Coated metallic base, production methdo therefor, composite including coated metallic base, and polymer for producing coated metallic base

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

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

[0001] The present invention relates to coated metal substrates and methods for their manufacture, composites containing the coated metal substrates, and polymers for producing the coated metal substrates.

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

[0003] When analyzing a specific analyte by specifically reacting a specific analyte with a specific specific binding substance to form a complex, there is a problem of poor signal-to-noise ratio (S / N ratio) due to nonspecific adsorption to the metal particle surface. Plasmon-excited fluorescence analysis using metal particles is no exception, and nonspecific adsorption to the metal particle surface can worsen the S / N ratio. Therefore, as described in Patent Document 1, for example, a polymer film is formed by binding the ends of one-dimensional polymers (polymer chains) to the surface of metal nanoparticles. A polymer film in which the polymer chains are arranged in a brush-like pattern is formed by arranging multiple polymer chains on the surface of a metal substrate so that they are approximately perpendicular to the surface of the metal substrate.

[0004] International Publication No. 2001 / 086301

[0005] However, the inventors of the present invention have conducted extensive research into the polymer described in Patent Document 1 and found that there is room for further improvement in detection sensitivity. Specifically, when the polymer described in Patent Document 1 is used, a polymer film is formed in which one-dimensional polymer chains are arranged in a brush-like configuration, which may prevent the polymer film formed on the surface of the metal substrate from being sufficiently thin. As a result, the separation distance between the metal substrates in the complex capturing the analyte cannot be sufficiently reduced, and fluorescence cannot be effectively enhanced. Furthermore, because the polymer film contains molecular chains containing hydrophobic alkyl groups arranged in a brush-like configuration, there is a risk that the adsorption of nonspecific adsorbed substances (nonspecific adsorption or nonspecific adsorption), which contributes to noise components, may not be effectively suppressed.

[0006] The present invention has been made in view of these problems. That is, a main object of the present invention is to provide a polymer that can reduce the film thickness, thereby sufficiently reducing the distance between metal substrates in a composite, and thereby increasing detection sensitivity. Another main object of the present invention is to provide a coated metal substrate having a polymer film formed thereon using such a polymer, a method for producing the same, and a composite including the coated metal substrate.

[0007] A polymer according to one embodiment of the present invention has hydrophilic groups at the ends of side chains that are bonded via disulfide groups.

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

[0009] A complex according to one embodiment of the present invention comprises two or more nanoparticles containing the metal nanoparticles, the two or more nanoparticles including a first nanoparticle and a second nanoparticle, and the first nanoparticle and the second nanoparticle are bound to each other via the test substance.

[0010] A complex according to one embodiment of the present invention comprises a nanoparticle body containing the metal nanoparticles and a coated metal thin film containing the metal thin film, wherein a first hydrophilic group is bonded to at least one of the surface of the metal nanoparticles and the surface of the metal thin film, and the nanoparticle body and the coated metal thin film are bonded via a test substance.

[0011] A method for producing a coated metal substrate according to one embodiment of the present invention comprises the steps of contacting the polymer with the surface of the metal substrate to bond the hydrophilic groups to the surface of the metal substrate while forming a polymer film on the surface of the metal substrate.

[0012] The present invention provides a polymer capable of reducing the thickness of the polymer film to sufficiently reduce the separation distance between metal substrates in a composite and enhance detection sensitivity, as well as a coated metal substrate having a polymer film formed thereon using such a polymer, a method for producing the same, and a composite including the coated metal substrate.

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

[0014] Hereinafter, the nanoparticles, their manufacturing method, composite, and measuring device according to the present invention will be described in detail with reference to the illustrated embodiments. Note that the drawings include schematic views and may not reflect actual dimensions or proportions.

[0015] Numerical ranges mentioned in this specification are intended to include the lower and upper limit values ​​themselves, unless otherwise specified, such as "less than," "greater than," or "smaller." For example, in the case of a numerical range such as 1 nm to 50 nm, unless otherwise specified, the numerical range is interpreted as including the lower limit value of "1 nm" and the upper limit value of "50 nm."

[0016] As used herein, the term "metal substrate" refers to a substrate that is substantially composed of metal and includes a metal thin film (also referred to as a metal plate or metal film) and metal nanoparticles. Metal nanoparticles and metal thin films differ primarily in shape. As used herein, "a target component substantially composed of a specific material" refers to the target component containing 95% by mass or more, 97% by mass or more, 99% by mass or more, or 100% by mass of the specific material. For example, "a metal substrate substantially composed of metal" refers to the metal substrate containing 95% by mass or more, 97% by mass or more, 99% by mass or more, or 100% by mass of metal.

[0017] As used herein, "metal nanoparticles" refer to particles that are substantially composed of metal, have a size on the order of nanometers (e.g., several nm to 100 nm), and are spherical or nearly spherical in shape. "Metal nanoparticles" induce localized surface plasmon resonance with other metal nanoparticles through interaction with light (e.g., visible light). As used herein, "metal thin film" refers to a thin film that is substantially composed of metal and has an original film thickness on the order of nanometers (e.g., several nm to 100 nm). "Metal thin films" induce 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 bonded via disulfide groups (disulfide bonds) (-S-S-). The polymer according to the first embodiment can contact the surface of a metal substrate to form a polymer film and hydrophilic groups on the surface of the metal substrate. This allows the production of a coated metal substrate (described in detail in the second embodiment).

[0019] [Mechanism of Action] A composite having a polymer film formed from the polymer according to the first embodiment has excellent detection sensitivity. Without being bound by any particular theory, the reason for this is presumed to be as follows. The polymer according to this embodiment is, for example, a polymer represented by the general formula (1): (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 in the formula (1), the polymer has a hydrophilic group at the end of the side chain bonded via a disulfide group (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 and subsequent embodiments). The present inventors have devised a method for cleaving the disulfide group, whereby one S atom contributes to reducing the thickness of the polymer film and the other S atom contributes to improving the ease of exposure of the hydrophilic group. More specifically, the present inventors have devised that cleavage of the disulfide group can realize the following two aspects: (First aspect): an aspect in which one S atom attached to the end of a side chain branching from the polymer main chain is bonded to a predetermined location on the surface of the metal substrate; and (Second aspect): an aspect in which the S atom connected to the other hydrophilic group X is bonded at a location different from the predetermined location on the surface of the metal substrate. As a result, in the present invention, as described above, it is possible to "reduce the thickness of the polymer film" and "improve the ease of exposing the hydrophilic groups."

[0020] 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 groups are cleaved, forming sulfur-atom-mediated bonding sites on the surface of the metal substrate. As a result, in the formed polymer film, the polymers constituting the polymer film are bonded to the metal substrate at the ends of their side chains. Because the polymer film has a structure in which the main chains of the constituting polymers two-dimensionally cover the surface of the metal substrate so that they are approximately parallel to the surface of the metal substrate, the film thickness of the polymer film can be reduced compared to a polymer film formed in which one-dimensional polymers are arranged approximately perpendicular to the surface of the metal substrate, as shown in Patent Document 1, for example. This allows the separation distance between the metal substrates in the composite to be sufficiently small.

[0021] In this specification, the term "main chain" refers to a hydrocarbon chain (e.g., an alkylene chain) that forms the main skeleton of the polymer and is a parallel hydrocarbon chain in the direction in which the multiple repeating units that make up the polymer are bonded to each other and extend. This hydrocarbon chain may have a divalent group containing a heteroatom, such as an ether group, an amide bond, or an ester bond. In this specification, the term "side chain" refers to a hydrocarbon chain branched from the main chain. Like the hydrocarbon chain of the main chain, the hydrocarbon chain of the side chain may also have a divalent group containing a heteroatom.

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

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

[0024] (Hydrophilic Group) The hydrophilic group includes a polar group and / or a charged 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. The zwitterionic group includes, for example, at least one selected from the group consisting of a phosphorylcholine group and a betaine group.

