Nanoparticles and electrochemiluminescence immunoassay methods using nanoparticles

Abstract

The present invention provides nanoparticles that can increase the intensity of electrochemiluminescence. [Solution] A nanoparticle body comprising metal nanoparticles, a polymer film coating the surface of the metal nanoparticles, a specific binding substance bonded to at least one of the metal nanoparticles and the polymer film and specifically binding to the test substance in the sample, and an electrochemiluminescent substance bonded to at least one of the polymer film and the specific binding substance.

Description

[Technical Field]

[0001] This disclosure relates to nanoparticles and an electrochemiluminescence immunoassay method using nanoparticles. [Background technology]

[0002] A biosensor detects a specific test substance by specifically reacting it with a specific binding substance to form a complex, and then detecting the test substance based on a signal derived from the specific binding in the complex.

[0003] For example, in an immunoassay method that utilizes electrochemiluminescence (electrochemiluminescence immunoassay), a primary antibody specifically binds to the test substance and captures it. Furthermore, a secondary antibody modified with a labeling substance specifically binds to the captured test substance to form a complex. Electrochemiluminescence can be of the co-actant type or annihilation type. In co-actant type electrochemiluminescence, electrochemiluminescence is detected by applying a voltage to the labeling substance of the complex in the presence of the co-actant.

[0004] In the measurement method described in Patent Document 1, the analyte (test substance) is sandwiched between an antibody bound to magnetic microparticles and a luminescently labeled antibody, forming a complex. This complex is captured by a magnetic component equipped with a working electrode through a channel. Co-reactants are supplied to the captured complex, and electrochemiluminescence is detected by applying a voltage. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2013-152215 [Overview of the project] [Problems that the invention aims to solve]

[0006] By the way, as a result of intensive studies by the present inventors, it has been found that there is room for improving the detection sensitivity in the measurement method using the above antibody.

[0007] The present disclosure has been made in view of such problems. That is, an object of the present disclosure is to provide a nanoparticle body capable of increasing the intensity of electrochemiluminescence. Another object of the present disclosure is to provide an electrochemiluminescence immunoassay method that is superior in the detection sensitivity of electrochemiluminescence using the above nanoparticle body.

Means for Solving the Problems

[0008] The nanoparticle body according to one embodiment of the present disclosure is metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, a specific binding substance that is bound to at least one of the metal nanoparticles and the polymer film and specifically binds to a test substance in a sample, and an electrochemiluminescent substance that is bound to at least one of the polymer film and the specific binding substance. It consists of.

[0009] [[ID=u25]]The electrochemiluminescence immunoassay method according to one embodiment of the present disclosure is an electrochemiluminescence immunoassay method using a plasmon-enhanced field, a contact step of bringing a first solution containing the above nanoparticle body and a test substance into contact with a working electrode having a specific binding substance, and binding the nanoparticle body and the working electrode via the test substance; a coreactant supply step of supplying a second solution containing a coreactant onto the working electrode; a voltage application step of applying a voltage to the working electrode to generate electrochemiluminescence, and the electrochemiluminescence being enhanced by plasmon; a detection step of detecting the plasmon-enhanced electrochemiluminescence It consists of.

Effects of the Invention

[0010] The nanoparticle body according to an embodiment of the present disclosure can increase the intensity of electrochemiluminescence. Further, the electrochemiluminescence immunoassay method according to another embodiment of the present disclosure is excellent in the detection sensitivity of electrochemiluminescence.

