Nanoparticle body, composite containing nanoparticle bodies, and method for forming polymer membrane containing nanoparticle body

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

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

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

[0002] A biosensor specifically reacts a specific analyte to be detected with a specific specific binding substance to form a complex, and detects the analyte based on a signal resulting from the specific binding in the complex. In plasmon excitation fluorescence analysis, the complex comprises, for example, an analyte, a specific binding substance, a fluorescent substance, and metal particles. When excitation light is irradiated onto the complex, surface plasmon resonance is induced in the metal particles within the complex, forming a near-field near the surface of the metal particles. This near-field increases the fluorescence intensity of the fluorescent substance.

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

[0004] JP 2011-220705 A

[0005] However, as a result of extensive research by the present inventors, it has been found that there is room for further improvement in the detection sensitivity of the above-mentioned sensor. Specifically, in the past, when selecting a fluorescent substance, the relationship between the absorption spectrum of the fluorescent substance and the emission wavelength of plasmon resonance was not taken into consideration, or even if such a relationship was mentioned, the relationship between the absorption spectrum of the fluorescent substance and the emission wavelength of plasmon resonance derived from a single metal nanoparticle was taken into consideration. As a result, the fluorescence emitted by the fluorescent substance in the composite was not sufficiently enhanced by plasmon resonance.

[0006] The present invention has been made in view of the above-mentioned problems, and its main object is to provide a nanoparticle that can enhance detection sensitivity by selecting an appropriate fluorescent substance and sufficiently enhancing the fluorescence emitted from the fluorescent substance in the complex through plasmon resonance.

[0007] A nanoparticle according to one embodiment of the present invention comprises a metal nanoparticle, a polymer membrane covering the surface of the metal nanoparticle, a specific binding substance that specifically binds to a test substance in a specimen, and a fluorescent substance labeled on the surface of the polymer membrane or on the specific binding substance, wherein the fluorescent substance is excited by light of an emission wavelength of plasmon resonance in a complex in which two or more of the nanoparticles are bound via the test substance.

[0008] In another embodiment of the present invention, the complex comprises two or more of the nanoparticles, the two or more nanoparticles including a first nanoparticle and a second nanoparticle, and the first nanoparticle and the second nanoparticle are bound to each other via the test substance.

[0009] A method for forming a polymer film according to another embodiment of the present invention comprises the steps of contacting a polymer having a disulfide bond in a side chain with metal nanoparticles to form a polymer film in which the polymer is bonded to the surface of the metal nanoparticles via sulfur atoms.

[0010] The present invention can provide a nanoparticle that can enhance detection sensitivity by selecting an appropriate fluorescent substance and sufficiently enhancing the fluorescence emitted from the fluorescent substance in the complex through plasmon resonance.

[0011] FIG. 1 is a conceptual diagram showing the plasmon resonance spectrum derived from metal nanoparticles in a single particle state and the absorption spectrum of a fluorescent substance. FIG. 2 is a conceptual diagram showing the plasmon resonance spectrum derived from metal nanoparticles in a single particle state, the plasmon resonance spectrum derived from metal nanoparticles in a composite, and the absorption spectrum of a fluorescent substance. FIG. 3 is a conceptual diagram showing the plasmon resonance spectrum derived from metal nanoparticles in a single particle state, the plasmon resonance spectrum derived from metal nanoparticles in a composite, and the absorption spectrum of a fluorescent substance. FIG. 4 is a cross-sectional view schematically showing a nanoparticle body according to the first embodiment. FIG. 5 is an enlarged cross-sectional view of part A in FIG. 4. FIG. 6 is a schematic diagram showing a method for forming a polymer film 3. FIG. 7 is a conceptual diagram showing the plasmon resonance spectrum derived from metal nanoparticles in a single particle state, the plasmon resonance spectrum derived from metal nanoparticles in a composite, and the fluorescence spectrum of a fluorescent substance. FIG. 8 is a conceptual diagram showing the plasmon resonance spectrum derived from metal nanoparticles in a single particle state, the plasmon resonance spectrum of metal nanoparticles in a composite, and the absorption spectrum and fluorescence spectrum of a fluorescent substance. FIG. 9 is a cross-sectional view schematically showing a composite. FIG. 10 is a cross-sectional view schematically showing a composite. FIG. 11 is a cross-sectional view schematically showing a complex composed of two nanoparticles. FIG. 12 is a cross-sectional view schematically showing a complex composed of three nanoparticles. FIG. 13 is a diagram showing a measuring apparatus. FIG. 14 is a schematic diagram showing a method for forming a polymer film in Example 1. FIG. 15 is a schematic diagram showing a method for forming a polymer film in Example 1. FIG. 16 is a schematic diagram showing the structure of a nanoparticle bound to a fluorescently labeled antibody in Example 1. FIG. 17 is a schematic diagram showing an immunochromatography strip. FIG. 18 is a diagram showing the plasmon resonance spectrum of Example 1 ((a) blank preparation and (b) measurement preparation). FIG. 19 is a diagram showing the plasmon resonance spectrum of Example 1, as well as the absorption spectrum and fluorescence spectrum of a Ru complex. FIG. 20 is a diagram showing the plasmon resonance spectrum of Example 2 ((a) blank preparation and (b) measurement preparation).Fig. 21 shows the plasmon resonance spectrum of Example 2, and the absorption and fluorescence spectra of the Ru complex. Fig. 22 shows the absorption and fluorescence spectra of the fluorescent substance of Example 3, and the plasmon resonance spectrum of Example 1. Fig. 23 shows the fluorescence spectrum of the test substance-nanoparticle system of Example 3. Fig. 24 shows the plasmon resonance spectrum 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 manner). Fig. 25 shows the plasmon resonance spectrum 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 the shape of an ozone molecule).

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

[0013] Numerical ranges mentioned in this specification are intended to include the lower and upper limit values ​​themselves, unless otherwise specified, such as "less than / less than" or "greater than / more than / greater than." That is, for example, if a numerical range is 1 nm to 10 nm, that numerical range is interpreted as including the lower limit of "1 nm" and the upper limit of "10 nm."

[0014] As used herein, a target component substantially composed of a specific material or a target component consisting of a specific material refers to the target component containing 95% by mass or more, 97% by mass or more, 99% by mass or more, or 100% by mass of the specific material. For example, nanoparticles made of gold refer to nanoparticles containing 95% by mass or more, 97% by mass or more, 99% by mass or more, or 100% by mass of gold.

[0015] <First embodiment: nanoparticle> [Basic configuration of nanoparticle] The nanoparticle according to the first embodiment comprises metal nanoparticles, a polymer membrane, a specific binding substance, and a fluorescent substance. The metal nanoparticles induce plasmon resonance when irradiated with excitation light. The polymer membrane covers the surface of the metal nanoparticles. The specific binding substance specifically binds to the test substance in the specimen to form a complex. The fluorescent substance is labeled on the surface of the polymer membrane or on the specific binding substance, and emits fluorescence derived from the plasmon resonance.

[0016] (Method for detecting an analyte using nanoparticles) First, for the convenience of explaining the nanoparticles according to this embodiment and to facilitate understanding thereof, a method for detecting an analyte using nanoparticles according to this embodiment will be described. The nanoparticles according to this embodiment comprise metal nanoparticles, a polymer membrane covering the surface of the metal nanoparticles, a specific binding substance that specifically binds to an analyte in a specimen, and a fluorescent substance labeled on the surface of the polymer membrane or on the specific binding substance. The nanoparticles according to this embodiment are dispersed in a specimen, and the analyte contained in the specimen is captured to form a complex (fourth embodiment). More specifically, the complex is formed by specific binding between the specific binding substance of the nanoparticles and the analyte. The complex has a structure in which, for example, two nanoparticles are bound via the analyte. In this complex, for example, two metal nanoparticles are spaced apart by binding to their respective specific binding substances for the same analyte.

[0017] In surface plasmon fluorescence spectroscopy (SPFS), when excitation light is irradiated onto the complex, localized surface plasmon resonance (LSPR) occurs, and a near-field is efficiently formed near the surface of the metal nanoparticle (particularly near the surface between two metal nanoparticles). The near-field and dipole-dipole mechanism efficiently excite the fluorescent substance in the complex, enhancing its fluorescence. The analyte in the sample can be detected by measuring the amount of fluorescence.

[0018] [Background to the Invention] Next, for the convenience of explaining the nanoparticle body according to this embodiment and to aid in its understanding, the background to the invention will be described in detail with reference to Figs. 1 to 3. Fig. 1 is a conceptual diagram showing the plasmon resonance spectrum derived from a metal nanoparticle in a single particle state and the absorption spectrum of a fluorescent substance. As shown in Fig. 1, the plasmon resonance spectrum 201 derived from a metal nanoparticle in a single particle state is in the second emission wavelength range WR Ｅ２ The absorption spectrum 202 of the fluorescent material has, for example, one peak in the absorption wavelength range WR Ａ For example, it has one peak as a plasmon resonance. When the fluorescent substance in the composite is excited by plasmon resonance, it is thought that the fluorescent substance is excited by a dipole-dipole mechanism (Förster mechanism (Förster Resonance Energy Transfer, Fluorescence Resonance Energy Transfer: FRET)) and a near-field. For this reason, as shown in FIG. 1 , there is an overlap between the plasmon resonance spectrum 201 derived from the metal nanoparticle in a single particle state and the absorption spectrum 202 of the fluorescent substance (absorption wavelength range WR Ａ and the second emission wavelength range WR Ｅ２ A fluorescent substance was selected so that the overlapping area 204 between the fluorescent substance and the target fluorescent substance was large. Conventionally, fluorescent substances in a complex have been selected in this manner from the viewpoint of increasing detection sensitivity.