[0025] The present inventors have found that, because nonspecifically adsorbed substances contain hydrophobic moieties, hydrophilic moieties are effective in suppressing nonspecific adsorption to polymer membranes. Furthermore, they have found that nonspecific adsorption can be effectively suppressed when the hydrophilic moieties can be exposed from the polymer membrane where they are formed. 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 present inventors have found that introducing a hydrophilic moiety into the main chain skeleton of the polymer constituting the polymer membrane, in addition to hydrophilic groups, is effective in suppressing nonspecific adsorption. From the viewpoint of further suppressing nonspecific adsorption, the main chain skeleton of polymer (1) preferably contains an amide bond. From the viewpoint of further suppressing nonspecific adsorption, the main chain skeleton of polymer (1) preferably contains 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 nonspecific adsorption, the main chain skeleton of polymer (1) preferably contains a biocompatible polymer as a block polymer. An example of such a biocompatible polymer is polymethyl methacrylate.

[0027] The term "one or more" as used herein more specifically refers to "one or more than one," and "one or more" (more specifically "one or more than one") is synonymous.

[0028] The polymer (1) may further include a positively charged group in a side chain. The side chain does not have a disulfide group. Such a positively charged group includes a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH ２ ＋ ) NH ２ When the polymer (1) contains such a positively charged group, the resulting polymer film can form an electrostatic bond with the surface of the metal substrate, thereby more firmly fixing the polymer film.

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

[0030] (Method for Producing Polymer) An example of a method for producing a polymer will be described. The method for producing a polymer includes, for example, a step of introducing a disulfide group into a side chain (disulfide group introduction step) and a step of introducing a hydrophilic group (hydrophilic group introduction step). The method is shown in Scheme 1 below:

[0031] (In Scheme 1, X represents a hydrophilic group, Y represents, for example, a pyridyl group, Z and T represent functional moieties, L represents, for example, a divalent bonding moiety, n represents the number of repeating units, and Z and T are different from each other and bond to each other to form L.) A method for producing polymer (1) will be described with reference to Scheme 1. Scheme 1 shows a reaction formula for explaining the method for producing polymer (1). Note that L, X, and n in Scheme 1 are synonymous with L, X, and n in general formula (1), respectively.

[0032] -Disulfide Group Introduction Step- As shown in Scheme 1, the disulfide group introduction step involves, for example, reacting a polymer having a functional moiety Z in a 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) having a disulfide group)) to obtain a polymer having a disulfide group (a polymer represented by formula (2) (hereinafter also referred to as polymer (2))). The functional moieties Z and T are different from each other and are, for example, reactive substituents such as an amino group, a carboxyl group, or an ester bond. This reaction is, for example, a nucleophilic substitution reaction in which the amino group attacks the carboxyl group or the 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, from 1 hour to 4 hours.

[0033] Examples of the functional moiety Z in the polymer (3) include an amino group, a carboxyl group, and an ester group. Examples include:

[0034] Examples of the compound (4) having a disulfide group include: Examples include:

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

[0036] Examples include:

[0037] <Second embodiment: Coated metal substrate> The coating film (i.e., polymer film) of the coated metal substrate according to the second embodiment can be formed from the polymer according to the first embodiment. That is, the polymer constituting the polymer film can be derived from the polymer according to the first embodiment. In the second embodiment, the same reference numerals as those in the first embodiment have the same configuration as those in the first embodiment, and therefore, the description thereof will be omitted.

[0038] The coated metal substrate according to the second embodiment comprises a metal substrate, a polymer film that coats the surface of the metal substrate, and a first hydrophilic group that suppresses nonspecific adsorption to the surface of the metal substrate, wherein the first hydrophilic group is bonded to the surface of the metal substrate, and the polymer film includes a bonding site between the polymer film and the surface of the metal substrate via a sulfur atom.

[0039] [Mechanism of Action] The coated metal substrate according to the second embodiment can improve detection sensitivity. Without being bound by any particular theory, the reason for this 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 nonspecific adsorption to the coated metal substrate, thereby suppressing a decrease in the S / N ratio during detection of an analyte and improving detection sensitivity. Furthermore, the coated metal substrate according to this embodiment includes, in addition to the first hydrophilic group, a polymer film containing a binding site between the metal substrate and the surface via a sulfur atom. In this way, the polymer film does not need to have the function of suppressing nonspecific adsorption, so the film thickness of the polymer film can be reduced. This allows the distance between the coated metal substrates to be reduced in the complex formed during detection of the analyte. From the above, it is believed that the coated metal substrate according to the second embodiment can improve detection sensitivity.

[0040] [Basic Structure of Coated Metal Substrate] The coated metal substrate will be described with reference to Fig. 1. Fig. 1 is a cross-sectional view schematically showing a coated metal substrate according to a second embodiment. The coated metal substrate 1 includes nanoparticles 1A and a coated metal thin film 1B.

[0041] As shown in Fig. 1(a), nanoparticle 1A comprises a metal substrate 2 which is a metal nanoparticle 2A, a polymer film 3 which covers the surface of the metal substrate 2, and first hydrophilic groups 7A (not shown in Fig. 1(a)) which suppress non-specific adsorption to the surface of the metal substrate 2. Nanoparticle 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 (LSPR, hereinafter simply referred to as "plasmon resonance") when irradiated with excitation light. The polymer film 3 covers the surface of the metal nanoparticles 2A and includes a bonding site between the metal nanoparticles 2A and the surface of the metal substrate 2 via a sulfur atom. The first hydrophilic groups 7 are bonded to the surface of the metal substrate 2.

[0043] As shown in Fig. 1(b), coated metal thin film 1B comprises metal substrate 2, which is metal thin film 2B, polymer film 3 covering the surface of metal substrate 2, and first hydrophilic groups 7 (not shown in Fig. 1(b)) that suppress nonspecific adsorption to the surface of metal substrate 2. Coated metal thin film 1B may further comprise specific binding substance 4.

[0044] The metal thin film 2B induces localized surface plasmon resonance when irradiated with excitation light. The polymer film 3 covers the surface of the metal thin film 2B and includes a bonding site between the polymer film 3 and the surface of the metal substrate 2 via a sulfur atom. The first hydrophilic groups 7 are bonded to the surface of the metal substrate 2.

[0045] The coated metal substrate 1 can be used for plasmon excitation fluorescence analysis. In other words, the coated metal substrate 1 can be used for surface plasmon excitation-enhanced fluorescence spectroscopic immunoassay. The coated metal substrate 1 can capture an analyte in a specimen, for example, to form a complex (described in detail in the third and fourth embodiments) containing two coated metal substrates 1 and one analyte. When excitation light is irradiated onto the complex, plasmon resonance occurs, forming a near-field. This near-field increases the fluorescence intensity. In other words, an electric field enhancement effect is obtained.

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

[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 heparin sodium.

[0048] (Metallic Substrate) The surface of the metallic substrate 2 is coated with a polymer film 3. The metallic substrate 2 preferably comprises gold or silver, more preferably silver. The metallic substrate 2 comprises metallic nanoparticles 2A and a metallic thin film 2B.

[0049] -Metal Nanoparticles- Metal nanoparticles 2A interact with light having a specific wavelength, causing localized surface plasmon resonance, although this varies depending on the type of metal constituting the metal nanoparticles 2A. Silver nanoparticles (nanoparticles consisting essentially of silver) have a localized surface plasmon resonance peak at 400 nm to 530 nm, while gold nanoparticles (nanoparticles consisting essentially of gold) have a localized surface plasmon resonance peak at 510 nm to 580 nm. This varies depending on the particle diameter of the metal nanoparticles 2A. For example, silver nanoparticles with a particle diameter of 20 nm resonate with light with a wavelength of 405 nm. Gold nanoparticles with a particle diameter of 20 nm resonate with light with a wavelength of 524 nm. The particle diameter (average primary particle diameter) of the metal nanoparticles 2A is, for example, 5 nm to 100 nm. The particle diameter of the metal nanoparticles 2 can be obtained by taking an image of the metal nanoparticles 2 using a scanning electron microscope (SEM) or a transmission electron microscope (TEM), measuring the particle diameters of the metal nanoparticles 2 in the image, and calculating the average value of multiple particle diameters (number of measurements: for example, at least 10 or more).

