Coated metal substrate and method for producing the same, composite containing the coated metal substrate, and polymer for producing the coated metal substrate

Abstract

A coated metallic base comprising a metallic base which is metal nanoparticles and / or a thin metal film, a polymer film covering the surface of the metallic base, and a first hydrophilic group, which inhibits non-specific adsorption onto the surface of the metallic base, wherein the first hydrophilic group has combined with the surface of the metallic base and the polymer film includes, between itself and the surface of the metallic base, a portion combined by a sulfur atom.

Description

[Technical Field]

[0001] The present invention relates to a coated metal substrate, a method for producing the same, a composite containing the coated metal substrate, and a polymer for producing the coated metal substrate. [Background technology]

[0002] A biosensor detects a specific test substance by specifically reacting it with a specific binding substance to form a complex, and then detecting the test substance based on the signal resulting from the specific binding in the complex. In plasmon-induced fluorescence analysis, the complex comprises, for example, a test substance, a specific binding substance, a fluorescent substance, and metal particles. When excitation light is shone on the complex, surface plasmon resonance is induced in the metal particles within the complex, creating a near-field near the surface of the metal particles. This near-field increases the fluorescence intensity of the fluorescent substance.

[0003] When analyzing a test substance by specifically reacting it with a specific specific binding substance to form a complex, there is a problem of deterioration of the signal-to-noise ratio (SNR) due to nonspecific adsorption to the metal particle surface. This is also true for plasmon-excited fluorescence analysis using metal particles, where nonspecific adsorption to the metal particle surface may worsen the SNR. For example, as described in Patent Document 1, a polymer film is formed by attaching the ends of one-dimensional polymers (polymer chains) to the surface of metal nanoparticles. By arranging multiple polymer chains on the metal substrate surface so as to be approximately perpendicular to the surface of the metal substrate, a polymer film is formed in which the polymer chains are arranged in a brush-like manner. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2001 / 086301 [Overview of the project] [Problems that the invention aims to solve]

[0005] Incidentally, after diligent research by the present inventors, it was found that there is room to further improve the detection sensitivity of the polymer described in Patent Document 1. Specifically, when using the polymer described in Patent Document 1, a polymer film is formed in which one-dimensional polymer chains are arranged in a brush-like manner, which may prevent the thickness of the polymer film formed on the metal substrate surface from being sufficiently thin. As a result, the separation distance between metal substrates in the composite that captures the test substance cannot be sufficiently reduced, and fluorescence cannot be effectively enhanced. Furthermore, in polymer films, because molecular chains containing hydrophobic alkyl groups are arranged in a brush-like manner, there is a risk that the adsorption of nonspecific adsorbent substances that contribute to noise components (nonspecific adsorption or non-specific adsorption) may not be effectively suppressed.

[0006] This invention has been made in view of the above problems. Specifically, the main objective of this invention is to provide a polymer that can reduce film thickness, sufficiently reduce the separation distance between metal substrates in a composite, and improve detection sensitivity. Another main objective is to provide a coated metal substrate having a polymer film formed using such a polymer, a method for producing the same, and a composite containing the coated metal substrate. [Means for solving the problem]

[0007] The polymer according to one embodiment of the present invention is It has a hydrophilic group at the end of its side chain, which is bonded via a disulfide group.

[0008] A coated metal substrate according to one embodiment of the present invention is It comprises a metal substrate which is metal nanoparticles and / or a metal thin film, a polymer film which covers the surface of the metal substrate, and a first hydrophilic group which suppresses nonspecific adsorption to the surface of the metal substrate. The first hydrophilic group is bonded to the surface of the metal substrate, The polymer film includes a bonding site between itself and the surface of the metal substrate via sulfur atoms.

[0009] A composite according to one embodiment of the present invention is The nanoparticle body comprising two or more of the aforementioned metal nanoparticles is comprised of The aforementioned two or more nanoparticles include a first nanoparticle and a second nanoparticle, The first nanoparticle and the second nanoparticle are bonded together via the test substance.

[0010] A composite according to one embodiment of the present invention is The nanoparticle body comprises the aforementioned metal nanoparticles and the coated metal thin film comprising the aforementioned metal thin film, The first hydrophilic group is bonded to at least one of the surface of the metal nanoparticle and the surface of the metal thin film. The nanoparticles and the coated metal thin film are bonded together via the test substance.

[0011] A method for manufacturing a coated metal substrate according to one embodiment of the present invention is: The method comprises the step of bringing the polymer into contact with the surface of a metal substrate, thereby bonding the hydrophilic groups to the surface of the metal substrate and forming a polymer film on the surface of the metal substrate. [Effects of the Invention]

[0012] The present invention provides a polymer capable of reducing the film thickness of the polymer film, thereby sufficiently reducing the separation distance between metal substrates in the composite, and improving detection sensitivity. Furthermore, the invention provides a coated metal substrate on which a polymer film is formed using such a polymer, a method for producing the same, and a composite containing the coated metal substrate. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a schematic cross-sectional view showing a coated metal substrate according to the second embodiment ((a) nanoparticles and (b) coated metal thin film). [Figure 2] Figure 2 is an enlarged schematic diagram of section A in Figure 1(a). [Figure 3] Figure 3 is a schematic cross-sectional view showing the composite according to the third embodiment. [Figure 4]FIG. 4 is a diagram schematically showing a measuring apparatus for detecting a test substance using the composite according to the third embodiment. [Figure 5] FIG. 5 is a cross-sectional view schematically showing the composite according to the fourth embodiment. [Figure 6] FIG. 6 is a diagram showing absorption spectra in each step of polymer synthesis. [Figure 7] FIG. 7 is a diagram showing the reaction related to the method for forming the polymer film of Example 1. [Figure 8] FIG. 8 is a SEM image of the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 1. [Figure 9] FIG. 9 is a diagram showing the distribution of the scattered light intensity ratio with respect to the zeta potential of the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 1. [Figure 10] FIG. 10 is a distribution of the scattered light intensity ratio with respect to the particle diameter of the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 1. [Figure 11] FIG. 11 is a diagram showing a SEM image of the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 2. [Figure 12] FIG. 12 is a diagram showing the distribution of the scattered light intensity ratio with respect to the zeta potential of the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 2.

Embodiments for Carrying Out the Invention

[0014] Hereinafter, the nanoparticle body, its manufacturing method, composite, and measuring apparatus, which are embodiments of the present invention, will be described in detail with reference to the illustrated embodiments. The drawings include schematic ones and may not reflect actual dimensions and ratios.

[0015] The numerical ranges referred to in this specification are intended to include the lower limit value and the upper limit value itself, unless special terms such as "less than", "greater than", and "smaller than" are added. For example, taking the numerical range of 1 nm to 50 nm as an example, unless special terms are added, the numerical range is interpreted as including the lower limit value "1 nm" and the upper limit value "50 nm".

[0016] In this specification, "metal substrate" means a substrate that is substantially composed of metal and contains metal thin films (also referred to as metal plates or metal films) and metal nanoparticles. Metal nanoparticles and metal thin films differ mainly in shape. In this specification, if a subject member is said to be substantially composed of a specific material or to consist of a specific material, it means that the subject member contains the specific material in proportions of 95% or more by mass, 97% or more by mass, 99% or more by mass, or 100% by mass. For example, if a metal substrate is said to be substantially composed of metal, it means that the metal substrate contains metal in proportions of 95% or more by mass, 97% or more by mass, 99% or more by mass, or 100% by mass.

[0017] In this specification, "metal nanoparticles" means particles substantially composed of metal, having a size on the order of nanometers (e.g., a few nm to 100 nm), and having a spherical or nearly spherical shape. "Metal nanoparticles" induce localized surface plasmon resonance with other metal nanoparticles through interaction with light (e.g., visible light). In this specification, "metallic thin film" refers to a thin film substantially composed of metal, with an initial film thickness on the order of nanometers (e.g., a few nm to 100 nm). The "metallic thin film" induces localized surface plasmon resonance with metal nanoparticles through interaction with light (e.g., visible light and near-infrared light).

[0018] <First Embodiment: Polymer> The polymer according to the first embodiment has hydrophilic groups at the ends of its side chains, which are linked via disulfide groups (disulfide bonds) (-SS-). The polymer according to the first embodiment can come into contact with the surface of a metal substrate to form a polymer film and hydrophilic groups on the surface of the metal substrate. This makes it possible to manufacture a coated metal substrate (detailed in the second embodiment).

[0019] [Mechanism of Action] The composite having a polymer film formed with the polymer according to the first embodiment exhibits excellent detection sensitivity. Although not bound by any particular theory, the reason is presumed to be as follows. The polymer according to this embodiment is, for example, general formula (1): [ka] (In general formula (1), X represents a hydrophilic group, L represents, for example, an amide bond, and n represents the number of repeating units.) As shown, the polymer has a hydrophilic group bonded via a disulfide group at the end of the side chain (hereinafter, the polymer represented by general formula (1) will also be referred to as "polymer (1)" in the first embodiment and as "polymer 3B" in the second embodiment and subsequent embodiments). The inventors have devised a method in which cleavage of the disulfide group allows one of the S atoms to contribute to reducing the thickness of the polymer film, and the other S atom to improve the ease of exposure of the hydrophilic group. More specifically, the inventors have devised that cleavage of the disulfide group can enable the following two embodiments; (First aspect): A method for bonding one of the S atoms attached to the end of a side chain branching from the polymer main chain to a predetermined location on the surface of a metal substrate, and (Second aspect): An embodiment in which the S atom connected to the other hydrophilic group X is connected at a location different from a predetermined location on the surface of the metal substrate. As described above, the present invention makes it possible to "reduce the film thickness of the polymer film" and "improve the ease of exposure of hydrophilic groups."

[0020] More specifically, when polymer (1) is brought into contact with a metal substrate, at least some of the bonds between the sulfur atoms of the disulfide group are cleaved, forming sulfur atom-mediated bonding sites on the surface of the metal substrate. As a result, in the polymer film formed, the polymers constituting the polymer film bond to the metal substrate at the ends of their side chains. Since the polymer film has a structure in which the main chains of the polymers constituting it cover the surface of the metal substrate in a two-dimensional manner, the thickness of the polymer film can be reduced compared to a polymer film formed by arranging one-dimensional polymers approximately perpendicular to the surface of the metal substrate, as shown in Patent Document 1, for example. This makes it possible to sufficiently reduce the separation distance between metal substrates in the composite.

