resonance and emits fluorescence. Examples of fluorescent substances include complexes (metal complexes) of metals such as europium and ruthenium. Examples of ruthenium complexes include tris(bipyridine)ruthenium(II), which may have a counter anion.

[0174] It is preferable that the fluorescent substance 6 has a large Stokes shift. Here, 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. When the Stokes shift of the fluorescent substance 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, allowing for more accurate measurement of the fluorescence intensity.

[0175] It is preferable that the fluorescence spectrum of the fluorescent substance 6 is sharp. If the fluorescence spectrum is sharp, it is less likely to overlap with the absorption spectrum of the fluorescent substance 6, and therefore (scattered light of) excitation light is less likely to enter the fluorescence to be detected, allowing for more accurate measurement of the fluorescence intensity.

[0176] (specific binding substance) The nanoparticle 1 may further comprise a specific binding substance 4. The specific binding substance 4 is a nano-sized substance (having a maximum size of 3 to 15 nm) that specifically binds to a test substance (described in the sixth embodiment) in a specimen.

[0177] As already mentioned, this embodiment allows for labeling of a larger number of fluorescent substances 6, resulting in superior detection sensitivity. While this description focuses on the fluorescent substances 6, let us now shift our perspective and focus on the specific binding substances 4. In this embodiment, the binding site of the specific binding substances 4 in the nanoparticle 1 is different from the labeling site of the fluorescent substance 6. For the same reason as for the fluorescent substance 6, this makes it possible to increase the number of specific binding substances 4 in the nanoparticle 1. Therefore, it is believed that the nanoparticle 1 according to this embodiment can bind to a larger number of specific binding substances 4.

[0178] By binding a larger number of specific-binding substances 4, the detection sensitivity can be further improved. Without being bound by any particular theory, the reason for this is presumed to be as follows: When the specific-binding nanoparticles are dissolved or dispersed in a specimen, the specific-binding nanoparticles collide (encounter) with the test substance in the specimen. This collision causes the specific-binding nanoparticles and the test substance to bond (specifically bond) at specific sites on each of them, resulting in the formation of a complex.

[0179] Here, specific binding is thought to occur at a constant rate relative to the number of collisions between the specific-binding nanoparticles and the test substance. This is because the specific-binding nanoparticles and the test substance do not necessarily have a favorable positional relationship for forming specific binding when they collide. In other words, for specific binding to occur, specific sites on the specific-binding nanoparticles and the test substance must come into contact with each other or at least be close to each other upon collision. For example, if the specific-binding substance is an antibody and the test substance is an antigen, the specific sites are the antigen-binding site of the antibody and the antigenic determinant (epitope) of the antigen. Considering these points, the probability of contact and proximity between specific sites during the collision increases if a larger number of specific binding substances 4 are bound to the specific-binding nanoparticle. Therefore, the nanoparticle according to this embodiment can bind a larger number of specific-binding substances 4, which is thought to facilitate complex formation and further improve detection sensitivity.

[0180] The specific binding substance 4 is, for example, at least one selected from the group consisting of an antibody (hereinafter referred to as a nanoantibody), a ligand, an enzyme, and a nucleic acid chain (more specifically, a DNA chain and an RNA chain). In this embodiment, the nanoparticle 1 to which such a specific binding substance 4 is bound exhibits superior detection sensitivity. For example, a nanoantibody as the specific binding substance 4 specifically binds to an antigen as a test substance at its tip (antigen-binding site) through an antigen-antibody reaction to form a complex. A ligand as the specific binding substance 4 forms a complex with a protein as a test substance through specific protein-ligand binding through a ligand-receptor reaction. A nucleic acid chain as the specific binding substance 4 forms a nucleic acid chain pair (double strand) with a complementary nucleic acid chain based on base pair complementarity. An enzyme as the specific binding substance 4 forms an enzyme-substrate complex with a substrate as a test substance at its active site (active center) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as those resulting from hydrogen bonds, as well as intermolecular forces, hydrophobic interactions and charge interactions.

[0181] The nanobody is, for example, at least one selected from the group consisting of a VHH (variable domain heavy chain antibody) antibody, a fragmented antibody (more specifically, a Fab (Fragment Antigen Binding) antibody, etc.), and variants thereof. A VHH antibody is a single domain antibody. A variant is an antibody in which part of the amino acid sequence has been modified or a substituent has been introduced, within the range that retains specific binding to an antigen. When the nanobody is at least one selected from the group consisting of a VHH antibody, a fragmented antibody, and variants thereof, these nanobodies have a relatively small volume, which can narrow the distance (separation) between two metal nanoparticles 2 in the complex, more efficiently form a near field, and further increase the fluorescence intensity.

[0182] 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. When the molecular mass is 60,000 Da or less (particularly 30,000 Da or less or 20,000 Da or less), the nanobody has a relatively small volume, which narrows the separation distance in the complex, more efficiently forms a near-field, and further increases the fluorescence intensity. Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.

[0183] The specific binding substance 4 may be directly bound to the polymer membrane 3, or may be bound to the polymer membrane 3 via a linker moiety derived from a crosslinking agent (more specifically, SM(PEG) n (where n is 4, 6, 8, etc.)) Examples of such crosslinkers include amino group-sulfhydryl group crosslinkers (more specifically, NHS-maleimide group crosslinkers, etc.).

[0184] [Method of manufacturing nanoparticles] The manufacturing method of the nanoparticle body 1 according to the fifth embodiment includes a step of mixing a polymer to which a fluorescent substance 6 is bonded via a disulfide bond with metal nanoparticles 2, labeling the surface of the metal nanoparticles 2 with the fluorescent substance 6, and forming a polymer film 3 on the surface of the metal nanoparticles 2 (hereinafter also referred to as the "fluorescent labeling film forming step"). The method for producing nanoparticles 1 according to the fifth embodiment includes a fluorescently labeled film formation step in which fluorescent labeling of the metal nanoparticle surface and polymer film formation can be carried out in parallel. This simplifies the fluorescent labeling process compared to conventional techniques, resulting in reduced costs. Furthermore, this production method allows the fluorescent substance 6 to be labeled at a site different from that of the specific binding substance 4, thereby increasing the fluorescent labeling density in the nanoparticles 1.

[0185] An example of a method for producing the nanoparticles 1 according to this embodiment will now be described. The method for producing the nanoparticle 1 may further comprise a fluorescent substance thiolation step, a polymer fluorescent labeling step, and a specific binding substance binding step in addition to the fluorescent labeling film formation step.