[0050] -Metal Thin Film- The metal thin film 2B interacts with light having a specific wavelength (for example, visible light), depending on the type of metal that constitutes the metal thin film 2B, and causes localized surface plasmon resonance between the metal thin film 2B and the metal nanoparticles.

[0051] (Polymer film) 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 dispose the fluorescent substance 6 at a distance from the surface of the metal substrate 2 by at least the thickness of the polymer film 3. This prevents the excited fluorescent substance 6 from coming into contact with the surface of the metal substrate 2 and quenching, thereby preventing a decrease in detection sensitivity. The presence of the polymer film 3 can be confirmed by capturing an image of the nanoparticles 1 using an 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 Fig. 2. Fig. 2 is an enlarged view of part A in Fig. 1(a), and is an enlarged schematic view of the vicinity of the interface between the polymer film 3 of the nanoparticle 1A and the surface of the metal nanoparticle 2A. Note that the polymer film 3 of the nanoparticle 1A has substantially the same configuration as the polymer film 3 of the coated metal thin film 1B, and therefore only the polymer film 3 of the nanoparticle 1A will be described below.

[0053] The polymer film 3 includes binding sites 3a between the surface of the metal nanoparticle 2 and the polymer film 3 via sulfur atoms (i.e., the polymer 3A constituting the polymer film 3 includes binding sites 3a between the surface of the metal substrate 2 (metal nanoparticle 2A) via sulfur atoms). The polymer film 3 may further include at least one selected from the group consisting of positively charged groups 3b and hydrophobic groups 3c (e.g., 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 the polymer 3A. This side chain does not have a disulfide group). More specifically, the polymer film 3 includes primary ammonium groups (—NH ３ ＋) and a hydrophobic group 3c. The binding site 3a bonds the surface of the metal nanoparticle 2 to the polymer film 3 via a sulfur atom. The positively charged group 3b forms an electrostatic bond (ionic bond) b with the surface of the negatively charged metal nanoparticle 2. The hydrophobic group 3c forms a hydrophobic bond c with the surface of the metal nanoparticle 2. In this specification, the polymer that constitutes the polymer film 3 is also referred to as "polymer 3A," and the polymer that serves as a raw material for forming the polymer film 3 by reaction is also referred to as "polymer 3B." These two polymers are written differently to make them easier to distinguish.

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

[0055] Furthermore, because the polymer film 3 can be configured to include polymer 3A, it is easier to chemically modify than a silica layer, and the need for surface modification, etc., is reduced. This allows the film thickness to be smaller than that of a silica layer, and the distance between the metal nanoparticles 2A in the composite to be appropriately reduced. This allows for more efficient formation of a near-field, further improving detection sensitivity.

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

[0057] The polymer 3A constituting the polymer membrane 3 can contain one or more second hydrophilic groups 7B per molecular chain of the polymer 3A, which are bonded to the side chain of the polymer 3A via disulfide groups. The inclusion of the second hydrophilic groups 7B in the polymer 3A constituting the polymer membrane 3 can suppress nonspecific adsorption to the polymer membrane 3. The second hydrophilic groups 7B are hydrophilic groups that bond to disulfide bonds that remain unreacted during the formation of the polymer membrane. For example, they are derived from some of the hydrophilic groups bonded via disulfide bonds in the raw material polymer 3B (the polymer according to the first embodiment). The polymer 3A containing the second hydrophilic groups 7B can be prepared by using milder reaction conditions in the method for forming the polymer membrane 3 (e.g., reaction conditions such as a lower reaction temperature and / or a shorter reaction time). Mild reaction conditions include, for example, a reaction time shorter than 12 to 36 hours and / or a reaction temperature lower than 20 to 30°C.

[0058] The second hydrophilic group 7B includes a polar group and / or a charged 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, a charged group, and a zwitterionic group, and the zwitterionic group includes, for example, at least one selected from the group consisting of a phosphorylcholine group and a betaine group.

[0059] The polymer 3A constituting the polymer membrane 3 preferably contains an amide bond in the main chain skeleton of the polymer 3A. When the polymer 3A has an amide bond in its main chain skeleton, the polymer 3A is hydrophilic, and therefore nonspecific adsorption to the polymer membrane 3 made of the polymer 3A is suppressed, further improving detection sensitivity.

[0060] The polymer 3A includes a structure derived from one or more amino acids in its main chain skeleton. 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. When the polymer 3A includes a structure derived from one or more amino acids in its main chain skeleton, the polymer 3A has hydrophilic properties, thereby suppressing nonspecific adsorption to the polymer membrane 3 composed of the polymer 3A, and further improving detection sensitivity.

[0061] The polymer 3A constituting the polymer membrane 3 preferably contains a biocompatible polymer as a block polymer in the main chain skeleton of the polymer 3A. When the main chain skeleton of the polymer 3A contains a biocompatible polymer as a block polymer, the polymer 3A has hydrophilic properties, thereby suppressing nonspecific adsorption to the polymer membrane 3 composed of the polymer 3A, and further improving detection sensitivity. An example of such a biocompatible polymer is polymethacrylate.

[0062] The polymer 3A constituting the polymer film 3 can two-dimensionally cover the surface of the metal substrate 2 so that the main chain of the polymer 3A is approximately parallel to the surface of the metal substrate 2. Such a coating mode with the polymer 3A can be achieved by bonding the ends of multiple side chains of the polymer 3A to the surface of the metal substrate 2 via sulfur atoms. Furthermore, since the polymer 3A covers the surface of the metal substrate 2 so that the main chain of the polymer 3A is approximately parallel to the surface of the metal substrate 2, the thickness of the polymer film 3 can be reduced compared to a polymer film formed in which polymer chains are arranged in a brush-like manner (prior art: WO 2001 / 086301).

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

[0064] -Positively Charged Group- The positively chargeable group 3b can form a relatively strong electrostatic bond b with the surface of the metal substrate 2 (metal nanoparticle 2A). In this specification, the positively chargeable group 3b is a group that has a valence of one or more and is completely positively ionized. When taking into consideration the multiple positively chargeable groups 3b contained in the polymer that constitutes the polymer film 3, the positively chargeable group 3b can be expressed by the following mathematical formula (1): [In formula (1), pKa is an electrically neutral group contained in the polymer 3A constituting the polymer film 3, and if it is positively charged, it is a positively charged group (more specifically, a primary ammonium group (—NH ３ ＋ ) etc.) a group which can be 3b (hereinafter also referred to as an electrically neutral group) (more specifically, a primary amino group (—NH ２ ) etc.), pH indicates the pH of the environment in which the test substance is detected (more specifically, the pH of the sample etc.), B indicates an electrically neutral group contained in the polymer 3A, BH ＋ indicates a positively charged group 3b contained in the polymer 3A]. That is, the positively charged group 3b is a group having a pKa of 7 or more, which is represented by the following chemical equilibrium formula (2): When an equilibrium state represented by the following formula is formed, the concentration of the positively charged group 3b ([BH ＋ ]) is 10 times or more greater than the concentration of electrically neutral groups ([B]).

[0065] The positively charged group 3b is preferably a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, or a guanidyl group (—NHC(═NH ２ ＋ ) NH ２ ) is at least one selected from the group consisting of

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

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

[0068] An aliphatic cyclic group is a group that does not contain an aromatic ring and contains a cyclic group consisting of a non-aromatic ring. Examples of the aliphatic cyclic group include an aliphatic carbocyclic group and an aliphatic heterocyclic group. An aliphatic carbocyclic group is a group that contains a non-aromatic ring whose ring atoms are all carbon atoms, such as a cycloalkyl group. An aliphatic heterocyclic group is a group that contains a non-aromatic ring whose ring atoms are at least one heteroatom.

