Nanoparticles, composites containing nanoparticles, and methods for forming polymer films contained in nanoparticles

Abstract

Provided is a nanoparticle body comprising: a metallic nanoparticle; a polymer membrane that covers the surface of the metallic nanoparticle; a specific binding substance that specifically binds to a target substance in a sample; and a fluorescent substance that labels the surface of the polymer membrane or the specific binding substance. The fluorescent substance is excited by the light of the luminescence emission wavelength from the plasmon resonance of a composite obtained by two or more of said nanoparticle bodies binding by means of the target substance.

Description

Technical Field

[0001] The present invention relates to nanoparticle bodies (particularly nanoparticle bodies used for plasmon-excited fluorescence analysis), composites containing nanoparticle bodies, and a method for forming a polymer film contained in nanoparticle bodies.

Background Art

[0002] A biosensor specifically reacts a specific test substance to be detected with a specific binding substance to form a complex, and detects the test substance based on a signal resulting from specific binding in the complex. In plasmon-excited fluorescence analysis, the complex includes, for example, a test substance, a specific binding substance, a fluorescent substance, and metal particles. When the excitation light irradiates the complex, surface plasmon resonance is induced in the metal particles in the complex, and a near-field is formed near the surface of the metal particles. The fluorescence intensity of the fluorescent substance is increased by this near-field.

[0003] The composite particles for an immunochromatogram described in Patent Document 1 have a structure in which the outside of fine particles made of metal is covered with at least one layer of silica containing at least one kind of fluorescent substance, and are composed of fine particles surface-modified with a labeling substance that specifically recognizes a target substance. The composite particles of Patent Document 1 cover the surface 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 contacting the metal particles. Thereby, quenching of the excited fluorescent substance is suppressed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] By the way, as a result of intensive studies by the present inventors on the above-described sensor, it has been found that there is room for further improving the detection sensitivity. Specifically, conventionally, in the selection of the fluorescent substance, the relationship between the absorption spectrum of the fluorescent substance and the emission wavelength of the plasmon resonance has not been considered, or even if the relationship has been mentioned, the relationship with the emission wavelength of the plasmon resonance derived from single-particle metal nanoparticles has been considered. Therefore, the fluorescence emitted by the fluorescent substance in the composite has not been sufficiently enhanced by the plasmon resonance.

[0006] The present invention has been made in view of such problems. That is, the main object of the present invention is to provide a nanoparticle body that selects an appropriate fluorescent substance, sufficiently enhances the fluorescence emitted by the fluorescent substance in the composite by plasmon resonance, and improves the detection sensitivity.

Means for Solving the Problems

[0007] The nanoparticle body according to one embodiment of the present invention is composed of metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, a specific binding substance that specifically binds to a test substance in a sample, and a fluorescent substance labeled on the surface of the polymer film or the specific binding substance, and is a nanoparticle body comprising the fluorescent substance is excited by light having the emission wavelength of the plasmon resonance in a composite in which two or more of the nanoparticle bodies are bound via the test substance.

[0008] The composite according to another embodiment of the present invention is composed of two or more of the nanoparticle bodies, the two or more nanoparticle bodies include a first nanoparticle body and a second nanoparticle body, and the first nanoparticle body and the second nanoparticle body are bound via the test substance.

[0009] The method for forming a polymer film according to another embodiment of the present invention is The method comprises the step of contacting a polymer having disulfide bonds in its side chains with metal nanoparticles to form a polymer film in which the polymer is bonded to the surface of the metal nanoparticles via sulfur atoms. [Effects of the Invention]

[0010] The present invention provides nanoparticles that enhance detection sensitivity by selecting an appropriate fluorescent material and sufficiently amplifying the fluorescence emitted by the fluorescent material in the complex through plasmon resonance. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a conceptual diagram showing the plasmon resonance spectrum originating from single-particle metal nanoparticles and the absorption spectrum of a fluorescent substance. [Figure 2] Figure 2 is a conceptual diagram showing the plasmon resonance spectra originating from single-particle metal nanoparticles, the plasmon resonance spectra originating from metal nanoparticles in a composite, and the absorption spectrum of a fluorescent substance. [Figure 3] Figure 3 is a conceptual diagram showing the plasmon resonance spectra originating from single-particle metal nanoparticles, the plasmon resonance spectra originating from metal nanoparticles in a composite, and the absorption spectrum of a fluorescent substance. [Figure 4] Figure 4 is a schematic cross-sectional view showing a nanoparticle body according to the first embodiment. [Figure 5] Figure 5 is an enlarged cross-sectional view of section A in Figure 4. [Figure 6] Figure 6 is a schematic diagram showing the method for forming the polymer film 3. [Figure 7] Figure 7 is a conceptual diagram showing the plasmon resonance spectra originating from single-particle metal nanoparticles, the plasmon resonance spectra originating from metal nanoparticles in a composite, and the fluorescence spectra of a fluorescent material. [Figure 8]Figure 8 is a conceptual diagram showing the plasmon resonance spectra originating from single-particle metal nanoparticles, the plasmon resonance spectra of metal nanoparticles in a composite, and the absorption and fluorescence spectra of a fluorescent substance. [Figure 9] Figure 9 is a schematic cross-sectional view of the composite. [Figure 10] Figure 10 is a schematic cross-sectional view showing the composite structure. [Figure 11] Figure 11 is a schematic cross-sectional view showing a composite composed of two nanoparticles. [Figure 12] Figure 12 is a schematic cross-sectional view showing a composite composed of three nanoparticles. [Figure 13] Figure 13 shows the measuring device. [Figure 14] Figure 14 shows a schematic diagram illustrating the method for forming the polymer film in Example 1. [Figure 15] Figure 15 shows a schematic diagram illustrating the method for forming the polymer film in Example 1. [Figure 16] Figure 16 is a schematic diagram showing the structure of the fluorescently labeled antibody-conjugated nanoparticles of Example 1. [Figure 17] Figure 17 is a schematic diagram showing an immunochromatography strip. [Figure 18] Figure 18 shows the plasmon resonance spectra of Example 1 ((a) blank slide and (b) measurement slide). [Figure 19] Figure 19 shows the plasmon resonance spectrum of Example 1, as well as the absorption and fluorescence spectra of the Ru complex. [Figure 20] Figure 20 shows the plasmon resonance spectra of Example 2 ((a) blank slide and (b) measurement slide). [Figure 21] Figure 21 shows the plasmon resonance spectrum of Example 2, as well as the absorption and fluorescence spectra of the Ru complex. [Figure 22]Figure 22 shows the absorption and fluorescence spectra of the fluorescent substance in Example 3, as well as the plasmon resonance spectrum of Example 1. [Figure 23] Figure 23 shows the fluorescence spectrum of the test substance-nanoparticle system in Example 3. [Figure 24] Figure 24 shows the plasmon resonance spectra of Comparative Example 1 ((a) silica-coated silver nanoparticles in the primary particle state and (b) aggregates in which three silica-coated silver nanoparticles are arranged in a substantially linear fashion). [Figure 25] Figure 25 shows the plasmon resonance spectra of Comparative Example 2 ((a) silica-coated silver nanoparticles in the primary particle state and (b) aggregates in which three silica-coated silver nanoparticles are arranged in an ozone molecular manner). [Modes for carrying out the invention]

[0012] Hereinafter, embodiments of the present invention, including nanoparticles, composites, and measuring devices, will be described in detail with reference to the illustrated embodiments. Note that the drawings include schematic representations and may not reflect actual dimensions or proportions.

[0013] Numerical ranges referred to herein are intended to include the lower and upper limits themselves, unless otherwise specified by terms such as "less than" and "greater than." For example, a numerical range of 1 nm to 10 nm is interpreted as including both a lower limit of "1 nm" and an upper limit of "10 nm."

[0014] In this specification, "substantially composed of a specific material" or "consisting of a specific material" means that the component contains the specific material in proportions of 95% or more by mass, 97% or more by mass, 99% or more by mass, or 100% by mass. For example, "nano-particles made of gold" means that the nano-particles contain gold in proportions of 95% or more by mass, 97% or more by mass, 99% or more by mass, or 100% by mass.

[0015] <First Embodiment: Nanoparticle Body> [Basic composition of nanoparticles] The nanoparticle body according to the first embodiment comprises metal nanoparticles, a polymer film, a specific binding substance, and a fluorescent substance. The metal nanoparticles induce plasmon resonance upon irradiation with excitation light. The polymer film coats the surface of the metal nanoparticles. The specific binding substance specifically binds to the test substance in the sample to form a complex. The fluorescent substance is labeled on the surface of the polymer film or the specific binding substance and emits fluorescence derived from plasmon resonance.

[0016] (Method for detecting test substances using nanoparticles) First, for the convenience of explaining the nanoparticles according to this embodiment and to aid in understanding them, a method for detecting a test substance using the nanoparticles according to this embodiment will be described. The nanoparticle body according to this embodiment comprises metal nanoparticles, a polymer film coating the surface of the metal nanoparticles, a specific binding substance that specifically binds to the test substance in the sample, and a fluorescent substance labeled on the surface of the polymer film or the specific binding substance. The nanoparticle body according to this embodiment is dispersed in the sample to capture the test substance contained in the sample and form a composite (fourth embodiment). More specifically, the composite is formed by the specific binding of the specific binding substance of the nanoparticle body to the test substance. The composite has a structure in which, for example, two nanoparticle bodies are bound together via the test substance. In this composite, for example, two metal nanoparticles are arranged spaced apart at a certain distance from each other by binding to their respective specific binding substances with respect to the same test substance.

[0017] In surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS), when excitation light is shone on the complex, localized surface plasmon resonance (LSPR), also simply referred to as "plasmon resonance," occurs, efficiently forming a near-field near the surface of the metal nanoparticles (particularly near the surface between two metal nanoparticles). The near-field and dipole-dipole mechanism efficiently excite the fluorescent material in the complex, enhancing its fluorescence. By measuring the amount of fluorescence, the test substance in the sample can be detected.

[0018] [Background to the present invention] Next, for the convenience of explaining the nanoparticles according to this embodiment and to aid in understanding them, the background to the present invention will be explained in detail with reference to Figures 1 to 3. Figure 1 is a conceptual diagram showing the plasmon resonance spectrum originating from single-particle metal nanoparticles and the absorption spectrum of a fluorescent substance. As shown in Figure 1, the plasmon resonance spectrum 201 originating from single-particle metal nanoparticles is in the second emission wavelength region WR. E2 For example, it has one peak. The absorption spectrum of the fluorescent substance 202 is in the absorption wavelength range WR. A For example, it has one peak. When the fluorescent material in the complex is excited by plasmon resonance, the fluorescent material is thought to be excited by a dipole-dipole mechanism (Förster mechanism (Fluorescence Resonance Energy Transfer, FRET)) and the near field. Therefore, as shown in Figure 1, there is an overlap between the plasmon resonance spectrum 201 originating from single-particle metal nanoparticles and the absorption spectrum 202 of the fluorescent material (absorption wavelength range WR). A and the second emission wavelength range WR E2 The fluorescent substance was selected in such a way that the overlap (204) would be large. Conventionally, from the viewpoint of improving detection sensitivity, the fluorescent substance in the complex was selected in this manner.