[0186] The method for producing the nanoparticles 1 will be described with reference to Fig. 26. Fig. 26 is a reaction scheme showing an example of the method for producing the nanoparticles 1.

[0187] (Fluorescent substance thiolation process) In the fluorescent substance thiolation step, the fluorescent substance 6 is thiolated. More specifically, as represented by reaction formula (R-1) in the reaction scheme, (A) a fluorescent substance having an ester bond (hereinafter also referred to as a fluorescent substance) is reacted with (B) a thiol having an amino group (hereinafter also referred to as an aminoalkanethiol) to form (C) an amide bond to synthesize a thiolated fluorescent substance (hereinafter also referred to as a thiolated fluorescent substance).

[0188] 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) of (A) the fluorescent substance to (B) the aminoalkanethiol is, for example, 2:1 to 1:2. In reaction (R-1), the reaction temperature is, for example, 20 to 30°C. The reaction time is, for example, 0.5 to 2 hours. Reaction (R-1) can also be carried out under stirring conditions.

[0189] (A) R in fluorescent material 1 represents an alkylene group. Examples of such alkylene groups include alkylene groups having 2 to 5 carbon atoms (more specifically, ethylene, propylene, butylene, and pentylene groups). (A) T in the fluorescent substance 1 indicates a terminal group. Such a terminal group T 1 Examples of the group include an NHS group.

[0190] (B) Aminoalkanethiol includes, for example, aminoethanethiol (cysteamine). 2 represents an alkylene group. 2 The alkylene group represented by the above-mentioned R 1 The (B) aminoalkanethiol may be in the form of a salt. Examples of such salts include hydrochlorides of aminoalkanethiols (more specifically, cysteamine hydrochloride, etc.).

[0191] (C) R in thiolated fluorescent substance 1 and R 2 is (A) R in the fluorescent material 1 and (B) R in aminoalkanethiol 2 and the same respectively.

[0192] (Polymer fluorescent labeling process) In the polymer fluorescent labeling step, a polymer is fluorescently labeled. More specifically, as shown in reaction formula (R-2) in the reaction scheme, (C) a thiolated fluorescent substance is reacted with (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 fluorescent-labeled polymer).

[0193] In the reaction represented by reaction formula (R-2) (hereinafter also referred to as reaction (R-2)), the molar ratio of (C) thiolated fluorescent substance to (D) disulfide bond in the polymer is, for example, 3:1 to 1:1. The reaction temperature is, for example, 30 to 40°C. The reaction time is, for example, 0.5 to 2 hours. Reaction (R-2) can also be carried out under stirring conditions.

[0194] (Fluorescent labeling film formation process) In the fluorescently labeled film formation process, (E) a fluorescently labeled polymer (a polymer to which fluorescent substance 6 is bonded via a disulfide bond) and (F) metal nanoparticles 2 are mixed to label the surface of the metallic nanoparticles 2 with fluorescent substance 6 while forming a polymer film 3 on the surface of the metallic nanoparticles 2. In other words, in the fluorescently labeled film formation process, labeling the surface of the (F) metallic nanoparticles 2 with fluorescent substance 6 and forming a polymer film 3 on the surface of the (F) metallic nanoparticles 2 can be performed simultaneously. This allows fluorescent labeling and film formation to be performed in a single process rather than two processes, which is preferable from the perspective of cost reduction. The formed polymer film 3 contains a binding site 3a between the surface of the (F) metallic nanoparticles 2 and the surface via a sulfur atom. Note that the reaction represented by reaction formula (R-3) in Figure 26 (hereinafter also referred to as reaction (R-3)) shows an enlarged schematic diagram of the interface between the polymer film 3 of the nanoparticles 1 and the surface of the metallic nanoparticles 2 as its product.

[0195] In reaction (R-3), the reaction time is, for example, 12 to 36 hours. The reaction temperature is, for example, 20 to 30°C. Reaction (R-3) can also be carried out under stirring conditions. In reaction (R-3), in order to label as much of the surface of (F) metal nanoparticles 2 as possible with fluorescent substance 6, an excess of (E) fluorescently labeled polymer is added to the surface of (F) metal nanoparticles 2 until reaction (R-3) substantially stops proceeding.

[0196] By carrying out the fluorescent substance thiolation step, the polymer fluorescent labeling step, and the fluorescent labeling film formation step, it is possible to produce nanoparticles 1 comprising metal nanoparticles 2, a polymer film 3 covering the surface of the metal nanoparticles 2, and a fluorescent substance 6 labeled on the surface of the metal nanoparticles 2.

[0197] (Specific binding substance binding process) In the specific binding substance binding step, a specific binding substance 4 is bound to the polymer membrane 3. The binding site of the polymer membrane 3 that binds to the specific binding substance 4 is, for example, a functional group of the fluorescently labeled polymer (E). Such a functional group is, for example, at least one functional group selected from the group consisting of an amino group, a carboxyl group, a thiol group, an N-hydroxysuccinimide group (NHS group), and a maleimide group.

[0198] In addition to the fluorescent substance thiolation step, polymer fluorescent labeling step, and fluorescent label film formation step, a specific binding substance binding step can be performed to produce nanoparticles 1 comprising metal nanoparticles 2, a polymer film 3 covering the surface of the metal nanoparticles 2, a fluorescent substance 6 labeled on the surface of the metal nanoparticles 2, and a specific binding substance 4 bound to the polymer film 3.

[0199] The production method of this embodiment may further include any step (more specifically, a purification step of removing impurities and purifying the product) as needed, in addition to the steps of carrying out reactions (R-1) to (R-3).

[0200] Sixth Embodiment: Composite The complex will be described with reference to FIG. 27. FIG. 27 is a cross-sectional view showing a schematic diagram of the complex. The complex 40 comprises an analyte 30, which is the detection target, and two nanoparticles 10, 20. In the complex 40, the two nanoparticles 10, 20 are bound via the analyte 30. That is, the nanoparticles 10, 20 according to the fifth embodiment are bound via the analyte 30 to form the complex according to the sixth embodiment. One of the two nanoparticles 10, 20 will be referred to as the first nanoparticle 10, and the other nanoparticle will be referred to as the second nanoparticle 20. In this way, the complex 40 comprises the first nanoparticle 10 and the second nanoparticle 20 as the nanoparticles 1.