[0069] An aliphatic chain group is a chain (more specifically, a straight-chain or branched-chain) group that does not contain an aromatic ring or a non-aromatic ring. Examples of the aliphatic chain group include an aliphatic carbon chain group (more specifically, an alkyl group, an alkylene group, etc.) and an aliphatic hetero chain group. An example of the alkyl group is a butyl group. An example of the alkylene group is an n-butylene group.

[0070] The film thickness of the polymer film 3 is preferably 1 nm to 50 nm, and more preferably 1 nm to 10 nm. When the film thickness of the polymer film 3 is 50 nm or less, the separation distance (separation distance) is such that a near-field is efficiently formed in the space between the two metal nanoparticles 2, thereby further improving detection sensitivity. Furthermore, when the thickness of the polymer film 3 is 1 nm or more, the metal nanoparticles 2 and the fluorescent substance are arranged at a predetermined distance, thereby suppressing quenching of the excited fluorescent substance during measurement, thereby further improving detection sensitivity. In this specification, the separation distance (separation distance) refers to the minimum distance (shortest distance) between the surfaces of the metal nanoparticles contained in the two nanoparticles that are bound to each other via the analyte in the complex.

[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 via a sulfur atom between the first hydrophilic group 7A and the surface of the metal substrate 2. Such a bonding site can be formed, for example, by mixing the metal substrate 2 (metal nanoparticles 2A) as a starting material with the 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 a charged 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, a charged group, and a zwitterionic group, and the zwitterionic group includes, for example, at least one selected from the group consisting of a phosphorylcholine group and a betaine group.

[0073] The first hydrophilic groups 7A are preferably exposed from the polymer membrane 3. In this case, non-specific adsorption to the polymer membrane 3 is further suppressed.

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

[0075] From the viewpoint of suppressing fluorescence quenching, the fluorescent substance 6 is preferably labeled on the surface of the metal substrate 2 via a linker moiety (for example, an alkylene group containing an amide group). When the fluorescent substance 6 is labeled on the surface of the metal substrate 2 via a linker moiety, the linker moiety is located between the fluorescent substance 6 and the surface of the metal substrate 2, which makes it easy 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] The fluorescent substance 6 is excited by light of a wavelength that resonates with the plasmon and emits fluorescence. Examples of the fluorescent substance include complexes of metals such as europium and ruthenium (metal complexes). Examples of ruthenium complexes include tris(bipyridine)ruthenium(II), which may have a counter anion.

[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 are less likely to overlap, and excitation light (scattered light) is less likely to enter the fluorescence to be detected, allowing for more accurate measurement of the fluorescence intensity.

[0078] It is preferable that the fluorescence spectrum of the fluorescent substance 6 is sharp. If the fluorescence spectrum is sharp, it is less likely to overlap with the absorption spectrum of the fluorescent substance 6, and therefore (scattered light of) excitation light is less likely to enter the fluorescence to be detected, allowing for more accurate measurement of the fluorescence intensity.

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

[0080] The specific binding substance 4 is, for example, at least one selected from the group consisting of an antibody (hereinafter referred to as a nanoantibody), a ligand, an enzyme, and a nucleic acid chain (more specifically, a DNA chain and an RNA chain). In this embodiment, the 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 an antigen as a test substance at its tip (antigen-binding site) through an antigen-antibody reaction to form a complex. A ligand as the specific binding substance 4 forms a complex with a protein as a test substance through a specific protein-ligand bond through a ligand-receptor reaction. A nucleic acid chain as the specific binding substance 4 forms a nucleic acid chain pair (double strand) with a complementary nucleic acid chain based on base pair complementarity. An enzyme as the specific binding substance 4 forms an enzyme-substrate complex with a substrate as a test substance at its active site (active center) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as those resulting from hydrogen bonds, as well as intermolecular forces, hydrophobic interactions and charge interactions.

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

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

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

[0084] [Method for Manufacturing Coated Metal Substrate] The method for manufacturing a coated metal substrate 1 according to the second embodiment includes a step (polymer film formation step) of contacting a polymer 3B having a hydrophilic group 7 at the end of a side chain bonded via a disulfide bond with the surface of a metal substrate 2, thereby bonding the hydrophilic group 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, bonding the hydrophilic group 7 to the metal substrate 2 and forming the polymer film can be carried out in parallel in one step. This simplification compared to conventional techniques allows for further cost reduction. The polymer used as the starting material in the method for manufacturing a coated metal substrate is the polymer according to the first embodiment.

[0085] In the reaction in which the 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 first side chain length from the disulfide group to the hydrophilic group 7 in the side chain is longer than the second side chain length from the disulfide group to the main chain of the polymer 3B. In this case, in the polymer 3A constituting the polymer membrane 3, the second side chain length corresponds to the length of the side chain bonded to the surface of the metal substrate 2 (i.e., the length of the side chain from the bonding site with the surface of the metal substrate 2 via a sulfur atom to the main chain of the polymer 3 constituting the polymer membrane 3). The first side chain length corresponds to the length from the bonding site with the surface of the metal substrate 2 via a sulfur atom 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 membrane 3. This further suppresses nonspecific adsorption to the polymer membrane 3.

[0087] Third Embodiment: Conjugate In the third embodiment, a conjugate in which two nanoparticles are bound via a test substance will be described as an example. In the third embodiment, the same reference numerals as those in the first and second embodiments have the same configurations as those in the first and second embodiments, and therefore, in principle, the description thereof will be omitted.

[0088] The composite (composite nanoparticle) according to the third embodiment can be formed by capturing an analyte by sandwiching it between the coated metal substrate (nanoparticle) according to the second embodiment when detecting the analyte. The composite comprises two or more nanoparticles. The composite according to the third embodiment comprises two or more nanoparticles. The coated metal substrate 1 capable of forming the composite according to the third embodiment is a nanoparticle 1A containing metal nanoparticles 2A as the metal substrate 2, where the nanoparticles 1A include first nanoparticles and second nanoparticles, and the first hydrophilic groups 7A are bonded to at least one of the surfaces of the metal nanoparticles 2A of the first nanoparticles and the surfaces of the second metal nanoparticles of the second nanoparticles, forming a composite in which the first nanoparticles and the second nanoparticles are bonded via the analyte.

[0089] The composite will be described with reference to FIG. 3 . FIG. 3 is a cross-sectional view schematically illustrating a composite according to the third embodiment. The composite 40A according to the third embodiment includes two nanoparticles 1A, each including a metal nanoparticle 2A. The two nanoparticles 1A include a first nanoparticle 10A and a second nanoparticle 20A, and the first nanoparticle 10A and the second nanoparticle 20A are bonded via an analyte 30. More specifically, the composite 40A includes an analyte 30 to be detected and two nanoparticles 10A, 20A. The two nanoparticles 10A, 20A are bonded via the analyte 30 in the composite 40A. In other words, the composite 40A according to the third embodiment can be formed by bonding the nanoparticles 1A according to the second embodiment via the analyte 30. One of the two nanoparticles 10A, 20A will be referred to as the first nanoparticle 10A, and the other will be referred to as the second nanoparticle 20A. In this way, the composite 40A includes, as the nanoparticles 1, first nanoparticles 10A and second nanoparticles 20A.