[0019] After extensive research, the present inventors have found that the selection of conventional fluorescent substances has the following problems. Plasmon resonance originating from single-particle metal nanoparticles (i.e., plasmon resonance induced in single-particle metal nanoparticles) is dipole resonance, while plasmon resonance induced in a composite is plasmon resonance (higher-order resonance, i.e., multipole resonance) resulting from dipole-dipole interactions. Examples of multipole resonance include quadrupole resonance. In other words, the plasmon resonance that primarily contributes to the detection of an analyte is multipole resonance induced by the proximity of metal nanoparticles in a composite. Therefore, in order to efficiently increase the detection sensitivity in the detection of an analyte, it is necessary to enhance fluorescence primarily resulting from multipole resonance, rather than fluorescence resulting from dipole resonance.

[0020] 2 is a conceptual diagram showing a plasmon resonance spectrum 201 derived from a metal nanoparticle in a single particle state, a plasmon resonance spectrum 206 derived from a metal nanoparticle in a composite, and an absorption spectrum 202 of a fluorescent substance. As shown in FIG. 2, the plasmon resonance spectrum (hereinafter also referred to as a multipole resonance spectrum) 206 of the metal nanoparticle in the composite is in the first emission wavelength range WR Ｅ１ , for example, has one peak and is located on the longer wavelength side compared to the peak of the plasmon resonance spectrum (hereinafter also referred to as dipole resonance spectrum) 201 derived from the metal nanoparticle in a single particle state. That is, the first emission wavelength range WR of the plasmon resonance Ｅ１ is the second emission wavelength range WR of the plasmon resonance originating from a metal nanoparticle in a single particle state (i.e., the plasmon resonance induced in a single metal nanoparticle). Ｅ２ In other words, the absorption spectrum 202 of the fluorescent substance overlaps with the plasmon resonance spectrum 206 derived from the metal nanoparticles in the composite (absorption wavelength range WR Ａ and the first light emission wavelength range WR Ｅ１ There is no overlap with

[0021] From the viewpoint of increasing the detection sensitivity of the test substance, the inventors believe that the spectral overlap 204 (also described in FIG. 1) is an apparent overlap. １ It has been found that it is important to select a fluorescent substance that allows the multipole resonance spectrum 206 to exist, and preferably to select a fluorescent substance that allows a large overlap 208 between the multipole resonance spectrum 206 and the absorption spectrum 202 of the fluorescent substance (see FIG. 3 described later). １ The overlapping region 208 refers to the region where the multipole resonance spectrum 206 and the absorption spectrum 202 of the fluorescent substance overlap (the region corresponding to the overlap 208), as shown in FIG. 3, which will be described later.

[0022] 3 is a conceptual diagram showing a plasmon resonance spectrum 201 derived from a metal nanoparticle in a single particle state, a plasmon resonance spectrum 206 derived from the metal nanoparticle in the composite, and an absorption spectrum 202 of a fluorescent substance. In this embodiment, the fluorescent substance is selected so that there is (preferably, a large) overlap 208 between the absorption spectrum 202 of the fluorescent substance and the resonance spectrum 206 derived from the metal nanoparticle in the composite. In this way, the absorption wavelength range WR of the fluorescent substance Ａ is the first emission wavelength range WR of the plasmon resonance Ｅ１ When the overlapping wavelengths are such that the fluorescent substance is sufficiently excited, the fluorescence is enhanced, and the detection sensitivity is greatly improved. Therefore, it is believed that the nanoparticle according to this embodiment can enhance the detection sensitivity.

[0023] Based on this technical knowledge, the present inventors have investigated specific means for improving detection sensitivity by focusing on adjusting the spectral properties of fluorescent substances, and as a result, have come to the conclusion that "a fluorescent substance is excited by light of the emission wavelength of localized surface plasmon resonance in a complex in which two or more nanoparticles are bound via an analyte."

[0024] The nanoparticle according to the first embodiment comprises a metal nanoparticle, a polymer membrane covering the surface of the metal nanoparticle, a specific binding substance that specifically binds to a test substance in a specimen, and a fluorescent substance labeled on the surface of the polymer membrane or on the specific binding substance, and the fluorescent substance is excited by light of the emission wavelength of localized surface plasmon resonance in a complex in which two or more of the nanoparticles are bound via the test substance.

[0025] [Mechanism of Action] The nanoparticles according to this embodiment can enhance detection sensitivity. Without being bound by any particular theory, the reason for this is presumed to be as follows: In the nanoparticles according to this embodiment, the fluorescent substance is excited by light of the emission wavelength of plasmon resonance in a complex in which two or more nanoparticles are bound via an analyte. This results in overlap between the absorption spectrum of the fluorescent substance and the plasmon resonance spectrum derived from the metal nanoparticles in the complex, and fluorescence for detecting the analyte is induced by the Förster mechanism and the near-field (hereinafter, such fluorescence is also referred to as "excitation-induced fluorescence"). Therefore, the fluorescent substance is sufficiently excited by light of the emission wavelength of plasmon resonance derived from the complex, thereby enhancing detection sensitivity. Furthermore, fluorescence for detecting the analyte is also induced by overlap between the fluorescence spectrum of the fluorescent substance and the plasmon resonance spectrum derived from the metal nanoparticles in the complex (hereinafter, such fluorescence is also referred to as "luminescence-induced fluorescence"). Luminescence-induced fluorescence will be described in detail in the second embodiment.

[0026] [Scheme 1] Scheme 1 of the process for detecting a test substance using the nanoparticles according to this embodiment: (In the elementary steps (1) to (3) of Scheme 1, S ０ denotes the ground state, and S １ denotes an excited singlet state, * denotes an excited state, Flu denotes a fluorescent substance (in the composite), and M denotes a metal nanoparticle (in the composite). The fluorescence enhancement in this embodiment will be further described with reference to the following:

[0027] Scheme 1 includes elementary steps (1) to (3). As shown in elementary step (1), the fluorescent substance is excited by light of the emission wavelength of plasmon resonance in a complex in which two or more nanoparticles are bound via an analyte. When the complex formed by capturing the analyte is irradiated with externally irradiated light (hereinafter also referred to as "external irradiation light") as excitation light, plasmon resonance (multipole resonance) is induced (i.e., plasmon resonance in the complex is induced by the externally irradiated light). Note that the analyte may be, for example, an analyte derived from a specimen such as blood, plasma, urine, or saliva. As shown in elementary step (2), the fluorescent substance is excited by a dipole-dipole mechanism and a near-field. The absorption wavelength range WR of the fluorescent substance Ａ is the first emission wavelength range WR of the plasmon resonance Ｅ１ When the excitation currents overlap, the fluorescent substance is excited efficiently. As shown in elementary process (3), the excited fluorescent substance relaxes and emits fluorescent light.

[0028] In one preferred embodiment, the first emission wavelength range WR Ｅ１ The maximum absorption wavelength of the fluorescent substance in the composite is located in the range of 500 to 700 nm (more preferably 550 to 700 nm). Ｅ１ The maximum absorption wavelength of the fluorescent substance is located in the range of 500 to 700 nm within the range. Examples of fluorescent substances include fluorescein derivatives, rhodamine derivatives, cyanine dyes, and Alexa Fluor (registered trademark) manufactured by Molecular Probes. Among these, fluorescent substances with a maximum absorption wavelength of 500 to 700 nm include Alexa Fluor (registered trademark) series 532, 546, 555, 568, 594, and 640. The maximum absorption wavelength can be determined as follows: the absorption spectrum of an aqueous solution of the fluorescent substance (solvent: deionized water) is measured, and the peak position of the obtained absorption spectrum is taken as the maximum absorption wavelength. In another preferred embodiment, when the composite comprises two nanoparticles 1, the fluorescent substance is located between the first nanoparticle and the second nanoparticle in the composite.

[0029] The structure of the nanoparticle will be described below. The nanoparticle will be described with reference to Fig. 4. Fig. 4 is a cross-sectional view schematically showing a nanoparticle according to this embodiment. The nanoparticle 1 according to this embodiment comprises a metal nanoparticle 2, a polymer film 3 covering the surface of the metal nanoparticle 2, a specific binding substance 4 that specifically binds to an analyte in a specimen, and a fluorescent substance 6 labeled on the surface of the polymer film 3.

[0030] The nanoparticle 1 can be used in plasmon excitation fluorescence analysis. In other words, the nanoparticle 1 can be used in surface plasmon excitation enhanced fluorescence spectroscopic immunoassay. The nanoparticle 1 can capture an analyte in a test specimen and form a complex containing two or more nanoparticles 1 and the analyte. When excitation light is irradiated onto the complex, plasmon resonance occurs, forming a near-field. Fluorescence is enhanced by the near-field and dipole-dipole mechanism. The nanoparticle 1, for example, captures one analyte in a test specimen and forms a complex containing two nanoparticles 1 and the analyte. In such a case, the nanoparticle 1 contains first and second nanoparticles as nanoparticles, and forms a complex in which the first and second nanoparticles are bound via the analyte.

[0031] The nanoparticle 1 may also have non-specific binding sites blocked by a blocking agent. The blocked nanoparticle 1 suppresses the formation of non-specific binding of the specific binding substance 4 to substances other than the detection target (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 proteins such as bovine serum albumin (BSA), skim milk, and casein, as well as 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 heparin sodium.

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

[0034] (Polymer Film) The polymer film 3 covers the surface of the metal nanoparticle 2. The polymer film 3 functions as a metal quenching molecular film. In the composite, the polymer film 3 can dispose the fluorescent substance 6 at a distance from the surface of the metal nanoparticle 2 by at least the thickness of the polymer film 3. This prevents the excited fluorescent substance 6 from coming into contact with the surface of the metal nanoparticle 2 and being quenched (quenching due to the Dexter mechanism (Dexter Electron Transfer)), thereby preventing a decrease in detection sensitivity. The presence of the polymer film 3 can be confirmed by capturing an image of the nanoparticle 1 using an SEM or TEM and observing the nanoparticle 1 in the image.