[0201] In the composite 40, the first nanoparticles 10 include first metal nanoparticles 12 as metal nanoparticles, a first polymer film 13 as a polymer film, and a first fluorescent substance 16 as a fluorescent substance, and the second nanoparticles 20 include second metal nanoparticles 22 as metal nanoparticles, a second polymer film 23 as a polymer film, and a second fluorescent substance 26 as a fluorescent substance. The fluorescent substances 16, 26 are labeled on at least one of the surfaces of the first metal nanoparticles 12 and the second metal nanoparticles 22. Specifically, in the composite 40, the first nanoparticles 10 comprise first metal nanoparticles 12, a first polymer film 13 covering the surfaces of the first metal nanoparticles 12, and a first fluorescent substance 16 labeled on the surfaces of the first metal nanoparticles 12. The second nanoparticles 20 comprise second metal nanoparticles 22, a second polymer film 23 covering the surfaces of the second metal nanoparticles 22, and a second fluorescent substance 26 labeled on the surfaces of the second metal nanoparticles 22.

[0202] The first nanoparticles 10 further include a first specific binding substance 14 as a specific binding substance, and the second nanoparticles 20 further include a second specific binding substance 24 as a specific binding substance. Specifically, the first nanoparticle 10 further comprises a first specific binding substance 14 bound to a first polymer film 13, and the second nanoparticle 20 further comprises a second specific binding substance 24 bound to a second polymer film 23.

[0203] From the viewpoint of further increasing the fluorescence intensity, it is preferable that the separation distance L is small, within a range in which the excited fluorescent substances 16, 26 are not easily quenched. More specifically, in a preferred embodiment, the two nanoparticles 10, 20 in the composite 40 are close to each other. In a more preferred embodiment, the two nanoparticles 10, 20 are close to each other so that the first polymer film 13 of the first nanoparticle 10 and the second polymer film 23 of the second nanoparticle 20 in the composite 40 come into contact. In an even more preferred embodiment, the two nanoparticles 10, 20 are close to each other so that at least one of the polymer films 13 of the first nanoparticle 10 and the second polymer film 23 of the second nanoparticle 20 in the composite 40 contracts and comes into contact.

[0204] In a further preferred embodiment, when at least one of polymer films 13, 23 shrinks and comes into contact with each other, for example, in complex 40 shown in Figure 27, it is believed that at least one of analyte 30, specific binding substances 14, 24 that bind to analyte 30, and fluorescent substances 16, 26 can be inserted into polymer films 13, 23. Furthermore, in a more preferred embodiment, when polymer films 13, 23 come into contact with each other, for example, in complex 40 shown in Figure 27, it is believed that at least one of analyte 30, specific binding substances 14, 24 that bind to analyte 30, and fluorescent substances 16, 26 can be inserted into polymer films 13, 23, as in the further preferred embodiment.

[0205] In this embodiment, the films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, which can increase the fluorescence intensity. The reason for this is presumed to be as follows: The films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, which have relatively high flexibility compared to inorganic films containing inorganic oxides. Therefore, in the composite 40, the polymer films 13 and 23 can contract, which allows the two metal nanoparticles 12 and 22 to be closer than a distance equivalent to two polymer film thicknesses (the film thickness of the polymer film 13 plus the film thickness of the polymer film 23). In other words, because the films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, the separation distance L can be less than two polymer film thicknesses. This makes it easier to obtain the plasmon enhancement effect, further increasing the fluorescence intensity. In this specification, the thickness of the polymer film in "two thicknesses of the polymer film" does not refer to the thickness of the polymer film 13, 23 in the contracted portion that is the target of the separation distance, but to the thickness of the polymer film 13, 23 in the non-contracted portion that is not the target of separation (for example, T1 in Figure 31 described later).

[0206] In this embodiment, the polymer films 13 and 23 contain binding sites 3a between themselves and the surfaces of the metal nanoparticles via sulfur atoms. For example, the polymer 3A constituting the polymer films 13 and 23 contains binding sites 3a via sulfur atoms. This can further increase the fluorescence intensity. The reason for this is presumed to be as follows. In this case, the binding sites 3a form bonds with the surfaces of the metal nanoparticles 12 and 22, and the polymer 3A has a mesh-like structure, which is thought to coat the surfaces of the metal nanoparticles 12 and 22 in a mesh-like manner. Because the polymer 3A has this mesh-like structure, it has relatively high flexibility. This allows the polymer films 13 and 23 to further contract in the composite 40, thereby enabling the two metal nanoparticles 12 and 22 to be closer than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two polymer film thicknesses, further enhancing the plasmon enhancement effect and further increasing the fluorescence intensity.

[0207] In a preferred embodiment, the polymer film 13, 23 includes at least one group selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c, in addition to the sulfur atom-mediated binding site 3a between the polymer film 13, 23 and the surface of the metal nanoparticle. This further increases the fluorescence intensity. The reason for this is presumed to be as follows: In this case, at least one group selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c forms a bond with the surface of the metal nanoparticle 12, 22, and the polymer 3A has a mesh-like structure, which is thought to coat the surface of the metal nanoparticle 12, 22 in a mesh-like manner. The mesh-like structure of the polymer 3A further enhances flexibility. This allows the polymer film 13, 23 to further contract in the composite 40, thereby enabling the two metal nanoparticles 12, 22 to be closer than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two polymer film thicknesses, further enhancing the plasmon enhancement effect and further increasing the fluorescence intensity.

[0208] In a preferred embodiment, the polymer 3A constituting the polymer film 13, 23 contains a binding site 3a via a sulfur atom in its side chain (more specifically, at the end of the side chain). In a preferred embodiment, the fluorescence intensity can be further increased. The reason for this is presumed to be as follows: In this case, the binding site 3a forms a bond with the surface of the metal nanoparticle 12, 22. Therefore, the polymer 3A has a mesh-like structure, and it is thought that the side chain serves as a binding site to coat the surface of the metal nanoparticle 12, 22 in a mesh-like manner. Because the polymer 3A has this mesh-like structure, it has relatively high flexibility. Therefore, in the composite 40, the polymer film 13, 23 can further contract, thereby enabling the two metal nanoparticles 12, 22 to be closer than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two polymer film thicknesses, further enhancing the plasmon enhancement effect and further increasing the fluorescence intensity.