[0090] In the composite 40A, the first nanoparticles 10A comprise first metal nanoparticles 12A as metal nanoparticles, a first polymer film 13A as a polymer film, and first hydrophilic groups 7A (not shown in FIG. 3 ). That is, in the composite 40A, the first nanoparticles 10A comprise first metal nanoparticles 12A, a first polymer film 13A covering the surfaces of the first metal nanoparticles 12A, and first hydrophilic groups 7A that suppress nonspecific adsorption to the surfaces of the first metal nanoparticles 12A, wherein the first hydrophilic groups 7A are bonded to the surfaces of the first metal nanoparticles 12A, and the first polymer film 13A is bonded to the surfaces of the first metal nanoparticles 12A via a sulfur atom. The first nanoparticles 10A further comprise 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 the composite 40A, the second nanoparticles 20A comprise second metal nanoparticles 22A as metal nanoparticles, a second polymer film 23A as a polymer film, and first hydrophilic groups 7A (not shown in FIG. 3 ). That is, in the composite 40A, the second nanoparticles 20A comprise first metal nanoparticles 22A, a second polymer film 23A covering the surfaces of the second metal nanoparticles 22A, and first hydrophilic groups 7A that suppress nonspecific adsorption to the surfaces of the second metal nanoparticles 22A, wherein the first hydrophilic groups 7A are bonded to the surfaces of the second metal nanoparticles 22A, and the second polymer film 23A is bonded to the surfaces of the second metal nanoparticles 22A via a sulfur atom. The second nanoparticles 20A further comprise 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 the fluorescence intensity, it is preferable that the separation distance L is small, within a range in which the excited fluorescent substances 16A, 26A are not easily quenched. More specifically, in a preferred embodiment, the two nanoparticles 10A, 20A in the composite 40A are close to each other. In a more preferred embodiment, the two nanoparticles 10A, 20A are close to each other so 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 come into contact. In an even more preferred embodiment, the two nanoparticles 10A, 20A are close to each other so that at least one of the polymer films 13A of the first nanoparticle 10A and the second polymer film 23A of the second nanoparticle 20A in the composite 40A contracts and comes into contact.

[0093] In a further preferred embodiment, when at least one of polymer films 13A, 23A shrinks and comes into contact with each other, for example, in complex 40A shown in Figure 3, it is believed that at least one of analyte 30, specific binding substances 14A, 24A that bind to analyte 30, and fluorescent substances 16A, 26A can be inserted into polymer films 13A, 23A. Furthermore, in a more preferred embodiment, when polymer films 13A, 23A come into contact with each other, for example, in complex 40A shown in Figure 3, it is believed that at least one of analyte 30, specific binding substances 14A, 24A that bind to analyte 30, and fluorescent substances 16A, 26A can be inserted into polymer films 13A, 23A, as in the further preferred embodiment.

[0094] In this embodiment, the films covering the surfaces of the metal nanoparticles 12A and 22A are polymer films 13A and 23A, respectively, which can increase the fluorescence intensity. The reason for this is presumed to be as follows: The films covering the surfaces of the metal nanoparticles 12A and 22A are polymer films 13A and 23A, which 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 than a distance equivalent to two polymer film thicknesses (the film thickness of the polymer film 13A plus the film thickness of the 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 achieve the plasmon enhancement effect, further increasing the fluorescence intensity. In this specification, the thickness of the polymer film in "two thicknesses of the polymer film" does not refer to the thickness of the polymer film 13A, 23A in the contracted portion that is the target of the separation distance, but to the thickness of the polymer film 13A, 23A in the non-contracted portion that is not the target of separation.

[0095] In this embodiment, the polymer 3A constituting the polymer films 13A and 23A includes a binding site 3a in its side chain (more specifically, at the end of the side chain) that is connected to the surface of the metal nanoparticles 12A and 22A via a sulfur atom. This can further increase the fluorescence intensity. Without being bound by any particular theory, the reason for this is presumed to be as follows. In this case, the binding site 3a forms a bond with the surface of the metal nanoparticles 12A and 22A, and the polymer 3A has a mesh-like structure, which is thought to coat the surface of the metal nanoparticles 12A and 22A in a mesh-like manner. Because the polymer 3A has this mesh-like structure, it has even greater flexibility. Therefore, in the composite 40A, the polymer films 13A and 23A can further contract, allowing the two metal nanoparticles 12A and 22A to be closer than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two polymer film thicknesses, further achieving a plasmon enhancement effect and further increasing the fluorescence intensity.

[0096] In a preferred embodiment, the polymer 3A constituting the polymer film 13A, 23A includes, in its side chain (more specifically, at the side chain end), a binding site 3a between the surface of the metal nanoparticle 12A, 22A and the polymer film 13A, 23A, and at least one selected from the group consisting of a positively charged group 3b and a hydrophobic group 3c, in addition to the binding site 3a via a sulfur atom. This further increases the fluorescence intensity. Without being bound by any particular theory, the reason for this is presumed to be as follows. In this case, at least one selected from the group consisting of the positively charged group 3b and the hydrophobic group 3c forms a bond with the surface of the metal nanoparticle 12A, 22A, so that the polymer 3A has a mesh-like structure and coats the surface of the metal nanoparticle 12A, 22A in a mesh-like manner. Because the polymer 3A has this mesh-like structure, it also has relatively high flexibility. Therefore, in the composite 40A, the polymer film 13A, 23A can further contract, thereby allowing the two metal nanoparticles 12A, 22A to be closer together than the distance equivalent to two polymer film thicknesses. Therefore, in this embodiment, the separation distance L can be less than two times the thickness of the polymer film, and the plasmon enhancement effect is further obtained, further increasing the fluorescence intensity.

[0097] The separation distance L between the first nanoparticles 10A and the second nanoparticles 20A is, for example, 12 nm to 52 nm, and preferably 12 nm to 27 nm. The separation distance L is the distance between the first metal nanoparticles 12A and the second metal nanoparticles 22A, and is the distance at which the line segment connecting a first point P1 on the surface of the first nanoparticles 10A and a second point P2 on the surface of the second nanoparticles 20A is the shortest. If 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 further increasing the fluorescence intensity.

[0098] The films covering the surfaces of the metal nanoparticles 12A and 22A are polymer films 13A and 23A, and the polymer films 13A and 23A include binding sites 3a via sulfur atoms between the surfaces of the metal nanoparticles 12A and 22A. Therefore, as described above, the separation distance L can be closer than the distance equivalent to twice the thickness of the polymer films covering the surfaces of the two metal nanoparticles 12A and 22A in the composite 40A. For example, when 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 to 9 nm, 3 to 8 nm, 4 to 7 nm, etc.).

[0099] 3, the fluorescent substances 16A, 26A are preferably 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, 22A is one in which a near-field is efficiently generated, and therefore, by positioning the fluorescent substances 16A, 26A in the space between the metal nanoparticles 12A, 22A, the fluorescence intensity is likely to be increased.

[0100] (Test Substance) The test substance 30 is a substance to be detected that is contained in a specimen. Examples of the test substance 30 include antigens, proteins, substrates, and nucleic acid chains. The test substance 30 specifically binds to the specific binding substances 14A and 24A. For example, an antigen has at least two antigenic determinants and forms specific bonds with the first and second specific binding substances 14A and 24A through the antigenic determinants. Antigens include proteins such as c-reactive protein, myoglobin, troponin T, troponin I, and BNP, as well as antigenic proteins of viruses such as influenza virus and respiratory syncytial virus. The test substance 30 is derived from a specimen such as blood, plasma, urine, or saliva. In other words, specimens containing the test substance 30 include, for example, blood, plasma, serum, urine, and saliva. The sample may further include a solvent and a buffer solution (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, MES buffer, etc.).

[0101] (Method for detecting an analyte using a complex) An example of a method for detecting an analyte using a complex according to the third embodiment will be described. The nanoparticles 1A according to the second embodiment are dispersed in a specimen, and the analyte 30 contained in the specimen is captured to form a complex 40A.

[0102] In surface plasmon-field enhanced fluorescence spectroscopy (SPFS), when excitation light is irradiated onto the composite 40A, localized surface plasmon resonance occurs, and a near-field is efficiently formed near the surfaces of the metal nanoparticles 12A and 22A (particularly near the surface between the two metal nanoparticles 12A and 22A). The near-field and dipole-dipole mechanism efficiently excite the fluorescent substances 16A and 26A in the composite 40A, enhancing their fluorescence. The test substance in the specimen can be detected by measuring the amount of fluorescence.