[0021] In this specification, the main chain of a polymer refers to the hydrocarbon chain (e.g., alkylene chain) that forms the main backbone of the polymer, and is a hydrocarbon chain parallel to the direction in which multiple repeating units constituting the polymer are bonded to each other and extend. This hydrocarbon chain may have divalent groups containing heteroatoms, such as ether groups, amide bonds, and ester bonds. Furthermore, in this specification, the side chain of a polymer refers to a hydrocarbon chain branched from the main chain. The hydrocarbon chain of the side chain may also have divalent groups containing heteroatoms, similar to the hydrocarbon chain of the main chain.

[0022] Furthermore, during the cleavage of the sulfur atoms in the disulfide group, hydrophilic groups are bonded to the surface of the metal substrate via sulfur atoms, in parallel with the formation of sulfur atom-mediated bonding sites on the surface of the metal substrate. As a result, the resulting polymer film can have a structure in which hydrophilic groups are easily exposed. This suppresses nonspecific adsorption and can improve the signal-to-noise ratio (S / N ratio) in plasmon-induced fluorescence analysis, for example. From the above, the composite having a polymer film formed with the polymer according to the first embodiment exhibits excellent detection sensitivity.

[0023] In general formula (1), n, which indicates the number of repeating units, is, for example, a number such that the weight-average molecular weight Mw of polymer (1) is greater than 12,000.

[0024] (hydrophilic group) Hydrophilic groups include polar groups and / or electrostatic groups. Polar groups include, for example, at least one selected from the group consisting of carboxyl groups, hydroxyl groups, amino groups, sulfonyl groups, phosphate groups, and alkylene oxide groups. Electrostatic groups include, for example, at least one selected from the group consisting of charged polar groups and zwitterionic groups. Zwitterionic groups include, for example, at least one selected from the group consisting of phosphorylcholine groups and betaine groups.

[0025] The inventors have found that since nonspecific adsorbent substances contain hydrophobic moieties, hydrophilic moieties are effective in suppressing nonspecific adsorption to polymer films. They have also found that nonspecific adsorption can be effectively suppressed when the hydrophilic moieties are exposed from the polymer film. Based on these technical findings, they have derived a polymer (1) having hydrophilic groups bonded to the side chain ends via disulfide bonds.

[0026] The inventors have found that, in addition to hydrophilic groups, introducing hydrophilic moieties into the main chain skeleton of the polymer constituting the polymer film is effective in suppressing nonspecific adsorption. From the viewpoint of further suppressing non-specific adsorption, it is preferable that the main chain skeleton of polymer (1) contains an amide bond. From the viewpoint of further suppressing non-specific adsorption, it is preferable that the main chain skeleton of the polymer (1) has a structure derived from one or more amino acids. Such amino acids include, for example, at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, serine, and threonine. From the viewpoint of further suppressing non-specific adsorption, it is preferable that the main chain skeleton of polymer (1) contains a biocompatible polymer as a block polymer. Examples of such biocompatible polymers include polymethyl methacrylate.

[0027] In this specification, the term "one or more" more specifically means "one or two or more," and "one or more" (more specifically "one or two or more") is synonymous.

[0028] Polymer (1) may further contain positively charged groups in its side chains. These side chains do not have disulfide groups. Such positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH2) + It is at least one selected from the group consisting of )NH2). When polymer (1) contains such positively charged groups, the resulting polymer film can form electrostatic bonds with the surface of the metal substrate. This allows the polymer film to be more firmly fixed.

[0029] Polymer (1) may further contain hydrophobic groups in its side chains. These side chains do not have disulfide bonds. Such hydrophobic groups are at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. When polymer (1) contains such hydrophobic groups, the polymer film that is formed can form hydrophobic bonds with the surface of the metal substrate. This allows the polymer film to be fixed more firmly.

[0030] (Method of manufacturing polymers) An example of a polymer manufacturing method is described below. The polymer manufacturing method includes, for example, a step of introducing disulfide groups into the side chains (disulfide group introduction step) and a step of introducing hydrophilic groups (hydrophilicity introduction step). The following scheme 1:

[0031] [ka] (In Scheme 1, X represents a hydrophilic group, Y represents, for example, a pyridyl group, Z and T represent functional sites, L represents, for example, a divalent bonding site, n represents the number of repeating units, Z and T are different from each other and bond to each other to form L.) The method for producing polymer (1) will be explained by referring to [reference]. Scheme 1 shows the reaction equation for explaining the method for producing polymer (1). Note that L, X, and n in Scheme 1 are equivalent to L, X, and n in general formula (1), respectively.

[0032] -Disulfide group introduction process- As shown in Scheme 1, the disulfide group introduction step involves reacting a polymer having a functional site Z in its side chain (a polymer represented by formula (3) (hereinafter also referred to as polymer (3))) with a compound having a disulfide group (a compound represented by formula (4) (hereinafter also referred to as compound (4))) to obtain a polymer having a disulfide group (a polymer represented by formula (2) (hereinafter also referred to as polymer (2))). Functional sites Z and T are different from each other and are reactive substituents such as an amino group, a carboxyl group, and an ester bond. This reaction is a nucleophilic substitution reaction in which, for example, an amino group attacks a carboxyl group or an ester bond to form an amide bond. The reaction temperature is, for example, room temperature (20°C to 25°C), and the reaction time is, for example, 1 hour or more and 4 hours or less.

[0033] Examples of functional sites Z in polymer (3) include amino groups, carboxyl groups, and ester groups. Examples of polymer (3) include: [ka] These are some examples.

[0034] Examples of compounds having a disulfide group (4) include, [ka] These are some examples.

[0035] -Hydrophilic introduction process- In the hydrophilicity introduction step, polymer (2) is reacted with a thiol compound having hydrophilic groups (a thiol compound represented by formula (5) (hereinafter also referred to as thiol compound (5))) to obtain polymer (1) having hydrophilic groups. This reaction is a thiol nucleophilic reaction in which the thiol group attacks the disulfide bond of polymer (2). The reaction temperature is, for example, 35-40°C (especially °C), and the reaction time is, for example, 30 minutes or more to 2 hours or more (especially 1 hour or more). Examples of thiol compound (5) are,

[0036] [ka] [ka] These are some examples.

[0037] <Second Embodiment: Coated Metal Substrate> In the second embodiment, the coating film (i.e., polymer film) of the coated metal substrate can be formed by the polymer according to the first embodiment. In other words, the polymer constituting the polymer film may originate from the polymer according to the first embodiment. Note that in the second embodiment, the same reference numerals as in the first embodiment have the same configuration as in the first embodiment, so their explanation is omitted.

[0038] The coated metal substrate according to the second embodiment comprises a metal substrate, a polymer film covering the surface of the metal substrate, and a first hydrophilic group that suppresses nonspecific adsorption to the surface of the metal substrate. The first hydrophilic group is bonded to the surface of the metal substrate. The polymer film contains bonding sites between itself and the surface of the metal substrate via sulfur atoms.

[0039] [Mechanism of Action] The coated metal substrate according to the second embodiment can enhance detection sensitivity. While not bound by any particular theory, the reason is presumed to be as follows: The coated metal substrate according to this embodiment includes a first hydrophilic group bonded to the surface of the metal substrate. The first hydrophilic group suppresses non-specific adsorption to the coated metal substrate, thereby suppressing a decrease in the signal-to-noise ratio in the detection of the test substance and improving detection sensitivity. Furthermore, the coated metal substrate according to this embodiment comprises a polymer film that includes a sulfur atom-mediated bonding site between the first hydrophilic group and the surface of the metal substrate. In this way, the polymer film does not need to have a non-specific adsorption suppression function, and the thickness of the polymer film can be reduced. This makes it possible to reduce the distance between the coated metal substrates in the composite formed during the detection of the test substance. From the above, it is considered that the coated metal substrate according to the second embodiment can improve detection sensitivity.

[0040] [Basic composition of coated metal substrate] The coated metal substrate will be described with reference to Figure 1. Figure 1 is a schematic cross-sectional view showing a coated metal substrate according to the second embodiment. The coated metal substrate 1 includes a nanoparticle body 1A and a coated metal thin film 1B.

[0041] As shown in Figure 1(a), the nanoparticle body 1A comprises a metal substrate 2 which is a metal nanoparticle 2A, a polymer film 3 which coats the surface of the metal substrate 2, and a first hydrophilic group 7A (not shown in Figure 1(a)) which suppresses nonspecific adsorption to the surface of the metal substrate 2. The nanoparticle body 1A may further comprise a fluorescent substance 6 and / or a specific binding substance 4.

[0042] The metal nanoparticles 2A induce localized surface plasmon resonance (or localized surface plasmon resonance; LSPR, hereinafter also simply referred to as "plasmon resonance") upon excitation light irradiation. The polymer film 3 coats the surface of the metal nanoparticles 2A and contains a sulfur atom-mediated bonding site between it and the surface of the metal substrate 2. The first hydrophilic group 7 is bonded to the surface of the metal substrate 2.

[0043] As shown in Figure 1(b), the coated metal thin film 1B comprises a metal substrate 2 which is a metal thin film 2B, a polymer film 3 that covers the surface of the metal substrate 2, and a first hydrophilic group 7 (not shown in Figure 1(b)) that suppresses nonspecific adsorption to the surface of the metal substrate 2. The coated metal thin film 1B may further contain a specific bonding substance 4.

[0044] The metal thin film 2B induces localized surface plasmon resonance upon excitation light irradiation. The polymer film 3 coats the surface of the metal thin film 2B and contains sulfur atom-mediated bonding sites between it and the surface of the metal substrate 2. The first hydrophilic group 7 is bonded to the surface of the metal substrate 2.