[0209] In a preferred embodiment, the polymer 3A constituting the polymer film 13, 23 contains at least one group selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c in its side chain (more specifically, at the side chain terminal). In a preferred embodiment, the fluorescence intensity can be further increased. The reason for this is presumed to be as follows: In such a case, the positively charged group 3b and / or the hydrophobic group 3c form bonds with the surfaces of the metal nanoparticles 12, 22. Therefore, the polymer 3A has a mesh-like structure, and it is thought that the side chains serve as binding sites to coat the surfaces of the metal nanoparticles 12, 22 in a mesh-like manner. Because of this mesh-like structure, the polymer 3A has relatively high flexibility. Therefore, in the composite 40, the polymer film 13, 23 can further contract, allowing the two metal nanoparticles 12, 22 to be closer than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two polymer film thicknesses, further enhancing the plasmon enhancement effect and further increasing the fluorescence intensity.

[0210] The separation distance L between the first nanoparticles 10 and the second nanoparticles 20 is, for example, 12 nm to 52 nm, and 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 at which the line segment connecting a first point P1 on the surface of the first nanoparticles 10 and a second point P2 on the surface of the second nanoparticles 20 is the shortest. If the separation distance L is 52 nm or less, when excitation light is irradiated onto the complex 40, a near-field is generated more efficiently in the space near the surfaces between the first and second metal nanoparticles 12, 22, thereby further increasing the fluorescence intensity.

[0211] The films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, and the polymer films 13 and 23 include binding sites 3a between the surfaces of the metal nanoparticles and the polymer films 13 and 23 via sulfur atoms. Therefore, as described above, the separation distance L can be closer than the distance equivalent to twice the thickness of the polymer films covering the surfaces of the two metal nanoparticles 12 and 22 in the composite 40. For example, when the thickness of the polymer films 13 and 23 is 5 nm, the separation distance L can be less than 10 nm (more specifically, 2 to 9 nm, 3 to 8 nm, 4 to 7 nm, etc.).

[0212] (fluorescent material) 27, it is preferable that the fluorescent substances 16, 26 are positioned at least between the first metal nanoparticle 12 and the second metal nanoparticle 22 in the composite 40. This is because the space between the metal nanoparticles 12, 22 is one in which a near-field is efficiently generated, and therefore, by positioning the fluorescent substances 16, 26 in the space between the metal nanoparticles 12, 22, the fluorescence intensity is likely to be increased.

[0213] (Test substance) The test substance 30 is a substance to be detected that is contained in a sample. Examples of the test substance 30 include antigens, proteins, substrates, and nucleic acid chains. The test substance 30 specifically binds to the specific binding substances 14, 24. For example, an antigen has at least two antigenic determinants and forms specific bonds with the first and second specific binding substances 14, 24 at the antigenic determinants. Examples of antigens include proteins such as c-reactive protein, myoglobin, troponin T, troponin I, and BNP, as well as antigenic proteins of viruses such as influenza virus and respiratory syncytial virus. The test substance 30 is derived from a sample such as blood, plasma, urine, or saliva. That is, examples of samples containing the test substance 30 include blood, plasma, serum, urine, and saliva. The sample may further contain a solvent and a buffer solution (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, MES buffer, etc.).

[0214] <Seventh embodiment: Measuring device> The measuring device will be described with reference to Fig. 28. Fig. 28 is a diagram schematically showing a measuring device according to a seventh embodiment. As shown in Fig. 28, the measuring device 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 of the excitation light 112, such as by focusing it, and outputs incident excitation light 122. The excitation light irradiation optical system 120 includes a lens 124 and a polarizing element (λ / 2 plate) 126. The incident excitation light 122 output from the excitation light irradiation optical system 120 enters a reagent container 130 and is irradiated onto the measurement sample in the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell, a slide, etc.) or a microchannel chip. The microchannel chip is a chip having a minute channel. When the reagent container 130 is a microchannel chip, for example, the nanoparticles (reagent) and the sample according to the fifth embodiment can be mixed and continuously supplied. This eliminates the need to prepare the measurement sample by mixing them in advance, enabling continuous measurement.

[0215] The measurement sample irradiated with the incident excitation light 122 emits fluorescence (detection light 132). The light-receiving optical system 140 is disposed perpendicular to the direction of travel of the incident excitation light 122 into the reagent container 130. The light-receiving optical system 140 adjusts the cross-sectional diameter of the detection light 132 emitted from the measurement sample, and can remove scattered light from the incident excitation light 122 or adjust the light intensity. The light-receiving optical system 140 includes a lens 144 and an optical filter 146. The optical filter 146 is, for example, a band-pass filter or a dichroic mirror.

[0216] The fluorescence 142 that passes through the light-receiving optical system 140 is detected by the light-receiving element 150. The light-receiving element 150 is, for example, a PD, an APD, a PMT, a CCD camera, or a spectrometer. The light-receiving element 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring a fluorescence spectrum, and creating a two-dimensional fluorescence image.

[0217] The present invention is not limited to the above-described embodiment, and design modifications are possible within the scope of the gist of the present invention.

[0218] In the fifth and sixth embodiments, the nanoparticles 1, 10, and 20 are labeled with four fluorescent substances 6, 16, and 26, respectively, but this is not limiting. For example, the number of fluorescent substances labeled on the nanoparticles 1, 10, and 20 may be five or more.

[0219] In the fifth and sixth embodiments, the nanoparticles 1, 10, and 20 are labeled with four specific binding substances 4, 14, and 24, respectively (FIGS. 24(b) and 26), but the present invention is not limited to this. For example, the number of specific binding substances 4, 14, and 24 labeled on the nanoparticles 1, 10, and 20 may be five or more.

[0220] In the seventh embodiment, the light-receiving optical system 140 in the measuring device 100 is disposed in a direction perpendicular to the traveling direction of the incident excitation light 122 into the reagent container 130, but is not limited to this. The light-receiving optical system 140 may be disposed, for example, in a direction parallel to the traveling direction of the incident excitation light 122, or in a direction forming an acute angle or an obtuse angle with respect to the traveling direction of the incident excitation light 122. (Example)

[0221] The present invention will be described in more detail below using examples. However, the present invention is not limited to the following examples. Furthermore, unless otherwise specified, parts and percentages in the examples are by mass.

[0222] In the examples, the concentration of metal nanoparticles in a dispersion liquid may be expressed as absorbance. The absorbance was measured using an ultraviolet-visible spectrophotometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.). The subscripted number in the absorbance notation OD indicates the absorption wavelength. For example, OD 455 =0.1 indicates that the absorbance at a wavelength of 455 nm is 0.1.