[0103] (Measuring Device for Detecting an Analyte Using a Complex) With reference to FIG. 4 , an example of a measuring method for detecting an analyte using a complex 40A according to the third embodiment will be described. FIG. 4 is a schematic diagram illustrating a measuring device for detecting an analyte using a complex according to the third embodiment. As shown in FIG. 4 , the measuring device 100 includes an excitation light source 110, an excitation light irradiation optical system 120, a reagent container 130, a light-receiving optical system 140, and a light-receiving element 150. The excitation light source 110 emits excitation light 112. The excitation light source 110 is, for example, a laser. The excitation light irradiation optical system 120 adjusts the cross-sectional diameter of the excitation light 112, such as by focusing the excitation light 112, and outputs incident excitation light 122. The excitation light irradiation optical system 120 includes a lens 124 and a polarizing element (λ / 2 plate) 126. The incident excitation light 122 output from the excitation light irradiation optical system 120 enters a reagent container 130 and is irradiated onto the measurement sample in the reagent container 130. The reagent container 130 is, for example, a removable container (more specifically, a cell, a preparation, or the like) and a microchannel chip. The microchannel chip is a chip having a minute channel. When the reagent container 130 is a microchannel chip, for example, the nanoparticle body (reagent) according to the first embodiment and a specimen can be mixed and continuously supplied. This eliminates the need to mix and prepare a measurement sample in advance, enabling continuous measurement.

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

[0105] The fluorescence 142 that passes through the light-receiving optical system 140 is detected by the light-receiving element 150. The light-receiving element 150 is, for example, a PD, an APD, a PMT, a CCD camera, or a spectroscope. The light-receiving element 150 is capable of measuring the amount of fluorescence at a single wavelength, measuring a fluorescence spectrum, and creating a two-dimensional fluorescence image.

[0106] <Fourth embodiment: composite> The composite (nanoparticle-attached metal thin film) 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 those in the first to third embodiments have the same configuration as those in the first to third embodiments, and therefore, in principle, their description will be omitted.

[0107] The composite according to the fourth embodiment can be formed by capturing an analyte between the coated metal substrate (coated metal thin film and two or more nanoparticles) according to the second embodiment when detecting the analyte. 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 capable of forming the composite according to the fourth embodiment comprises a nanoparticle 1A containing metal nanoparticles 2A as the metal substrate 2, and a coated metal thin film 1B containing a metal thin film 2B as the metal substrate 2, in which 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, forming a composite in which the coated metal thin film 1B and the nanoparticles 1A are bonded via the analyte.

[0108] The complex comprises a nanoparticle 1A according to the second embodiment shown in Fig. 1(a) and a coated metal thin film 1B according to the third embodiment shown in Fig. 1(b), and the nanoparticle 1A and the coated metal thin film 1B are bonded to each other via an analyte 30. The first hydrophilic group 7A is bonded to at least one of the surface of the metal nanoparticle 2A and the surface of the metal thin film 2B.

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

[0110] In composite 40B, third nanoparticles 10B comprise third metal nanoparticles 12B as metal nanoparticles, a third polymer film 13B as a polymer film, and first hydrophilic groups 7A (not shown in FIG. 5 ). That is, in composite 40B, third nanoparticles 10B comprise third metal nanoparticles 12B, a third polymer film 13B covering the surfaces of the third metal nanoparticles 12B, and first hydrophilic groups 7A (not shown in FIG. 5 ) that suppress nonspecific adsorption to the surfaces of the third metal nanoparticles 12B. The first hydrophilic groups 7A are bonded to the surfaces of the third metal nanoparticles 12B, and the third polymer film 13B is bonded to the surfaces of the third metal nanoparticles 12B via sulfur atoms. Third nanoparticles 10B further comprise 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, first coated metal substrate 20B comprises first metal thin film 22B as a metal substrate, fourth polymer film 23B as a polymer film, and first hydrophilic groups 7A (not shown in FIG. 5 ). That is, in composite 40B, first coated metal substrate 20B comprises first metal thin film 22B, fourth polymer film 23B coating the surface of first metal thin film 22B, and first hydrophilic groups 7A (not shown in FIG. 5 ) that suppress nonspecific adsorption to the surface of first metal thin film 22B, wherein first hydrophilic groups 7A are bonded to the surface of first metal thin film 22B, and fourth polymer film 23B includes a bond via a sulfur atom between itself and the surface of first metal thin film 22B. First coated metal substrate 20B further comprises fourth specific binding substance 24B bonded to fourth polymer film 23B.

[0112] (Method for detecting an analyte using a complex) An example of a method for detecting an analyte using a complex according to the fourth embodiment will be described. The measuring device shown in FIG. 4 is used. A first coated metal substrate 20B is placed in a reagent container 130 with the fourth polymer film 23B exposed so as to come into contact with an analyte 30 and third nanoparticles 10B (described later). A dispersion liquid containing the analyte 30 is flowed into the reagent container 130 and brought into contact with the fourth polymer film 23B. This causes the analyte 30 to specifically bind to the fourth specific binding substance 24B bound to the fourth polymer film 23B. Next, a dispersion liquid containing the third nanoparticles 10B is flowed into the reagent container 130 and brought into contact with the fourth polymer film 23B. This causes the third nanoparticles 10B to specifically bind to the analyte 30 bound to the fourth specific binding substance 24B of the first coated metal substrate 20B. As a result, a complex 40B is formed. Fluorescence is measured in the same manner as in the measurement method of the third embodiment. This allows the analyte to be detected.

[0113] The present invention is not limited to the above-described embodiments, and design modifications are possible within the scope of the present invention, and 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 to a metal thin film 22B coated with a polymer film 23B via one analyte 30, but the present invention is not limited to this. For example, the composite (metal thin film with composite nanoparticles) may be formed by bonding one composite 40A to a metal thin film 22B coated with a polymer film 23B via one analyte 30.

[0115] In the second embodiment, the nanoparticle 1A and the coated metal thin film 1B are labeled with three and two fluorescent substances 6, respectively, as shown in Fig. 1, but this is not limiting. The number of specific binding substances 4 (Fig. 1) is also similar. The number of fluorescent substances 16A, 26A and specific binding substances 14A, 24A in the composite 40A according to the third embodiment (Fig. 3) is also similar. The number of fluorescent substances 16B and specific binding substances 14B, 24B in the composite 40B according to the fourth embodiment (Fig. 5) is also similar.

[0116] In the second to fourth embodiments, the fluorescent substance is bound to a specific binding substance, but this is not limiting. 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 disposed in a direction perpendicular to the traveling direction of the incident excitation light 122 into the reagent container 130, but is not limited to this. The light-receiving optical system 140 may be disposed, for example, in a direction parallel to the traveling direction of the incident excitation light 122, or in a direction forming an acute angle or an obtuse angle with respect to the traveling direction of the incident excitation light 122.

[0118] The present invention will be described in more detail below using examples. However, the present invention is not limited to the following examples. Furthermore, unless otherwise specified, parts and percentages in the examples are by mass.

[0119] In the examples, the concentration of metal nanoparticles in a dispersion liquid may be expressed as absorbance. The absorbance was measured using an ultraviolet-visible spectrophotometer ("infinite M200 PRO" manufactured by TECAN Japan Co., Ltd.). The subscript number in the absorbance notation OD indicates the absorption wavelength. For example, OD ４５５ = 0.1 indicates that the absorbance at a wavelength of 455 nm is 0.1.

[0120] Example 1 Preparation of Polymer The polymer of Example 1 was prepared as follows.

[0121] (Introduction of Disulfide Bonds into Polymers: Disulfide Group Introduction Step) (3a-1) Poly-L-lysine (Peptide Institute, 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)propionamido-PEG4-NHS (Thermo Fisher Scientific, serial 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 (Tatec Corporation, "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 a "polymer having a disulfide group") was obtained. This synthesis reaction is represented by reaction formula (r-1): As shown in Figure 1, this is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamido-PEG4-NHS.