[0045] The coated metal substrate 1 can be used for plasmon-excited fluorescence analysis. In other words, the coated metal substrate 1 can be used for surface plasmon-excited enhanced fluorescence spectroscopy immunoassay. The coated metal substrate 1 can capture the test substance in the sample and form a complex (detailed in the third and fourth embodiments) containing, for example, two coated metal substrates 1 and one test substance. When the complex is irradiated with excitation light, plasmon resonance occurs and a near-field is formed. This near-field increases the fluorescence intensity; that is, an electric field enhancement effect is obtained.

[0046] The coated metal substrate 1 may also have its nonspecific binding sites blocked by a blocking agent. A blocked coated metal substrate 1 suppresses the formation of nonspecific binding of the specific binding substance 4 to substances other than the target substance (i.e., substances other than the test substance), thereby reducing background and false positive signals and improving the signal-to-noise ratio. In such cases, detection sensitivity can be further improved. Examples of blocking agents include bovine serum albumin (BSA), proteins such as skim milk and casein, and chemically synthesized polymers.

[0047] When the nanoparticles 1A are present in a solvent, the dispersion of the nanoparticles 1A may further contain a dispersant to improve the dispersibility of the nanoparticles 1A. Examples of such dispersants include sodium heparin.

[0048] (metal base material) The metal substrate 2 is coated on its surface with a polymer film 3. The metal substrate 2 preferably contains gold or silver, and more preferably contains silver. The metal substrate 2 consists of metal nanoparticles 2A and a metal thin film 2B.

[0049] -Metal nanoparticles- Metal nanoparticles 2A interact with light of specific wavelengths, causing localized surface plasmon resonance, although this varies depending on the type of metal that constitutes them. Silver nanoparticles (nanoparticles substantially composed of silver) exhibit localized surface plasmon resonance peaks at 400nm to 530nm, while gold nanoparticles (nanoparticles substantially composed of gold) exhibit peaks at 510nm to 580nm. This varies depending on the particle size of the metal nanoparticles 2A. For example, silver nanoparticles with a particle size of 20nm resonate with light of 405nm wavelength. Gold nanoparticles with a particle size of 20nm resonate with light of 524nm wavelength. The particle size (average primary particle size) of metal nanoparticles 2 is, for example, 5nm to 100nm. The particle size of metal nanoparticles 2 can be obtained by imaging the metal nanoparticles 2 using a scanning electron microscope (SEM) or transmission electron microscope (TEM), measuring the particle size of the metal nanoparticles 2 in the image, and calculating the average value of multiple particle sizes (number of measurements: e.g., at least 10).

[0050] -Metal Thin Film- The metal thin film 2B interacts with light of a specific wavelength (e.g., visible light), depending on the type of metal that makes up the metal thin film 2B, and undergoes localized surface plasmon resonance with the metal nanoparticles.

[0051] (polymer membrane) The polymer film 3 covers the surface of the metal substrate 2. The polymer film 3 functions as a metal quenching film. In the composite, the polymer film 3 can position the fluorescent substance 6 at least by the thickness of the polymer film 3 away from the surface of the metal substrate 2. This suppresses the quenching of the excited fluorescent substance 6 by contact with the surface of the metal substrate 2, thereby suppressing a decrease in detection sensitivity. The presence of the polymer film 3 can be confirmed by taking an image of the nanoparticle body 1 using SEM or TEM and observing the coated metal substrate 1 in the image.

[0052] The polymer film 3 will be described in detail with reference to Figure 2. Figure 2 is an enlarged view of part A in Figure 1(a), and is a schematic enlarged view of the vicinity of the interface between the polymer film 3 of nanoparticle body 1A and the surface of metal nanoparticles 2A. Since the polymer film 3 of nanoparticle body 1A has substantially the same structure as the polymer film 3 of the coated metal thin film 1B, the polymer film 3 of nanoparticle body 1A will be described below.

[0053] The polymer film 3 includes a sulfur atom-mediated bonding site 3a between itself and the surface of the metal nanoparticles 2 (i.e., the polymer 3A constituting the polymer film 3 includes a sulfur atom-mediated bonding site 3a between itself and the surface of the metal substrate 2 (metal nanoparticles 2A)). The polymer film 3 may further include at least one selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c (for example, the polymer 3A constituting the polymer film 3 may further include at least one selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c in the side chain of polymer 3A. This side chain does not have a disulfide group). More specifically, in addition to the sulfur atom-mediated bonding site 3a between itself and the surface of the metal nanoparticles 2, the polymer film 3 includes a primary ammonium group (-NH3) as the positively charged group 3b. + The polymer may further contain a hydrophobic group 3c. The bonding site 3a bonds the surface of the metal nanoparticle 2 and the polymer film 3 via a sulfur atom. The positively charged group 3b forms an electrostatic bond (ionic bond) b with the negatively charged surface of the metal nanoparticle 2. The hydrophobic group 3c forms a hydrophobic bond c with the surface of the metal nanoparticle 2. In this specification, the polymer constituting polymer film 3 is also referred to as "polymer 3A," and the polymer used as a raw material to form polymer film 3 by reaction is also referred to as "polymer 3B." This distinction is made to facilitate differentiation between these two polymers.

[0054] All three of the above bonds are relatively strong bonds to the surface of the metal nanoparticles 2. The polymer film 3 is stably fixed to the surface of the metal nanoparticles 2 by the bonding site 3a. Furthermore, the polymer film 3 can be even more stably fixed to the surface of the metal nanoparticles 2 by the hydrophobic bond c and the electrostatic bond b formed by the positively charged group 3b. In this way, the polymer film 3 is stably fixed to the surface of the metal nanoparticles 2, and the fluorescent substance 6 and the surface of the metal nanoparticles 2 can be stably separated by a predetermined distance. Therefore, in this embodiment, the quenching of the excited fluorescent substance 6 is suppressed, and the decrease in detection sensitivity can be suppressed.

[0055] Furthermore, since the polymer film 3 may be composed of polymer 3A, it is easier to chemically modify than the silica layer, and the need for surface modification is reduced. This allows for a smaller film thickness compared to the silica layer, and a moderate reduction in the distance between metal nanoparticles 2A in the composite. Therefore, a near-field can be formed more efficiently, and detection sensitivity can be further improved.

[0056] As shown in Figure 2, the polymer 3A that can constitute the polymer film 3 may include, in addition to a sulfur atom-mediated bonding site 3a between itself and the surface of the metal nanoparticles 2, at least one selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c. The presence of the sulfur atom-mediated bonding site 3a, the positively charged group 3b, and the hydrophobic group 3c can be confirmed by measuring the signals originating from them using infrared spectroscopy, energy-dispersive X-ray spectroscopy (TEM-EDS), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), and nuclear magnetic resonance spectroscopy.

[0057] The polymer 3A constituting the polymer film 3 may contain one or more secondary hydrophilic groups 7B per molecular chain of polymer 3A, which are bonded to the side chains of polymer 3A via disulfide groups. The inclusion of secondary hydrophilic groups 7B in polymer 3A constituting the polymer film 3 suppresses non-specific adsorption to the polymer film 3. The secondary hydrophilic groups 7B are hydrophilic groups that bond to the remaining disulfide bonds that did not react completely during the formation of the polymer film. For example, they originate from a portion of the hydrophilic groups bonded via disulfide bonds in polymer 3B (the polymer according to the first embodiment) used as a raw material. Polymer 3A containing secondary hydrophilic groups 7B can be prepared by making the reaction conditions in the method for forming the polymer film 3 milder (for example, a lower reaction temperature and / or a shorter reaction time). Mild reaction conditions include, for example, a reaction time shorter than 12-36 hours and / or a reaction temperature lower than 20-30°C.

[0058] The second hydrophilic group 7B includes a polar group and / or an electrostatic group. The polar group includes, for example, at least one selected from the group consisting of a carboxyl group, a hydroxyl group, an amino group, a sulfonyl group, a phosphate group, and an alkylene oxide group. The electrostatic group includes, for example, at least one selected from the group consisting of a charged polar group and a zwitterionic group, wherein the zwitterionic group includes at least one selected from the group consisting of a phosphorylcholine group and a betaine group.

[0059] The polymer 3A constituting the polymer film 3 preferably contains an amide bond in its main chain skeleton. When polymer 3A has an amide bond in its main chain skeleton, polymer 3A is hydrophilic, which suppresses nonspecific adsorption to the polymer film 3 composed of polymer 3A, further improving detection sensitivity.

[0060] Polymer 3A contains a structure in its main chain skeleton derived from one or more amino acids. Such amino acids include, for example, at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, glutamine aspartate, asparagine, serine, and threonine. Because Polymer 3A has a structure in its main chain skeleton derived from one or more amino acids, Polymer 3A is hydrophilic, nonspecific adsorption to the polymer membrane 3 composed of Polymer 3A is suppressed, and detection sensitivity is further improved.

[0061] The polymer 3A constituting the polymer film 3 preferably contains a biocompatible polymer as a block polymer in its main chain skeleton. When a biocompatible polymer is included as a block polymer in the main chain skeleton of polymer 3A, polymer 3A becomes hydrophilic, which suppresses nonspecific adsorption to the polymer film 3 composed of polymer 3A, and further improves detection sensitivity. Examples of such biocompatible polymers include polymethacrylate.

[0062] The polymer 3A constituting the polymer film 3 can cover the surface of the metal substrate 2 in a two-dimensional manner, such that the main chains of polymer 3A are substantially parallel to the surface of the metal substrate 2. This type of coating by polymer 3A can be achieved by bonding the ends of multiple side chains of polymer 3A to the surface of the metal substrate 2 via sulfur atoms. Furthermore, because polymer 3A covers the surface of the metal substrate 2 with its main chains substantially parallel to the surface of the metal substrate 2, the film thickness of polymer film 3 can be reduced compared to polymer films formed by arranging polymer chains in a brush-like manner (prior art: International Publication No. 2001 / 086301).

[0063] -Bonding site via sulfur atom- The sulfur atom-mediated bonding site 3a is formed, for example, by mixing a polymer having a site containing a disulfide bond as a side chain with a metal substrate 2 (metal nanoparticle 2A).