[0223] Example 6: Preparation of nanoparticles The nanoparticles of Example 6 were prepared as follows. (Introduction of thiol groups into ruthenium complex derivatives) (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.) were mixed by stirring using a small rotary incubator ("RT-30mini" manufactured by Taitec Corporation) at room temperature (e.g., 25°C) for 1 hour. As a result, a thiol group was introduced at the end of the ligand of the Ru complex. This synthesis reaction is shown in reaction formula (r-1): (chemical 2) As shown in TIFF0007811942000008.tif97149, the primary amino group of (b) cysteamine hydrochloride attacks the NHS ester group of (a) NHS-labeled Ru complex derivative through a nucleophilic substitution reaction. This resulted in the preparation of (c) a Ru complex derivative bearing a thiol group (hereinafter referred to as "Ru complex-SH"). The Ru complex moiety in the resulting (c) Ru complex-SH is a fluorescent substance.

[0224] (Introduction of disulfide bonds into polymers) On the other hand, poly-L-lysine (Peptide Institute, Inc., "3075") and 3-(2-pyridyldithio)propionamido-PEG4-NHS (Thermo Fisher Scientific, serial number "26128," "NHS-PEG4-SPDP") were mixed at room temperature for 4 hours using a small rotary incubator (Tatec Corporation, "RT-30mini"). As a result, (d) a polymer having a pyridyl group (pyridinyl group) in the side chain via an SPDP linker and a disulfide bond (hereinafter also referred to as a "polymer having a disulfide group") was obtained. This synthesis reaction is represented by reaction formula (r-4): (C3) As shown in TIFF0007811942000009.tif108164, this is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamido-PEG4-NHS.

[0225] (Introduction of ruthenium complex derivatives into polymers) (c) Ru complex-SH and (d) a polymer having a disulfide group were stirred and mixed at 37°C for 1 hour. As a result, (e) a polymer in which a Ru complex was bound to the side chain via an SPDP linker and a disulfide bond (hereinafter referred to as "Ru complex-labeled polymer") 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 shown in reaction formula (r-2): (C4) As shown in TIFF0007811942000010.tif108164, this is a thiol nucleophilic reaction in which the thiol group of (c) the Ru complex-SH attacks the disulfide group of (d) a polymer having a disulfide group.

[0226] (Fluorescent substance labeling and polymer membrane formation: nanoparticle preparation) The resulting (e) Ru complex-labeled polymer was then transferred to (f) silver nanoparticles ("AGCB80-1M" manufactured by nanocomposix, diameter 80 nm, OD 455The solution was added to 1 mL of a dispersion of silver nanoparticles (pH 7.0) containing 100% ruthenium (K=0.1) and stirred overnight at room temperature using a small rotary incubator (RT-30mini, manufactured by Taitec Corporation). As a result, a dispersion of silver nanoparticles coated with a polymer film and labeled with a fluorescent substance was obtained. In this reaction, the disulfide bond of the (e) Ru complex-labeled polymer is cleaved by the reduction of silver. The poly-L-lysine moiety and the ruthenium complex moiety generated by the cleavage are each bonded to the surface of the silver nanoparticles via sulfur atoms. As a result, as shown in Figure 29, the Ru complex as a fluorescent substance is labeled on the surface of the silver nanoparticles via sulfur atoms, and the polymer film is bonded to the surface of the silver nanoparticles via sulfur atoms. In other words, the labeling of the fluorescent substance and the formation of the polymer film proceeded in parallel. Figure 29 is a schematic diagram illustrating the method for producing the nanoparticles of Example 6.

[0227] [Measurement and evaluation methods] The nanoparticles of Example 6 were confirmed to be labeled with a fluorescent substance by the following method. The obtained nanoparticles were placed in a measurement container. The measurement container was placed in a fluorometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.) and the fluorescence spectrum was measured. The measurement conditions were an excitation light wavelength of 430 nm and a detection wavelength of 490 to 700 nm. The measured fluorescence spectrum is shown in Figure 30.

[0228] 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 obtained fluorescence spectrum had a peak at approximately 620 nm. The shape of this fluorescence spectrum was almost identical to that of the Ru complex. This result confirmed that the nanoparticles of Example 6 were labeled with the Ru complex.

[0229] Example 7: Preparation of nanoparticles In preparing the nanoparticles of Example 7, first, the polymer membrane of polymer-coated silver nanoparticles was labeled with a fluorescent substance to prepare the nanoparticles. Next, a polyanionic polymer was added to the nanoparticles to prepare a nanoparticle composition containing the nanoparticles of Example 7. (polymer-coated silver nanoparticles) Poly-L-lysine (Peptide Institute, Inc., product number "3075") and 3-(2-pyridyldithio)propionamido-PEG4-NHS (Thermo Fisher Scientific, product number "26128" and "NHS-PEG4-SPDP") were mixed at room temperature for 4 hours using a small rotating incubator (Tatec Corporation, product number "RT-30mini"). This resulted in a polymer. This synthesis reaction was a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamido-PEG4-NHS. The resulting polymer was then mixed with a dispersion of silver nanoparticles (nanocomposix, product number "AGCB80-1M," diameter 80 nm, OD 1000). 430 The mixture was added to 1 mL of 1% ethanol (pH 7.0) and stirred overnight at room temperature using a small rotary incubator (RT-30mini, manufactured by Taitec Co., Ltd.) to obtain a dispersion of silver nanoparticles coated with a polymer membrane (hereinafter also referred to as polymer-coated silver nanoparticles).

[0230] (Binding of crosslinkers to polymer-coated silver nanoparticles) Next, to 1 mL of the prepared dispersion of polymer-coated silver nanoparticles, the crosslinker SM(PEG)6 (PEGylated, long-chain SMCC crosslinker) (ThermoFisher Scientific, "22105") and heparin sodium (Fujifilm Wako Pure Chemical Industries, "081-00136") were added, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator (Titec Corporation, "RT-30mini"). As a result, a dispersion of silver nanoparticles with the crosslinker SM(PEG)6 bound to the polymer membrane (hereinafter referred to as polymer-coated silver nanoparticles bound with SM(PEG)6 linkers) was obtained. The SM(PEG)6 linkers bound to the polymer-coated silver nanoparticles contained maleimide groups.