[0035] The polymer film 3 will be described with reference to Fig. 5. Fig. 5 is an enlarged view of part A in Fig. 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 nanoparticle 2. The polymer film 3 includes at least one selected from the group consisting of a binding site 3a, which is bonded to the surface of the metal nanoparticle 2 via a sulfur atom, a positively charged group 3b, and a hydrophobic group 3c. More specifically, the polymer film 3 includes a binding site 3a, which is bonded to the surface of the metal nanoparticle 2 via a sulfur atom, and a primary ammonium group (-NH ３ ＋ ) and hydrophobic groups 3c. The binding sites 3a bond the surfaces of the metal nanoparticles 2 to the polymer film 3 via sulfur atoms. The positively charged groups 3b form electrostatic bonds (ionic bonds) b with the surfaces of the negatively charged metal nanoparticles 2. The hydrophobic groups 3c form hydrophobic bonds c with the surfaces of the metal nanoparticles 2.

[0036] Because all three of these bonds provide a relatively strong bond between the surface of the metal nanoparticle 2 and the polymer film 3, the polymer film 3 is stably fixed to the surface of the metal nanoparticle 2 by at least one of these three bonds. This prevents the polymer film 3 from peeling off from the surface of the metal nanoparticle 2. As a result, the detachment of the specific binding substance 4 due to peeling off of the polymer film 3 is suppressed, thereby suppressing a decrease in detection sensitivity. In addition, exposure of the surface of the metal nanoparticle 2 due to peeling off of the polymer film 3 is suppressed, suppressing quenching due to contact with the excited fluorescent substance 6 and suppressing a decrease in detection sensitivity. Furthermore, because the polymer film 3 contains polymer 3A, it is easier to chemically modify than a silica layer and requires less surface modification. This allows the film thickness to be smaller than that of a silica layer, thereby reducing the distance between the metal nanoparticles 2 in the composite. Therefore, a near-field is formed more efficiently, improving detection sensitivity. As a result, the nanoparticle 1 according to this embodiment has superior detection stability.

[0037] As shown in FIG. 5 , the polymer 3A constituting the polymer film 3 can include at least one selected from the group consisting of a sulfur atom-mediated binding site 3a, a positively charged group 3b, and a hydrophobic group 3c between the surface of the metal nanoparticle 2 and the polymer 3A. The presence of the sulfur atom-mediated binding site 3a, the positively charged group 3b, and the hydrophobic group 3c can be confirmed by measuring signals derived therefrom using infrared spectroscopy, 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 can have a site containing a disulfide bond (—S—S—) as a side chain. The site containing the disulfide bond can have a positively charged group 3b and / or a hydrophobic group 3c.

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

[0039] -Positively Charged Group- The polymer film 3 can contain at least a positively charged group 3b in the side chain of a polymer 3A constituting the polymer film 3. The positively charged group 3b forms a relatively strong electrostatic bond with the surface of the metal nanoparticle 2. In this specification, the positively charged group is a group that has a valence of one or more and is completely positively ionized. When considering the multiple positively charged groups 3b contained in the polymer 3A constituting the polymer film 3, the positively charged group 3b can be expressed by the following mathematical formula (1): [In formula (1), pKa is an electrically neutral group contained in the polymer 3A constituting the polymer film 3, and if it is positively charged, it is a positively charged group (more specifically, a primary ammonium group (—NH ３ ＋ ) etc.) a group which can be 3b (hereinafter also referred to as an electrically neutral group) (more specifically, a primary amino group (—NH ２) etc.), pH indicates the pH of the environment in which the test substance is detected (more specifically, the pH of the sample etc.), B indicates an electrically neutral group contained in the polymer 3A, BH ＋ indicates a positively charged group 3b contained in the polymer 3A]. That is, the positively charged group 3b is a group having a pKa of 7 or more, which is represented by the following chemical equilibrium formula (2): [In chemical equilibrium equation (2), H ＋ indicates a proton, and B and BH ＋ is B and BH in formula (1). ＋ and are synonymous with each other], and the concentration of positively charged groups ([BH ＋ ]) is 10 times or more greater than the concentration of electrically neutral groups ([B]).

[0040] The positively charged group may be, for example, a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, or a guanidyl group (—NHC(═NH ２ ＋ ) NH ２ ) is at least one selected from the group consisting of

[0041] -Hydrophobic Group- The polymer membrane 3 can include at least a hydrophobic group 3c in the side chain of a polymer 3A constituting the polymer membrane 3. The hydrophobic group 3c is, for example, at least one type selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group.

[0042] Examples of aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups. Aromatic carbocyclic groups are groups that do not include aromatic heterocyclic rings and contain aromatic rings in which all ring atoms are carbon atoms. Examples of aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups, etc.) and arylalkyl groups (more specifically, benzyl groups, etc.). Aromatic heterocyclic groups are groups that contain aromatic rings in which at least one ring atom is a heteroatom (more specifically, oxygen atom, sulfur atom, nitrogen atom, etc.). Examples of aromatic heterocyclic groups include nitrogen-containing aromatic heterocyclic groups (more specifically, imidazolyl groups and pyridyl groups (pyridinyl groups), etc.), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups.

[0043] An aliphatic cyclic group is a group that does not contain an aromatic ring and contains a cyclic group consisting of a non-aromatic ring. Examples of the aliphatic cyclic group include an aliphatic carbocyclic group and an aliphatic heterocyclic group. An aliphatic carbocyclic group is a group that contains a non-aromatic ring whose ring atoms are all carbon atoms, such as a cycloalkyl group. An aliphatic heterocyclic group is a group that contains a non-aromatic ring whose ring atoms are at least one heteroatom.

[0044] The aliphatic chain group is a chain (more specifically, a linear or branched chain) group that does not contain an aromatic ring or a non-aromatic ring. Examples of the aliphatic chain group include aliphatic carbon chain groups (more specifically, alkyl groups, alkylene groups, etc.) and aliphatic hetero chain groups.

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

[0046] The polymer 3A constituting the polymer membrane 3 may form a bond via a sulfur atom via a moiety (linker moiety) derived from a crosslinking agent. Examples of such a crosslinking agent include an amino group-sulfhydryl group crosslinking agent (more specifically, an NHS-maleimide group crosslinking agent, etc.).

[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 comprising two metal nanoparticles 2, the separation distance (separation distance) is such that a near-field is efficiently formed in the space between the two metal nanoparticles 2, thereby further improving detection sensitivity. Furthermore, when the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance 6 in the composite are arranged at a predetermined distance, thereby suppressing quenching of fluorescence emitted from the excited fluorescent substance 6 during measurement, thereby further improving detection sensitivity. Note that, in this specification, the separation distance (separation distance) refers to the minimum distance (shortest distance) between the surfaces of the metal nanoparticles 2 contained in each of the two nanoparticles bound via the analyte in the composite.

[0048] (Method for Forming a Polymer Film) The method for forming a polymer film 3 includes a step of contacting a polymer having a disulfide bond in its side chain with a metal nanoparticle 2 to form a polymer film 3 in which the polymer is bonded to the surface of the metal nanoparticle 2 via a sulfur atom. The polymer as a raw material includes, for example, at least one group selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c bonded via a disulfide bond. In this case, the step of forming the polymer film 3 also bonds at least one group selected from the group consisting of the positively charged group 3b and the hydrophobic group 3c to the surface of the metal nanoparticle 2. Note that when a polymer having a positively charged group 3b and / or a hydrophobic group 3c in its side chain via a disulfide group is used as a raw material in the method for forming the polymer film 3, the polymer 3A constituting the polymer film 3 may have a positively charged group 3b and / or a hydrophobic group 3c in its side chain via a disulfide group in the formed polymer film 3. The positively charged groups 3 b and / or hydrophobic groups 3 c in the polymer 3 A are groups that remain after not completely reacting in the method for forming the polymer film 3 .

[0049] A more detailed description will be given with reference to FIG. 6 . FIG. 6 is a schematic diagram illustrating a method for forming a polymer film 3. A polymer 3B has positively charged groups 3b and hydrophobic groups 3c on its side chains, which are bonded via a disulfide bond. When the polymer 3B is brought into contact with a metal nanoparticle 2, the bond between the sulfur atoms of the disulfide bond is cleaved, and the polymer 3B is bonded to the surface of the metal nanoparticle 2 via the sulfur atom. At the same time, the positively charged groups 3b and the hydrophobic groups 3c are each bonded to the surface of the metal nanoparticle 2 via the sulfur atom. The method for forming the polymer film 3 can simultaneously form the polymer film 3 on the surface of the metal nanoparticle 2 and modify the surface of the metal nanoparticle 2 (e.g., surface modification with 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) The specific binding substance 4 is a nano-sized substance (having a maximum size of 3 to 15 nm) that specifically binds to a test substance (described in the fourth embodiment) in a sample. Examples of the specific binding substance 4 include antibodies (hereinafter referred to as nanoantibodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA and RNA chains). For example, a nanoantibody serving as the specific binding substance 4 specifically binds to an antigen serving as a test substance at its tip (antigen binding site) through an antigen-antibody reaction to form a complex. A ligand serving as the specific binding substance 4 forms a complex with a protein serving as a test substance through a specific protein-ligand bond through a ligand-receptor reaction. The nucleic acid chain serving as the specific binding substance 4 forms a nucleic acid chain pair (double strand) with a complementary nucleic acid chain based on base pair complementarity. The enzyme serving as the specific binding substance 4 forms an enzyme-substrate complex with a substrate serving as a test substance at its active site (active center) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as those resulting from hydrogen bonds, as well as intermolecular forces, hydrophobic interactions and charge interactions.