[0019] As a result of intensive studies, the inventors of the present application have found that there are the following problems in the selection of conventional fluorescent substances. The plasmon resonance derived from single-particle metal nanoparticles (i.e., the plasmon resonance induced in single-particle metal nanoparticles) is a dipole resonance, and the plasmon resonance induced in the composite is a plasmon resonance (higher-order resonance, i.e., multipole resonance) derived from dipole-dipole interaction. Examples of multipole resonance include quadrupole resonance. That is, the plasmon resonance that mainly contributes to the detection of the test substance is the multipole resonance induced by the proximity between metal nanoparticles in the composite. Therefore, in order to efficiently increase the detection sensitivity in the detection of the test substance, it is necessary to enhance the fluorescence mainly caused by multipole resonance rather than the fluorescence caused by dipole resonance.

[0020] Figure 2 is a conceptual diagram showing the plasmon resonance spectrum 201 derived from single-particle metal nanoparticles, the plasmon resonance spectrum 206 derived from metal nanoparticles in the composite, and the absorption spectrum 202 of the fluorescent substance. The plasmon resonance spectrum (hereinafter also referred to as the multipole resonance spectrum) 206 of the metal nanoparticles in the composite, as shown in Figure 2, has, for example, one peak and is located on the longer wavelength side compared to the peak of the plasmon resonance spectrum (hereinafter also referred to as the dipole resonance spectrum) 201 derived from single-particle metal nanoparticles. That is, the first emission wavelength range WR E1 of plasmon resonance is located on the longer wavelength side compared to the second emission wavelength range WR E1 of the plasmon resonance derived from single-particle metal nanoparticles (i.e., the plasmon resonance induced in single-particle metal nanoparticles). That is, the first emission wavelength range WR E2 of plasmon resonance is located on the longer wavelength side compared to the second emission wavelength range WR A of the plasmon resonance derived from single-particle metal nanoparticles (i.e., the plasmon resonance induced in single-particle metal nanoparticles). That is, there is no overlap between the absorption spectrum 202 of the fluorescent substance and the plasmon resonance spectrum 206 derived from metal nanoparticles in the composite (the overlap between the absorption wavelength range WR E1 and the first emission wavelength range WR

[0021] The inventors have found that, from the perspective of increasing the detection sensitivity of the test substance, the spectral overlap 204 (as also mentioned in Figure 1) can be considered an apparent overlap. From the viewpoint of increasing the detection sensitivity of the test substance, it is important to select a fluorescent substance that gives rise to a first region R1, and preferably a fluorescent substance that increases the overlap 208 between the multipole resonance spectrum 206 and the absorption spectrum 202 of the fluorescent substance (see Figure 3 below). Here, the first region R1 refers to the region where the multipole resonance spectrum 206 and the absorption spectrum 202 of the fluorescent substance overlap (the region corresponding to overlap 208), as shown in Figure 3 below.

[0022] Figure 3 is a conceptual diagram showing the plasmon resonance spectrum 201 originating from single-particle metal nanoparticles, the plasmon resonance spectrum 206 originating from metal nanoparticles in the composite, and the absorption spectrum 202 of the fluorescent material. In this embodiment, the fluorescent material is selected such that there is an overlap 208 between the absorption spectrum 202 of the fluorescent material and the resonance spectrum 206 originating from metal nanoparticles in the composite (preferably large). Thus, the absorption wavelength range WR of the fluorescent material is A This is the first emission wavelength range of plasmon resonance WR E1 When this coincides, the fluorescent substance is sufficiently excited, the fluorescence is enhanced, and the detection sensitivity is greatly improved. Therefore, it is believed that the nanoparticles according to this embodiment can increase detection sensitivity.

[0023] Based on this technical knowledge, the inventors focused on adjusting the spectral characteristics of fluorescent materials and investigated specific means to improve detection sensitivity. As a result, the inventors came to the conclusion that "fluorescent materials are excited by light of the emission wavelength of localized surface plasmon resonance in a composite in which two or more nanoparticles are bound via the test material."

[0024] The nanoparticle material according to the first embodiment is Metal nanoparticles and A polymer film that coats the surface of the metal nanoparticles, A specific binding substance that specifically binds to the test substance in the sample, A fluorescent substance labeled on the surface of the polymer film or the specific bonding substance and A nanoparticle body comprising, The fluorescent substance is excited by light of the emission wavelength of localized surface plasmon resonance in a composite in which two or more of the nanoparticles are bonded via the test substance.

[0025] [Mechanism of Action] The nanoparticles according to this embodiment can enhance detection sensitivity. Although not bound by any particular theory, the reason is presumed to be as follows. In the nanoparticles according to this embodiment, the fluorescent substance is excited by light at the emission wavelength of the plasmon resonance in a complex formed by two or more nanoparticles bound together via the test substance. This causes an overlap between the absorption spectrum of the fluorescent substance and the plasmon resonance spectrum originating from the metal nanoparticles in the complex, inducing fluorescence for detecting the test substance via the Förster mechanism and the near field (hereinafter, such fluorescence will also be referred to as "excitation-induced fluorescence"). Therefore, since the fluorescent substance is sufficiently excited by light at the emission wavelength of the plasmon resonance originating from the complex, detection sensitivity can be enhanced. Furthermore, fluorescence for detecting the test substance can also be induced by the overlap between the fluorescence spectrum of the fluorescent substance and the plasmon resonance spectrum derived from metal nanoparticles in the complex (hereinafter, this type of fluorescence will also be referred to as "emission-induced fluorescence"). Emission-induced fluorescence will be described in detail in the second embodiment.

[0026] [Scheme 1] Scheme 1 of the process for detecting the test substance using nanoparticles according to this embodiment: [ka] (In the elementary processes (1) to (3) of Scheme 1, S0 represents the ground state, S1 represents the excited singlet state, * represents the excited state, Flu represents the fluorescent substance (in the complex), and M represents the metal nanoparticle (in the complex)) Refer to the following for further explanation of fluorescence enhancement in this embodiment.

[0027] Scheme 1 consists of elementary processes (1) to (3). As shown in elementary process (1), the fluorescent substance is excited by light at the emission wavelength of plasmon resonance in a complex formed by the bonding of two or more nanoparticles via the test substance. When the complex formed by trapping the test substance is irradiated with externally irradiated light as excitation light (hereinafter also referred to as "external irradiation light"), plasmon resonance (multipole resonance) is induced (that is, plasmon resonance in the complex is induced by externally irradiated light). Examples of the test substance include a sample derived from blood, plasma, urine, or saliva. As shown in elementary process (2), the fluorescent material is excited by a dipole-dipole mechanism and a near-field. Absorption wavelength range WR of the fluorescent material A This is the first emission wavelength range of plasmon resonance WR E1 When these conditions overlap, the fluorescent substance is efficiently excited. As shown in elementary process (3), the excited-state fluorescent substance relaxes and emits fluorescence.

[0028] In one preferred embodiment, the first emission wavelength range WR E1 The maximum absorption wavelength of the fluorescent substance in this complex is located in the 500-700 nm (more preferably 550-700 nm) range. In other words, the first emission wavelength region WR of the plasmon resonance spectrum in the complex is... E1 The maximum absorption wavelength of a fluorescent substance lies between 500 and 700 nm. Examples of fluorescent substances include fluorescein derivatives, rhodamine derivatives, cyanine dyes, and Alexa Flour® manufactured by Molecular Probes. Among these, fluorescent substances with a maximum absorption wavelength of 500 to 700 nm include, for example, Alexa Flour® series 532, 546, 555, 568, 594, and 640. The maximum absorption wavelength can be determined as follows: Measure the absorption spectrum of an aqueous solution of the fluorescent substance (solvent: deionized water), and the peak position of the obtained absorption spectrum is defined as the maximum absorption wavelength. In another preferred embodiment, the composite comprises two nanoparticles 1, wherein the fluorescent substance is positioned between the first nanoparticle and the second nanoparticle in the composite.

[0029] The composition of the nanoparticle body will be described below. The nanoparticle body will be described with reference to Figure 4. Figure 4 is a schematic cross-sectional view showing the nanoparticle body according to this embodiment. The nanoparticle body 1 according to this embodiment comprises metal nanoparticles 2, a polymer film 3 that covers the surface of the metal nanoparticles 2, a specific binding substance 4 that specifically binds to the test substance in the sample, and a fluorescent substance 6 labeled on the surface of the polymer film 3.

[0030] Nanoparticle 1 can be used in plasmon-excited fluorescence analysis. In other words, nanoparticle 1 can be used in surface plasmon-excited enhanced fluorescence spectroscopy immunoassay. Nanoparticle 1 can capture a test substance in a sample and form a complex containing two or more nanoparticles 1 and the test substance. When the complex is irradiated with excitation light, plasmon resonance occurs and a near-field is formed. Fluorescence is enhanced by the near-field and a dipole-dipole mechanism. For example, nanoparticle 1 can capture one test substance in a sample and form a complex containing two nanoparticles 1 and the test substance. In this case, nanoparticle 1 includes a first nanoparticle and a second nanoparticle, and the first and second nanoparticles form a complex bound to the test substance.

[0031] The nanoparticles 1 may also have their nonspecific binding sites blocked by a blocking agent. Blocked nanoparticles 1 suppress the formation of nonspecific binding of the specific binding substance 4 to substances other than the target substance (i.e., substances other than the test substance), thereby reducing background and false positive signals and improving the signal-to-noise ratio (S / N ratio). Examples of blocking agents include bovine serum albumin (BSA), skim milk, proteins such as casein, and chemically synthesized polymers.

[0032] 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 sodium heparin.

[0033] (Metal nanoparticles) The metal nanoparticles 2 are coated with a polymer film 3 on their surface. Depending on the type of metal, the metal nanoparticles 2 interact with light of a specific wavelength, causing plasmon resonance. Silver nanoparticles have plasmon resonance peaks at 400 nm to 530 nm, while gold nanoparticles have peaks at 510 nm to 580 nm. This varies depending on the particle size. For example, silver nanoparticles with a particle size of 20 nm resonate with light at a wavelength of 405 nm, and gold nanoparticles with a particle size of 20 nm resonate with light at a wavelength of 524 nm. The particle sizes (average primary particle size) of the metal nanoparticles 2 are, for example, 5 nm to 100 nm, 40 nm to 90 nm, and 50 nm to 80 nm. The particle size of the metal nanoparticles 2 can be obtained by taking images of the metal nanoparticles 2 using a scanning electron microscope (SEM) or transmission electron microscope (TEM), measuring the particle size of the metal nanoparticles 2 in the images, and calculating the average value of multiple particle sizes (number of measurements: for example, at least 10 or more). The metal nanoparticles 2 preferably consist of gold or silver, and more preferably consist of silver.

[0034] (polymer membrane) The polymer film 3 covers the surface of the metal nanoparticles 2. The polymer film 3 functions as a metal quenching molecular film. In the composite, the polymer film 3 can position the fluorescent substance 6 at least by the thickness of the polymer film 3 away from the surface of the metal nanoparticles 2. This suppresses the quenching of the excited fluorescent substance 6 by contact with the surface of the metal nanoparticles 2 (quenching by the Dexter Electron Transfer mechanism), thereby suppressing a decrease in detection sensitivity. The presence of the polymer film 3 can be confirmed by imaging the nanoparticles 1 using SEM or TEM and observing the nanoparticles 1 in the image.

[0035] The polymer film 3 will be described with reference to Figure 5. Figure 5 is an enlarged view of part A in Figure 4, and is an enlarged cross-sectional view of the vicinity of the interface between the polymer film 3 of the nanoparticle body 1 and the surface of the metal nanoparticles 2. The polymer film 3 includes 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 between it and the surface of the metal nanoparticles 2. More specifically, the polymer film 3 includes a sulfur atom-mediated bonding site 3a between it and the surface of the metal nanoparticles 2, and a primary ammonium group (-NH3) as the positively charged group 3b. + The polymer contains a 3c hydrophobic group. The bonding 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 negatively charged surface of the metal nanoparticle 2. The hydrophobic group 3c forms a hydrophobic bond c with the surface of the metal nanoparticle 2.