Brief Description of Drawings

[0011] [Figure 1] FIG. 1 is a cross-sectional view schematically showing a nanoparticle body according to the first embodiment. [Figure 2] FIG. 2 is a cross-sectional view schematically showing a metal thin film (metal thin film with nanoparticle body) on which the nanoparticle body according to the first embodiment is captured. [Figure 3] FIG. 3 is a diagram schematically showing a plasmon enhancement mechanism of electrochemiluminescence. [Figure 4] FIG. 4 is a cross-sectional view schematically showing a composite nanoparticle body in which the nanoparticle body according to the first embodiment is bound via a test substance. [Figure 5] FIG. 5 is a cross-sectional view schematically showing a structure in which the composite nanoparticle body is captured on a metal thin film. [Figure 6] FIG. 6 is an enlarged schematic view of part A in FIG. 1. [Figure 7] FIG. 7 is a flowchart showing an example of an electrochemiluminescence immunoassay method according to the second embodiment. [Figure 8] FIG. 8 is a reaction scheme showing an example of a method for producing a nanoparticle body according to the first embodiment. [Figure 9] FIG. 9 is a schematic view for explaining a polymer coating form of the nanoparticle body of Example 1. [Figure 10] FIG. 10 is a schematic view showing the nanoparticle body of Example 1. [Figure 11] FIG. 11 is a plan view showing an electrochemiluminescence sensor. [Figure 12] FIG. 12 is a cross-sectional view (cross-sectional view taken along line I-I in FIG. 11) showing an electrochemiluminescence sensor. [Figure 13] FIG. 13 is a perspective view showing a cell for an electrochemiluminescence sensor equipped with an electrochemiluminescence sensor. [Figure 14]Figure 14 is a perspective view showing an electrochemiluminescence immunoassay analyzer. [Figure 15] Figure 15 is a cross-sectional view (section II-II of Figure 13) showing an electrochemiluminescence immunoassay apparatus. [Modes for carrying out the invention]

[0012] The nanoparticle body and electrochemiluminescence measurement method, which are embodiments of this disclosure, will be described in detail below with reference to the illustrated embodiments. Note that the drawings include schematic representations and may not reflect actual dimensions or proportions.

[0013] Numerical ranges referred to herein are intended to include the lower and upper limits themselves, unless otherwise specified by terms such as "less than," "greater than," and "less than." For example, a numerical range of 1 nm to 50 nm is interpreted as including the lower limit "1 nm" and the upper limit "50 nm."

[0014] In this specification, "above" broadly refers to a position in one direction from the surface of the member in question toward the outside of that member (for example, on the working electrode), and narrowly refers to a position in the opposite direction to the direction in which gravity acts on the member in question (vertical direction).

[0015] <First Embodiment: Nanoparticle Body> The first embodiment relates to a nanoparticle body. The nanoparticle body according to the first embodiment is Metal nanoparticles and A polymer film that coats the surface of metal nanoparticles, A specific binding substance is bonded to a polymer membrane and specifically binds to the test substance in the sample, Electrochemiluminescent material bonded to a polymer film and It consists of including.

[0016] [Mechanism of Action] (Plasmon enhancement of electrochemiluminescence by a metal thin film with nanoparticles) The nanoparticles according to the first embodiment can increase the intensity of electrochemiluminescence. Although not bound by any particular theory, the reason can be inferred as follows by referring to Figures 1 and 2. Figure 1 is a schematic cross-sectional view showing the nanoparticles according to the first embodiment. Figure 2 is a schematic cross-sectional view showing a metal thin film in which the nanoparticles are trapped (metal thin film with nanoparticles).

[0017] Electrochemiluminescence is plasmon-enhanced by a nanoparticle-attached metal thin film formed from the nanoparticle body according to the first embodiment. Specifically, as shown in Figure 1, the nanoparticle body 10 according to the first embodiment comprises metal nanoparticles 12, a polymer film 13 covering the surface of the metal nanoparticles 12, a specific binding substance 14 bound to the polymer film 13 and specifically binding to the test substance in the sample, and an electrochemiluminescent substance 16 bound to the polymer film 13. Therefore, as shown in Figure 2, the specific binding substance 14 of the nanoparticle body 10 according to the first embodiment specifically binds to the test substance 70 captured by the third specific binding substance 254 of the metal thin film (working electrode) 25. As a result, the nanoparticle body 10 is captured by the metal thin film 25, and a nanoparticle-attached metal thin film 70A is formed. In other words, the nanoparticle body 10 forms a nanoparticle-attached metal thin film 70A that is bound to the metal thin film 25, on which the third specific binding substance 254 is bound, via the test substance 70. Here, the metal thin film 25 is, for example, the working electrode of an electrochemiluminescence immunoassay analyzer. When a voltage is applied to a metal thin film 70A with nanoparticles in the presence of a coreactant, an electrochemical reaction proceeds between the electrochemiluminescent substance 16 in the nanoparticles 10 and the coreactant, resulting in electrogenerated chemiluminescence (ECL).