[0051] Nanobodies include, for example, VHH (variable domain heavy chain antibody) antibodies, Fab (fragment antigen binding) antibodies, and their variants. VHH antibodies are single-domain antibodies. Variants are antibodies with partially modified amino acid sequences or antibodies with introduced substituents, within the range of specific binding to an antigen. Nanobodies are preferably VHH antibodies. When the nanobody is a VHH antibody, the VHH antibody has a relatively small volume, which can narrow the distance (separation distance) between two metal nanoparticles 2 in the complex, form a near field more efficiently, and further increase the fluorescence intensity.

[0052] The molecular mass of the nanobody is preferably 60,000 Da or less, more preferably 30,000 Da or less, and even more preferably 20,000 Da or less. When the molecular mass is 60,000 Da or less (particularly 30,000 Da or less or 20,000 Da or less), the nanobody has a relatively small volume, which narrows the separation distance in the complex, more efficiently forms a near-field, and further increases the fluorescence intensity. Methods for measuring the 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 membrane 3, or may be bound to the polymer membrane 3 via a linker moiety (more specifically, SM(PEG)) derived from a crosslinking agent (more specifically, an NHS-maleimide group crosslinking agent, etc.). ６ The polymer film 3 may be indirectly bonded via a bonding agent such as a fluorine-containing polymer.

[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 complexes of metals such as europium and ruthenium (metal complexes), and dyes in 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 are less likely to overlap, and excitation light (scattered light) is less likely to enter the fluorescence to be detected, allowing for more accurate measurement of the fluorescence intensity.

[0056] The fluorescent substance preferably has a sharp fluorescence spectrum. A sharp fluorescence spectrum is less likely to overlap with the absorption spectrum, making it less likely for excitation light (scattered light) to enter the fluorescence to be detected, allowing for more accurate measurement of fluorescence intensity.

[0057] (Measuring Device for Detecting Test Substance) A measuring device using the above-described method for detecting a test substance will be described with reference to FIG. 13 . FIG. 13 is a diagram illustrating the measuring device. As shown in FIG. 13 , the measuring device 100 includes an excitation light source 110, an excitation light irradiation optical system 120, a reagent container 130, a light-receiving optical system 140, and a light-receiving element 150. The excitation light source 110 emits excitation light 112. The excitation light source 110 is, for example, a laser light source. The excitation light irradiation optical system 120 adjusts the cross-sectional diameter of the excitation light 112, such as by focusing the excitation light, and outputs incident excitation light 122. The excitation light irradiation optical system 120 includes a lens 124 and a polarizing element (λ / 2 plate) 126. The incident excitation light 122 output from the excitation light irradiation optical system 120 enters the reagent container 130 and is irradiated onto the measurement sample in the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell, a preparation, or the like) and a microchannel chip. The microchannel chip is a chip having a minute channel. When the reagent container 130 is a microchannel chip, for example, the nanoparticle body (reagent) according to the first embodiment and a specimen can be mixed and continuously supplied. This eliminates the need to mix and prepare a measurement sample in advance, enabling continuous measurement.

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

[0059] The fluorescence 142 that passes through the light-receiving optical system 140 is detected by the light-receiving element 150. The light-receiving element 150 is, for example, a PD, an APD, a PMT, a CCD camera, or a spectroscope. The light-receiving element 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring a fluorescence spectrum, and creating a two-dimensional fluorescence image.

[0060] Second Embodiment: Nanoparticles The second embodiment differs from the first embodiment in the spectral characteristics of the fluorescent substance. This difference will be mainly described below. Note that in the second embodiment, the same reference numerals as in the first embodiment represent the same configuration as in the first embodiment, and therefore their description will be omitted.

[0061] In the first embodiment, the fluorescence used to detect the test substance is "excitation-induced fluorescence" induced by the overlap 208 between the absorption spectrum 202 and the plasmon resonance spectrum 206 of the fluorescent substance 6. In the second embodiment, the fluorescence used to detect the test substance is "luminescence-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 nanoparticles according to the second embodiment is also referred to as "fluorescent substance 6A" to indicate that it has different spectral characteristics from and to distinguish it from the fluorescent substance 6 in the nanoparticles according to the first embodiment.

[0062] The nanoparticles according to this embodiment can enhance detection sensitivity. Without being bound by any particular theory, the reason for this 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 plasmon resonance in a complex in which two or more nanoparticles are bound via an analyte. This results in 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, and fluorescence for detecting the analyte is induced by a dipole-dipole mechanism due to multipole resonance and the Purcell effect. Therefore, the fluorescent substance 6A is sufficiently excited by light at the emission wavelength of plasmon resonance derived from the complex, thereby enhancing detection sensitivity.

[0063] The following description will be made with reference to Fig. 7. Fig. 7 is a conceptual diagram showing a plasmon resonance spectrum 201 derived from the metal nanoparticle 2 in a single particle state, a plasmon resonance spectrum 206 derived from the metal nanoparticle 2 in the composite, and a fluorescence spectrum 210 of the fluorescent substance 6A. In this embodiment, the fluorescent substance 6A is selected so that there is (preferably, a large) overlap 212 between the fluorescence spectrum 210 of the fluorescent substance 6A and the resonance spectrum 206 derived from the metal nanoparticle in the composite. In this way, the fluorescence wavelength range WR of the fluorescent substance 6A Ｅ is the first emission wavelength range WR of the plasmon resonance Ｅ１ When the second region R overlaps with the first region R, the fluorescent substance 6A is sufficiently excited, the fluorescence is enhanced, and the detection sensitivity is greatly improved. Therefore, it is considered that the nanoparticle according to this embodiment can improve the detection sensitivity. In other words, from the viewpoint of improving the detection sensitivity of the test substance, ２ It is important to select the fluorescent material 6A so that the second region R ２ 7, the second region R 1 is 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). ２ It is important to select a fluorescent material 6A that has a large value.

[0064] [Scheme 2] Scheme 2 of the process for detecting a test substance using the nanoparticles according to this embodiment: (In the elementary steps (1) to (3) of Scheme 2, S ０ denotes the ground state, and S １ indicates an excited singlet state, * indicates an excited state, Flu indicates a fluorescent substance 6A (in the composite), and M indicates a metal nanoparticle 2 (in the composite)).

[0065] Scheme 2 includes elementary steps (1) to (3). As shown in elementary step (1), for example, the fluorescent substance 6A is excited by external irradiation light. As shown in elementary step (2), the excited fluorescent substance 6A relaxes and emits fluorescence. As shown in elementary step (3), the emitted fluorescence induces plasmon resonance (multipole resonance) on the surface of the metal nanoparticle 2 (i.e., the plasmon resonance in the composite is induced by the fluorescence emitted by the fluorescent substance 6A). This plasmon resonance (multipole resonance) occurs in the emission wavelength range WR of the fluorescent substance 6A. Ｅ is the first emission wavelength range WR of the plasmon resonance Ｅ１ When these overlap, fluorescent emission from the excited 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 substance 6A in the composite is located in the range of 500 to 700 nm (more preferably, 550 to 700 nm, and even more preferably, 600 to 700 nm). That is, the first emission wavelength region WR of the plasmon resonance spectrum originating from the metal nanoparticles 2 in the composite Ｅ１The maximum fluorescence wavelength of fluorescent substance 6A is located in the range of 500 to 700 nm within the range. Examples of fluorescent substances include fluorescein derivatives, rhodamine derivatives, cyanine dyes, and Alexa Fluor (registered trademark) manufactured by Molecular Probes. Among these, examples of fluorescent substance 6A having a maximum fluorescence wavelength of 500 to 700 nm include "Ruthenium(II) tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd., and Alexa Fluor (registered trademark) series 430, 488, 532, 546, 555, 568, 594, and 640. The maximum fluorescence wavelength can be determined as follows: The absorption spectrum of an aqueous solution of the fluorescent substance (solvent: deionized water) is measured, and the peak position of the obtained absorption spectrum is determined. The fluorescent substance 6A is irradiated with excitation light having a wavelength at the peak position, the fluorescence spectrum of an aqueous solution of the fluorescent substance is measured, and the peak position of the obtained fluorescence spectrum is taken as the maximum fluorescence wavelength.

[0067] Third Embodiment: Nanoparticles The third embodiment differs from the first and second embodiments in the spectral characteristics of the fluorescent substance. This difference will be mainly described below. Note that in the third embodiment, the same reference numerals as those in the first and second embodiments represent the same configuration as those in the first and second embodiments, and therefore their description will be omitted.

[0068] In the third embodiment, the fluorescence for detecting a test substance is induced by an overlap 208 between the absorption spectrum 202 and the plasmon resonance spectrum 206 of the fluorescent substance 6 shown in the first embodiment, and is induced by an 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 composite is excitation-induced fluorescence induced by external irradiation light, and emission-induced fluorescence induced by fluorescence. In this specification, the fluorescent substance in the nanoparticles 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 nanoparticles according to the first embodiment and the fluorescent substance 6A in the nanoparticles according to the second embodiment, which have different spectral characteristics.

[0069] The following description will be made with reference to Fig. 8. Fig. 8 is a conceptual diagram showing a plasmon resonance spectrum 201 derived from the metal nanoparticle 2 in a single particle state, a plasmon resonance spectrum 206 of the metal nanoparticle 2 in the composite, and an absorption spectrum 202 and a fluorescence spectrum 210 of the fluorescent substance 6B. In this embodiment, from the viewpoint of enhancing the detection sensitivity of the test substance, the overlap between the absorption spectrum 202 and the fluorescence spectrum 210 of the fluorescent substance 6B and the resonance spectrum 206 derived from the metal nanoparticle 2 in the composite (respectively, the absorption wavelength range WR Ａ and the first emission wavelength range WR Ｅ１ and the fluorescence wavelength range WR Ｅ and the first emission wavelength range WR Ｅ１ The fluorescent substance 6B is selected so that there is an overlap 212 with the fluorescent substance 6B. Furthermore, in a preferred embodiment, from the viewpoint of further increasing the detection sensitivity of the test substance, the fluorescent substance 6B can be selected so that the overlap 208 and the overlap 212 are large. 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 nanoparticle according to this embodiment can increase the detection sensitivity.