[0036] Since all three of the above bonds create relatively strong bonds between the surface of the metal nanoparticles 2 and the polymer film 3, the polymer film 3 is stably fixed to the surface of the metal nanoparticles 2 by at least one of the three bonds. This prevents the polymer film 3 from peeling off from the surface of the metal nanoparticles 2. As a result, the detachment of the specific binding substance 4 due to the peeling of the polymer film 3 is suppressed, and the decrease in detection sensitivity is suppressed. In addition, the exposure of the surface of the metal nanoparticles 2 due to the peeling of the polymer film 3 is suppressed, and quenching due to contact with the excited fluorescent substance 6 is suppressed, thus suppressing the decrease in detection sensitivity. Furthermore, since the polymer film 3 is composed of polymer 3A, it is easier to chemically modify than the silica layer, and the need for surface modification is lower. As a result, the film thickness can be reduced compared to the silica layer, and the distance between the metal nanoparticles 2 in the composite can be reduced. Therefore, a near field can be formed more efficiently, and the detection sensitivity can be improved. From the above, the nanoparticle body 1 according to this embodiment has superior detection stability.

[0037] As shown in Figure 5, the polymer 3A constituting the polymer film 3 may contain 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 between itself and the surface of the metal nanoparticles 2. The presence of the sulfur atom-mediated bonding site 3a, the positively charged group 3b, and the hydrophobic group 3c can be confirmed by measuring the signals originating from them using infrared spectroscopy, nuclear magnetic resonance spectroscopy, energy-dispersive X-ray spectroscopy (TEM-EDS), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Furthermore, the polymer 3A constituting the polymer film 3 may have a site containing a disulfide bond (-SS-) as a side chain. The site containing the disulfide bond may have a positively charged group 3b and / or a hydrophobic group 3c.

[0038] -Bonding site via sulfur atom- The sulfur atom-mediated bonding site 3a is formed, for example, by mixing a polymer having a site containing a disulfide bond as a side chain with metal nanoparticles 2. If the polymer used as a raw material has a hydrophobic group 3c via a disulfide bond in its side chain, as shown in Figure 5, it forms a sulfur atom-mediated bonding site between the polymer and the surface of the metal nanoparticles 2 (see Figure 5 and Figure 15 described later).

[0039] -Positively charged group- The polymer film 3 may contain at least one positively charged group 3b in the side chains of the polymer 3A constituting the polymer film 3. The positively charged group 3b forms a relatively strong electrostatic bond with the surface of the metal nanoparticles 2. In this specification, a positively charged group is a group having a valency of 1 or more and being completely positively ionized. When considering multiple positively charged groups 3b contained in the polymer 3A constituting the polymer film 3, the positively charged group 3b is expressed by the following formula (1):

number

[0040] Positively charged groups include, for example, primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH2) + It is at least one selected from the group consisting of )NH2).

[0041] -Hydrophobic group- Polymer film 3 may contain at least one hydrophobic group 3c in the side chain of polymer 3A constituting polymer film 3. The hydrophobic group 3c is, for example, at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups.

[0042] Examples of aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups. Aromatic carbocyclic groups are groups that do not contain aromatic heterocyclic groups but contain aromatic rings in which all member 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 groups that contain aromatic rings in which at least one of the member atoms is a heteroatom (more specifically, oxygen atoms, sulfur atoms, and nitrogen atoms, etc.). Examples of aromatic heterocyclic groups include nitrogen-containing aromatic heterocyclic groups (more specifically, imidazoyl groups and pyridyl groups (pyridinyl groups), etc.), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups.

[0043] Aliphatic cyclic groups are groups that contain a cyclic group consisting of a non-aromatic ring, without an aromatic ring. Examples of aliphatic cyclic groups include aliphatic carbocyclic groups and aliphatic heterocyclic groups. Aliphatic carbocyclic groups are groups that contain a non-aromatic ring in which all ring member atoms are carbon atoms, and examples include cycloalkyl groups. Aliphatic heterocyclic groups are groups that contain a non-aromatic ring in which at least one of the ring member atoms is a heteroatom.

[0044] Aliphatic chain groups are chain-like (more specifically, linear and branched) groups that do not contain aromatic or non-aromatic rings. Examples of aliphatic chain groups include aliphatic carbon chain groups (more specifically, alkyl and alkylene groups, etc.) and aliphatic heterochain groups.

[0045] The polymer 3A constituting the polymer film 3 can form hydrophobic bonds c between the hydrophobic groups 3c that polymer 3A may possess and the surface of the metal nanoparticles 2. The polymer 3A constituting the polymer film 3 can also form other types of hydrophobic bonds c. For example, a hydrophobic bond c can be formed between a hydrophobic group 3c that is bonded to the surface of the metal nanoparticles 2 via a sulfur atom (more specifically, a pyridyl group bonded to the surface of the metal nanoparticles 2 via a sulfur atom in Figure 15, described later) and a hydrophobic group 3c that polymer 3A constituting the polymer film 3 may possess (more specifically, an alkylene group possessed by polymer 3A in Figure 15) 3c. When such a hydrophobic bond c is formed, the polymer film 3 is more stably fixed to the surface of the metal nanoparticles 2. The hydrophobic group 3c that is bonded to the surface of the metal nanoparticles 2 via a sulfur atom is formed as follows. As previously described, the sulfur atom-mediated bonding site 3a can be formed, for example, by mixing a polymer having a hydrophobic group 3c via a disulfide bond in its side chain with the metal nanoparticles 2. Here, the hydrophobic group 3c bonded to the sulfur atom also bonds to the surface of the metal nanoparticle 2. In this way, a hydrophobic group 3c is formed on the surface of the metal nanoparticle 2, bonded via the sulfur atom.

[0046] The polymer 3A constituting the polymer film 3 may form bonds via sulfur atoms through a linker portion derived from the crosslinking agent. Examples of such crosslinking agents include amino group-sulfhydryl group crosslinking agents (more specifically, NHS-maleimide group crosslinking agents, etc.).

[0047] The thickness of the polymer film 3 is preferably 1 nm to 50 nm, and more preferably 1 nm to 10 nm. When the thickness of the polymer film 3 is 50 nm or less, for example, in a composite composed of two metal nanoparticles 2, the separation distance (separation distance) becomes such that a near field is efficiently formed in the space between the two metal nanoparticles 2, thus further improving the detection sensitivity. Furthermore, when the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance 6 in the composite are arranged at a predetermined distance from each other, so the quenching of fluorescence emitted from the excited fluorescent substance 6 during measurement is suppressed, further improving the detection sensitivity. In this specification, the separation distance (or separation distance) refers to the minimum distance (shortest distance) between the surfaces of two metal nanoparticles contained in each of the two nanoparticle bodies that are bonded together via the test substance in the composite.

[0048] (Method for forming polymer films) The method for forming the polymer film 3 comprises the step of contacting a polymer having disulfide bonds in its side chains with metal nanoparticles 2 to form a polymer film 3 in which the polymer is bonded to the surface of the metal nanoparticles 2 via sulfur atoms. The polymer used as a raw material includes, for example, at least one group selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c that are bonded via disulfide bonds. In this case, the step involves forming the polymer film 3 and bonding at least one group selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c to the surface of the metal nanoparticles 2. Furthermore, in the method for forming the polymer film 3, if a polymer having a positively charged group 3b and / or a hydrophobic group 3c via a disulfide group in its side chain is used as a raw material, the polymer 3A constituting the polymer film 3 may also have a positively charged group 3b and / or a hydrophobic group 3c via a disulfide group in its side chain. The positively charged group 3b and / or hydrophobic group 3c in polymer 3A are groups that remained unreacted in the method for forming the polymer film 3.

[0049] The method will be explained in detail with reference to Figure 6. Figure 6 is a schematic diagram showing the method for forming the polymer film 3. Polymer 3B has positively charged groups 3b and hydrophobic groups 3c in its side chains, which are linked via disulfide bonds. When polymer 3B is brought into contact with metal nanoparticles 2, the bonds between the sulfur atoms of the disulfide bonds are broken, and polymer 3B bonds to the surface of metal nanoparticles 2 via sulfur atoms. In parallel, the positively charged groups 3b and hydrophobic groups 3c bond to the surface of metal nanoparticles 2 via sulfur atoms, respectively. The method for forming the polymer film 3 can perform both the formation of the polymer film 3 on the surface of metal nanoparticles 2 and the modification of the surface of metal nanoparticles 2 (for example, surface modification of the functional groups positively charged groups 3b and / or hydrophobic groups 3c) in a single step. Therefore, this method is cost-effective.

[0050] (specific binding substance) Specific binding substance 4 is a nano-sized substance (with a maximum size of 3 to 15 nm) that specifically binds to the test substance (described in the fourth embodiment) in the sample. Examples of specific binding substance 4 include antibodies (hereinafter referred to as nano-antibodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA and RNA chains). For example, a nano-antibody as specific binding substance 4 specifically binds to the antigen (test substance) at its tip (antigen binding site) through an antigen-antibody reaction, forming a complex. A ligand as specific binding substance 4 forms a complex with a protein (test substance) through a specific protein-ligand binding reaction. A nucleic acid chain as specific binding substance 4 forms a pair (double helix) of complementary nucleic acid chains based on base pair complementarity. An enzyme as specific binding substance 4 forms an enzyme-substrate complex with the substrate (test substance) at its active site (active site) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as hydrogen bonds, as well as bonds resulting from intermolecular forces, hydrophobic interactions, and charge interactions.

[0051] Nanoantibodies include, for example, VHH (variable domain of heavy chain antibody) antibodies, Fab (Fragment Antigen Binding) antibodies, and their variants. VHH antibodies are single-domain antibodies. Variants are antibodies in which a portion of the amino acid sequence has been rearranged or a substituent has been introduced, within a range that maintains specific binding to the antigen. Nanoantibodies are preferably VHH antibodies. When the nanoantibody is a VHH antibody, because VHH antibodies have a relatively small volume, the distance (separation distance) between the two metal nanoparticles 2 in the complex can be narrowed, more efficiently forming a near-field and further increasing the fluorescence intensity.

[0052] The molecular mass of the nanoantibody 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 volume of the nanoantibody is relatively small, which narrows the separation distance in the complex, allows for more efficient formation of the near field, and further increases the fluorescence intensity. Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.

[0053] The specific binding substance 4 may be directly bound to the polymer film 3, or it may be a linker portion derived from a crosslinking agent (more specifically, an NHS-maleimide group crosslinking agent, etc.) (more specifically, SM(PEG) 6 It may also be indirectly bonded to the polymer film 3 via (etc.).

[0054] (Fluorescent substance) The fluorescent substance 6 is labeled on the surface of the polymer film 3 and / or the specific binding substance 4. The fluorescent substance 6 is excited by the near-field formed by plasmon resonance and emits fluorescence. Examples of the fluorescent substance 6 include metal complexes such as europium and ruthenium, as well as dyes from the Alexsa Fluor series (registered trademark) (manufactured by Molecular Probes).

[0055] 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. When the Stokes shift of the fluorescent substance 6 is large, the absorption spectrum and the fluorescence spectrum do not overlap easily, so that the excitation light (scattered light) does not easily enter the fluorescence to be detected, and the fluorescence intensity can be measured more accurately.