[0064] -Positively charged group- The positively charged group 3b can form a relatively strong electrostatic bond b with the surface of the metal substrate 2 (metal nanoparticles 2A). In this specification, the positively charged group 3b is a group having a valency of 1 or more and that is completely positively ionized. When considering multiple positively charged groups 3b contained in the polymer constituting the polymer film 3, the positively charged group 3b is expressed by the following formula (1):

number

[0065] The positively charged group 3b is preferably a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH2) + It is at least one selected from the group consisting of )NH2).

[0066] -Hydrophobic group- The hydrophobic group 3c can form a hydrophobic bond c with the surface of the metal substrate 2 (metal nanoparticles 2A). The hydrophobic group 3c is, for example, at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups.

[0067] Examples of aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups. Aromatic carbocyclic groups are groups that do not contain aromatic heterocyclic groups but contain aromatic rings in which all member atoms are carbon atoms. Examples of aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups, etc.) and arylalkyl groups (more specifically, benzyl groups, etc.). Aromatic heterocyclic groups are groups that contain aromatic rings in which at least one of the member atoms is a heteroatom (more specifically, oxygen atoms, sulfur atoms, and nitrogen atoms, etc.). Examples of aromatic heterocyclic groups include nitrogen-containing aromatic heterocyclic groups (more specifically, pyridyl groups (pyridinyl groups, etc.), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups.

[0068] Aliphatic cyclic groups are groups that contain a cyclic group consisting of a non-aromatic ring, without an aromatic ring. Examples of aliphatic cyclic groups include aliphatic carbocyclic groups and aliphatic heterocyclic groups. Aliphatic carbocyclic groups are groups that contain a non-aromatic ring in which all ring member atoms are carbon atoms, and examples include cycloalkyl groups. Aliphatic heterocyclic groups are groups that contain a non-aromatic ring in which at least one of the ring member atoms is a heteroatom.

[0069] Aliphatic chain groups are chain-like (more specifically, linear and branched) groups that do not contain aromatic or non-aromatic rings. Examples of aliphatic chain groups include aliphatic carbon chain groups (more specifically, alkyl and alkylene groups, etc.) and aliphatic heterochain groups. An example of an alkyl group is the butyl group. An example of an alkylene group is the n-butylene group.

[0070] 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, the separation distance (spacing distance) between the two metal nanoparticles 2 is such that a near field is efficiently formed in the space between them, thus further improving the detection sensitivity. Also, when the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance are arranged at a predetermined distance from each other, so the quenching of the excited fluorescent substance during measurement is suppressed, and the detection sensitivity is further improved. In this specification, the separation distance (or separation distance) refers to the minimum distance (shortest distance) between the surfaces of the metal nanoparticles contained in each of the two nanoparticle bodies that are bonded together via the test substance in the composite.

[0071] (first hydrophilic group) The first hydrophilic group 7A is bonded to the surface of the metal substrate 2. More specifically, the first hydrophilic group 7A includes a bonding site between itself and the surface of the metal substrate 2 via a sulfur atom. Such a bonding site can be formed, for example, by mixing the metal substrate 2 (metal nanoparticles 2A) as a starting material with a polymer 3B or a compound having a disulfide bond (see the first embodiment).

[0072] The first hydrophilic group 7A includes a polar group and / or an electrostatic group. The polar group includes, for example, at least one selected from the group consisting of a carboxyl group, a hydroxyl group, an amino group, a sulfonyl group, a phosphate group, and an alkylene oxide group. The charged group includes, for example, at least one selected from the group consisting of a polar group that is charged and a zwitterionic group, and the zwitterionic group includes at least one selected from the group consisting of a phosphorylcholine group and a betaine group.

[0073] The first hydrophilic group 7A is preferably exposed from the polymer film 3. In this case, nonspecific adsorption to the polymer film 3 is further suppressed.

[0074] (Fluorescent substance) The fluorescent substance 6 is labeled on the surface of the metal substrate 2 and / or on the polymer film 3.

[0075] From the viewpoint of suppressing fluorescence quenching, it is preferable that the fluorescent substance 6 is labeled to the surface of the metal substrate 2 via a linker portion (for example, an alkylene group containing an amide group). When the fluorescent substance 6 is labeled to the surface of the metal substrate 2 via a linker portion, the linker portion is located between the fluorescent substance 6 and the surface of the metal substrate 2, making it easier to prevent the excited fluorescent substance 6 from coming into contact with the surface of the metal substrate 2. This is thought to further suppress fluorescence quenching.

[0076] Fluorescent material 6 is excited at a light wavelength that resonates with plasmon energy and emits fluorescence. Examples of fluorescent materials include metal complexes such as europium and ruthenium. An example of a ruthenium complex is tris(bipyridine)ruthenium(II), which may have a counteranion.

[0077] It is preferable that the fluorescent substance 6 has a large Stokes shift. Here, the Stokes shift is the difference between the absorption peak wavelength (maximum excitation wavelength) in the absorption spectrum of the fluorescent substance 6 and the fluorescence peak wavelength (maximum fluorescence wavelength) in the fluorescence spectrum of the fluorescent substance 6. When the Stokes shift of the fluorescent substance 6 is large, the absorption spectrum and the fluorescence spectrum do not overlap easily, so that the excitation light (scattered light) does not easily enter the fluorescence to be detected, and the fluorescence intensity can be measured more accurately.

[0078] The fluorescence spectrum of the fluorescent substance 6 is preferably sharp. A sharp fluorescence spectrum is less likely to overlap with the absorption spectrum of the fluorescent substance 6, thus reducing the interference of excitation light (and its scattered light) with the fluorescence to be detected, allowing for more accurate measurement of fluorescence intensity.

[0079] (specific binding substance) The specific binding substance 4 is a nano-sized substance (with a maximum size of 3 to 15 nm) that specifically binds to the test substance (described in detail in the third embodiment) in the sample. The test substance may be, for example, a test substance derived from a sample such as blood, plasma, urine, or saliva.

[0080] The specific binding substance 4 is, for example, at least one selected from the group consisting of antibodies (hereinafter referred to as nanoantibodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA chains and RNA chains). In this embodiment, the coated metal substrate 1 to which such a specific binding substance 4 is bound exhibits superior detection sensitivity. For example, a nanoantibody as the specific binding substance 4 specifically binds to the antigen (test substance) at its tip (antigen binding site) through an antigen-antibody reaction, forming a complex. A ligand as the specific binding substance 4 forms a complex with a protein (test substance) through a specific protein-ligand binding reaction. A nucleic acid chain as the specific binding substance 4 forms a pair (double helix) of nucleic acid chains that are complementary to each other based on base pair complementarity. An enzyme as the specific binding substance 4 forms an enzyme-substrate complex with the substrate (test substance) at its active site (active site) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as hydrogen bonds, as well as bonds resulting from intermolecular forces, hydrophobic interactions, and charge interactions.

[0081] A nanoantibody is, for example, at least one selected from the group consisting of VHH (variable domain of heavy chain antibody) antibodies, fragmentation antibodies (more specifically, Fab (Fragment Antigen Binding) antibodies, etc.) and their variants. A VHH antibody is a single-domain antibody. A variant is an antibody in which a portion of the amino acid sequence has been rearranged or a substituent has been introduced, within a range that maintains specific binding to the antigen. Because the nanoantibody is at least one selected from the group consisting of VHH antibodies, fragmentation antibodies, and their variants, these nanoantibodies have a relatively small volume, for example, they can narrow the distance (separation distance) between the two metal substrates 2 in the complex, more efficiently form a near field, and further increase the fluorescence intensity.

[0082] The molecular mass of the nanoantibody is preferably 60,000 Da or less, more preferably 30,000 Da or less, and even more preferably 20,000 Da or less. When the molecular mass is 60,000 Da or less (particularly 30,000 Da or less, or 20,000 Da or less), the volume of the nanoantibody is relatively small, which narrows the separation distance in the complex, allows for more efficient formation of the near field, and further increases the fluorescence intensity. Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.

[0083] The specific binding substance 4 may be directly bound to the polymer film 3, or it may be a linker portion derived from the crosslinking agent (more specifically, SM(PEG) n The crosslinking agent may be indirectly bonded to the polymer film 3 via (where n is 4, 6, and 8, etc.). Examples of such crosslinking agents include amino group-sulfhydryl group crosslinking agents (more specifically, NHS-maleimide group crosslinking agents, etc.).

[0084] [Method for manufacturing coated metal substrates] The method for manufacturing the coated metal substrate 1 according to the second embodiment includes a step (polymer film formation step) in which a polymer 3B having hydrophilic groups 7 linked via disulfide bonds at the ends of its side chains is brought into contact with the surface of the metal substrate 2, thereby bonding the hydrophilic groups 7 to the surface of the metal substrate 2 and forming a polymer film 3 on the surface of the metal substrate 2. In the polymer film formation step, the bonding of the hydrophilic groups 7 to the metal substrate 2 and the formation of the polymer film can be carried out in parallel in step 1. In this way, the method can be simplified compared to the conventional technology, and costs can be further reduced. The polymer used as a starting material in the method for manufacturing the coated metal substrate is the polymer according to the first embodiment.

[0085] In the reaction in which polymer 3B is brought into contact with the surface of the metal substrate 2, the reaction time is, for example, 12 to 36 hours. The reaction temperature is, for example, 20 to 30°C. This reaction can also be carried out under stirring conditions.

[0086] In a preferred embodiment, the length of the first side chain from the disulfide group to the hydrophilic group 7 is longer than the length of the second side chain from the disulfide group to the main chain of polymer 3B. In this case, in polymer 3A constituting the polymer film 3, the second side chain length corresponds to the length of the side chain that binds to the surface of the metal substrate 2 (i.e., the length of the side chain from the binding site via sulfur atoms to the surface of the metal substrate 2 to the main chain of polymer 3 constituting the polymer film 3). The first side chain length corresponds to the length from the binding site via sulfur atoms to the surface of the metal substrate 2 to the tip of the first hydrophilic group 7A. Therefore, in this case, the first hydrophilic group 7A is more likely to be exposed from the polymer film 3. This further suppresses nonspecific adsorption to the polymer film 3.