[0018] The nanoparticle-attached metal thin film (working electrode with nanoparticles) 70A has a sandwich structure in which the nanoparticles 10 and the metal thin film (working electrode) 25 sandwich the test substance 70, and in the nanoparticle-attached metal thin film 70A, the metal nanoparticles 12 and the metal thin film 25 are arranged in close proximity. Therefore, when the generated electrochemiluminescence irradiates the metal nanoparticles 12 and the metal thin film 25, localized surface plasmon resonance (LSPR; hereinafter also simply referred to as "plasmon resonance") occurs, and a near-field is efficiently formed near the surfaces of the metal nanoparticles 12 and the metal thin film 25 (particularly near the space between the metal nanoparticles 12 and the metal thin film 25). This near-field enhances the electrochemiluminescence. From the above, the nanoparticles 10 according to the first embodiment can increase the intensity of electrochemiluminescence (plasmon enhancement).

[0019] -Mechanisms of plasmon enhancement in electrochemiluminescence- Thus, the nanoparticle body 10 according to the first embodiment can plasmon-enhance the luminescence (electrochemiluminescence) generated by the electrochemical reaction of the electrochemiluminescent material 16. Furthermore, the plasmon enhancement mechanism of electrochemiluminescence will be explained with reference to Scheme 1 and Figure 3. Scheme 1 and Figure 3 show the plasmon enhancement mechanism of electrochemiluminescence (co-reaction type electrochemiluminescence) in the first embodiment.

[0020] [ka] [In the elementary processes (1) to (6) of Scheme 1, ECLM (Electrogenerated Chemiluminescence Material) represents electrochemiluminescent material 16, ECLM + This shows an oxide of electrochemiluminescent material 16. * ECLM indicates the electrochemiluminescent material 16 in the excited state, and CR (coreactant) indicates the co-reactant. ·+ This indicates the radical cation of the coreactate, CR · represents the radical of the co-reactant, and M represents the metal nanoparticles 12 and metal thin film 25 in the metal thin film 70A with nanoparticles.* M represents the metal nanoparticles 12 for plasmon resonance excitation and the metal thin film 25 for plasmon resonance excitation in the metal thin film 70A with nanoparticle bodies. Note that the symbols in FIG. 3 also indicate the same chemical species as the symbols in Scheme 1.

[0021] As shown in Scheme 1 and FIG. 3, the plasmon enhancement mechanism of electrochemiluminescence in the first embodiment is composed of elementary processes (1) to (6). In elementary process (1), the electrochemiluminescent material (ECLM) 16 in the metal thin film 70A with nanoparticle bodies is oxidized by voltage application, and an oxide of the electrochemiluminescent material 16 (ECLM + ) is generated. In elementary process (2), the co-reactant (CR) is oxidized by voltage application, and an oxide of the co-reactant (the radical cation of the co-reactant CR ·+ ) is generated. In elementary process (3), a proton dissociates from the oxide of the co-reactant to generate a radical of the co-reactant (CR · ). In elementary process (4), the oxide of the electrochemiluminescent material 16 (ECLM + ) reacts (electron exchange reaction) with the radical of the co-reactant (CR · ) to generate an excited-state electrochemiluminescent material ( * ECLM) 16 and a product of the co-reactant (CR product). In elementary process (5), a part of the excited-state electrochemiluminescent material 16 relaxes to emit electrochemiluminescence. In elementary process (6), the emitted electrochemiluminescence is irradiated onto the metal nanoparticles 12 and the metal thin film 25 in the metal thin film 70A with nanoparticle bodies. Thereby, plasmon resonance is induced on the surfaces of the metal nanoparticles 12 and the metal thin film 25 (particularly, the space between the metal nanoparticles 12 and the metal thin film 25), and the metal nanoparticles 12 and the metal thin film 25 for plasmon resonance excitation ( * M) are generated. The plasmon resonance induces relaxation of other excited-state electrochemiluminescent materials 16 (induced relaxation), and the luminescence quantum efficiency (luminescence quantum yield) of electrochemiluminescence is improved. (That is, the electrochemiluminescence is plasmon-enhanced).