[0056] The fluorescence spectrum of a fluorescent substance is preferably sharp. A sharp fluorescence spectrum is less likely to overlap with the absorption spectrum, which means that excitation light (and its scattered light) is less likely to interfere with the fluorescence being detected, allowing for more accurate measurement of fluorescence intensity.

[0057] (Measuring device for detecting the test substance) A measuring device using the above-described method for detecting the test substance will be explained with reference to Figure 13. Figure 13 is a diagram of the measuring device. As shown in Figure 13, the measuring device 100 comprises 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 light source. The excitation light irradiation optical system 120 adjusts the cross-sectional diameter, such as focusing the excitation light 112, and outputs incident excitation light 122. The excitation light irradiation optical system 120 consists of 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 is incident on the reagent container 130 and irradiates the sample to be measured inside the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell and a slide, etc.) and a microfluidic chip. The microfluidic chip is a chip having minute channels. When the reagent container 130 is a microfluidic chip, for example, the nanoparticle body (reagent) and the sample according to the first embodiment can be mixed and continuously supplied. Therefore, it is not necessary to mix and prepare the sample to be measured in advance, and continuous measurement becomes possible.

[0058] The sample to be measured, when irradiated with incident excitation light 122, emits fluorescence (detection light 132). The light-receiving optical system 140 is positioned perpendicular to the direction of propagation of the incident excitation light 122 into the reagent container 130. The light-receiving optical system 140 can adjust the cross-sectional diameter of the detection light 132 emitted from the sample to remove scattered light from the incident excitation light 122 or adjust the light intensity. The light-receiving optical system 140 consists of a lens 144 and an optical filter 146. The optical filter 146 is, for example, a bandpass filter and a dichroic mirror.

[0059] The fluorescence 142 that has passed through the photodetector optical system 140 is detected by the photodetector 150. The photodetector 150 is, for example, a PD, APD, PMT, CCD camera, and spectrometer. The photodetector 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring the fluorescence spectrum, and creating a two-dimensional planar fluorescence image.

[0060] <Second Embodiment: Nanoparticle Body> The second embodiment differs from the first embodiment in the spectral characteristics of the fluorescent material. These differing configurations will be primarily described below. Note that in the second embodiment, reference numerals identical to those in the first embodiment indicate the same configuration, and therefore their explanation will be omitted.

[0061] In the first embodiment, the fluorescence used to detect the test substance was "excitation-induced fluorescence" induced by the overlap 208 between the absorption spectrum 202 of the fluorescent substance 6 and the plasmon resonance spectrum 206. In the second embodiment, the fluorescence used to detect the test substance is "emission-induced fluorescence" induced by the overlap between the fluorescence spectrum of the fluorescent substance 6A and the plasmon resonance spectrum 206. In this specification, the fluorescent substance in the nanoparticle body according to the second embodiment is also referred to as "fluorescent substance 6A" to indicate and distinguish it from the fluorescent substance 6 in the nanoparticle body according to the first embodiment, as its spectral characteristics differ.

[0062] The nanoparticles according to this embodiment can enhance detection sensitivity. While not bound by any particular theory, the reason is presumed to be as follows: In the nanoparticles according to this embodiment, the fluorescent substance 6A is excited by light at the emission wavelength of the plasmon resonance in a complex formed by two or more nanoparticles bound together via the test substance. This causes an overlap between the fluorescence spectrum of the fluorescent substance 6A and the plasmon resonance spectrum 206 derived from the metal nanoparticles in the complex, inducing fluorescence for detecting the test substance through a dipole-dipole mechanism and the Purcell effect due to multipole resonance. Therefore, since the fluorescent substance 6A is sufficiently excited by light at the emission wavelength of the plasmon resonance derived from the complex, detection sensitivity can be enhanced.

[0063] This will be explained with reference to Figure 7. Figure 7 is a conceptual diagram showing the plasmon resonance spectrum 201 originating from single-particle metal nanoparticles 2, the plasmon resonance spectrum 206 originating from metal nanoparticles 2 in the composite, and the fluorescence spectrum 210 of fluorescent material 6A. In this embodiment, fluorescent material 6A is selected such that there is an overlap 212 between the fluorescence spectrum 210 of fluorescent material 6A and the resonance spectrum 206 originating from metal nanoparticles in the composite (preferably large). Thus, the fluorescence wavelength range WR of fluorescent material 6A is E This is the first emission wavelength range of plasmon resonance WR E1 When this coincides, the fluorescent substance 6A is sufficiently excited, the fluorescence is enhanced, and the detection sensitivity is greatly improved. Therefore, it is considered that the nanoparticles according to this embodiment can increase detection sensitivity. In other words, from the viewpoint of increasing the detection sensitivity of the test substance, it is important to select a fluorescent substance 6A that creates a second region R2. Here, the second region R2 refers to the region where the multipole resonance spectrum 206 and the fluorescence spectrum 210 of the fluorescent substance overlap (the region corresponding to the overlap 212), as shown in Figure 7. From the viewpoint of further increasing the detection sensitivity of the test substance, it is preferably important to select a fluorescent substance 6A that has a large second region R2.

[0064] [Scheme 2] Scheme 2 of the process for detecting the test substance using nanoparticles according to this embodiment: [ka] (In the elementary processes (1) to (3) of Scheme 2, S0 represents the ground state, S1 represents the excited singlet state, * represents the excited state, Flu represents the fluorescent substance 6A (in the complex), and M represents the metal nanoparticle 2 (in the complex)) The following is shown.

[0065] Scheme 2 consists of elementary processes (1) to (3). As shown in elementary process (1), for example, the fluorescent substance 6A is excited by external light. As shown in elementary process (2), the excited fluorescent substance 6A relaxes and emits fluorescence. As shown in elementary process (3), the emitted fluorescence induces plasmon resonance (multipole resonance) on the surface of the metal nanoparticle 2 (that is, plasmon resonance in the composite is induced by the fluorescence emitted by the fluorescent substance 6A). This plasmon resonance (multipole resonance) is in the emission wavelength range WR of the fluorescent substance 6A. E This is the first emission wavelength range of plasmon resonance WR E1 When these conditions overlap, fluorescence emission from the excited-state fluorescent substance 6A is efficiently induced.

[0066] In one preferred embodiment, the first emission wavelength range WR E1 The maximum fluorescence wavelength of the fluorescent material 6A in the composite is located in the range of 500-700 nm (more preferably 550-700 nm, and even more preferably 600-700 nm). In other words, the first emission wavelength region WR of the plasmon resonance spectrum originating from the metal nanoparticles 2 in the composite is E1 The maximum fluorescence wavelength of fluorescent substance 6A is located in the 500-700 nm range. Examples of fluorescent substances include fluorescein derivatives, rhodamine derivatives, cyanine dyes, and Alexa Flour® manufactured by Molecular Probes. Among these, examples of fluorescent substance 6A with a maximum fluorescence wavelength of 500-700 nm include "Ruthenium(II)tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd., and Alexa Flour® series 430, 488, 532, 546, 555, 568, 594, and 640. The maximum fluorescence wavelength can be determined as follows: Measure the absorption spectrum of an aqueous solution of the fluorescent substance (solvent: deionized water) and determine the peak position of the obtained absorption spectrum. Irradiate the fluorescent substance 6A with excitation light at the wavelength of the peak position, measure the fluorescence spectrum of the aqueous solution of the fluorescent substance, and set the peak position of the obtained fluorescence spectrum as the maximum fluorescence wavelength.

[0067] <Third Embodiment: Nanoparticle Body> The third embodiment differs from the first and second embodiments in its spectral characteristics of the fluorescent material. These differing configurations will be primarily described below. Note that in the third embodiment, reference numerals identical to those in the first and second embodiments correspond to the same components as in the first and second embodiments, and therefore their descriptions are omitted.

[0068] In the third embodiment, the fluorescence for detecting the test substance is induced at the overlap 208 between the absorption spectrum 202 and the plasmon resonance spectrum 206 of the fluorescent substance 6 shown in the first embodiment, and at the overlap 212 between the fluorescence spectrum 210 and the plasmon resonance spectrum 206 of the fluorescent substance 6A shown in the second embodiment. In other words, the plasmon resonance in the complex is excitation-induced fluorescence induced by external light, and emission-induced fluorescence induced by fluorescence. In this specification, the fluorescent substance in the nanoparticle body according to the third embodiment is also referred to as "fluorescent substance 6B" to indicate and distinguish it from the fluorescent substance 6 in the nanoparticle body according to the first embodiment and the fluorescent substance 6A in the nanoparticle body according to the second embodiment, as these have different spectral characteristics.

[0069] This will be explained with reference to Figure 8. Figure 8 is a conceptual diagram showing the plasmon resonance spectrum 201 originating from single-particle metal nanoparticles 2 and the plasmon resonance spectrum 206 of metal nanoparticles 2 in the composite, as well as the absorption spectrum 202 and fluorescence spectrum 210 of fluorescent substance 6B. In this embodiment, from the viewpoint of increasing the detection sensitivity of the test substance, the overlap between the absorption spectrum 202 and fluorescence spectrum 210 of fluorescent substance 6B and the resonance spectrum 206 originating from metal nanoparticles 2 in the composite (each in the absorption wavelength range WR) is considered. A and the first emission wavelength range WR E1 Overlap with 208 and fluorescence wavelength range WR E and the first emission wavelength range WR E1The fluorescent substance 6B is selected such that there is an overlap (212) with the other element. Furthermore, in a preferred embodiment, the fluorescent substance 6B may be selected such that the overlaps 208 and 212 are large, from the viewpoint of further increasing the detection sensitivity of the test substance. As a result, the fluorescent substance 6B is sufficiently excited, the fluorescence is enhanced, and the detection sensitivity is greatly improved. Therefore, it is considered that the nanoparticles according to this embodiment can increase the detection sensitivity.

[0070] [Scheme 3] Scheme 3 of the process for detecting the test substance using nanoparticles according to this embodiment: [ka] (In the elementary processes (1) to (4) of Scheme 3, S0 represents the ground state, S1 represents the excited singlet state, * represents the excited state, Flu represents the fluorescent substance 6B (in the complex), and M represents the metal nanoparticle 2 (in the complex)) The following is shown.

[0071] Scheme 3 consists of elementary processes (1) to (4). As shown in elementary process (1), when the composite formed by trapping the test substance is irradiated with external light, plasmon resonance (multipole resonance) is induced on the surface of the metal nanoparticles 2 (that is, plasmon resonance in the composite is induced by external light). As shown in elementary process (2), the fluorescent substance 6B is excited by a dipole-dipole mechanism and a near-field. Absorption wavelength range WR of fluorescent substance 6B A This is the first emission wavelength range of plasmon resonance WR E1 When this coincides, the fluorescent substance 6B is efficiently excited. As shown in elementary process (3), the excited fluorescent substance 6B relaxes and emits fluorescence. As shown in elementary process (4), the emitted fluorescence induces plasmon resonance (multipole resonance) on the surface of the metal nanoparticle 2 (that is, plasmon resonance in the composite is induced by the fluorescence emitted by the fluorescent substance 6B). This plasmon resonance (multipole resonance) is in the emission wavelength range WR of the fluorescent substance 6B.E This is the first emission wavelength range of plasmon resonance WR E1 When these conditions overlap, fluorescence emission from the excited fluorescent substance 6B is efficiently induced.

[0072] The amount of light from the external illumination source is greater than the amount of light from the fluorescence source. Therefore, from the viewpoint of further improving detection sensitivity, it is preferable to use the first emission wavelength range WR. E1 and the absorption wavelength range WR of fluorescent substance 6B A The first region R1 where these overlap is the second emission wavelength region WR. E2 and the absorption wavelength range WR of fluorescent substance 6B A The fluorescent substance 6B is selected so that it is larger than the second region R2 where it overlaps (see Figure 8).