[0231] (Fluorescent labeling of VHH antibodies) An NHS-labeled Ru complex derivative (Ruthenium(II) tris(Bipyridyl)-C5-NHS ester, Tokyo Chemical Industry Co., Ltd.) was added to 100 μg of a VHH antibody (RePHAGEN, molecular mass 18,000 Da), and the mixture was stirred at room temperature for 1 hour using a small rotary incubator (RT-30mini, Taitec Co., Ltd.). As a result, a fluorescently-conjugated VHH antibody (hereinafter also referred to as a fluorescently-labeled VHH antibody) was obtained.

[0232] (Binding of cross-linking agent to fluorescently labeled VHH antibody) Next, 3-(2-pyridyldithio)propionamido-PEG4-NHS (Thermo Fisher Scientific, product number "26128," "NHS-PEG4-SPDP"), an NHS-bipyridyl disulfide crosslinker, was added to the fluorescently labeled VHH antibody at an 8-fold molar ratio, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator (Tatec Co., Ltd., "RT-30mini") to obtain a VHH antibody bound to a fluorescent substance and an SPDP linker (hereinafter referred to as an SPDP linker-bound fluorescently labeled VHH antibody).

[0233] (Thiolation of fluorescently labeled VHH antibodies bound to crosslinkers) Next, a 2-fold molar equivalent of the reducing agent TCEP (ThermoFisher Scientific, "77720") was added to the SPDP linker-conjugated fluorescently labeled VHH antibody, and the mixture was stirred and mixed using a mixer (BioSan, "TS-100") at 37°C for 1 hour. As a result, a VHH antibody (hereinafter also referred to as a fluorescently labeled VHH antibody conjugated with a reduced SPDP linker) was obtained, in which a fluorescent substance and a reduced SPDP linker (hereinafter also referred to as a reduced SPDP linker) were conjugated. The reduced SPDP linker contained a thiol group (-SH group) generated by reduction of the disulfide bond.

[0234] (Binding of fluorescently labeled VHH antibodies to silver nanoparticles) Next, a dispersion of polymer-coated silver nanoparticles (OD 430 A fluorescently labeled VHH antibody bound to a reduced SPDP linker was added to the suspension (pH 7.0, pH ...

[0235] [Polymer membrane shrinkage] (Preparation of the complex) 35 μL of the prepared nanoparticle composition dispersion was added to a phosphate buffer solution (Fujifilm Wako Pure Chemical Industries, Ltd.). Company 65 μL of "PBS-T" and C Reactive Protein (manufactured by ADVY CHEMICAL Co., Ltd., "00-AGN-AP-CRP-00") (hereinafter also referred to as CRP antigen) as a test substance were added, and the cells were incubated in a small rotary incubator (Taitec Co., Ltd.) at room temperature for 5 minutes. Company The mixture was stirred using an RT-30mini to prepare a measurement sample containing a sandwich-type complex.

[0236] (SEM image capture) Using a scanning electron microscope (Regulus 8220 manufactured by Hitachi High-Technologies Corporation), an SEM image (magnification 100K) of the composite in the measurement sample obtained in Example 7 was taken. Figure 31 shows an SEM image of the composite prepared using the nanoparticles of Example 7. In the SEM image obtained as shown in Figure 31, the separation distance L1 between the metal nanoparticles of the nanoparticles in the composite and the thickness T1 of the polymer film other than between the metal nanoparticles of the nanoparticles were measured and compared. Note that the thickness T1 of the polymer film was the thickness of the unshrunk portion of the polymer film that was not subject to the separation distance. As a result, the separation distance L1 was smaller than the thickness (T1 × 2) equivalent to twice the thickness of the polymer film. Therefore, in the composite prepared with the nanoparticles of Example 7, the polymer film contracted, and the two metal nanoparticles of the composite were closer than the separation distance equivalent to twice the thickness of the polymer film (T1 × 2). This strongly suggests that the plasmon enhancement effect was further obtained, further increasing the fluorescence intensity.

[0237] [Reference Example 1: Shrinkage of inorganic film] In order to clarify the technical significance of the shrinkage of the polymer film shown in Example 7, an aggregate of metal nanoparticles coated with an inorganic film (Reference Example 1) was examined as a comparative example.

[0238] (Preparation of measurement sample) Silica-coated silver nanoparticles (manufactured by nanoComposix, silver nanoparticle particle diameter (core particle diameter) 50 nm, silica film thickness 20 nm) were diluted with water to prepare an aqueous dispersion of silica-coated silver nanoparticles. The resulting aqueous dispersion was used as the measurement sample. Specifically, in addition to the primary particles of silica-coated silver nanoparticles, the measurement sample also contained aggregates of two particles. These aggregates were identified and evaluated. As in Example 7, SEM images (500K magnification) were taken, and the thickness T2 of the inorganic film in the aggregate and the separation distance L2 between two metal nanoparticles were measured and compared from the SEM images. As a result, the separation distance L2 was approximately twice the thickness T2 of the inorganic film. Note that the thickness T2 of the inorganic film was the thickness of the inorganic film in the portion not subject to the separation distance. (Explanation of symbols)

[0239] 1. Nanoparticles 2. Metal nanoparticles 3...Polymer membrane 3A: Polymers that make up the polymer membrane 3a: Sulfur atom-mediated binding site 3b: Positively charged group 3c...Hydrophobic group 4...specific binding substance 6. Fluorescent materials 10. First nanoparticle 12. First metal nanoparticles 13...first polymer membrane 14...first specific binding substance 16. First fluorescent material 20 Second nanoparticle 22 Secondary metal nanoparticles 23...Second polymer membrane 24...Second specific binding substance 26 Second fluorescent material 30 Test substance 40 Complex L...Separation distance (separation distance)

[0240] The embodiments of the present invention also include the following aspects. [1] The method comprises: a metal nanoparticle; a polymer film covering the surface of the metal nanoparticle; and a nano-sized specific binding substance bound to the surface of the polymer film, the specific binding substance binding specifically to a test substance in a specimen; The nanoparticle body, wherein the polymer membrane comprises at least one selected from the group consisting of a bonding site between the polymer membrane and the surface of the metal nanoparticle via a sulfur atom, a positively charged group, and a hydrophobic group. [2] The nanoparticle according to [1], wherein the polymer constituting the polymer membrane contains at least one group selected from the group consisting of the binding site, the positively charged group, and the hydrophobic group in its side chain. [3] The nanoparticle according to [1] or [2], wherein the polymer membrane contains at least the binding site via the sulfur atom. [4] The nanoparticle according to any one of [1] to [3], wherein the polymer film contains at least the positively charged group. [5] The positively charged group includes a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH2 +)NH2). [6] The nanoparticle according to any one of [1] to [5], wherein the polymer membrane contains at least the hydrophobic group. [7] The nanoparticle according to [6], 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. [8] The nanoparticle according to any one of [1] to [7], wherein the polymer constituting the polymer membrane has a moiety containing a disulfide bond as a side chain. [9] The nanoparticle according to [8], which is dependent on [3] or [4], wherein the disulfide bond-containing moiety has the positively charged group.