[0070] [Scheme 3] Scheme 3 of the process of detecting a test substance using the nanoparticles according to this embodiment: (In the elementary steps (1) to (4) of Scheme 3, S ０ denotes the ground state, and S １ indicates an excited singlet state, * indicates an excited state, Flu indicates the fluorescent substance 6B (in the composite), and M indicates the metal nanoparticle 2 (in the composite)) are shown below.

[0071] Scheme 3 includes elementary steps (1) to (4). As shown in elementary step (1), when the complex formed by capturing the test substance is irradiated with external irradiation light, plasmon resonance (multipole resonance) is induced on the surface of the metal nanoparticle 2 (i.e., the plasmon resonance in the complex is induced by the external irradiation light). As shown in elementary step (2), the fluorescent substance 6B is excited by the dipole-dipole mechanism and the near field. The absorption wavelength range WR of the fluorescent substance 6B Ａ is the first emission wavelength range WR of the plasmon resonance Ｅ１When the excitation wavelengths of the fluorescent substance 6B overlap, the fluorescent substance 6B is excited efficiently. 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 (i.e., the plasmon resonance in the composite is induced by the fluorescence emitted by the fluorescent substance 6B). This plasmon resonance (multipole resonance) occurs in the emission wavelength range WR of the fluorescent substance 6B. Ｅ is the first emission wavelength range WR of the plasmon resonance Ｅ１ When these overlap, fluorescent emission from the excited fluorescent substance 6B is efficiently induced.

[0072] The amount of external irradiation light is greater than the amount of fluorescence. Therefore, from the viewpoint of further improving the detection sensitivity, it is preferable that the first emission wavelength range WR Ｅ１ and the absorption wavelength range WR of the fluorescent material 6B Ａ The first region R １ is the second light emitting wavelength range WR Ｅ２ and the absorption wavelength range WR of the fluorescent material 6B Ａ The second region R ２ The fluorescent material 6B is selected so that the luminance is larger than that of the fluorescent material 6B (see FIG. 8).

[0073] (First area R １ and the second region R ２ Method for determining the magnitude relationship with the first region R １ and the second region R ２ The method for determining the magnitude relationship between the normalized multipole resonance spectrum 206 and the normalized fluorescence spectrum 210 of the fluorescent substance 6B is performed as follows: The plasmon resonance spectrum of the composite is measured using a fluorescence microspectrometer (described in detail 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 (a measurement sample of the fluorescent substance 6B is prepared using deionized water as a solvent). An overlap 208 between the normalized multipole resonance spectrum 206 and the normalized absorption spectrum 202 of the fluorescent substance 6B is created. The integral value of the overlap 208 is calculated. An overlap 212 between the normalized multipole resonance spectrum 206 and the normalized fluorescence spectrum 210 of the fluorescent substance 6B is created. The integral value of the overlap 212 is calculated. Based on the magnitude relationship of the obtained integral values, the first region R１ and the second region R ２ The magnitude relationship with is determined.

[0074] <Fourth Embodiment: Conjugate> The conjugate will be described with reference to Fig. 9 . Fig. 9 is a cross-sectional view schematically illustrating a conjugate according to the fourth embodiment. The conjugate according to the fourth embodiment comprises two nanoparticles 1 according to the first embodiment, each of which comprises a first nanoparticle 10 and a second nanoparticle 20, and the first nanoparticle 10 and the second nanoparticle 20 are bound via an analyte 30. The conjugate 40 comprises an analyte 30 to be detected, and two nanoparticles 10, 20. The two nanoparticles 10, 20 are bound via the analyte 30 in the conjugate 40. In other words, the nanoparticles 10, 20 according to the first embodiment are bound via the analyte 30 to form a conjugate according to the fourth embodiment. One of the two nanoparticles 10, 20 will be referred to as the first nanoparticle 10, and the other nanoparticle will be referred to as the second nanoparticle 20.

[0075] In the complex 40, the first nanoparticles 10 comprise first metal nanoparticles 12, a first polymer film 13 coating the surfaces of the first metal nanoparticles 12, a first specific binding substance 14 that specifically binds to an analyte 30 in a specimen, 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. That is, the first nanoparticles 10 comprise first metal nanoparticles 12 as metal nanoparticles, the first polymer film 13 as a polymer film, the first specific binding substance 14 as a specific binding substance, and the first fluorescent substance 16 as a fluorescent substance. In the complex 40, the second nanoparticles 20 comprise second metal nanoparticles 22, a second polymer film 23 coating the surfaces of the second metal nanoparticles 22, a second specific binding substance 24 that specifically binds to an analyte 30 in a specimen, 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. That is, the second nanoparticle body 20 includes 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 the fluorescence intensity, it is preferable that the separation distance L is small, within a range in which the excited fluorescent substances 16, 26 are not easily quenched. More specifically, in a preferred embodiment, the two nanoparticles 10, 20 in the composite 40 are close to each other. In a more preferred embodiment, the two nanoparticles 10, 20 are close to each other so that the first polymer film 13 of the first nanoparticle 10 and the second polymer film 23 of the second nanoparticle 20 in the composite 40 come into contact. In an even more preferred embodiment, the two nanoparticles 10, 20 are close to each other so that at least one of the polymer films 13 of the first nanoparticle 10 and the second polymer film 23 of the second nanoparticle 20 in the composite 40 contracts and comes into contact.

[0077] In a further preferred embodiment, when at least one of the polymer films 13, 23 shrinks and comes into contact with each other, for example, in the complex 40 shown in Figure 9, it is believed that at least one of the analyte 30, the specific binding substances 14, 24 that bind to the analyte 30, and the fluorescent substances 16, 26 can be inserted into the polymer films 13, 23. Furthermore, in a more preferred embodiment, when the polymer films 13, 23 come into contact with each other, for example, in the complex 40 shown in Figure 9, it is believed that at least one of the analyte 30, the specific binding substances 14, 24 that bind to the analyte 30, and the fluorescent substances 16, 26 can be inserted into the polymer films 13, 23, as in the further preferred embodiment (the same applies to the complex shown in Figure 10 described below).

[0078] In this embodiment, the films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, respectively, which can increase the fluorescence intensity. Without being bound by any particular theory, the reason for this is presumed to be as follows: The films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, which have relatively high flexibility compared to inorganic films containing inorganic oxides. Therefore, in the composite 40, the polymer films 13 and 23 can contract, which allows the two metal nanoparticles 12 and 22 to be closer than a distance equivalent to two polymer film thicknesses (the film thickness of the polymer film 13 plus the film thickness of the polymer film 23). In other words, because the films covering the surfaces of the metal nanoparticles 12 and 22 are polymer films 13 and 23, the separation distance L can be less than two polymer film thicknesses. This facilitates the plasmon enhancement effect, further increasing the fluorescence intensity. In this specification, the thickness of the polymer film in "two thicknesses of the polymer film" does not refer to the thickness of the polymer film 13, 23 in the contracted portion that is the target of the separation distance, but to the thickness of the polymer film 13, 23 in the non-contracted portion that is not the target of separation.

[0079] In this embodiment, the polymer films 13, 23 contain at least one selected from the group consisting of sulfur-atom-mediated binding sites 3a, positively charged groups 3b, and hydrophobic groups 3c (e.g., the polymer 3A constituting the polymer films 13, 23 contains at least one selected from the group consisting of sulfur-atom-mediated binding sites 3a, positively charged groups 3b, and hydrophobic groups 3c), thereby further increasing the fluorescence intensity. Without being bound by any particular theory, the reason for this is presumed to be as follows. In this case, at least one of the binding sites 3a, positively charged groups 3b, and hydrophobic groups 3c forms a bond with the surface of the metal nanoparticles 12, 22. Therefore, the polymer 3A is thought to have a mesh-like structure and coat the surfaces of the metal nanoparticles 12, 22 in a mesh-like manner. Because the polymer 3A has this mesh-like structure, it also has relatively high flexibility. Therefore, in the composite 40, the polymer films 13, 23 can further contract, thereby allowing the two metal nanoparticles 12, 22 to be closer together than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two times the thickness of the polymer film, and the plasmon enhancement effect is further obtained, further increasing the fluorescence intensity.

[0080] In a preferred embodiment, the polymer 3A constituting the polymer film 13, 23 includes at least one selected from the group consisting of a binding site 3a, a positively charged group 3b, and a hydrophobic group 3c via a sulfur atom in its side chain (more specifically, at the side chain terminal). In a preferred embodiment, the fluorescence intensity can be further increased. Without being bound by any particular theory, the reason for this is presumed to be as follows. In such a case, at least one of the binding site 3a, the positively charged group 3b, and the hydrophobic group 3c forms a bond with the surface of the metal nanoparticle 12, 22. Therefore, the polymer 3A has a mesh-like structure, and it is thought that the side chain serves as a binding site to coat the surface of the metal nanoparticle 12, 22 in a mesh-like manner. Because of this mesh-like structure, the polymer 3A has relatively high flexibility. Therefore, in the composite 40, the polymer film 13, 23 can further contract, thereby allowing the two metal nanoparticles 12, 22 to be closer together than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two times the thickness of the polymer film, and the plasmon enhancement effect is further obtained, further increasing the fluorescence intensity.