[0073] (Method for determining the relative size of the first region R1 and the second region R2) The relative magnitudes of the first region R1 and the second region R2 are determined as follows: The plasmon resonance spectrum of the complex is measured using a fluorescence microspectroscopy apparatus (detailed in the examples). The multipole resonance spectrum 206 in the plasmon resonance spectrum is identified. The absorption spectrum 202 and fluorescence spectrum 210 of the fluorescent substance 6B are measured (the sample of fluorescent substance 6B is prepared with deionized water as the solvent). An overlap 208 is created between the normalized multipole resonance spectrum 206 and the normalized absorption spectrum 202 of the fluorescent substance 6B. The integral value of the overlap 208 is calculated. An overlap 212 is created between the normalized multipole resonance spectrum 206 and the normalized fluorescence spectrum 210 of the fluorescent substance 6B. The integral value of the overlap 212 is calculated. Based on the relative magnitudes of the obtained integral values, the relative magnitudes of the first region R1 and the second region R2 are determined.

[0074] <Fourth Embodiment: Composite> The composite will be described with reference to Figure 9. Figure 9 is a schematic cross-sectional view showing the composite according to the fourth embodiment. The composite according to the fourth embodiment comprises two nanoparticles 1 according to the first embodiment, the two nanoparticles 1 comprising a first nanoparticle 10 and a second nanoparticle 20, and the first nanoparticle 10 and the second nanoparticle 20 are bound together via the test substance 30. The composite 40 comprises the test substance 30, which is the substance to be detected, and the two nanoparticles 10 and 20. The two nanoparticles 10 and 20 are bound together in the composite 40 via the test substance 30. In other words, the nanoparticles 10 and 20 according to the first embodiment are bound together via the test substance 30 to form the composite according to the fourth embodiment. Of the two nanoparticles 10 and 20, one will be referred to as the first nanoparticle 10 and the other as the second nanoparticle 20.

[0075] In the composite 40, the first nanoparticle body 10 comprises first metal nanoparticles 12, a first polymer film 13 covering the surface of the first metal nanoparticles 12, a first specific binding substance 14 that specifically binds to the test substance 30 in the sample, and a first fluorescent substance 16 labeled on the first polymer film 13. The first specific binding substance 14 is bound to the surface of the first polymer film 13. In other words, the first nanoparticle body 10 comprises first metal nanoparticles 12 as metal nanoparticles, a first polymer film 13 as a polymer film, a first specific binding substance 14 as a specific binding substance, and a first fluorescent substance 16 as a fluorescent substance. Furthermore, in the composite 40, the second nanoparticle body 20 comprises second metal nanoparticles 22, a second polymer film 23 covering the surface of the second metal nanoparticles 22, a second specific binding substance 24 that specifically binds to the test substance 30 in the sample, and a second fluorescent substance 26 labeled on the second polymer film 23. The second specific binding substance 24 is bound to the surface of the second polymer film 23. In other words, the second nanoparticle body 20 comprises second metal nanoparticles 22 as metal nanoparticles, a second polymer film 23 as a polymer film, a second specific binding substance 24 as a specific binding substance, and a second fluorescent substance 26 as a fluorescent substance.

[0076] From the viewpoint of further increasing fluorescence intensity, it is preferable that the separation distance L be small in a range in which the excited fluorescent substances 16 and 26 are not easily quenched. More specifically, in a preferred embodiment, the two nanoparticles 10 and 20 in the composite 40 are in close proximity to each other. In a more preferred embodiment, the two nanoparticles 10 and 20 are in close proximity to each other such 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 are in contact. In an even more preferred embodiment, the two nanoparticles 10 and 20 are in close proximity to each other such that at least one of the polymer films of 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 is contracted and in contact with each other.

[0077] In a more preferred embodiment, if at least one of the polymer films 13 and 23 shrinks and comes into contact with each other, for example, in the composite 40 shown in Figure 9, it is considered that the test substance 30, at least one of the specific binding substances 14 and 24 that bind to the test substance 30, and the fluorescent substances 16 and 26 can be incorporated into the polymer films 13 and 23. Furthermore, in a more preferred embodiment, if the polymer films 13 and 23 come into contact with each other, for example, in the composite 40 shown in Figure 9, it is considered that at least one of the test substance 30, the specific binding substances 14 and 24 that bind to the test substance 30, and the fluorescent substances 16 and 26 can be incorporated into the polymer films 13 and 23, similar to the more preferred embodiment (the same applies to the composite shown in Figure 10, which will be described later).

[0078] In this embodiment, the fluorescence intensity can be increased because the film covering the surface of the metal nanoparticles 12 and 22 is a polymer film 13 and 23. Although not bound by any particular theory, the reason is presumed to be as follows: The film covering the surface of the metal nanoparticles 12 and 22 is a polymer film 13 and 23, and the polymer film 13 and 23 have relatively high flexibility compared to inorganic films containing inorganic oxides. Therefore, in the composite 40, the polymer film 13 and 23 can contract, which allows the two metal nanoparticles 12 and 22 to be closer together than a distance equivalent to two polymer film thicknesses (the thickness of polymer film 13 + the thickness of polymer film 23). In other words, because the film covering the surface of the metal nanoparticles 12 and 22 is a polymer film 13 and 23, the separation distance L can be less than two polymer film thicknesses. This makes it easier to obtain a plasmon enhancement effect and further increases the fluorescence intensity. In this specification, the "thickness of the polymer film" in "two times the thickness of the polymer film" refers to the thickness of the non-shrinking portion of the polymer film 13, 23 that is not subject to separation, not the thickness of the shrinking portion of the polymer film 13, 23 that is subject to separation distance.

[0079] In this embodiment, the fluorescence intensity can be further increased because the polymer films 13 and 23 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 (for example, the polymer 3A constituting the polymer films 13 and 23 includes 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). Although not bound by any particular theory, the reason 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 nanoparticles 12 and 22. Therefore, it is thought that polymer 3A has a network structure and coats the surface of the metal nanoparticles 12 and 22 in a network-like manner. Because polymer 3A has such a network structure, it also has relatively high flexibility. Therefore, in the composite 40, the polymer films 13 and 23 can contract further, which allows the two metal nanoparticles 12 and 22 to come closer together than a distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two layers of polymer film thickness, further enhancing the plasmon effect and increasing the fluorescence intensity.

[0080] In a preferred embodiment, the polymer 3A constituting the polymer films 13,23 includes 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 in its side chain (more specifically, at the end of the side chain). In a preferred embodiment, the fluorescence intensity can be further increased. Although not bound by any particular theory, the reason 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 nanoparticles 12,22. Therefore, the polymer 3A is thought to have a network structure, and to coat the surface of the metal nanoparticles 12,22 in a network-like manner using the side chain as a binding site. Because the polymer 3A has a network structure in this way, it also has relatively high flexibility. Therefore, in the composite 40, the polymer films 13,23 can contract further, which allows the two metal nanoparticles 12,22 to come closer together than a distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two layers of polymer film thickness, further enhancing the plasmon effect and increasing the fluorescence intensity.

[0081] The separation distance L between the first nanoparticle 10 and the second nanoparticle 20 is, for example, 12 nm to 52 nm, preferably 12 nm to 27 nm. The separation distance L is the distance between the first metal nanoparticle 12 and the second metal nanoparticle 22, and is the distance at which the line segment connecting a first point P1 on the surface of the first nanoparticle 10 and a second point P2 on the surface of the second nanoparticle 20 is minimized. When the separation distance L is 52 nm or less, when the composite 40 is irradiated with excitation light, a near-field is generated more efficiently in the space near the surfaces between the first and second metal nanoparticles 12 and 22, thereby increasing the fluorescence intensity.

[0082] The film covering the surface of the metal nanoparticles 12 and 22 is a polymer film 13 and 23, and the polymer 3A constituting the polymer film 13 and 23 has at least one selected from the group consisting of a sulfur atom-mediated bonding site 3a (for example, on the side chains (more specifically, at the ends of the side chains)), a positively charged group 3b, and a hydrophobic group 3c. Therefore, as described above, the separation distance L can be closer than the distance equivalent to two times the thickness of the polymer film covering the surface of the two metal nanoparticles 12 and 22 in the composite 40. For example, if the thickness of the polymer film 13 and 23 is 5 nm, the separation distance L can be less than 10 nm (more specifically, 2-9 nm, 3-8 nm, and 4-7 nm, etc.).

[0083] The composite 40 may consist of two nanoparticles 10 and 20, as shown in Figures 9 to 11, or it may consist of three nanoparticles 10, 20, and 60, for example, as shown in Figure 12. The composite may also consist of four or more nanoparticles. Figure 11 shows a composite 40 composed of two nanoparticles. Figure 12 shows a composite 40 composed of three nanoparticles.

[0084] In the first to third embodiments, the multipole resonance was described as plasmon resonance originating from the metal nanoparticles 12 and 22 in the composite 40. This will be explained in more detail with reference to Figures 11 and 12. In Figure 11, the binding sites between the nanoparticles 10 and 20 in the composite 40 (more specifically, the test substance 30 and the specific binding substances 14 and 24) are denoted by binding points 50 (the same applies to Figure 12). In the composite 40 composed of the two nanoparticles 10 and 20, the direction of vibration of the electric field component of the excitation light (the polarization direction of the electric field component of the excitation light) is along the long axis D of the composite 40. LA When parallel to the long axis, the free electrons on the surface of metal nanoparticles 12,22 are aligned along the long axis D LA Collective oscillations can occur. Therefore, multipole resonance is caused by dipole-dipole interaction. On the other hand, the direction of oscillation of the electric field component of the excitation light is the short axis direction D of the complex 40. SA When parallel to the direction, the free electrons on the surface of metal nanoparticles 12,22 are aligned along the short axis D. SAThis can induce collective vibrations. Therefore, dipole resonance is triggered, similar to that of single-particle metal nanoparticles.

[0085] In the complex 40 composed of three nanoparticles 10, 20, and 60, the number of vibration directions D1, D2, and D3 of the electric field components of the excitation light that are permissible for multipole resonance is 3, which is more than in the complex 40 composed of two nanoparticles 10 and 20. Furthermore, the complex 40 composed of three nanoparticles 10, 20, and 60 has 3 binding points 50, which is more than in the complex 40 composed of two nanoparticles 10 and 20. Thus, it is possible to include multiple binding points 50 (where two nanoparticles 10 and 20 are bound via one test substance 30). Therefore, the more nanoparticles 10 and 20 that make up the complex 40, the easier it is for fluorescence to be enhanced, and the potentially better the detection sensitivity.

[0086] (Fluorescent substance) It is preferable that the fluorescent materials 16 and 26 are positioned between the first metal nanoparticle 12 and the second metal nanoparticle 22, as shown in Figures 9 and 10. This is because the space between the metal nanoparticles 12 and 22 is a space where a near-field is efficiently generated, and by positioning the fluorescent materials 16 and 26 in this space, the fluorescence intensity is easily increased by the Parcell effect.

[0087] (Test substance) The test substance 30 is a substance to be detected contained in the sample. Examples of the test substance 30 include antigens, proteins, substrates, and nucleic acid chains. The test substance 30 specifically binds to specific binding substances 14 and 24. For example, an antigen has at least two antigenic determinants (epitopes), which form specific bindings with the first and second specific binding substances 14 and 24. Antigens include proteins such as c-reactive proteins, myoglobin, troponin T, troponin I, and BNP, as well as antigenic proteins of viruses such as influenza virus and RSV. The test substance 30 is a test substance derived from a sample such as blood, plasma, urine, or saliva. That is, examples of samples containing the test substance 30 are blood, plasma, serum, urine, and saliva. The sample may further contain a solvent and buffer (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, and MES buffer, etc.).