[0122] (Introduction of Hydrophilic Groups into Polymer: Hydrophilic Group Introduction Step) (2-1) A polymer having a disulfide group and (5j-1) a thiol compound having a hydrophilic group (sulfobetaine 3-undecanetiol, Dojindo Chemical Industries "S350") were stirred and mixed at 37°C for 1 hour. As a result, (1-1) a polymer in which betaine was bound to the side chain via an SPDP linker and a disulfide bond (hereinafter also referred to as "betaine-introduced polymer" (PLL-S-S-betaine)) was obtained. The synthesized (1-1) betaine-introduced polymer had a hydrophobic group (n-butylene group) and a positively charged group (primary ammonium group). This synthesis reaction is represented by reaction formula (r-2): As shown in Figure 1, (5j-1) is a thiol nucleophilic reaction in which the thiol group of a thiol compound having a hydrophilic group attacks the disulfide group of a polymer having a disulfide group (2-1).

[0123] The identification of (1-1) PLL-S-S-betaine will be explained with reference to Figure 6. Figure 6 shows the absorption spectra at each step of polymer synthesis. The solid line shows the absorption spectrum after reaction (r-1), the dashed line shows the absorption spectrum after reaction (r-2), and the dashed line shows the absorption spectrum after ultrafiltration treatment was performed after reaction (r-2).

[0124] The absorption peak around 280 nm in the absorption spectrum (solid line) was assigned to the pyridyl group of the (2-1) polymer having a disulfide group. The absorption peak around 343 nm in the absorption spectrum (dashed line) was assigned to the following resonance structure: The product was assigned to 3-mercaptopyridine having the formula: 3-mercaptopyridine is a by-product produced in reaction (r-2). The absorption spectrum (dash-dotted line) did not have a maximum peak in the visible range. From these results, it was confirmed that the above reactions (r-1) and (r-2) had progressed, resulting in the production of (1-1) PPL-S—S-betaine.

[0125] [Formation of polymer film and labeling of hydrophilic groups on the surface of metal nanoparticles] The obtained (1-1) PLL-S-S-betaine was used as a metal nanoparticle, and silver nanoparticles ("AGCB80-1M" manufactured by nanocomposix, diameter 80 nm, OD ４５５ The solution was added to 1 mL of a dispersion of silver nanoparticles (pH 7.0) containing 1 mL of hydroxybenzoates (pH 7.0, pH ...

[0126] [Evaluation Method] (SEM Image) Using a scanning electron microscope ("Regulus 8220" manufactured by Hitachi High-Technologies Corporation), an SEM image (magnification 300K) of the obtained hydrophilic group-labeled polymer-coated silver nanoparticles of Example 1 was taken. Figure 8 shows an SEM image of the hydrophilic group-labeled polymer-coated silver nanoparticles of Example 1. As shown in Figure 8, the obtained SEM image confirmed that the silver nanoparticles were coated with a polymer film.

[0127] (Measurement Evaluation by 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 measurement device ("ZETA SIZER Nanoseries nano-ZS" manufactured by MALVERN) (number of measurements for each: n = 3). The zeta potential measurement results will be explained with reference to FIG. 9. FIG. 9 shows the distribution of the scattered light intensity ratio versus zeta potential (zeta potential distribution) for the polymer-coated, hydrophilic-group-labeled silver nanoparticles of Example 1 (specifically, this is one distribution out of three obtained by measurements). The distribution of the scattered light intensity ratio versus zeta potential had a peak between -5.77 and -2.33 mV. The polymer-coated, hydrophilic-group-labeled silver nanoparticles were slightly negatively charged overall.

[0128] The results of particle size distribution measurements will be explained with reference to Fig. 10. Fig. 10 shows the distribution of the scattered light intensity ratio versus particle size (particle size distribution) for the polymer-coated, hydrophilic-group-labeled silver nanoparticles of Example 1 (specifically, this is one of three distributions obtained by measuring the particle size). The distribution of the scattered light intensity ratio versus particle size was 97.93 nm (expected value M) ± 0.163 nm (polydispersity index (PDI)).

[0129] Example 2 Thiol compounds having a hydrophilic group in reaction (r-2) were replaced with thiol compounds having a phosphorcholine group represented by (5j-1) to (5k-1) (manufactured by Ikagaku Soyaku Co., Ltd., product code: MCP-03-0001-PC): (2-1) A polymer having disulfide groups (PLL-SS-PC) was obtained in the same manner as in Example 1, except that (5k-1) was used. The same absorption spectrum as in Example 1 was obtained.

[0130] SEM images (300K magnification) of the hydrophilic-group-labeled, polymer-coated silver nanoparticles of Example 2 were taken, and the zeta potential distribution and particle size distribution were obtained. Figure 11 shows an SEM image of the hydrophilic-group-labeled, polymer-coated silver nanoparticles of Example 2. Figure 11 confirms that the silver nanoparticles are 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). The results confirmed the presence of phosphate in the area where the silver nanoparticles were present. It was concluded that this phosphate was a fragment derived from the phosphorocholine group bound to the silver nanoparticle surface. Figure 12 shows the zeta potential distribution of the polymer-coated, hydrophilic-group-labeled silver nanoparticles of Example 2 (specifically, one of three distributions obtained by measurement). The zeta potential distribution showed that the peaks were located between +20 and +27 mV. The polymer-coated, hydrophilic-group-labeled silver nanoparticles were slightly positively charged overall. Table 1: 1 shows 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.

[0131] Comparative Example 1 In Comparative Example 1, polymer-coated silver nanoparticles were produced in the same manner as in Example 1, except that the polymer for coating the silver nanoparticles was changed from (1-1) PLL-S-S-betaine to PLL ("3075" manufactured by Peptide Institute, Inc.). In Comparative Example 1, the zeta potential distribution was measured in the same manner as in Example 1. The zeta potential distribution in Comparative Example 1 had a peak near +50 mV. 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 ("AGCB80-1M" manufactured by nanocomposix, diameter 80 nm) were used instead of the polymer-coated hydrophilic group-labeled silver nanoparticles. The zeta potential distribution in Comparative Example 2 had a peak near -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 1-2, the absolute value of the zeta potential at the peak position was smaller than the absolute value of the zeta potential at the peak position of Comparative Examples 1-2. Normally, when the zeta potential approaches 0 mV, aggregation occurs, making it difficult to disperse the particles in the primary particle state. Despite this, the particle size distribution results of Examples 1-2 showed that the expected value was only slightly larger than the particle size of silver nanoparticles (80 nm). This increase is thought to be due to the film thickness of the polymer film. Furthermore, the PDI was not particularly large. Taking these factors into consideration, it is believed that the hydrophilic group-labeled polymer-coated silver nanoparticles (coated metal substrate) are dispersed in the 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 believed that the adsorption of nonspecific adsorbed substances that contribute to noise components can be effectively suppressed.