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

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

[0083] The composite 40 may be configured to include two nanoparticles 10, 20 as shown in Figures 9 to 11, or may be configured to include three nanoparticles 10, 20, 60 as shown in Figure 12. The composite may also be configured to include four or more nanoparticles. Figure 11 is a diagram showing a composite 40 configured from two nanoparticles. Figure 12 is a diagram showing a composite 40 configured from three nanoparticles.

[0084] In the first to third embodiments, the multipole resonance was described as plasmon resonance originating from the metal nanoparticles 12, 22 in the composite 40. This will be described in more detail with reference to Fig. 11 and Fig. 12. In Fig. 11, the binding site between the nanoparticles 10, 20 in the composite 40 (more specifically, the test substance 30 and the specific binding substances 14, 24) is represented by a binding point 50 (the same applies to Fig. 12). In the composite 40 formed of two nanoparticles 10, 20, the vibration direction of the electric field component of the excitation light (the polarization direction of the electric field component of the excitation light) is aligned with the long axis direction D of the composite 40. ＬＡ When the free electrons on the surfaces of the metal nanoparticles 12 and 22 are parallel to the long axis D ＬＡ Therefore, multipole resonance is induced by dipole-dipole interaction. On the other hand, if the vibration direction of the electric field component of the excitation light is in the short axis direction D of the composite 40, ＳＡ When the free electrons on the surfaces of the metal nanoparticles 12 and 22 are parallel to the minor axis D ＳＡThis can cause collective vibrations, which leads to dipole resonance, similar to that of single-particle metal nanoparticles.

[0085] In the composite 40 composed of the three nanoparticles 10, 20, and 60, the vibration direction D of the electric field component of the excitation light that is allowed for multipole resonance is １ , D ２ , D ３ The number of binding points 50 in the complex 40 is three, which is more than that in the complex 40 composed of two nanoparticles 10, 20. Furthermore, the complex 40 composed of three nanoparticles 10, 20, 60 has three binding points 50, which is more than that in the complex 40 composed of two nanoparticles 10, 20. In this way, the complex 40 may include a plurality of binding points 50 (where two nanoparticles 10, 20 are bound via one analyte 30). Therefore, the greater the number of nanoparticles 10, 20 constituting the complex 40, the more likely it is that fluorescence will be enhanced, and the greater the detection sensitivity may be.

[0086] 9 and 10, the fluorescent substances 16, 26 are preferably positioned between the first metal nanoparticles 12 and the second metal nanoparticles 22. This is because the space between the metal nanoparticles 12, 22 is one in which a near-field is efficiently generated, and therefore, by positioning the fluorescent substances 16, 26 in the space between the metal nanoparticles 12, 22, the fluorescence intensity is likely to be increased by the Purcell effect.

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

[0088] Other Embodiments The present invention is not limited to the above-described embodiments, and design changes are possible without departing from the gist of the present invention.

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

[0090] In the fourth embodiment, the first and second fluorescent substances 16 and 26 are labeled on the first and second polymer films 13 and 23, respectively. However, this is not limiting. For example, FIG. 10 is a cross-sectional view schematically illustrating a complex according to a modified example of the fourth embodiment. As shown in FIG. 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. This is more preferable because it makes it easier to position the first and second fluorescent substances 16 and 26 between the first metal nanoparticle 12 and the second metal nanoparticle 22, improving detection intensity. Alternatively, one of the first and second fluorescent substances 16 and 26 may be labeled on the polymer films 13 and 23, and the other may be labeled on the specific binding substances 14 and 24.

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

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

[0093] The present invention will be described in more detail below using examples. However, the present invention is not limited to the following examples. Unless otherwise specified, parts and percentages in the examples are by mass. Unless otherwise specified, the examples and comparative examples were carried out in the atmosphere at room temperature (1 atmosphere, 25°C).

[0094] In the examples and comparative examples, the concentration of metal nanoparticles in a dispersion liquid is sometimes expressed as absorbance. The absorbance was measured using an ultraviolet-visible spectrophotometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.). The absorption wavelength differs depending on the sample, so it is listed for each sample. The number subscripted to the absorbance notation OD indicates the absorption wavelength. For example, OD ４５５= 0.1 indicates that the absorbance at a wavelength of 455 nm is 0.1.

[0095] Example 1 [1. Formation of polymer film] A method for forming a polymer film will be described with reference to Figures 14 and 15. Figures 14 and 15 are schematic diagrams showing the method for forming a polymer film in Example 1. As shown in Figure 14, poly-L-lysine ("3075" manufactured by Peptide Institute, Inc.) and 3-(2-pyridyldithio)propionamide-PEG ４ -NHS (Thermo Fisher Scientific, serial number "26128", "NHS-PEG ４ The mixture was stirred and mixed with 3-(2-pyridyldithio)propionamide-PEG (poly-L-lysine) at room temperature for 4 hours using a small rotary incubator (RT-30min manufactured by Taitec Co., Ltd.). As a result, a polymer was obtained. This synthesis reaction was carried out by converting the primary amino group of poly-L-lysine to 3-(2-pyridyldithio)propionamide-PEG. ４ This is a nucleophilic substitution reaction in which NHS attacks the NHS ester group. The synthesized polymer 3B had a disulfide bond in the side chain. More specifically, the synthesized polymer had a hydrophobic group (pyridyl group) 3c and a positively charged group (primary ammonium group) 3b bonded via a disulfide bond.

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

[0097] As shown in Figure 15, the polymer film 3 contains a sulfur atom-mediated binding 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. That is, the polymer 3A constituting the polymer film 3 contains a sulfur atom-mediated binding 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. SEM images (magnification: 500,000x) of the resulting silver nanoparticles 2 confirmed that the surfaces of the silver nanoparticles 2 were continuously coated with the polymer film 3 (hereinafter, silver nanoparticles coated with the polymer film 3 are referred to as "polymer-coated silver nanoparticles"). The thickness of the polymer film 3 coating the silver nanoparticles 2 was also measured from the SEM image.

[0098] 2. Preparation of Nanoparticles Figure 16 is a schematic diagram showing the structure of nanoparticle 1 bound to a fluorescently labeled antibody in Example 1. The nanoparticles shown in Figure 16 were prepared by first binding a crosslinker to the surface of polymer-coated metal nanoparticles, separately binding a fluorescent substance and a crosslinker to a nanoantibody, and then binding the crosslinker bound to the polymer-coated metal nanoparticles with the crosslinker bound to the nanoantibody. Details of the preparation of nanoparticles bound to a fluorescently labeled antibody are described below.

[0099] (2-1. Bonding of crosslinker to polymer-coated silver nanoparticles) To 1 mL of the prepared dispersion of polymer-coated silver nanoparticles, the crosslinker SM(PEG)2 (PEGylated, long-chain SMCC crosslinker) (ThermoFisher SCIENTIFIC "22105") and heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd. "081-00136") were added, and the mixture was stirred and mixed using a small rotary incubator (Taitec Co., Ltd. "RT-30mini") at room temperature for 1 hour. As a result, a dispersion of silver nanoparticles in which the crosslinker SM(PEG)2 was bonded to the polymer membrane 3 (hereinafter also referred to as polymer-coated silver nanoparticles bonded to the SM(PEG)2 linker) was obtained. The SM(PEG)2 linker bonded to the polymer-coated silver nanoparticles had a maleimide group.

[0100] (2-2. Fluorescent labeling of VHH antibody) A fluorescent label of the chemical formula [Chemical Formula 5] was used for 100 μg of a VHH antibody (manufactured by RePHAGEN, molecular mass 18,000 Da). An NHS-labeled Ru complex derivative represented by the formula ("Ruthenium(II) tris(bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator ("RT-30mini" manufactured by Taitec Corporation). As a result, a VHH antibody bound to a fluorescent substance (hereinafter also referred to as a fluorescent-labeled VHH antibody) was obtained. Note that the NHS-labeled Ru complex derivative is a fluorescent substance having a maximum fluorescence wavelength in the range of 500 to 700 nm.

[0101] (2-3. Binding of a crosslinker to a fluorescently labeled VHH antibody) Next, an 8-fold molar equivalent of 3-(2-pyridyldithio)propionamide-PEG4-NHS (manufactured by Thermo Fisher Scientific, product number "26128," "NHS-PEG4-SPDP") serving as an NHS-bipyridyl disulfide crosslinker was added to the fluorescently labeled VHH antibody, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator (manufactured by Taitec Corporation, "RT-30mini"). As a result, a VHH antibody bound to a fluorescent substance and an SPDP linker (hereinafter also referred to as an SPDP linker-bound fluorescently labeled VHH antibody) was obtained.

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

[0103] (2-5. Binding of fluorescently labeled VHH antibodies to silver nanoparticles) Next, a dispersion of polymer-coated silver nanoparticles to which SM(PEG)6 linkers having maleimide groups were bound (OD ４３０ A fluorescently labeled VHH antibody bound to a reduced SPDP linker was added to the SM(PEG)6 solution (pH 7.0, pH ...

[0104] [3. Preparation of Complex] To the obtained phosphate buffer solution of the nanoparticles, C Reactive Protein (manufactured by ADVY CHEMICAL Sigma-Aldrich Co., Ltd., "00-AGN-AP-CRP-00") (hereinafter also referred to as CRP antigen) as the test substance 30 was added, and the mixture was stirred at room temperature for 5 minutes using a small rotary incubator (manufactured by Taitec Co., Ltd., "RT-30mini") to obtain a measurement sample (OD 455 = 0.4) was prepared. As a blank, a phosphate buffer solution of the nanoparticles not containing the CRP antigen 30 was prepared separately.