[0088] <Other Embodiments> The present invention is not limited to the embodiments described above, and design modifications are possible without departing from the spirit of the invention.

[0089] In the first embodiment, polymer 3B had one positively charged group 3b and one hydrophobic group 3c bonded via disulfide bonds, but is not limited to this. Polymer 3B may have two or more positively charged groups 3b and hydrophobic groups 3c independently. That is, the number of side chains to which positively charged groups 3b and hydrophobic groups 3c are each bonded in polymer 3B may be two or more.

[0090] In the fourth embodiment, the first and second fluorescent substances 16 and 26 were labeled on the first and second polymer films 13 and 23, respectively, but the invention is not limited to this. For example, Figure 10 is a schematic cross-sectional view showing a composite according to a modified example of the fourth embodiment. As shown in Figure 10, the first and second fluorescent substances 16 and 26 may be labeled on the first and second specific binding substances 14 and 24, respectively. In this case, it is easier to position the first and second fluorescent substances 16 and 26 between the first metal nanoparticles 12 and the second metal nanoparticles 22, and detection intensity is improved, which is therefore more preferable. 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 on the specific binding substances 14 and 24.

[0091] In the fourth embodiment, the complex 40 was labeled with two fluorescent substances 16,26, but is not limited thereto. For example, the number of fluorescent substances labeled on the complex 40 may be one or three or more.

[0092] In the fifth embodiment, the light-receiving optical system 140 in the measuring device 100 is arranged perpendicular to the direction of propagation of the incident excitation light 122 into the reagent container 130, but is not limited thereto. The light-receiving optical system 140 may be arranged, for example, parallel to the direction of propagation of the incident excitation light 122, or at an acute or obtuse angle with respect to the direction of propagation of the incident excitation light 122. [Examples]

[0093] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited in any way by the following examples. Unless otherwise specified, parts and percentages in the examples are based on mass. The examples and comparative examples were carried out under ambient air and room temperature (1 atmosphere, 25°C) unless otherwise specified.

[0094] Furthermore, in the examples and comparative examples, the concentration of metal nanoparticles in the dispersion is sometimes expressed in terms of absorbance. Absorbance was measured using a UV-Vis spectrophotometer (TECAN Japan Co., Ltd. "infinite M200 PRO"). Since the absorption wavelength differs for each sample, it is listed for each sample. The number appended 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.

[0095] <Example 1> [1. Formation of polymer films] The method for forming the polymer film will be explained with reference to Figures 14-15. Figures 14-15 show schematic diagrams illustrating the polymer film formation method of Example 1. As shown in Figure 14, poly-L-lysine (Peptide Laboratories, Inc., "3075") and 3-(2-pyridyldithio)propionamide-PEG4-NHS (Thermo Fisher Scientific, Ltd., lot number "26128", "NHS-PEG4-SPDP") were mixed and stirred at room temperature for 4 hours using a small rotary incubator (Tytec Co., Ltd., "RT-30mini"). As a result, a polymer was obtained. This synthesis reaction is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamide-PEG4-NHS. The synthesized polymer 3B had a disulfide bond in its side chain. More specifically, the synthesized polymer had a hydrophobic group (pyridyl group) 3c and a positively charged group (first ammonium group) 3b linked via a disulfide bond.

[0096] The obtained polymer 3B was brought into contact with metal nanoparticles 2 to form a polymer film 3. More specifically, the obtained polymer 3B was used as the metal nanoparticle 2, which was silver nanoparticle (nanocomposix "AGCB80-1M", diameter 80 nm, OD 455 The mixture was added to 1 mL of a dispersion of (=0.1) and stirred and mixed at room temperature and overnight using a small rotary incubator (RT-30mini, manufactured by Taitec Co., Ltd.). As a result, a dispersion of silver nanoparticles coated with a polymer membrane was obtained.

[0097] As shown in Figure 15, the polymer film 3 contains a sulfur atom-mediated bonding site 3a on the surface of the silver nanoparticles 2, a hydrophobic group (pyridyl group) 3c that forms a hydrophobic bond with the surface of the silver nanoparticles 2, and a positively charged group (primary ammonium group) 3b that forms an electrostatic bond b with the surface of the silver nanoparticles 2. In other words, the polymer 3A constituting the polymer film 3 has a sulfur atom-mediated bonding site 3a on the surface of the silver nanoparticles 2, a hydrophobic group (pyridyl group) 3c that forms a hydrophobic bond c with the surface of the silver nanoparticles 2, and a positively charged group (primary ammonium group) 3b that forms an electrostatic bond with the surface of the silver nanoparticles 2. An SEM image (magnification 500,000x) of the obtained silver nanoparticles 2 was created, and it was confirmed that the surface of the silver nanoparticles 2 was continuously coated by the polymer film 3 (hereinafter, silver nanoparticles coated with polymer film 3 will be referred to as "polymer-coated silver nanoparticles"). In addition, the film thickness of the polymer film 3 coating the silver nanoparticles 2 was measured from the SEM image.

[0098] [2. Fabrication of Nanoparticles] Figure 16 is a schematic diagram showing the structure of the fluorescently labeled antibody-conjugated nanoparticle 1 of Example 1. The nanoparticle shown in Figure 16 was prepared by first conjugating a crosslinking agent to the surface of polymer-coated metal nanoparticles, separately conjugating a fluorescent substance and a crosslinking agent to a nano-antibody, and then conjugating the crosslinking agent conjugated to the polymer-coated metal nanoparticles with the crosslinking agent conjugated to the nano-antibody. The details of the preparation of the fluorescently labeled antibody-conjugated nanoparticles will be described below.

[0099] (2-1. Binding of crosslinking agents to polymer-coated silver nanoparticles) To 1 mL of the dispersion of the prepared polymer-coated silver nanoparticles, further add the crosslinking agent SM(PEG) 6(PEGylated, long-chain SMCC crosslinker) (ThermoFisher SCIENTIFIC "22105") and heparin sodium (Fujifilm Wako Pure Chemical Corporation "081-00136") were added and mixed using a small rotary incubator (Tytec Corporation "RT-30mini") at room temperature for 1 hour. As a result, the crosslinking agent SM(PEG) was found to be present on polymer membrane 3. 6 Silver nanoparticles (hereinafter referred to as SM(PEG)) bonded to them 6 A dispersion of polymer-coated silver nanoparticles (also called linker-bound silver nanoparticles) was obtained. SM(PEG) 6 The linker had a maleimide group.

[0100] (2-2. Fluorescent labeling of VHH antibody) [Chemical Formula 5] is obtained for 100 μg of VHH antibody (RePHAGEN, molecular weight 18,000 Da): [ka] An NHS-labeled Ru complex derivative represented by ("Ruthenium(II)tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd.) was added and mixed using a small rotary incubator ("RT-30mini" manufactured by Taitec Co., Ltd.) at room temperature for 1 hour. As a result, a VHH antibody conjugated with a fluorescent substance (hereinafter also referred to as a fluorescently labeled VHH antibody) was obtained. The NHS-labeled Ru complex derivative is a fluorescent substance with a maximum fluorescence wavelength of 500-700 nm.

[0101] (2-3. Binding of crosslinking agents to fluorescently labeled VHH antibodies) Next, 3-(2-pyridyldithio)propionamide-PEG4-NHS (Thermo Fisher SCIENTIFIC, product number "26128", "NHS-PEG4-SPDP"), used as an NHS-bipyridyl disulfide crosslinking agent, was added in an 8-fold molar equivalent volume. The mixture was then stirred and mixed at room temperature for 1 hour using a small rotary incubator (Tytec Co., Ltd., "RT-30mini"). As a result, a VHH antibody conjugated with the fluorescent substance and SPDP linker (hereinafter also referred to as the SPDP linker-conjugated fluorescently labeled VHH antibody) was obtained.

[0102] (2-4. Thiolation of fluorescently labeled VHH antibody bound to a crosslinking agent) Next, the fluorescently labeled VHH antibody bound to the SPDP linker was mixed with a reducing agent TCEP (ThermoFisher SCIENTIFIC "77720") in a molar ratio of 2 equivalents, and stirred using a stirrer (BioSan "TS-100") at 37°C for 1 hour. As a result, a VHH antibody bound to the fluorescent substance and the reduced SPDP linker (hereinafter also referred to as the reduced SPDP linker) was obtained (hereinafter also referred to as the fluorescently labeled VHH antibody bound to the reduced SPDP linker). The reduced SPDP linker had a thiol group (-SH group) generated by the reduction of the disulfide bond.

[0103] (2-5. Binding of fluorescently labeled VHH antibody to silver nanoparticles) Next, a dispersion of polymer-coated silver nanoparticles to which a maleimide group-containing SM(PEG)6 linker is attached (OD 430 To a solution of 0.1, a fluorescently labeled VHH antibody conjugated to a reduced SPDP linker was added and mixed using a small rotary incubator (RT-30mini, manufactured by Taitec Co., Ltd.) at room temperature and overnight. As a result, the maleimide group of the SM(PEG)6 linker reacted with the thiol group of the reduced SPDP linker to obtain nanoparticles to which the fluorescently labeled VHH antibody was conjugated via the linker (see Figure 16).

[0104] [3. Formation of the complex] To the phosphate buffer solution of the obtained nanoparticles, C Reactive Protein (ADVY CHEMICAL Sigma-Aldrich "00-AGN-AP-CRP-00") (hereinafter also referred to as CRP antigen) was added as test substance 30, and the mixture was stirred using a small rotary incubator (Tytec Co., Ltd. "RT-30mini") at room temperature for 5 minutes to prepare the measurement sample (OD 455 A solution with a concentration of 0.4 was prepared. As a blank, a phosphate-buffered solution of nanoparticles without CRP antigen 30 was prepared separately.

[0105] [4. Evaluation Method] (4-1. Detection of test substances by immunochromatography) Figure 17 is a schematic diagram showing an immunochromatography strip (hereinafter also simply referred to as "strip"). This strip 300 is rectangular in shape and has a sample pad 301 at one end for dropping the sample to be measured, and a determination line 303 located in the center. The determination line 303 is for C as the test substance 30. RP The antigen is immobilized. When the measurement sample is dropped onto the sample pad 301, the complex 40 in the measurement sample moves toward the detection line 303 (along the direction 305) due to capillary action. When the complex 40 reaches the detection line 303, it reacts with the CRP antigen in an antigen-antibody reaction, and the complex 40 is captured.

[0106] The sample to be measured was dropped onto the sample pad 301 of strip 300 and allowed to stand for a predetermined time. The standing strip 300 was then placed in a fluorometer (PHC Corporation's "In-house Experimental Fluorescence Meter"). Excitation light (wavelength: 415-455 nm) was shone onto the determination line 303, and the amount of fluorescence (detection wavelength 573 nm or 613 nm) was measured. The blank measurement value was subtracted from the obtained measurement value to determine the presence or absence of fluorescence from the fluorescent substance. As a result, in the preparation of the measurement sample, C RP The antigen concentration is 42 pM (unit: ×10⁻¹⁰ -12 Fluorescence was detected at concentrations of mol / L or higher.