[0022] As described above, the nanoparticle-attached metal thin film 70A is formed by the specific bonding of the nanoparticle body 10's specific bonding material 14 and the third specific bonding material 254 bonded to the surface of the metal thin film 25, as shown in Figure 2, to the (same) test substance 70. The nanoparticle-attached metal thin film 70A comprises the nanoparticle body 10 according to the first embodiment, the metal thin film 25, and the test substance 70. In the nanoparticle-attached metal thin film 70A, the nanoparticle body 10 and the metal thin film 25 are bonded via the test substance 70.

[0023] In a preferred embodiment, from the viewpoint of further increasing the intensity of electrochemiluminescence, the separation distance L1 between the metal nanoparticles 12 of the nanoparticle body 10 and the metal thin film 25 in the nanoparticle-attached metal thin film 70A is small, in a range in which the excited-state electrochemiluminescent material 16 is less likely to be quenched. In this specification, the separation distance L1 refers to the shortest distance between the surface of the metal nanoparticles 12 in the nanoparticle-attached metal thin film 70A and the surface of the electrode portion 251 of the metal thin film 25. When the separation distance L1 is small, the metal nanoparticles 12 and the metal thin film 25 (the electrode portion 251) are arranged in close proximity in the nanoparticle-attached metal thin film 70A. Such close arrangement effectively forms a near field and contributes to increasing the intensity of electrochemiluminescence. Therefore, in the nanoparticle-attached metal thin film 70A, the intensity of electrochemiluminescence can be further increased when the separation distance L1 is small.

[0024] In a preferred embodiment, from the viewpoint of further increasing the intensity of electrochemiluminescence, the electrochemiluminescent material 16 is arranged at least between the metal nanoparticles 12 and the metal thin film 25 in the nanoparticle-attached metal thin film 70A, as shown in Figure 2. The space between the metal nanoparticles 12 and the metal thin film 25 in the nanoparticle-attached metal thin film 70A is a space where a near field is efficiently generated. When the electrochemiluminescent material 16 is arranged at least between the metal nanoparticles 12 and the metal thin film 25 in the nanoparticle-attached metal thin film 70A, the intensity of electrochemiluminescence can be further increased.

[0025] (Plasmon enhancement of electrochemiluminescence by a metal thin film with composite nanoparticles) As described above, electrochemiluminescence is plasmon-enhanced by a nanoparticle-attached metal thin film 70A formed from the nanoparticle body 10 according to the first embodiment (Figures 1-3 and Scheme 1). In addition, electrochemiluminescence can also be plasmon-enhanced by a composite nanoparticle-attached metal thin film formed from the nanoparticle body 10 according to the first embodiment. The composite nanoparticle-attached metal thin film can be formed from the nanoparticle body 10. Refer to Figures 1-3 and Scheme 1, as well as Figures 4-5, to explain the plasmon enhancement of electrochemiluminescence by a composite nanoparticle-attached metal thin film. Figure 4 is a schematic cross-sectional view showing a composite nanoparticle body in which the nanoparticle body 10 is bound via the test substance 70. Figure 5 is a schematic cross-sectional view showing a structure in which the composite nanoparticle body is trapped in the metal thin film 25.