[0087] <Third Embodiment: Composite> In the third embodiment, a composite in which two nanoparticles are bound together via a test substance will be described as an example. In the third embodiment, reference numerals that are the same as those in the first and second embodiments have the same configuration as those in the first and second embodiments, respectively, and therefore, their descriptions will be omitted in principle.

[0088] Furthermore, the composite (composite nanoparticle) according to the third embodiment can be formed when the coated metal substrate (nanoparticle) according to the second embodiment captures the test substance by sandwiching it during detection. The composite is composed of two or more nanoparticles. The composite according to the third embodiment is composed of two or more nanoparticles. The coated metal substrate 1 that can form the composite according to the third embodiment is a nanoparticle 1A containing metal nanoparticles 2A as the metal substrate 2, wherein the nanoparticle 1A includes a first nanoparticle and a second nanoparticle, and the first hydrophilic group 7A is bonded to at least one of the surfaces of the metal nanoparticles 2A of the first nanoparticle and the surface of the second metal nanoparticle of the second nanoparticle, and the coated metal substrate 1 forms a composite in which the first nanoparticle and the second nanoparticle are bonded via the test substance.

[0089] The composite will be described with reference to Figure 3. Figure 3 is a schematic cross-sectional view showing the composite according to the third embodiment. The composite 40A according to the third embodiment comprises two nanoparticle bodies 1A containing metal nanoparticles 2A, and the two nanoparticle bodies 1A contain a first nanoparticle body 10A and a second nanoparticle body 20A, with the first nanoparticle body 10A and the second nanoparticle body 20A being bonded via the test substance 30. More specifically, the composite 40A comprises the test substance 30 to be detected and two nanoparticles 10A and 20A. The two nanoparticles 10A and 20A are bound together in the composite 40A via the test substance 30. In other words, the composite 40A according to the third embodiment can be formed by binding the nanoparticle 1A according to the second embodiment via the test substance 30. Of the two nanoparticles 10A and 20A, one will be referred to as the first nanoparticle 10A and the other as the second nanoparticle 20A. Thus, the composite 40A includes the first nanoparticle 10A and the second nanoparticle 20A as nanoparticle 1.

[0090] In composite 40A, the first nanoparticle body 10A comprises a first metal nanoparticle 12A as a metal nanoparticle, a first polymer film 13A as a polymer film, and a first hydrophilic group 7A (not shown in Figure 3). That is, in composite 40A, the first nanoparticle body 10A comprises a first metal nanoparticle 12A, a first polymer film 13A that covers the surface of the first metal nanoparticle 12A, and a first hydrophilic group 7A that suppresses nonspecific adsorption to the surface of the first metal nanoparticle 12A, wherein the first hydrophilic group 7A is bonded to the surface of the first metal nanoparticle 12A, and the first polymer film 13A contains a bond between itself and the surface of the first metal nanoparticle 12A via a sulfur atom. The first nanoparticle body 10A further comprises a first specific binding substance 14A bonded to the first polymer film 13A and a first fluorescent substance 16A bonded to the first specific binding substance 14A.

[0091] In composite 40A, the second nanoparticle body 20A comprises a second metal nanoparticle 22A as a metal nanoparticle, a second polymer film 23A as a polymer film, and a first hydrophilic group 7A (not shown in Figure 3). In other words, in composite 40A, the second nanoparticle body 20A comprises a first metal nanoparticle 22A, a second polymer film 23A that covers the surface of the second metal nanoparticle 22A, and a first hydrophilic group 7A that suppresses nonspecific adsorption to the surface of the second metal nanoparticle 22A, wherein the first hydrophilic group 7A is bonded to the surface of the second metal nanoparticle 22A, and the second polymer film 23A contains a bond between itself and the surface of the second metal nanoparticle 22A via a sulfur atom. The second nanoparticle body 20A further comprises a second specific binding substance 24A bonded to the second polymer film 23A, and a second fluorescent substance 26A bonded to the second specific binding substance 24A.

[0092] From the viewpoint of further increasing fluorescence intensity, it is preferable that the separation distance L be small in a range in which the excited fluorescent substances 16A and 26A are less likely to be quenched. More specifically, in a preferred embodiment, the two nanoparticles 10A and 20A in the composite 40A are in close proximity to each other. In a more preferred embodiment, the two nanoparticles 10A and 20A are in close proximity to each other such that the first polymer film 13A of the first nanoparticle 10A and the second polymer film 23A of the second nanoparticle 20A in the composite 40A are in contact. In an even more preferred embodiment, the two nanoparticles 10A and 20A are in close proximity to each other such that at least one of the polymer films of the first polymer film 13A of the first nanoparticle 10A and the second polymer film 23A of the second nanoparticle 20A in the composite 40A is contracted and in contact.

[0093] In a more preferred embodiment, if at least one of the polymer films 13A and 23A shrinks and comes into contact with each other, for example, in the composite 40A shown in Figure 3, it is considered that the test substance 30, at least one of the specific binding substances 14A and 24A that bind to the test substance 30, and the fluorescent substances 16A and 26A can be incorporated into the polymer films 13A and 23A. Furthermore, in a more preferred embodiment, if the polymer films 13A and 23A come into contact with each other, for example, in the composite 40A shown in Figure 3, it is considered that, similar to the more preferred embodiment, at least one of the test substance 30, the specific binding substances 14A and 24A that bind to the test substance 30, and the fluorescent substances 16A and 26A can be incorporated into the polymer films 13A and 23A.

[0094] In this embodiment, the fluorescence intensity can be increased because the films covering the surfaces of the metal nanoparticles 12A and 22A are polymer films 13A and 23A. The reason for this is presumed to be as follows: The films covering the surfaces of the metal nanoparticles 12A and 22A are polymer films 13A and 23A, and polymer films 13A and 23A have relatively high flexibility compared to inorganic films containing inorganic oxides. Therefore, in the composite 40A, the polymer films 13A and 23A can contract, which allows the two metal nanoparticles 12A and 22A to be closer together than a distance equivalent to two polymer film thicknesses (the thickness of polymer film 13A + the thickness of polymer film 23A). In other words, because the films covering the surfaces of the metal nanoparticles 12A and 22A are polymer films 13A and 23A, the separation distance L can be less than two polymer film thicknesses. This makes it easier to obtain a plasmon enhancement effect and further increases the fluorescence intensity. In this specification, the "thickness of the polymer film" in "two times the thickness of the polymer film" refers to the thickness of the non-shrinking portion of the polymer film 13A, 23A that is not subject to separation, not the thickness of the shrinking portion of the polymer film 13A, 23A that is subject to separation distance.

[0095] In this embodiment, the polymer 3A constituting the polymer films 13A and 23A contains a sulfur atom-mediated binding site 3a in its side chain (more specifically, at the end of the side chain) between it and the surface of the metal nanoparticles 12A and 22A. This allows for a further increase in fluorescence intensity. While not bound by any particular theory, the reason is presumed to be as follows: In this case, the binding site 3a forms a bond with the surface of the metal nanoparticles 12A and 22A, so the polymer 3A has a network structure and is thought to cover the surface of the metal nanoparticles 12A and 22A in a network-like manner. Because the polymer 3A has such a network structure, it also has relatively high flexibility. Therefore, in the composite 40A, the polymer films 13A and 23A can contract further, allowing the two metal nanoparticles 12A and 22A to come into closer proximity than a distance equivalent to two layers of polymer film thickness. Thus, in this embodiment, the separation distance L can be less than two layers of polymer film thickness, further enhancing the plasmon effect and further increasing the fluorescence intensity.

[0096] In a preferred embodiment, the polymer 3A constituting the polymer films 13A and 23A further includes, in addition to the sulfur atom-mediated bonding sites 3a between its side chains (more specifically, the end of the side chains) and the surfaces of the metal nanoparticles 12A and 22A, at least one selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c. This further increases the fluorescence intensity. While not bound by any particular theory, the reason is presumed to be as follows: In this case, at least one selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c forms a bond with the surfaces of the metal nanoparticles 12A and 22A, so the polymer 3A has a network structure and is thought to coat the surfaces of the metal nanoparticles 12A and 22A in a network-like manner. Because the polymer 3A has such a network structure, it also has relatively high flexibility. Therefore, in the composite 40A, the polymer films 13A and 23A can further contract, which allows the two metal nanoparticles 12A and 22A to come into closer proximity than a distance equivalent to two layers of polymer film thickness. Therefore, in this embodiment, the separation distance L can be less than two layers of polymer film thickness, further enhancing the plasmon effect and increasing the fluorescence intensity.

[0097] The separation distance L between the first nanoparticle 10A and the second nanoparticle 20A is, for example, 12 nm to 52 nm, preferably 12 nm to 27 nm. The separation distance L is the distance between the first metal nanoparticle 12A and the second metal nanoparticle 22A, and is the distance at which the line segment connecting a first point P1 on the surface of the first nanoparticle 10A and a second point P2 on the surface of the second nanoparticle 20A is minimized. When the separation distance L is 52 nm or less, when excitation light is irradiated onto the composite 40A, a near-field is generated more efficiently in the space near the surfaces between the first and second metal nanoparticles 12A and 22A, thereby increasing the fluorescence intensity.

[0098] The polymer films 13A and 23A cover the surfaces of the metal nanoparticles 12A and 22A, and the polymer films 13A and 23A contain sulfur atom-mediated bonding sites 3a between themselves and the surfaces of the metal nanoparticles 12A and 22A. Therefore, the separation distance L can be closer than the distance equivalent to two times the thickness of the polymer films covering the surfaces of the two metal nanoparticles 12A and 22A in the composite 40A, as described above. For example, if the thickness of the polymer films 13A and 23A is 5 nm, the separation distance L can be less than 10 nm (more specifically, 2-9 nm, 3-8 nm, and 4-7 nm, etc.).

[0099] (Fluorescent substance) As shown in Figure 3, it is preferable that the fluorescent materials 16A and 26A are positioned at least between the first metal nanoparticle 12A and the second metal nanoparticle 22A in the composite 40A. This is because the space between the metal nanoparticles 12A and 22A is a space where a near-field is efficiently generated, and therefore, positioning the fluorescent materials 16A and 26A in this space tends to increase their fluorescence intensity.