[0107] The standing strip 300 was placed in a spectrophotometer (PHC Corporation's "In-house Experimental Spectrophotometer"). Visible light (wavelength 415-455 nm) was shone onto the detection line 303, and the reflected light was measured. The blank measurement value was subtracted from the obtained measurement value to determine the presence or absence of reflected light originating from metal nanoparticles. As a result, reflected light was detected when the concentration of CRP antigen was 670 pM or higher during the preparation of the measurement sample.

[0108] The test substance 30, CRP antigen, was detected by immunography. This confirmed the formation of complex 40 in the measurement sample and the presence of complex 40 in the detection line.

[0109] (4-2. Analysis of plasmon resonance wavelengths in the presence of the test substance) First, the sample for measurement was prepared. The sample was prepared in the same manner as in 4-1. The sample was dropped onto a glass slide, and a glass cover plate was placed on top of the droplet to sandwich it, thereby creating a slide for measurement. The slide was used to measure the plasmon resonance spectrum while the droplet was still present. A blank slide was prepared in the same manner as the slide for measurement, except that the CRP antigen was not added.

[0110] Next, the plasmon resonance spectrum of the target was measured using a fluorescence microspectroscopy device. The target (complex) on the measurement slide was continuously irradiated with finely focused excitation light (wavelength: 400-650 nm), and the plasmon resonance spectrum was measured. The target (complex) in Example 1 was a complex composed of two nanoparticles linked together in a diatomic molecular manner via the test substance (CRP antigen). The measurement results are shown in Figure 18(b). Figure 18(b) shows the plasmon resonance spectrum of Example 1 for the measurement slide used as the measurement sample.

[0111] Similarly, measurements were taken on blank slides. In this measurement, monodisperse (primary particle state) nanoparticles were used as the target. The measurement results are shown in Figure 18(a). Figure 18(a) shows the plasmon resonance spectrum of Example 1 for the blank slide used as the measurement sample.

[0112] (Measurement results) As shown in Figure 18(a), the plasmon resonance spectrum of the blank slide exhibited a spectral shape with a single peak around 460–470 nm. This peak was attributed to plasmon resonance (dipole resonance) caused by monodisperse Ag nanoparticles.

[0113] As shown in Figure 18(b), the plasmon resonance spectrum of the measurement slide exhibited a spectral shape with a peak around 460-470 nm (short-wavelength peak) and a peak around 590-600 nm (long-wavelength peak). The short-wavelength peak was attributed to plasmon resonance (dipole resonance) induced by excitation light having a polarization direction of the electric field component parallel to the short axis direction of the composite (a sandwich-type composite consisting of two nanoparticles). The long-wavelength peak was attributed to plasmon resonance (multipole resonance) induced by excitation light having a polarization direction of the electric field component parallel to the long axis direction of the composite.

[0114] Separately, the absorption and fluorescence spectra of a fluorescent substance (Ru-complex "Ruthenium(II)tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd.) were measured using a spectrophotometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.). The absorption spectrum was measured at a wavelength of 230-500 nm in an aqueous solvent at an arbitrary concentration. The fluorescence spectrum was measured at an excitation wavelength of 450 nm, a measurement wavelength of 500-700 nm, and the same sample as the absorption spectrum was used. The obtained absorption spectra and fluorescence spectra of the fluorescent substance were superimposed on the plasmon resonance spectra of the measurement slides shown in Figure 18(b) to create Figure 19.

[0115] Figure 19 shows the plasmon resonance spectrum of Example 1, as well as the absorption spectrum 202 and fluorescence spectrum 210 of the Ru complex. As shown in Figure 19, the fluorescence wavelength range WR of the fluorescence spectrum 210 of the Ru complex E And the first emission wavelength region WR of the long-wavelength peak of the plasmon resonance spectrum (plasmon resonance spectrum 206) E1 An overlap of 212 (second region R2) was confirmed. Therefore, it was concluded that the fluorescence detected in the system in Example 1 is emission-induced fluorescence.

[0116] <Example 2> [3. Fabrication of Nanoparticles] In the preparation of the nanoparticles in Example 2, metal nanoparticles were used as silver nanoparticles (nanocomposix "AGCB80-1M", diameter 80 nm, OD). 455 From 1 mL of a dispersion of (=0.1), silver nanoparticles (nanocomposix "AGCB50-1M", diameter 50 nm, OD) were extracted. 455 Nanoparticles were prepared in the same manner as in Example 1, except that the value was changed to 0.1).

[0117] (4-2. Analysis of plasmon resonance wavelengths in the presence of the test substance) The plasmon resonance wavelength was analyzed in the same manner as in Example 1 in the presence of the test substance. The measurement results are shown in Figure 20. Figure 20 shows the plasmon resonance spectra of Example 2 for (a) a blank slide and (b) a measurement slide. The object measured in Example 2 was a composite composed of 15 nanoparticles.

[0118] As shown in Figure 20(a), the plasmon resonance spectrum of the blank slide exhibited a spectral shape with a single peak around 460–470 nm. This peak was attributed to plasmon resonance (dipole resonance) caused by monodisperse Ag nanoparticles.

[0119] As shown in Figure 20(b), the plasmon resonance spectrum of the measurement slide exhibited a spectral shape with a peak around 460-470 nm (short-wavelength peak) and a peak around 590-600 nm (long-wavelength peak). The short-wavelength peak was attributed to plasmon resonance (dipole resonance) induced by excitation light having a polarization direction of the electric field component parallel to the short axis of the complex. The long-wavelength peak was attributed to plasmon resonance (multipole resonance) induced by excitation light having a polarization direction of the electric field component parallel to the long axis of the complex.

[0120] Figure 21 was created by superimposing the absorption and fluorescence spectra of the Ru complex obtained in Example 1 with the plasmon resonance spectra of the measurement slide shown in Figure 20(b). As shown in Figure 21, the fluorescence wavelength range WR of the Ru complex fluorescence spectrum 210 E And the first emission wavelength region WR of the long-wavelength peak of the plasmon resonance spectrum (plasmon resonance spectrum 206) E1 An overlap of 212 (second region R2) was confirmed. Therefore, it was concluded that the fluorescence detected in the system in Example 2 is emission-induced fluorescence.

[0121] <Example 3> [3. Fabrication of Nanoparticles] In the preparation of the nanoparticles in Example 3, the nanoparticles were prepared in the same manner as in Example 1, except that the fluorescent substance was changed from an NHS-labeled Ru complex derivative ("Ruthenium(II)tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd.) to Alexa Fluor 594 carboxylic acid, succinimidyl ester ("A10169" manufactured by Invitrogen). Alexa Fluor 594 is a fluorescent substance having maximum absorption and maximum fluorescence wavelengths in the 500-700 nm range.

[0122] (4-1. Detection of test substances by immunochromatography) Similar to Example 1, the test substance was detected by immunochromatography. The wavelength of the excitation light irradiated onto the detection line 303 was 600 nm. As a result of measuring the fluorescence intensity, it was found that C was present in the preparation of the measurement sample. RP Fluorescence was detected at antigen concentrations of 42 pM or higher. The results of measuring reflected light indicated that C was present in the preparation of the measurement sample. RP Reflected light was detected at antigen concentrations of 670 pM or higher. The test substance, CRP antigen, was detected by immunography. This confirmed the formation of complex 40 in the measurement sample and the presence of complex 40 in the detection line.

[0123] (4-2. Analysis of plasmon resonance wavelengths in the presence of the test substance) Separately, the absorption and fluorescence spectra of a fluorescent substance (Alexa Fluor 594 carboxylic acid, succinimidyl ester (Invitrogen A10169 20004)) were measured using a spectrophotometer (TECAN Japan Co., Ltd. "infinite M200 PRO"). The measurement conditions for the absorption spectrum were a measurement wavelength of 450-650 nm, and the solvent for the sample was deionized water.

[0124] Figure 22 was created by superimposing the absorption and fluorescence spectra of the obtained fluorescent substance with the plasmon resonance spectrum of the measurement slide shown in Figure 20(b). Figure 22 shows the absorption and fluorescence spectra of the fluorescent substance in Example 3, and the plasmon resonance spectrum of Example 1. The fluorescence spectrum 210 of the fluorescent substance in Example 3 has a peak around 584 nm. The fluorescence spectrum 210 of the fluorescent substance in Example 3 has a peak around 613 nm. Furthermore, the plasmon resonance spectrum of the system in Example 1 was used as a substitute for the system in Example 3. This is because, in the composite of Example 3, the only difference from the composite of Example 1 is the type of fluorescent substance; the material and particle size of the metal nanoparticles that primarily characterize the plasmon resonance spectrum are the same (it is thought that this is the only difference, as the absorption of the fluorescent substance is slightly added to the plasmon resonance spectrum).

[0125] As shown in Figure 22, the absorption wavelength range WR of the absorption spectrum 202 of the fluorescent substance A And the first emission wavelength region WR of the long-wavelength peak (multipole resonance spectrum) 206 in the plasmon resonance spectrum E1 An overlap of 208 (first region R1) was confirmed. Therefore, it was concluded that the fluorescence detected in the system in Example 3 is mainly excitation-induced fluorescence.

[0126] (4-3. Fabrication of the composite) The obtained nanoparticle phosphate buffer solution was mixed with CRP antigen (test substance 30) to a final concentration of 90 ng / mL and reacted (antibody-antigen reaction) at room temperature for 10 minutes. This prepared a mixture of the nanoparticle and the test substance. The prepared mixture was used as the measurement sample. A phosphate buffer solution of nanoparticles without CRP antigen 30 was prepared separately as a blank.

[0127] (4-4. Fluorescence analysis of the test substance-nanoparticle system) Fluorescence analysis was performed on the test substance-nanoparticle system. Using a spectrofluorometer (FP-8550, manufactured by JASCO Corporation), the fluorescence spectrum after composite formation was measured under conditions of an excitation wavelength of 580 nm and a measurement wavelength range of 600-720 nm. The measurement results are shown in Figure 23. Figure 23 shows the fluorescence spectrum of the test substance-nanoparticle system. The solid line shows the fluorescence spectrum of the test substance-nanoparticle system, and the dashed line shows the fluorescence spectrum of the blank sample. The fluorescence spectrum of the test substance-nanoparticle system has a peak located around 614 nm. The peak intensity around 614 nm is more than four times greater than the peak intensity around 614 nm in the fluorescence spectrum of the blank sample.

[0128] As shown in Figure 22, the fluorescent material of Example 3 has a maximum absorption wavelength around 586 nm. Furthermore, the absorption spectrum 202 of the fluorescent material of Example 3 largely overlaps with the multipole resonance spectrum 206. By using a fluorescent material that has absorption matched to the plasmon resonance wavelength of the complex as shown in Figure 22, we confirmed that fluorescence is effectively enhanced as shown in Figure 23.

[0129] <Comparative Example 1> Silica-coated silver nanoparticles (manufactured by nanoComposix, with a particle size (core particle size) of 50 nm and a silica film thickness of 20 nm) were diluted with water (deionized 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 particle state of silica-coated silver nanoparticles (hereinafter also referred to as silica-coated silver nanoparticles), aggregates of two or more silver nanoparticles were also present in the measurement sample.

[0130] The plasmon resonance wavelength was analyzed in the same manner as in Example 1 in the presence of the test substance. The measurement results are shown in Figure 24. Figure 24 shows the plasmon resonance spectrum of Comparative Example 1 ((a) silica-coated silver nanoparticles in primary particle state and (b) aggregates of three silica-coated silver nanoparticles arranged in a substantially linear fashion). In Figure 24(a), silica-coated silver nanoparticles in primary particle state were used as the measurement target, and in Figure 24(b), aggregates of three silica-coated silver nanoparticles arranged in a substantially linear fashion were used as the measurement target. The plasmon resonance spectrum shown in Figure 24(a) showed a spectral shape with one peak attributed to dipole resonance around 450 nm. The plasmon resonance spectrum shown in Figure 24(b) showed a spectral shape with one peak attributed to dipole resonance around 470 nm, similar to Figure 24(a). In Comparative Example 1, no peaks in the plasmon resonance spectrum attributable to multipoles were observed.