[10] the site containing the disulfide bond has the hydrophobic group; [5] or [6] subordinate to [8] The nanoparticle according to claim 1.

[11] The nanoparticle according to any one of [1] to

[10] , wherein the polymer film has a thickness of 1 nm to 10 nm.

[12] The nanoparticle according to any one of [1] to

[11] , which is a nanoparticle used in plasmon excitation fluorescence analysis.

[13] The nanoparticle according to any one of [1] to

[12] , wherein the specific binding substance is a nanobody.

[14] The nanoparticle according to any one of [1] to

[13] , wherein the specific binding substance is a VHH antibody.

[15] The nanoparticle according to any one of [1] to

[14] , wherein the metal nanoparticles comprise gold or silver.

[16] The nanoparticle according to any one of [1] to

[15] , wherein at least one of the surface of the polymer membrane and the specific binding substance is labeled with a fluorescent substance.

[17] the nanoparticles include first nanoparticles and second nanoparticles; the first nanoparticle bodies include first metal nanoparticles as the metal nanoparticles, a first polymer film as the polymer film, and a first specific binding substance as the specific binding substance; the second nanoparticles include second metal nanoparticles as the metal nanoparticles, a second polymer film as the polymer film, and a second specific binding substance as the specific binding substance; The nanoparticle according to any one of [1] to

[16] , wherein a fluorescent substance is labeled to at least one of the first polymer membrane, the second polymer membrane, the first specific binding substance, and the second specific binding substance.

[18] The nanoparticle according to

[17] , wherein the first nanoparticle and the second nanoparticle form a complex bound to each other via the test substance.

[19] The nanoparticle according to

[18] , wherein the test substance is derived from the specimen, which is blood, plasma, urine, or saliva.

[20] The nanoparticle according to

[18] or

[19] , wherein in the complex, the fluorescent substance is located between the first nanoparticle and the second nanoparticle.

[0241] The embodiments of the present invention also include the following aspects. [1] The present invention relates to a method for manufacturing a fluorescent material comprising: a metal nanoparticle; a polymer film covering the surface of the metal nanoparticle; and a fluorescent substance. the fluorescent substance is labeled on the surface of the metal nanoparticle; The nanoparticle body has a bonding site between the polymer film and the surface of the metal nanoparticle via a sulfur atom. [2] The nanoparticle according to [1], wherein the polymer constituting the polymer membrane contains the binding site in its side chain. [3] The nanoparticle according to [1] or [2], wherein the fluorescent substance has a binding site between the fluorescent substance and the surface of the metal nanoparticle via a sulfur atom. [4] The nanoparticle according to any one of [1] to [3], further comprising a specific binding substance bound to the polymer membrane that specifically binds to an analyte in a specimen. [5] The nanoparticle according to any one of [1] to [4], wherein the polymer membrane further comprises at least one group selected from the group consisting of a positively charged group and a hydrophobic group. [6] The nanoparticle according to [5], wherein the polymer constituting the polymer membrane further comprises at least one group selected from the group consisting of the positively charged group and the hydrophobic group in its side chain. [7] the polymer film contains at least the positively charged group, The positively charged group includes a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH2 + )NH2). [8] 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. [9] The nanoparticle according to any one of [1] to [8], wherein the polymer film has a thickness of 1 nm to 10 nm.

[10] The nanoparticle according to any one of [1] to [9], which is a nanoparticle used in plasmon excitation fluorescence analysis.

[11] The nanoparticle according to any one of [5] to

[10] dependent on [4], wherein the specific binding substance is at least one selected from the group consisting of a nanobody, a ligand, an enzyme, and a nucleic acid chain.

[12] The nanoparticle according to any one of [5] to

[11] according to [4], wherein the specific binding substance is at least one nanobody selected from the group consisting of VHH antibodies, antibody fragments, and variants thereof.

[13] the metal nanoparticles comprise gold or silver; [1]~

[12] One of t The nanoparticle according to claim 1.

[14] the nanoparticles include first nanoparticles and second nanoparticles; the first nanoparticle bodies include first metal nanoparticles as the metal nanoparticles and a first polymer film as the polymer film, the second nanoparticle bodies include second metal nanoparticles as the metal nanoparticles and a second polymer film as the polymer film, The nanoparticle according to any one of [1] to

[13] , wherein the fluorescent substance is labeled on at least one of the surfaces of the first metal nanoparticles and the surfaces of the second metal nanoparticles.

[15] The nanoparticle according to

[14] , wherein the first nanoparticle and the second nanoparticle form a complex bound to each other via the test substance.

[16] The nanoparticle according to

[15] , wherein the test substance is derived from a specimen that is blood, plasma, urine, or saliva.

[17] The nanoparticle according to

[15] or

[16] , wherein in the complex, at least the fluorescent substance is located between the first nanoparticle and the second nanoparticle.

[18] A method for producing nanoparticles, comprising the steps of mixing a polymer to which a fluorescent substance is bonded via a disulfide bond with metal nanoparticles, labeling the surface of the metal nanoparticles with the fluorescent substance, and forming a polymer film on the surface of the metal nanoparticles.

[0242] The embodiments of the present invention also include the following aspects. [1] 1. A nanoparticulate composition comprising a nanoparticulate object and a solvent containing the nanoparticulate object, the nanoparticle body comprises a metal nanoparticle, a coating film that coats the surface of the metal nanoparticle, and a specific binding substance that binds to the surface of the coating film or the surface of the metal nanoparticle and specifically binds to a test substance in a specimen; A nanoparticle composition, wherein the solvent contains a polyanionic polymer in addition to the nanoparticles. [2] The nanoparticle composition according to [1], wherein 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. [3] The nanoparticle composition according to [1] or [2], wherein 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. [4] The coating film is an inorganic film containing an inorganic oxide, or a polymer film containing at least one selected from the group consisting of a bonding site between the surface of the metal nanoparticle and the polymer film via a sulfur atom, a positively charged group, and a hydrophobic group; The nanoparticulate composition according to any one of [1] to [3], wherein [5] the coating film is the polymer film, The nanoparticle composition according to [4], wherein the polymer constituting the polymer film has at least one selected from the group consisting of the sulfur atom-mediated bonding site, the positively charged group, and the hydrophobic group at the end of the side chain. [6] The nanoparticulate composition according to any one of [1] to [5], wherein the specific binding substance is a nanobody. [7] The 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 a polyalkylene ether chain and an alkyl chain. [8] The nanoparticle composition according to any one of [1] to [7], wherein the polyanionic polymer is present so as to surround each of the nanoparticles. [9] The nanoparticulate composition according to any one of [1] to [8], wherein the solvent comprises an aqueous solvent.