[0105] [4. Evaluation Method] (4-1. Detection of Test Substance by Immunochromatography) Figure 17 is a schematic diagram showing an immunochromatography strip (hereinafter also simply referred to as "strip"). This strip 300 is in the shape of a rectangular strip and includes a sample pad 301 located at one end thereof, onto which a measurement sample is dropped, and a test line 303 located in the center. A CRP antigen is immobilized on the test line 303 as the test substance 30. When a measurement sample is dropped onto the sample pad 301, a complex 40 in the measurement sample moves toward the test line 303 (along a direction 305) due to capillary action. When the complex 40 reaches the test line 303, an antigen-antibody reaction occurs with the CRP antigen, and the complex 40 is captured.

[0106] A measurement sample was dropped onto the sample pad 301 of the strip 300 and allowed to stand for a predetermined time. The left-standing strip 300 was set in a fluorometer ("fluorometer for in-house experiments" manufactured by PHC Corporation). Excitation light (wavelength: 415 to 455 nm) was irradiated onto the test 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 whether or not there was an amount of fluorescence derived from the fluorescent substance. As a result, it was found that the concentration of CPR antigen in the preparation of the test sample was 42 pM (unit: × 10 －１２ Fluorescence was detected at concentrations of 0.01% mol / L or higher.

[0107] The left-standing strip 300 was set in an absorptiometer (PHC Corporation's "in-house experimental absorptiometer"). Visible light (wavelength 415 to 455 nm) was irradiated onto the test 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 derived from metal nanoparticles. As a result, in the preparation of the test sample, reflected light was detected when the CRP antigen concentration was 670 pM or higher.

[0108] The CRP antigen, which is the test substance 30, was detected by immunography, confirming that the complex 40 had been formed in the measurement sample and that the complex 40 was present at the test line.

[0109] (4-2. Analysis of plasmon resonance wavelength in the presence of a test substance) First, a measurement sample was prepared. The measurement sample was prepared in the same manner as in 4-1. A measurement preparation was prepared as a measurement sample by dropping the measurement sample onto a glass slide and placing a glass cover plate on the droplet to sandwich the droplet. The measurement preparation was used to measure the plasmon resonance spectrum in the presence of the droplet. A blank preparation was prepared in the same manner as the measurement preparation, except that no CRP antigen was added.

[0110] Next, the plasmon resonance spectrum of the measurement object was measured using a fluorescence microspectrometer. The measurement object (complex) on the measurement preparation was continuously irradiated with finely focused excitation light (wavelength: 400 to 650 nm), and the plasmon resonance spectrum was measured. The measurement object (complex) in Example 1 was a complex formed by two nanoparticles bound in a diatomic molecular form 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 preparation used as the measurement sample.

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

[0112] 18(a), the plasmon resonance spectrum of the blank preparation exhibited a spectral shape with a single peak around 460 to 470 nm. This peak was attributed to plasmon resonance (dipole resonance) resulting from monodisperse Ag nanoparticles.

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

[0114] Separately, the absorption spectrum and fluorescence spectrum 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 measurement wavelength of 230 to 500 nm in an aqueous solvent at an arbitrary concentration. The fluorescence spectrum was measured using an excitation wavelength of 450 nm and a measurement wavelength of 500 to 700 nm, using the same measurement sample as used for the absorption spectrum. The obtained absorption spectrum and fluorescence spectrum of the fluorescent substance were superimposed on the plasmon resonance spectrum of the measurement preparation in FIG. 18(b) to produce FIG. 19.

[0115] 19 is a diagram showing 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 FIG. 19, the fluorescence wavelength range WR Ｅ and the first emission wavelength range WR of the peak on the long wavelength side of the plasmon resonance spectrum (plasmon resonance spectrum 206). Ｅ１ overlap 212 (second region R ２ ) was confirmed. Therefore, it was concluded that the fluorescence detected in the system of Example 1 was luminescence-induced fluorescence.

[0116] Example 2 [3. Preparation of nanoparticles] In the preparation of nanoparticles in Example 2, the metal nanoparticles were replaced with silver nanoparticles ("AGCB80-1M" manufactured by nanocomposix, diameter 80 nm, OD ４５５ Silver nanoparticles (NANOCOMPOSIX "AGCB50-1M", diameter 50 nm, OD = 0.1) were extracted from 1 mL of the dispersion. ４５５ The nanoparticles were prepared in the same manner as in Example 1, except that the saturation temperature was changed to 0.1.

[0117] (4-2. Analysis of plasmon resonance wavelength in the presence of a test substance) Analysis of plasmon resonance wavelength in the presence of a test substance was carried out in the same manner as in Example 1. The measurement results are shown in Figure 20. Figure 20 shows the plasmon resonance spectra of Example 2 for (a) a blank preparation and (b) a measurement preparation. The measurement subject in Example 2 was a complex composed of 15 nanoparticles.

[0118] 20(a), the plasmon resonance spectrum of the blank preparation exhibited a spectral shape with a single peak around 460 to 470 nm, which was attributed to plasmon resonance (dipole resonance) resulting from monodisperse Ag nanoparticles.

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

[0120] The absorption spectrum and fluorescence spectrum of the Ru complex obtained in Example 1 were superimposed on the plasmon resonance spectrum of the measurement preparation in FIG. 20(b) to create FIG. 21. As shown in FIG. 21, the fluorescence wavelength range WR of the fluorescence spectrum 210 of the Ru complex Ｅand the first emission wavelength range WR of the peak on the long wavelength side of the plasmon resonance spectrum (plasmon resonance spectrum 206). Ｅ１ overlap 212 (second region R ２ ) was confirmed. Therefore, it was concluded that the fluorescence detected in the system of Example 2 was luminescence-induced fluorescence.

[0121] Example 3 3. Preparation of Nanoparticles In the preparation of 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 fluorescence wavelengths in the range of 500 to 700 nm.

[0122] (4-1. Detection of Test Substance by Immunochromatography) As in Example 1, the test substance was detected by immunochromatography. The wavelength of the excitation light irradiated onto the test line 303 was 600 nm. As a result of measuring the amount of fluorescent light, fluorescence was detected when the concentration of CPR antigen in the preparation of the measurement sample was 42 pM or higher. As a result of measuring reflected light, reflected light was detected when the concentration of CPR antigen in the preparation of the measurement sample was 670 pM or higher. The test substance, CRP antigen, was detected by immunography. This confirmed that complex 40 had been formed in the measurement sample and that complex 40 was present at the test line.

[0123] (4-2. Analysis of Plasmon Resonance Wavelength in the Presence of Test Substance) Separately, the absorption spectrum and fluorescence spectrum of a fluorescent substance (Alexa Fluor 594 carboxylic acid, succinimidyl ester (Invitrogen Corporation, "A10169" 20004)) were measured using a spectrophotometer (TECAN Japan Corporation, "infinite M200 PRO"). The absorption spectrum was measured at a wavelength of 450 to 650 nm, and the solvent for the measurement sample was deionized water.

[0124] The absorption spectrum and fluorescence spectrum of the obtained fluorescent substance were superimposed on the plasmon resonance spectrum of the measurement preparation in FIG. 20(b) to produce FIG. 22. FIG. 22 shows the absorption spectrum and fluorescence spectrum of the fluorescent substance of Example 3 and the plasmon resonance spectrum of Example 1. The fluorescence spectrum 210 of the fluorescent substance of Example 3 has a peak near 584 nm. The fluorescence spectrum 210 of the fluorescent substance of Example 3 has a peak near 613 nm. Note that the plasmon resonance spectrum of the system of Example 1 was used as a substitute for the system of Example 3. This is because the composite of Example 3 differs from the composite of Example 1 only in the type of fluorescent substance, and the material and particle size of the metal nanoparticles that primarily characterize the plasmon resonance spectrum are the same (it is thought that the difference is due to the slight addition of the absorption of the fluorescent substance to the plasmon resonance spectrum).

[0125] As shown in FIG. 22, the absorption wavelength range WR of the absorption spectrum 202 of the fluorescent substance Ａ and the first emission wavelength range WR of the peak (multipole resonance spectrum) 206 on the long wavelength side of the plasmon resonance spectrum. Ｅ１ overlap 208 (first region R １ ) was confirmed. Therefore, it was concluded that the fluorescence detected in the system of Example 3 was mainly excitation-induced fluorescence.

[0126] (4-3. Preparation of Complex) CRP antigen as test substance 30 was added to the obtained phosphate buffer solution of the nanoparticles, and the reaction (antibody-antigen reaction) was carried out at room temperature for 10 minutes at a final concentration of 90 ng / mL. In this way, a mixture of a complex composed of the nanoparticles and the test substance was prepared. The prepared mixture was used as a measurement sample. A phosphate buffer solution of the nanoparticles not containing the CRP antigen 30 was separately prepared as a blank.

[0127] (4-4. Fluorescence Analysis of Test Substance-Nanoparticle System) Fluorescence analysis of the test substance-nanoparticle system was performed. Using a spectrofluorometer (JASCO Corporation, "FP-8550"), the fluorescence spectrum after complex formation was measured under conditions of an excitation wavelength of 580 nm and a measurement wavelength range of 600 to 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 near 614 nm. The peak intensity near 614 nm is approximately four times higher than the peak intensity near 614 nm in the fluorescence spectrum of the blank sample.

[0128] As shown in Fig. 22, the fluorescent substance of Example 3 has a maximum absorption wavelength near 586 nm. Furthermore, the absorption spectrum 202 of the fluorescent substance of Example 3 largely overlaps with the multipole resonance spectrum 206. It was confirmed that by using a fluorescent substance having absorption matching the plasmon resonance wavelength of the composite as shown in Fig. 22, fluorescence was effectively enhanced, as shown in Fig. 23.