[0131] <Comparative Example 2> Silica-coated silver nanoparticles (manufactured by nanoComposix, with a core particle size of 50 nm and a silica film thickness of 7 nm) were diluted with water (deionized 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 particle state of silica-coated silver nanoparticles, aggregates of two or more silver nanoparticles were also present in the measurement sample.

[0132] The plasmon resonance wavelength was analyzed in the same manner as in Example 1 in the presence of the test substance. The measurement results are shown in Figure 25. Figure 25 shows the plasmon resonance spectrum of Comparative Example 2 ((a) silica-coated silver nanoparticles in primary particle state and (b) aggregates in which three silica-coated silver nanoparticles are arranged in a manner similar to ozone molecules). In Figure 25(a), silica-coated silver nanoparticles in primary particle state were used as the measurement target, and in Figure 25(b), aggregates in which three silica-coated silver nanoparticles are arranged in a manner similar to ozone molecules were used as the measurement target. The plasmon resonance spectrum shown in Figure 25(a) showed a spectral shape with one peak attributed to dipole resonance around 450 nm. The plasmon resonance spectrum shown in Figure 25(b) showed a spectral shape with one peak attributed to dipole resonance around 450 nm, similar to Figure 25(a). In Comparative Example 2, no peaks in the plasmon resonance spectrum attributable to multipoles were observed.

[0133] The embodiments of the nanoparticles, composites containing nanoparticles, and methods for forming polymer films contained in the nanoparticles relating to this disclosure are as follows. <1> Metal nanoparticles and A polymer film that coats the surface of the metal nanoparticles, A specific binding substance that specifically binds to the test substance in the sample, A fluorescent substance labeled on the surface of the polymer film or the specific bonding substance and A nanoparticle body comprising, The fluorescent substance is a nanoparticle body which is excited by light of the emission wavelength of plasmon resonance in a composite in which two or more of the nanoparticle bodies are bonded via the test substance. <2> Plasmon resonance in the aforementioned complex is induced by light irradiated from the outside. <1> The nanoparticles described above. <3> Plasmon resonance in the aforementioned complex is induced by the fluorescence emitted by the fluorescent substance. <1> or <2> The nanoparticles described above. <4> The absorption wavelength range of the fluorescent substance overlaps with the first emission wavelength range of the plasmon resonance. <1> ~ <3> A nanoparticle body as described in any one of the items. <5> The first emission wavelength range of the plasmon resonance is located at a longer wavelength than the second emission wavelength range of the plasmon resonance induced in the single-particle metal nanoparticle. <1> ~ <4> A nanoparticle body as described in any one of the items. <6> The fluorescent substance is selected such that the first region where the first emission wavelength range and the absorption wavelength range of the fluorescent substance overlap is larger than the second region where the second emission wavelength range and the absorption wavelength range of the fluorescent substance overlap. <5> The nanoparticles described above. <7> The maximum absorption wavelength of the fluorescent substance in the first emission wavelength range is located at 500 to 700 nm. <5> or <6> The nanoparticles described above. <8> The maximum fluorescence wavelength of the fluorescent substance in the first emission wavelength range is located at 500 to 700 nm. <5> ~ <7> A nanoparticle body as described in any one of the items. <9> The plasmon resonance induced in the aforementioned complex is a multipole resonance. <1> ~ <8> A nanoparticle body as described in any one of the items. <10> The plasmon resonance induced in the aforementioned single-particle metal nanoparticle is a dipole resonance. <5> ~ <8> A nanoparticle body as described in any one of the items. <11> The polymer film includes at least one selected from the group consisting of a sulfur atom-mediated bonding site, a positively charged group, and a hydrophobic group, between itself and the surface of the metal nanoparticles. <1> ~ <10> A nanoparticle body as described in any one of the items. <12> The polymer film contains at least the positively charged group in the side chains of the polymer constituting the polymer film, The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH2 +At least one selected from the group consisting of )NH2), <11> The nanoparticles described above. <13> The polymer film contains at least the hydrophobic group in the side chains of the polymer constituting the polymer film, The hydrophobic group is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. <11> The nanoparticles described above. <14> The film thickness of the polymer film is 1 nm to 10 nm. <1> ~ <13> A nanoparticle body as described in any one of the items. <15> These are nanoparticles used in plasmon-induced fluorescence analysis. <1> ~ <14> A nanoparticle body as described in any one of the items. <16> The aforementioned specific binding substance is a nanoantibody. <1> ~ <15> A nanoparticle body as described in any one of the items. <17> The aforementioned specific binding substance is a VHH antibody. <1> ~ <16> A nanoparticle body as described in any one of the items. <18> The aforementioned metal nanoparticles contain gold or silver. <1> ~ <17> A nanoparticle body as described in any one of the items. <19> The particle size of the aforementioned metal nanoparticles is 5 to 100 nm. <1> ~ <18> A nanoparticle body as described in any one of the items. <20> The aforementioned nanoparticles include a first nanoparticle and a second nanoparticle. The first nanoparticle and the second nanoparticle form a composite by being bound together via the test substance. <1> ~ <19> A nanoparticle body as described in any one of the items. <21> The test substance is a test substance derived from the specimen, which is blood, plasma, urine, or saliva. <1> ~ <20> A nanoparticle body as described in any one of the items. <22> In the composite, a fluorescent substance is positioned between the first nanoparticle and the second nanoparticle. <20> The nanoparticles described above. <23> <1> ~ <22> A composite comprising two or more nanoparticles as described in any one of the above, wherein the two or more nanoparticles comprise a first nanoparticle and a second nanoparticle, and the first nanoparticle and the second nanoparticle are bonded together via the test substance. <24> The nanoparticles include multiple binding points through which two of the nanoparticles are bonded via one of the test substances. <23> The composite described above. <25> A method for forming a polymer film, comprising the step of contacting a polymer having disulfide bonds in its side chains with metal nanoparticles to form a polymer film in which the polymer is bonded to the surface of the metal nanoparticles via sulfur atoms. <26> The polymer comprises at least one group selected from the group consisting of positively charged groups and hydrophobic groups, which are bonded via the disulfide bond. The above step involves forming the polymer film and bonding at least one group selected from the group consisting of the positively charged group and the hydrophobic group to the surface of the metal nanoparticles. <25> A method for forming a polymer film as described above. [Explanation of Symbols]

[0134] 1. Nanoparticles 2 ···metal nanoparticles 3...polymer membrane 3A... Polymers that make up polymer films 3B... Polymers that form polymer films 3a ···Bonding site via sulfur atom 3b ···Positively charged group 3c...Hydrophobic group 4...specific binding substance 6. Fluorescent substances 10 ···First Nanoparticle 12 ···First metal nanoparticles 13...first polymer membrane 14...first specific binding substance 16 ···First fluorescent substance 20 ···Second Nanoparticle 22 ···Second Metal Nanoparticles 23...Second polymer membrane 24...Second specific binding substance 26 ···Second fluorescent substance 30 ···Test substance 40 ···complex L...Separation distance (separation distance) R1...1st area R2...Second area WR A ...Absorption wavelength range of fluorescent substances WR E ...Emission wavelength range of fluorescent substances WR E1 ...First emission wavelength range WR E2 ...Second emission wavelength range

Claims

1. Metal nanoparticles and A polymer film covering the surface of the metal nanoparticles, A specific binding substance that specifically binds to the test substance in the sample, A fluorescent substance labeled on the surface of the polymer film or the specific bonding substance and A nanoparticle body comprising, The fluorescent substance is a nanoparticle body which is excited by light of the emission wavelength of plasmon resonance in a composite in which two or more of the nanoparticle bodies are bonded via the test substance.

2. The nanoparticle body according to claim 1, wherein plasmon resonance in the composite is induced by light irradiated from an external source.

3. The nanoparticle body according to claim 1, wherein plasmon resonance in the composite is induced by fluorescence emitted by the fluorescent substance.

4. The nanoparticle body according to claim 1, wherein the absorption wavelength range of the fluorescent substance overlaps with the first emission wavelength range of the plasmon resonance.

5. The nanoparticle body according to claim 1, wherein the first emission wavelength range of the plasmon resonance is located on the longer wavelength side compared to the second emission wavelength range of the plasmon resonance induced in the single-particle metal nanoparticle.

6. The nanoparticle body according to claim 5, wherein the fluorescent substance is selected such that the first region in which the first emission wavelength range and the absorption wavelength range of the fluorescent substance overlap is larger than the second region in which the second emission wavelength range and the absorption wavelength range of the fluorescent substance overlap.

7. The nanoparticle body according to claim 5, wherein the maximum absorption wavelength of the fluorescent substance in the first emission wavelength range is located at 500 to 700 nm.

8. The nanoparticle body according to claim 5, wherein the maximum fluorescence wavelength of the fluorescent substance in the first emission wavelength range is located at 500 to 700 nm.

9. The nanoparticle material according to claim 1, wherein the plasmon resonance induced in the composite is a multipole resonance.

10. The nanoparticle body according to claim 5, wherein the plasmon resonance induced in the single-particle metal nanoparticle is a dipole resonance.

11. The nanoparticle body according to claim 1, wherein the polymer film comprises at least one selected from the group consisting of a sulfur atom-mediated bonding site, a positively charged group, and a hydrophobic group between itself and the surface of the metal nanoparticle.

12. The polymer film contains at least the positively charged group in the side chains of the polymer constituting the polymer film, The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH ２ ＋ ) NH ２ The nanoparticle body according to claim 11, which is at least one selected from the group consisting of ).

13. The polymer film contains at least the hydrophobic group in the side chains of the polymer constituting the polymer film, The nanoparticle material according to claim 11, wherein the hydrophobic group is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups.

14. The nanoparticle body according to claim 1, wherein the thickness of the polymer film is 1 nm to 10 nm.

15. The nanoparticle body according to claim 1, which is a nanoparticle body used for plasmon excitation fluorescence analysis.

16. The nanoparticle material according to claim 1, wherein the specific binding substance is a nanoantibody.

17. The nanoparticle body according to claim 1, wherein the specific binding substance is a VHH antibody.

18. The nanoparticle body according to claim 1, wherein the metal nanoparticles comprise gold or silver.

19. The nanoparticle body according to claim 1, wherein the particle size of the metal nanoparticles is 5 to 100 nm.

20. The aforementioned nanoparticle body includes a first nanoparticle body and a second nanoparticle body. The nanoparticle material according to claim 1, wherein the first nanoparticle material and the second nanoparticle material form a composite by being bound together via the test substance.

21. The nanoparticle body according to claim 1, wherein the test substance is a test substance derived from the specimen, which is blood, plasma, urine, or saliva.

22. The nanoparticle body according to claim 20, wherein a fluorescent substance is positioned between the first nanoparticle body and the second nanoparticle body in the composite.

23. A composite comprising two or more nanoparticles according to any one of claims 1 to 22, wherein the two or more nanoparticles include a first nanoparticle and a second nanoparticle, and the first nanoparticle and the second nanoparticle are bonded together via the test substance.

24. The composite according to claim 23, comprising a plurality of binding points in which two nanoparticles are bonded via one of the test substances.

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