[10] The polyanionic polymer and the nanoparticles are included in the aggregate. [1]~[9] One of t The nanoparticulate composition according to claim 1.

[11] The nanoparticle composition according to any one of [1] to

[10] , which is a nanoparticle used in plasmon excitation fluorescence analysis.

[12] Any one of [1] to

[11] , wherein the metal nanoparticles comprise gold or silver. t The nanoparticulate composition according to claim 1.

[13] The nanoparticle composition according to any one of [1] to

[12] , wherein at least one of the surface of the coating film and the specific binding substance is labeled with a fluorescent substance.

[14] the nanoparticles include first nanoparticles and second nanoparticles; the first nanoparticles include first metal nanoparticles as the metal nanoparticles, a first coating film as the coating film, and a first specific binding substance as the specific binding substance; the second nanoparticles include second metal nanoparticles as the metal nanoparticles, a second coating film as the coating film, and a second specific binding substance as the specific binding substance; The nanoparticle composition according to any one of [1] to

[13] , wherein a fluorescent substance is labeled on at least one of the first coating film, the second coating film, the first specific binding substance, and the second specific binding substance.

[15] The nanoparticle composition according to

[14] , wherein the first nanoparticle and the second nanoparticle form a complex bound to each other via the test substance.

[16] The nanoparticle composition according to

[15] , wherein in the complex, the fluorescent substance is located between the first nanoparticle and the second nanoparticle.

[17] The nanoparticle composition according to any one of [1] to

[16] , wherein the test substance is derived from blood, plasma, urine, or saliva.

Claims

1. A nanoparticle body comprising metal nanoparticles, a polymer membrane covering the surface of the metal nanoparticles, and a nano-sized specific binding substance bound to the surface of the polymer membrane that specifically binds to a test substance in a specimen, wherein the polymer membrane contains binding sites between the surface of the metal nanoparticles and the polymer membrane via sulfur atoms, positively charged groups that form electrostatic bonds with the surface of the metal nanoparticles, and hydrophobic groups that form hydrophobic bonds with the metal nanoparticles.

2. further comprising a fluorescent material, The nanoparticle according to claim 1 , wherein the fluorescent substance is labeled on at least one selected from the group consisting of the surface of the metal nanoparticle, the surface of the polymer membrane, and the specific binding substance.

3. the fluorescent substance is labeled on the surface of the metal nanoparticle; The nanoparticle according to claim 2 , wherein the fluorescent substance has a binding site between the fluorescent substance and the surface of the metal nanoparticle via a sulfur atom.

4. The nanoparticle according to claim 1 or 2, wherein the polymer constituting the polymer membrane comprises at least one group selected from the group consisting of the binding site, the positively charged group, and the hydrophobic group in its side chain.

5. The positively charged group includes a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (—NHC(═NH ２ ＋ ) NH ２ 2. The nanoparticle according to claim 1, wherein the nanoparticle is at least one selected from the group consisting of:

6. The nanoparticle according to claim 1 , 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.

7. The nanoparticle according to claim 1 or 2, wherein the polymer constituting the polymer membrane has a moiety containing a disulfide bond as a side chain.

8. the polymer constituting the polymer membrane has a moiety containing a disulfide bond as a side chain, The nanoparticle according to claim 1 , wherein the disulfide bond-containing moiety has the positively charged group.

9. the polymer constituting the polymer membrane has a moiety containing a disulfide bond as a side chain, The nanoparticle according to claim 1 , wherein the disulfide bond-containing moiety has the hydrophobic group.

10. 3. The nanoparticle according to claim 1, wherein the polymer film has a thickness of 1 nm to 10 nm.

11. The nanoparticle according to claim 1 or 2, which is used in plasmon excitation fluorescence analysis.

12. The nanoparticle according to claim 1 or 2, wherein the specific binding substance is at least one selected from the group consisting of a nanobody, a ligand, an enzyme, and a nucleic acid chain.

13. The nanoparticle according to claim 1 or 2, wherein the specific binding substance is at least one nanobody selected from the group consisting of a VHH antibody, an antibody fragment, and a variant thereof.

14. 3. The nanoparticle of claim 1 or 2, wherein the metal nanoparticles comprise gold or silver.

15. The nanoparticles include first nanoparticles and second nanoparticles, the first nanoparticle bodies include first metal nanoparticles as the metal nanoparticles, a first polymer film as the polymer film, and a first specific binding substance as the specific binding substance; the second nanoparticles include second metal nanoparticles as the metal nanoparticles, a second polymer film as the polymer film, and a second specific binding substance as the specific binding substance; The nanoparticle body described in claim 1 or 2, wherein a fluorescent substance is labeled on at least one of the surfaces of the first metal nanoparticles and the second metal nanoparticles, the first polymer film and the second polymer film, and the first specific binding substance and the second specific binding substance.

16. The nanoparticle according to claim 15, wherein the first nanoparticle and the second nanoparticle are bound to each other via the test substance to form a complex.

17. The nanoparticle according to claim 15, wherein the test substance is derived from the specimen, which is blood, plasma, urine, or saliva.

18. further comprising a fluorescent material; The nanoparticle according to claim 15, wherein the fluorescent substance is positioned between the first nanoparticle and the second nanoparticle in a complex in which the first nanoparticle and the second nanoparticle are bound via the test substance.

19. A method for producing nanoparticles, comprising the steps of: mixing a polymer to which a fluorescent substance is bonded via a disulfide bond and which contains a positively charged group and a hydrophobic group with metal nanoparticles; labeling the surface of the metal nanoparticles with the fluorescent substance; and forming a polymer film between the surface of the metal nanoparticles and the polymer film, the polymer film including a binding site via a sulfur atom, the electrostatically bonded positively charged group, and the hydrophobicly bonded hydrophobic group.

Images