[0100] (Test substance) The test substance 30 is a substance to be detected contained in the sample. Examples of the test substance 30 include antigens, proteins, substrates, and nucleic acid chains. The test substance 30 specifically binds to the specific binding substances 14A and 24A. For example, an antigen has at least two antigenic determinants, which form specific bindings with the first and second specific binding substances 14A and 24A. Antigens include proteins such as c-reactive proteins, myoglobin, troponin T, troponin I, and BNP, as well as antigenic proteins of viruses such as influenza virus and RSV. The test substance 30 is a test substance derived from a sample such as blood, plasma, urine, or saliva. That is, examples of samples containing the test substance 30 are blood, plasma, serum, urine, and saliva. The sample may further contain a solvent and buffer (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, and MES buffer, etc.).

[0101] (Method for detecting test substances using a complex) An example of a method for detecting a test substance using a composite according to the third embodiment will be described. The nanoparticle body 1A according to the second embodiment is dispersed in the sample to capture the test substance 30 contained in the sample and form a composite 40A.

[0102] In surface plasmon-field enhanced fluorescence spectroscopy (SPFS), when excitation light is irradiated onto complex 40A, localized surface plasmon resonance occurs, efficiently forming a near-field near the surface of metal nanoparticles 12A and 22A (particularly near the surface between the two metal nanoparticles 12A and 22A). Through this near-field and dipole-dipole mechanism, the fluorescent substances 16A and 26A in complex 40A are efficiently excited, enhancing fluorescence. By measuring the amount of fluorescence, the test substance in the sample can be detected.

[0103] (A measuring device that detects test substances using a composite material.) Referring to Figure 4, an example of a measurement method for detecting a test substance using the composite 40A according to the third embodiment will be described. Figure 4 is a schematic diagram showing a measurement device for detecting a test substance using the composite according to the third embodiment. As shown in Figure 4, the measurement device 100 comprises an excitation light source 110, an excitation light irradiation optical system 120, a reagent container 130, a light receiving optical system 140, and a light receiving element 150. The excitation light source 110 emits excitation light 112. The excitation light source 110 is, for example, a laser. The excitation light irradiation optical system 120 adjusts the cross-sectional diameter, such as focusing the excitation light 112, and outputs incident excitation light 122. The excitation light irradiation optical system 120 consists of a lens 124 and a polarizing element (λ / 2 plate) 126. The incident excitation light 122 output from the excitation light irradiation optical system 120 is incident on the reagent container 130 and irradiates the sample to be measured inside the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell and a slide, etc.) and a microfluidic chip. The microfluidic chip is a chip having minute channels. When the reagent container 130 is a microfluidic chip, for example, the nanoparticle body (reagent) and the sample according to the first embodiment can be mixed and continuously supplied. Therefore, it is not necessary to mix and prepare the sample to be measured in advance, and continuous measurement becomes possible.

[0104] The sample to be measured, when irradiated with incident excitation light 122, emits fluorescence (detection light 132). The light-receiving optical system 140 is positioned perpendicular to the direction of propagation of the incident excitation light 122 into the reagent container 130. The light-receiving optical system 140 can adjust the cross-sectional diameter of the detection light 132 emitted from the sample to remove scattered light from the incident excitation light 122 or adjust the light intensity. The light-receiving optical system 140 consists of a lens 144 and an optical filter 146. The optical filter 146 is, for example, a bandpass filter and a dichroic mirror.

[0105] The fluorescence 142 that has passed through the photodetector optical system 140 is detected by the photodetector 150. The photodetector 150 is, for example, a PD, APD, PMT, CCD camera, and spectrometer. The photodetector 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring the fluorescence spectrum, and creating a two-dimensional planar fluorescence image.

[0106] <Fourth Embodiment: Composite> The composite (metal thin film with nanoparticles) according to the fourth embodiment differs from the composite according to the third embodiment, which is composed of the same type of coated metal substrate (nanoparticles), in that it is composed of different types of coated metal substrates (coated metal thin film and nanoparticles). In the fourth embodiment, the same reference numerals as in the first to third embodiments have the same configuration as in the first to third embodiments, and therefore, in principle, their descriptions are omitted.

[0107] Furthermore, the composite according to the fourth embodiment can be formed by capturing the test substance between the coated metal substrate (coated metal thin film and two or more nanoparticles) according to the second embodiment when detecting the test substance. The composite according to the fourth embodiment comprises a coated metal thin film 1B and (one or more) nanoparticles 1A. The coated metal substrate 1 that can form the composite according to the fourth embodiment comprises a nanoparticle body 1A containing metal nanoparticles 2A as a metal substrate 2, and a coated metal thin film 1B containing a metal thin film 2B as a metal substrate 2, wherein the first hydrophilic group 7A is bonded to at least one of the surfaces of the metal nanoparticles 2A and the metal thin film 2B, and the coated metal substrate 1 forms a composite in which the coated metal thin film 1B and the nanoparticles 1A are bonded via the test substance.

[0108] The composite comprises a nanoparticle body 1A according to the second embodiment shown in Figure 1(a) and a coated metal thin film 1B according to the third embodiment shown in Figure 1(b), with the nanoparticle body 1A and the coated metal thin film 1B being bonded via the test substance 30. The first hydrophilic group 7A is bonded to at least one of the surfaces of the metal nanoparticle 2A and the metal thin film 2B.

[0109] The composite will be described with reference to Figure 5. Figure 5 is a schematic cross-sectional view showing the composite according to the fourth embodiment. Specifically, the composite 40B comprises the test substance 30 to be detected, two or more third nanoparticles 10B, and one first coated metal substrate 20B. The third nanoparticles 10B and the first coated metal substrate 20B are bonded together in the composite 40B via the test substance 30. In other words, the composite 40B according to the fourth embodiment can be formed by bonding the nanoparticles 1A and the coated metal thin film 1B according to the second embodiment via the test substance 30.

[0110] In composite 40B, the third nanoparticle body 10B comprises a third metal nanoparticle 12B as a metal nanoparticle, a third polymer film 13B as a polymer film, and a first hydrophilic group 7A (not shown in Figure 5). In other words, in composite 40B, the third nanoparticle body 10B comprises a third metal nanoparticle 12B, a third polymer film 13B that covers the surface of the third metal nanoparticle 12B, and a first hydrophilic group 7A (not shown in Figure 5) that suppresses nonspecific adsorption to the surface of the third metal nanoparticle 12B, wherein the first hydrophilic group 7A is bonded to the surface of the third metal nanoparticle 12B, and the third polymer film 13B contains a bond between itself and the surface of the third metal nanoparticle 12B via a sulfur atom. The third nanoparticle body 10B further comprises a third specific binding substance 14B bonded to the third polymer film 13B, and a third fluorescent substance 16B bonded to the third specific binding substance 14B.

[0111] In composite 40B, the first coated metal substrate 20B comprises a first metal thin film 22B as a metal substrate, a fourth polymer film 23B as a polymer film, and a first hydrophilic group 7A (not shown in Figure 5). In other words, in composite 40B, the first coated metal substrate 20B comprises a first metal thin film 22B, a fourth polymer film 23B that coats the surface of the first metal thin film 22B, and a first hydrophilic group 7A (not shown in Figure 5) that suppresses nonspecific adsorption to the surface of the first metal thin film 22B, the first hydrophilic group 7A is bonded to the surface of the first metal thin film 22B, and the fourth polymer film 23B contains a bond between itself and the surface of the first metal thin film 22B via a sulfur atom. The first coated metal substrate 20B further comprises a fourth specific bonding substance 24B bonded to the fourth polymer film 23B.

[0112] (Method for detecting test substances using a complex) An example of a method for detecting a test substance using a composite according to the fourth embodiment will be described. The measuring apparatus shown in Figure 4 is used. The first coated metal substrate 20B is placed in the reagent container 130 with the fourth polymer membrane 23B exposed so as to be in contact with the test substance 30 and the third nanoparticle 10B, which will be described later. A dispersion containing the test substance 30 is flowed into the reagent container 130 and brought into contact with the fourth polymer membrane 23B. This causes the test substance 30 to specifically bind to the fourth specific binding substance 24B bonded to the fourth polymer membrane 23B. Next, a dispersion containing the third nanoparticle 10B is flowed into the reagent container 130 and brought into contact with the fourth polymer membrane 23B. This causes the third nanoparticle 10B to specifically bind to the test substance 30 bonded to the fourth specific binding substance 24B on the first coated metal substrate 20B. As a result, a composite 40B is formed. Fluorescence is measured using the same method as in the third implementation. This detects the test substance.

[0113] The present invention is not limited to the embodiments described above, and design modifications are possible without departing from the spirit of the invention. At least two of the first to fourth embodiments may be combined.

[0114] In the fourth embodiment, the composite (metal thin film with nanoparticles) 40B is formed by bonding one nanoparticle 10B and a metal thin film 22B coated with a polymer film 23B via one test substance 30, but is not limited thereto. The composite (metal thin film with composite nanoparticles) may be formed, for example, by bonding one composite 40A and a metal thin film 22B coated with a polymer film 23B via one test substance 30.

[0115] In the second embodiment, the fluorescent substances 6 labeled on the nanoparticles 1A and the coated metal thin film 1B were labeled in quantities of three and two, respectively, as shown in Figure 1, but are not limited to this. The same applies to the number of specific binding substances 4 (Figure 1). Similarly, the number of fluorescent substances 16A, 26A and specific binding substances 14A, 24A in the composite 40A according to the third embodiment (Figure 3) is also similar. Furthermore, the number of fluorescent substances 16B and specific binding substances 14B, 24B in the composite 40B according to the fourth embodiment (Figure 5) is also similar.

[0116] In the second to fourth embodiments, the fluorescent substance was bound to a specific binding substance, but this is not limited to that. For example, the fluorescent substance may be bound to a polymer film.