[0129] Comparative Example 1 Silica-coated silver nanoparticles (manufactured by nanoComposix, silver nanoparticle particle diameter (core particle diameter) 50 nm, silica film thickness 20 nm) were diluted with water (deionized water) to prepare an aqueous dispersion of silica-coated silver nanoparticles. The resulting aqueous dispersion was used as a measurement sample. Specifically, in addition to the primary particle state of silica-coated silver nanoparticles (hereinafter also referred to as silica-coated silver nanoparticles), the measurement sample also contained aggregates of two or more silver nanoparticles.

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

[0131] Comparative Example 2 Silica-coated silver nanoparticles (manufactured by nanoComposix, silver nanoparticle particle diameter (core particle diameter) 50 nm, silica film thickness 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 a measurement sample. Specifically, in addition to the primary particle state of the silica-coated silver nanoparticles, the measurement sample also contained aggregates of two or more silver nanoparticles.

[0132] Analysis of the plasmon resonance wavelength in the presence of the test substance was performed in the same manner as in Example 1. The measurement results are shown in Figure 25. Figure 25 shows the plasmon resonance spectra of Comparative Example 2 ((a) silica-coated silver nanoparticles in a primary particle state and (b) aggregates in which three silica-coated silver nanoparticles are arranged in a roughly ozone molecule-like configuration). In Figure 25(a), silica-coated silver nanoparticles in a primary particle state were used as the measurement sample, while in Figure 25(b), aggregates in which three silica-coated silver nanoparticles are arranged in a roughly ozone molecule-like configuration were used as the measurement sample. The plasmon resonance spectrum shown in Figure 25(a) exhibited a spectral shape with one peak attributable to dipole resonance near 450 nm. The plasmon resonance spectrum shown in Figure 25(b) exhibited a spectral shape with one peak attributable to dipole resonance near 450 nm, similar to Figure 25(a). In Comparative Example 2, no plasmon resonance spectrum peaks due to multipoles were observed.

[0133] The nanoparticles, composites containing the nanoparticles, and methods for forming polymer films contained in the nanoparticles according to the present disclosure are as follows: <1> A nanoparticle comprising metal nanoparticles, a polymer film covering the surfaces of the metal nanoparticles, a specific binding substance that specifically binds to an analyte in a specimen, and a fluorescent substance labeled on the surface of the polymer film or on the specific binding substance, wherein the fluorescent substance is excited by light having an emission wavelength of plasmon resonance in a composite in which two or more nanoparticles are bound via the analyte. <2> The nanoparticle according to <1>, wherein the plasmon resonance in the composite is induced by external light. <3> The nanoparticle according to <1> or <2>, wherein the plasmon resonance in the composite is induced by fluorescence emitted by the fluorescent substance. <4> The nanoparticle according to any one of <1> to <3>, wherein the absorption wavelength range of the fluorescent substance overlaps with the first emission wavelength range of the plasmon resonance. <5> The nanoparticle according to any one of <1> to <4>, wherein the first emission wavelength range of the plasmon resonance is longer than the second emission wavelength range of the plasmon resonance induced in the metal nanoparticle as a single particle. <6> The nanoparticle according to <5>, wherein the fluorescent substance is selected so that a first region where the first emission wavelength range and the absorption wavelength range of the fluorescent substance overlap is larger than a second region where the second emission wavelength range and the absorption wavelength range of the fluorescent substance overlap. <7> The nanoparticle according to <5> or <6>, wherein the maximum absorption wavelength of the fluorescent substance in the first emission wavelength range is 500 to 700 nm. <8> The nanoparticle according to any one of <5> to <7>, wherein the maximum fluorescence wavelength of the fluorescent substance in the first emission wavelength range is 500 to 700 nm. <9> The nanoparticle according to any one of <1> to <8>, wherein the plasmon resonance induced in the complex is a multipole resonance. <10> The nanoparticle according to any one of <5> to <8>, wherein the plasmon resonance induced in the single metal nanoparticle is a dipole resonance.<11> The nanoparticle according to any one of <1> to <10>, wherein the polymer film comprises at least one selected from the group consisting of a bonding site between the surface of the metal nanoparticle and the polymer film via a sulfur atom, a positively charged group, and a hydrophobic group. <12> The polymer film comprises at least the positively charged group in a side chain of a polymer constituting the polymer film, and the positively charged group is selected from the group consisting of a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH). ２ ＋ ) NH ２<13> The nanoparticle according to <11>, wherein the specific binding substance is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. <14> The nanoparticle according to any one of <1> to <13>, wherein the polymer membrane contains at least the hydrophobic group in a side chain of a polymer constituting the polymer membrane, and the hydrophobic group is at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group. <15> The nanoparticle according to any one of <1> to <14>, wherein the polymer membrane has a thickness of 1 nm to 10 nm. <16> The nanoparticle according to any one of <1> to <15>, wherein the specific binding substance is a nanoantibody. <17> The nanoparticle according to any one of <1> to <16>, wherein the specific binding substance is a VHH antibody. <18> The nanoparticle according to any one of <1> to <17>, wherein the metal nanoparticles comprise gold or silver. <19> The nanoparticle according to any one of <1> to <18>, wherein the particle diameter of the metal nanoparticles is 5 to 100 nm. <20> The nanoparticle according to any one of <1> to <19>, wherein the nanoparticles comprise first nanoparticles and second nanoparticles, and wherein the first nanoparticles and the second nanoparticles form a complex bound via the test substance. <21> The nanoparticle according to any one of <1> to <20>, wherein the test substance is derived from the specimen which is blood, plasma, urine, or saliva. <22> The nanoparticle according to <20>, wherein a fluorescent substance is positioned between the first nanoparticles and the second nanoparticles in the complex. <23> A complex comprising two or more nanoparticles according to any one of <1> to <22>, wherein the two or more nanoparticles comprise first nanoparticles and second nanoparticles, and the first nanoparticles and the second nanoparticles are bound via the test substance. <24> The complex according to <23>, wherein the two nanoparticles contain a plurality of binding points where the two nanoparticles are bound via one of the test substances.<25> A method for forming a polymer film, comprising the step of bringing a polymer having a disulfide bond in a side chain into contact with a metal nanoparticle to form a polymer film in which the polymer is bonded to the surface of the metal nanoparticle via a sulfur atom. <26> The method for forming a polymer film according to <25>, wherein the polymer includes at least one group selected from the group consisting of a positively charged group and a hydrophobic group bonded via the disulfide bond, and the step of bonding the at least one group selected from the group consisting of the positively charged group and the hydrophobic group to the surface of the metal nanoparticle while forming the polymer film.

[0134] REFERENCE SIGNS LIST 1 Nanoparticle 2 Metal nanoparticle 3 Polymer film 3A Polymer constituting polymer film 3B Polymer forming polymer film 3a Binding site via sulfur atom 3b Positively charged group 3c Hydrophobic group 4 Specific binding substance 6 Fluorescent substance 10 First nanoparticle 12 First metal nanoparticle 13 First polymer film 14 First specific binding substance 16 First fluorescent substance 20 Second nanoparticle 22 Second metal nanoparticle 23 Second polymer film 24 Second specific binding substance 26 Second fluorescent substance 30 Test substance 40 Complex L Separation distance (separation distance) R １ ...First area R ２ ...Second area WR Ａ ...Absorption wavelength range of fluorescent material WR Ｅ ...Emission wavelength range of fluorescent material WR Ｅ１ ...First light emission wavelength range WR Ｅ２ ...Second emission wavelength range

Claims

1. Metal nanoparticles; A polymer film that covers the surface of the metal nanoparticles; A specific binding substance that specifically binds to a test substance in a sample; a fluorescent substance labeled on the surface of the polymer membrane or on the specific binding substance; A nanoparticle comprising: The nanoparticles, wherein the fluorescent substance is excited by light having an emission wavelength of plasmon resonance in a complex to which two or more of the nanoparticles are bound via the test substance.

2. The nanoparticle according to claim 1 , wherein the plasmon resonance in the complex is induced by externally applied light.

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

4. The nanoparticle of claim 1 , wherein an absorption wavelength range of the fluorescent material overlaps with a first emission wavelength range of the plasmon resonance.

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

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

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

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

9. The nanoparticle of claim 1 , wherein the plasmon resonance induced in the complex is a multipole resonance.

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

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

12. the polymer membrane contains at least the positively charged group in a side chain of a polymer constituting the polymer membrane, The positively charged group includes a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH ２ ＋ ) N.H. ２ 12. The nanoparticle according to claim 11, which is at least one selected from the group consisting of:

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

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

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

16. The nanoparticle of claim 1 , wherein the specific binding agent is a nanobody.

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

18. 10. The nanoparticle of claim 1, wherein the metal nanoparticles comprise gold or silver.

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

20. The nanoparticles include a first nanoparticle and a second nanoparticle, The nanoparticle according to claim 1 , wherein the first nanoparticle and the second nanoparticle are bound to each other via the test substance to form a complex.

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

22. 21. The nanoparticle of claim 20, wherein in the composite, a fluorescent material is located between the first nanoparticle and the second nanoparticle.

23. A complex comprising two or more nanoparticle bodies according to any one of claims 1 to 22, the two or more nanoparticle bodies comprising a first nanoparticle body and a second nanoparticle body, the first nanoparticle body and the second nanoparticle body being bound to each other via the test substance.

24. 24. The complex of claim 23, comprising multiple binding points where two of the nanoparticles are bound via one of the test substances. a 25. A method for forming the polymer film of the nanoparticle body according to claim 1, comprising the steps of: A method for forming a polymer film, comprising the steps of contacting a polymer having a disulfide bond in a side chain with a metal nanoparticle to form a polymer film in which the polymer is bonded to the surface of the metal nanoparticle via a sulfur atom.

26. the polymer includes at least one group selected from the group consisting of a positively charged group and a hydrophobic group bonded via the disulfide bond; The method for forming a polymer film according to claim 25 , wherein the step comprises 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 while forming the polymer film.