[0134] The following are embodiments of the coated metal substrate, its manufacturing method, composite including the coated metal substrate, and polymer for manufacturing the coated metal substrate according to the present disclosure. <1> A coated metal substrate comprising a metal substrate that is a metal nanoparticle and / or a metal thin film, a polymer film coating the surface of the metal substrate, and first hydrophilic groups that suppress nonspecific adsorption to the surface of the metal substrate, the first hydrophilic groups being bonded to the surface of the metal substrate, and the polymer film containing a bonding site between the metal substrate and the surface via a sulfur atom. <2> The coated metal substrate according to <1>, wherein the first hydrophilic groups contain a bonding site between the metal substrate and the surface via a sulfur atom. <3> The coated metal substrate according to <1> or <2>, wherein a polymer forming the polymer film contains, in a side chain of the polymer, the bonding site between the metal substrate and the surface via the sulfur atom. <4> The coated metal substrate according to any one of <1> to <3>, wherein the polymer constituting the polymer film contains, per molecular chain, one or more second hydrophilic groups that are bonded to a side chain of the polymer via a disulfide group. <5> The coated metal substrate according to <4>, wherein the polymer further comprises the second hydrophilic group in a side chain of the polymer, wherein the first hydrophilic group and the second hydrophilic group each independently comprise a polar group and / or an electrostatically charged group, wherein 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, wherein the electrostatically charged group comprises at least one selected from the group consisting of a charged group and a zwitterionic group, and wherein the zwitterionic group comprises at least one selected from the group consisting of a phosphorylcholine group and a betaine group. <6> The coated metal substrate according to any one of <1> to <5>, wherein the polymer constituting the polymer film comprises an amide bond in the main chain skeleton of the polymer. <7> The polymer constituting the polymer film further contains a positively charged group in a side chain of the polymer, the side chain does not have a disulfide bond, and the positively charged group is selected from the group consisting of a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (—NHC(═NH ２ ＋ ) NH ２<8> The coated metal substrate according to any one of <1> to <6>, wherein the polymer constituting the polymer film further comprises a hydrophobic group in a side chain of the polymer, the side chain does not have a disulfide bond, and the hydrophobic group is at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group. <9> The coated metal substrate according to any one of <3> to <8>, wherein the polymer comprises a structure derived from one or more amino acids in its main chain skeleton, the amino acid comprising at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, aspartic acid glutamine, asparagine, serine, and threonine. <10> The coated metal substrate according to any one of <1> to <9>, wherein the polymer constituting the polymer film comprises a biocompatible polymer as a block polymer in the main chain skeleton of the polymer. <11> The coated metal substrate according to any one of <1> to <10>, further comprising a fluorescent substance and / or a specific binding substance that specifically binds to an analyte in a specimen. <12> The coated metal substrate according to <11>, wherein the specific binding substance is a nanobody. <13> The coated metal substrate according to any one of <1> to <12>, wherein the polymer film has a thickness of 1 nm to 10 nm. <14> The coated metal substrate according to any one of <1> to <13>, which is a coated metal substrate used in plasmon-excited fluorescence analysis. <15> The coated metal substrate according to any one of <1> to <14>, wherein the metal substrate comprises gold or silver. <16> The coated metal substrate according to any one of <1> to <15>, wherein the metal substrate is a nanoparticle body containing metal nanoparticles, wherein the nanoparticle bodies include first nanoparticles and second nanoparticles, the first hydrophilic group is bonded to at least one of a surface of the metal nanoparticle of the first nanoparticle and a surface of the metal nanoparticle of the second nanoparticle, and the first nanoparticle and the second nanoparticle are bonded to each other via the test substance to form a complex.<17> The coated metal substrate according to any one of <1> to <16>, comprising: a nanoparticle body containing the metal nanoparticles as the metal substrate; and a coated metal thin film containing the metal thin film as the metal substrate, wherein 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, and wherein the coated metal thin film and the nanoparticle body form a complex bound via the test substance. <18> The coated metal substrate according to <16> or <17>, wherein the test substance is derived from a specimen that is blood, plasma, urine, or saliva. <19> The coated metal substrate according to any one of <1> to <18>, wherein the molecules constituting the polymer film two-dimensionally cover the surface of the metal substrate such that the main chains of the polymers are approximately parallel to the surface of the metal substrate. <20> A composite comprising two or more nanoparticles each containing the metal nanoparticles according to any one of <1> to <19>, wherein the two or more nanoparticles comprise a first nanoparticle and a second nanoparticle, and the first nanoparticle and the second nanoparticle are bonded via a test substance. <21> A composite comprising a nanoparticle each containing the metal nanoparticles according to any one of <1> to <19> and a coated metal thin film comprising the metal thin film according to any one of <1> to <19>, wherein 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, and the nanoparticle and the coated metal thin film are bonded via a test substance. <22> A polymer having a hydrophilic group at the end of a side chain bonded via a disulfide group. <23> The polymer according to <22>, wherein the hydrophilic group comprises a polar group and / or a charged 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 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.<24> The polymer further includes a positively charged group in a side chain thereof, wherein the side chain does not have a disulfide bond, and the positively charged group is selected from the group consisting of a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH. ２ ＋ ) NH ２ <25> The polymer according to any one of <22> to <24>, further comprising a hydrophobic group in a side chain of the polymer, wherein the side chain does not have a disulfide bond, and the hydrophobic group is at least one selected from the group consisting of an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group. <26> The polymer according to any one of <22> to <25>, comprising an amide bond in the main chain skeleton of the polymer. <27> The polymer according to any one of <22> to <26>, comprising a structure derived from one or more amino acids in the main chain skeleton of the polymer, wherein the amino acids comprise at least one selected from the group consisting of lysine, histidine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, serine, and threonine. <28> The polymer according to any one of <22> to <27>, comprising a biocompatible polymer as a block polymer in the main chain skeleton of the polymer. <29> A method for producing a coated metal substrate, comprising the step of bringing the polymer according to any one of <22> to <28> into contact with a surface of a metal substrate, to bond the hydrophilic groups to the surface of the metal substrate and form a polymer film on the surface of the metal substrate. <30> The method for producing a coated metal substrate according to <29>, wherein, in the side chains, a first side chain length from the disulfide group to the hydrophilic group is longer than a second side chain length from the disulfide group to the main chain of the polymer.

[0135] LIST OF SYMBOLS 1 Coated metal substrate 1A Nanoparticle 1B Coated metal thin film 2 Metal substrate 2A Metal nanoparticle 2B Metal thin film 3 Polymer film 3A Polymer (constituting polymer film) 3B Polymer (forming polymer film) 4 Specific binding substance 6 Fluorescent substance 7 Hydrophilic group 7A First hydrophilic group 7B Second hydrophilic group 10A First nanoparticle 10B Third nanoparticle 12A First metal nanoparticle 12B Third metal nanoparticle 13A First polymer film 13B Third polymer film 14A First specific binding substance 14B Third specific binding substance 16A First fluorescent substance 16B Third fluorescent substance 20A Second nanoparticle 20B First coated metal substrate 22A Second metal nanoparticle 22B: First metal thin film 23A: Second polymer film 23B: Fourth polymer film 24A: Second specific binding substance 24B: Fourth specific binding substance 26A: Second fluorescent substance 30: Test substance 40A, 40B: Complex L: Separation distance (separation distance)

Claims

1. A polymer that has hydrophilic groups at the ends of its side chains that are linked via disulfide groups.

2. the hydrophilic group comprises a polar group and / or a charged group, the polar group includes 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; 2. The polymer of claim 1, wherein the charged group comprises at least one selected from the group consisting of a polar group, a charged 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.

3. The polymer further comprises a positively charged group on a side chain thereof, the side chain does not have a disulfide bond; The positively charged group includes a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH ２ ＋ ) N.H. ２ The polymer according to claim 1, which is at least one selected from the group consisting of:

4. The polymer further comprises a hydrophobic group on a side chain thereof, the 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 an aromatic cyclic group, an aliphatic cyclic group, and an aliphatic chain group.

5. The polymer according to claim 1 , comprising an amide bond in the main chain of the polymer.

6. The polymer has a structure derived from one or more amino acids in its main chain skeleton, The polymer of 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.

7. The polymer according to claim 1 , comprising a biocompatible polymer as a block polymer in the main chain backbone of the polymer.

8. 13. A method for producing a coated metal substrate, comprising the step of contacting the polymer of claim 1 with a surface of the metal substrate to bond the hydrophilic groups to the surface of the metal substrate and form a polymer film on the surface of the metal substrate.

9. 9. The method for producing a coated metal substrate according to claim 8, wherein in the side chains, a first side chain length from the disulfide group to the hydrophilic group is longer than a second side chain length from the disulfide group to a main chain of the polymer.