[0117] In the third embodiment, the light-receiving optical system 140 in the measuring device 100 is arranged perpendicular to the direction of propagation of the incident excitation light 122 into the reagent container 130, but is not limited thereto. The light-receiving optical system 140 may be arranged, for example, parallel to the direction of propagation of the incident excitation light 122, or at an acute or obtuse angle with respect to the direction of propagation of the incident excitation light 122. [Examples]

[0118] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited in any way by the following examples. Unless otherwise specified, parts and percentages in the examples are based on mass.

[0119] In addition, in the examples, the concentration of metal nanoparticles in the dispersion is sometimes expressed in terms of absorbance. Absorbance was measured using a UV-Vis spectrophotometer (TECAN Japan Co., Ltd. "infinite M200 PRO"). The number appended to the absorbance notation OD indicates the absorption wavelength. For example, OD 455 =0.1 indicates that the absorbance at a wavelength of 455 nm is 0.1.

[0120] <Example 1> [Preparation of polymers] The polymer of Example 1 was prepared as follows.

[0121] (Introduction of disulfide bonds into polymers: Disulfide group introduction process) (3a-1) Poly-L-lysine (manufactured by Peptide Laboratories, Inc., "3075", weight-average molecular weight Mw>12,000, hereinafter also referred to as "PLL") as a polymer having a functional site in its side chain, and (4-1) 3-(2-pyridyldithio)propionamide-PEG4-NHS (manufactured by Thermo Fisher Scientific, lot number "26128", "NHS-PEG4-SPDP") as a compound having a disulfide group, were mixed by stirring at room temperature for 4 hours using a small rotary incubator (manufactured by Taitec Co., Ltd., "RT-30mini"). As a result, (2-1) a polymer having a pyridyl group (pyridinyl group) in its side chain via an SPDP linker and a disulfide bond (hereinafter also referred to as "polymer having a disulfide group") was obtained. The reaction formula for this synthesis is (r-1): [ka] As shown, this is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamide-PEG4-NHS.

[0122] (Introduction of hydrophilic groups into polymers: hydrophilic group introduction process) (2-1) A polymer having a disulfide group and (5j-1) a thiol compound having a hydrophilic group (sulfobetaine 3-undecanetiol, Dojin Chemical "S350") were stirred and mixed at 37°C for 1 hour. As a result, (1-1) a polymer in which betaine was bonded to the side chain via an SPDP linker and a disulfide bond (hereinafter also referred to as "betaine-introduced polymer" (PLL-SS-betaine)) was obtained. The synthesized (1-1) betaine-introduced polymer had a hydrophobic group (n-butylene group) and a positively charged group (first ammonium group). The reaction formula for this synthesis is (r-2): [ka] As shown in FIG. 4, it is a thiol nucleophilic reaction in which the thiol group of the thiol compound having (5j-1) hydrophilic groups attacks the disulfide group of the polymer having (2-1) disulfide groups.

[0123] Referring to FIG. 6, the identification of (1-1) PLL-S-S-betaine will be described. FIG. 6 is a diagram showing absorption spectra in each step of polymer synthesis. The solid line represents the absorption spectrum after reaction (r-1), the broken line represents the absorption spectrum after reaction (r-2), and the dashed-dotted line represents the absorption spectrum after ultrafiltration treatment after reaction (r-2).

[0124] The absorption peak near 280 nm in the absorption spectrum (solid line) was attributed to the pyridyl group of the polymer having (2-1) disulfide groups. The absorption peak near 343 nm in the absorption spectrum (broken line) has the following resonance structure: [Chemical formula] was attributed to 3-mercaptopyridine having. 3-Mercaptopyridine is a by-product generated in reaction (r-2). The absorption spectrum (dashed-dotted line) had no maximum peak in the visible region. From these results, it was confirmed that the above reactions (r-1) and (r-2) proceeded and (1-1) PPL-S-S-betaine was generated.

[0125] [Formation of Polymer Membrane and Labeling of Hydrophilic Groups on the Surface of Metal Nanoparticles] The obtained (1-1) PLL-S-S-betaine was used as silver nanoparticles as metal nanoparticles (Nanocomposix's "AGCB80-1M", diameter 80 nm, OD 4551 mL of the dispersion of (0.1) was added and stirred and mixed at room temperature and overnight using a small rotary incubator ("RT-30mini" manufactured by Taitec Co., Ltd.). As a result, a dispersion of silver nanoparticles coated with a polymer film and labeled with a hydrophilic substance (betaine) (hereinafter also referred to as "hydrophilic group-labeled polymer-coated silver nanoparticles") was obtained. In this reaction, the disulfide groups of the polymer having hydrophilic groups are cleaved by the reducing action of silver. The resulting poly-L-lysine moiety and the hydrophilic groups bond to the surface of the silver nanoparticles via sulfur atoms. As a result, as shown in Figure 7 (a diagram showing the reaction for the polymer film formation method of Example 1), betaine as a hydrophilic substance is labeled to the surface of the silver nanoparticles via sulfur atoms, and the polymer film is bonded to the surface of the silver nanoparticles via sulfur atoms. In other words, in this process, the labeling of hydrophilic groups and the formation of a polymer film proceed in parallel.

[0126] [Evaluation Method] (SEM image) A scanning electron microscope (Regulus8220, Hitachi High-Tech Corporation) was used to capture SEM images (magnification 300K) of the hydrophilic group-labeled polymer-coated silver nanoparticles obtained in Example 1. Figure 8 shows the SEM image of the hydrophilic group-labeled polymer-coated silver nanoparticles in Example 1. As shown in Figure 8, the obtained SEM image confirmed that the silver nanoparticles were coated with a polymer film.

[0127] (Measurement and evaluation based on zeta potential and particle size distribution) The zeta potential and particle size distribution of the hydrophilic group-labeled polymer-coated silver nanoparticles of Example 1 were measured using a zeta potential measuring device (MALVERN "ZETA SIZER Nanoseries nano-ZS"). (n=3 measurements for each). The zeta potential measurement results are explained with reference to Figure 9. Figure 9 shows the distribution of the scattered light intensity ratio to the zeta potential (zeta potential distribution) for the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 1 (more specifically, it is one of the distributions obtained from three measurements). The distribution of the scattered light intensity ratio to the zeta potential showed peaks located between -5.77 and -2.33 mV. The polymer-coated hydrophilic group-labeled silver nanoparticles were slightly negatively charged overall.

[0128] The measurement results of the particle size distribution will be explained with reference to Figure 10. Figure 10 shows the distribution of the scattered light intensity ratio to particle size (particle size distribution) for polymer-coated hydrophilic group-labeled silver nanoparticles of Example 1 (more specifically, it is one of the distributions obtained from three measurements). The distribution of the scattered light intensity ratio to particle size was 97.93 nm (expected value M) ± 0.163 nm (Polydispersity Index (PDI)).

[0129] <Example 2> The thiol compound having a hydrophilic group in reaction (r-2) is a thiol compound having a phosphoroline group represented from (5j-1) to (5k-1) (manufactured by Medical Science Pharmaceutical Co., Ltd., "Product Code: MCP-03-0001-PC"): [ka] (5k-1) A polymer having a disulfide group (PLL-SS-PC) was obtained in the same manner as in Example 1, except that a change was made. The same absorption spectrum as in Example 1 was obtained.

[0130] SEM images (magnification 300K) of the hydrophilic group-labeled polymer-coated silver nanoparticles of Example 2 were acquired, and the zeta potential distribution and particle size distribution were obtained. Figure 11 shows the SEM image of the hydrophilic group-labeled polymer-coated silver nanoparticles of Example 2. From Figure 11, it was confirmed that the silver nanoparticles were coated with a polymer film. The hydrophilic group-labeled polymer-coated silver nanoparticles of Example 2 were measured using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As a result, the presence of phosphate was confirmed in the region where the silver nanoparticles were present. It was concluded that this phosphate is a fragment derived from a phosphorolcholine group bound to the surface of the silver nanoparticles. Figure 12 shows the zeta potential distribution of the polymer-coated hydrophilic group-labeled silver nanoparticles of Example 2 (more specifically, it is the distribution of one of the distributions obtained from three measurements). From the zeta potential distribution, the peaks were located in the +20 to +27 mV range. The polymer-coated hydrophilic group-labeled silver nanoparticles were slightly positively charged overall. Table 1: [Table 1] The expected value M and polydispersity index PDI for the particle size distribution of the hydrophilic group-labeled polymer-coated silver nanoparticles of Example 2 are shown below.

[0131] <Comparative Example 1> In Comparative Example 1, polymer-coated silver nanoparticles were prepared in the same manner as in Example 1, except that the polymer used to coat the silver nanoparticles was changed from (1-1)PLL-SS-betaine to PLL (Peptide Laboratories, Inc., "3075"). In Comparative Example 1, the zeta potential distribution was measured in the same manner as in Example 1. The zeta potential distribution of Comparative Example 1 had a peak around +50mV. The polymer-coated silver nanoparticles were positively charged overall.

[0132] <Comparative Example 2> In Comparative Example 2, the particle size distribution and zeta potential distribution were measured in the same manner as in Example 1, except that silver nanoparticles (nanocomposix "AGCB80-1M", 80 nm in diameter) were used instead of polymer-coated hydrophilic group-labeled silver nanoparticles. The zeta potential distribution of Comparative Example 2 had a peak located around -50 mV. The silver nanoparticles were negatively charged overall.

[0133] (Comparison of Examples 1-2 and Comparative Examples 1-2) In the zeta potential distributions of Examples 1 and 2, the absolute value of the zeta potential at the peak position was smaller than that of Comparative Examples 1 and 2. Normally, when the zeta potential approaches 0 mV, aggregation occurs, making it difficult to disperse in a primary particle state. Nevertheless, in the particle size distribution results of Examples 1 and 2, the expected value was only slightly larger than the particle size of the silver nanoparticles (80 nm). This increase is thought to be due to the thickness of the polymer film. Furthermore, the PDI was not very large. Considering these factors, it is thought that the hydrophilic group-labeled polymer-coated silver nanoparticles (coated metal substrate) are dispersed in a primary particle state. This is thought to be due to the presence of hydrophilic groups on the surface of the metal substrate. From the above, it is thought that the adsorption of nonspecific adsorbed substances that contribute to noise components can be effectively suppressed.

[0134] The coated metal substrate and its manufacturing method, the composite including the coated metal substrate, and the polymer for manufacturing the coated metal substrate relating to this disclosure are as follows. <1> It comprises a metal substrate which is metal nanoparticles and / or a metal thin film, a polymer film which covers the surface of the metal substrate, and a first hydrophilic group which suppresses nonspecific adsorption to the surface of the metal substrate. The first hydrophilic group is bonded to the surface of the metal substrate, A coated metal substrate wherein the polymer film includes a bonding site between the polymer film and the surface of the metal substrate via sulfur atoms. <2> The first hydrophilic group includes a bonding site between itself and the surface of the metal substrate via a sulfur atom. <1> The coated metal substrate described above. <3> The polymer forming the polymer film includes the sulfur atom-mediated bonding site in the side chain of the polymer between it and the surface of the metal substrate. <1> or <2> The coated metal substrate described above. <4> The polymer constituting the polymer film contains one or more secondary hydrophilic groups per molecular chain of the polymer, which are bonded to the side chains of the polymer via disulfide groups. <1> ~ <3> A coated metal substrate as described in any one of the items. <5> The polymer further contains the second hydrophilic group in the side chain of the polymer, The first hydrophilic group and the second hydrophilic group each independently include a polar group and / or an electrostatic group, The polar group comprises at least one selected from the group consisting of a carboxyl group, a hydroxyl group, an amino group, a sulfonyl group, a phosphate group, and an alkylene oxide group. The charged group comprises at least one selected from the group consisting of a charged polar group and a zwitterionic group, and the zwitterionic group comprises at least one selected from the group consisting of a phosphorylcholine group and a betaine group. <4> The coated metal substrate described above. <6> The polymer constituting the polymer film contains an amide bond in the main chain skeleton of the polymer. <1> ~ <5> A coated metal substrate as described in any one of the items. <7> The polymer constituting the polymer film further contains positively charged groups in the side chains of the polymer, The aforementioned side chain does not have a disulfide bond. The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH2) + At least one selected from the group consisting of )NH2), <1> ~ <6> A coated metal substrate as described in any one of the items. <8> The polymer constituting the polymer film further contains hydrophobic groups in the side chains of the polymer, The aforementioned side chain does not have a disulfide bond. The hydrophobic group is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. <1> ~ <7> A coated metal substrate as described in any one of the items. <9> The polymer comprises a main chain skeleton containing a structure derived from one or more amino acids. The aforementioned amino acid comprises at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, glutamine aspartate, asparagine, serine, and threonine. <3> ~ <8> A coated metal substrate as described in any one of the items. <10> The polymer constituting the polymer film includes a biocompatible polymer as a block polymer in the main chain skeleton of the polymer. <1> ~ <9> A coated metal substrate as described in any one of the items. <11> The present invention further comprises a fluorescent substance and / or a specific binding substance that specifically binds to the test substance in the sample, <1> ~ <10> A coated metal substrate as described in any one of the items. <12> The aforementioned specific binding substance is a nanoantibody. <11> The coated metal substrate described above. <13> The film thickness of the polymer film is 1 nm to 10 nm. <1> ~ <12> A coated metal substrate as described in any one of the items. <14> A coated metal substrate used in plasmon-induced fluorescence analysis, <1> ~ <13> A coated metal substrate as described in any one of the items. <15> The aforementioned metal substrate contains gold or silver. <1> ~ <14> A coated metal substrate as described in any one of the items. <16> The aforementioned metal substrate is a nanoparticle body comprising the aforementioned metal nanoparticles, The aforementioned nanoparticles include a first nanoparticle and a second nanoparticle. The first hydrophilic group is bonded to at least one of the surfaces of the metal nanoparticles in the first nanoparticle body and the surfaces of the metal nanoparticles in the second nanoparticle body. The first nanoparticle and the second nanoparticle form a composite by being bound together via the test substance. <1> ~ <15> A coated metal substrate as described in any one of the items. <17> The metal substrate comprises a nanoparticle body containing the metal nanoparticles and a coated metal thin film containing the metal thin film. The first hydrophilic group is bonded to at least one of the surface of the metal nanoparticles and the surface of the metal thin film. The coated metal thin film and the nanoparticles are bonded together via the test substance to form a composite. <1> ~ <16> A coated metal substrate as described in any one of the items. <18> The test substance is derived from a sample that is blood, plasma, urine, or saliva. <16> or <17> The coated metal substrate described above. <19> The molecules constituting the polymer film cover the surface of the metal substrate in a two-dimensional manner, such that the main chain of the polymer is substantially parallel to the surface of the metal substrate. <1> ~ <18> A coated metal substrate as described in any one of the items. <20> <1> ~ <19> It comprises two or more nanoparticle bodies containing metal nanoparticles as described in any one of the items, The aforementioned two or more nanoparticles include a first nanoparticle and a second nanoparticle, A composite in which the first nanoparticle and the second nanoparticle are bound together via the test substance. <21> <1> ~ <19> A nanoparticle body comprising metal nanoparticles as described in any one of the items, <1> ~ <19> It comprises a coated metal thin film containing a metal thin film as described in any one of the items, The first hydrophilic group is bonded to at least one of the surface of the metal nanoparticles and the surface of the metal thin film. A composite in which the nanoparticles and the coated metal thin film are bonded together via the test substance. <22> A polymer having hydrophilic groups at the ends of its side chains, linked via disulfide groups. <23> The hydrophilic group includes a polar group and / or an electrostatic group, The polar group comprises at least one selected from the group consisting of a carboxyl group, a hydroxyl group, an amino group, a sulfonyl group, a phosphate group, and an alkylene oxide group. The charged group comprises at least one selected from the group consisting of a charged polar group and a zwitterionic group, and the zwitterionic group comprises at least one selected from the group consisting of a phosphorylcholine group and a betaine group. <22> The polymers described above. <24> The polymer further contains positively charged groups in its side chains, The aforementioned side chain does not have a disulfide bond. The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH2) + At least one selected from the group consisting of )NH2), <22> or <23> The polymers described above. <25> The polymer further contains hydrophobic groups in its side chains, The aforementioned side chain does not have a disulfide bond. The hydrophobic group is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. <22> ~ <24> A polymer as described in any one of the items. <26> The main chain skeleton of the polymer contains an amide bond. <22> ~ <25> A polymer as described in any one of the items. <27> The main chain skeleton of the polymer has a structure derived from one or more amino acids, The aforementioned amino acid comprises at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, serine, and threonine. <22> ~ <26> A polymer as described in any one of the items. <28> The main chain skeleton of the aforementioned polymer contains a biocompatible polymer as a block polymer. <22> ~ <27> A polymer as described in any one of the items. <29> <22> ~ <28> A method for producing a coated metal substrate, comprising the step of bringing a polymer described in any one of the above items into contact with the surface of a metal substrate, thereby bonding the hydrophilic groups to the surface of the metal substrate and forming a polymer film on the surface of the metal substrate. <30> In the aforementioned side chain, the length of the first side chain from the disulfide group to the hydrophilic group is longer than the length of the second side chain from the disulfide group to the main chain of the polymer. <29> A method for manufacturing a coated metal substrate as described above. [Explanation of symbols]

[0135] 1. Coated metal substrate 1A ···Nanoparticles 1B ···Coated metal thin film 2...Metal base material 2A ···metal nanoparticles 2B...metal thin film 3...polymer membrane 3A ···(polymer that constitutes a polymer film) 3B... (polymers that form polymer films) 4...specific binding substance 6. Fluorescent substances 7...Hydrophilic group 7A...first hydrophilic group 7B...Second hydrophilic group 10A ···First Nanoparticle 10B ···Third Nanoparticle 12A ···First metal nanoparticle 12B ···Third metal nanoparticles 13A...First polymer membrane 13B...Third polymer membrane 14A...First specific binding substance 14B...Third specific binder 16A ···First fluorescent substance 16B ···Third fluorescent substance 20A ···Second Nanoparticle 20B ···First coated metal substrate 22A ···Second metal nanoparticles 22B...First metal thin film 23A...Second polymer membrane 23B...Fourth polymer membrane 24A...Second specific binding substance 24B...Fourth specific binding substance 26A ···Second fluorescent substance 30 ···Test substance 40A, 40B composite L...Separation distance (separation distance)

Claims

1. It has a hydrophilic group at the end of its side chain, which is bonded via a disulfide group. The hydrophilic group includes a polar group and / or an electrostatic group, The polar group comprises at least one selected from the group consisting of a carboxyl group, a hydroxyl group, an amino group, a sulfonyl group, a phosphate group, and an alkylene oxide group. The charged group comprises at least one selected from the group consisting of a charged polar group and a zwitterionic group, and the zwitterionic group comprises at least one selected from the group consisting of a phosphorylcholine group and a betaine group. A polymer having an amide bond in its main chain skeleton.

2. The polymer further contains positively charged groups in its side chains, The aforementioned side chain does not have a disulfide bond. The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH ２ ＋ ) NH ２ The polymer according to claim 1, which is at least one selected from the group consisting of ).

3. The polymer further contains hydrophobic groups in its side chains, The aforementioned side chain does not have a disulfide bond. The polymer according to claim 1, wherein the hydrophobic group is at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups.

4. The polymer has a main chain skeleton that is derived from one or more amino acids, The polymer according to claim 1, wherein the amino acid comprises at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, serine, and threonine.

5. The polymer according to claim 1, wherein the main chain skeleton of the polymer contains a biocompatible polymer as a block polymer.

6. A method for producing a coated metal substrate, comprising the steps of bringing the polymer described in claim 1 into contact with the surface of a metal substrate, thereby bonding the hydrophilic groups to the surface of the metal substrate, and forming a polymer film on the surface of the metal substrate.

7. The method for producing a coated metal substrate according to claim 6, wherein in the side chain, the length of the first side chain from the disulfide group to the hydrophilic group is longer than the length of the second side chain from the disulfide group to the main chain of the polymer.

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