[0026] When the nanoparticle body 10 according to the first embodiment (which includes a first nanoparticle body 10A and a second nanoparticle body 10B) comes into contact with the test substance 70, the first and second specific binding materials 14A and 14B specifically bind to the (same) test substance 70, for example, as shown in Figure 4. This allows the formation of a composite nanoparticle body 70B in which the first nanoparticle body 10A and the second nanoparticle body 10B are bound via the (same) test substance 70. The first specific binding material 14A in the formed composite nanoparticle body 70B specifically binds to the test substance 70 captured by the third specific binding material 254 of the metal thin film 25, as shown in Figure 5 (similarly, in other embodiments, the second specific binding material 14B in the composite nanoparticle body 70B can also specifically bind to the test substance 70 captured by the third specific binding material 254 of the metal thin film 25). As a result, the composite nanoparticle body 70B is captured by the metal thin film 25, and a composite nanoparticle-attached metal thin film 70C is formed. In other words, the composite nanoparticles 70B form a composite nanoparticle-attached metal thin film 70C, which is bonded to the metal thin film 25 on which a third specific bonding material 254 is bonded via the test material 70.

[0027] The composite nanoparticle-attached metal thin film 70C has a sandwich structure in which the composite nanoparticle-attached metal thin film 25 sandwiches the test substance 70. In the composite nanoparticle-attached metal thin film 70C, the first metal nanoparticle 12A and the metal thin film 25 are arranged in close proximity. Therefore, when the generated electrochemiluminescence is irradiated onto the first metal nanoparticle 12A and the metal thin film 25, which are in close proximity to each other, plasmon resonance occurs, and a near-field is efficiently formed near the surfaces of the first metal nanoparticle 12A and the metal thin film 25 (particularly near the space between the first metal nanoparticle 12A and the metal thin film 25).

[0028] Furthermore, the composite nanoparticle-attached metal thin film 70C has a sandwich structure in which the first metal nanoparticle 12A and the second metal nanoparticle 12B sandwich the test substance 70 within the composite nanoparticle 70B. In the composite nanoparticle-attached metal thin film 70C, the first metal nanoparticle 12A and the second metal nanoparticle 12B are arranged in close proximity. Therefore, when the generated electrochemiluminescence is irradiated onto the first metal nanoparticle 12A and the second metal nanoparticle 12B, which are in close proximity to each other, plasmon resonance occurs, and a near-field is efficiently formed near the surfaces of the first metal nanoparticle 12A and the second metal nanoparticle 12B (particularly near the space between the first metal nanoparticle 12A and the second metal nanoparticle 12B).

[0029] These near-fields increase the intensity of electrochemiluminescence. Therefore, the nanoparticle body 10 according to the first embodiment can have its electrochemiluminescence plasmon-enhanced by the metal thin film 70C with the composite nanoparticle body.

[0030] The plasmon enhancement mechanism of electrochemiluminescence by the composite nanoparticle-attached metal thin film 70C is explained in the same manner as in Figure 3 and Scheme 1, which show the plasmon enhancement mechanism of electrochemiluminescence by the nanoparticle-attached metal thin film 70A, except that ECLM is changed from the electrochemiluminescent substance 16 in the nanoparticle-attached metal thin film 70A to the first and second electrochemiluminescent substances 16A and 16B in the composite nanoparticle-attached metal thin film 60, and M is changed from the metal nanoparticles 12 and metal thin film 25 in the nanoparticle-attached metal thin film 70A to the first and second metal nanoparticles 12A and 12B and metal thin film 25 in the composite nanoparticle-attached metal thin film 70C.

[0031] As described above, the composite nanoparticle-attached metal thin film 70C can be formed by the specific bonding of the first specific bonding material 14A of the composite nanoparticle-attached metal thin film 70B and the third specific bonding material 254 bonded to the surface of the metal thin film 25 to the same test substance 70. The composite nanoparticle-attached metal thin film 70C comprises the composite nanoparticle-attached metal thin film 70B, the metal thin film 25, and the test substance 70. The composite nanoparticle-attached metal thin film 70B comprises the first and second nanoparticle-attached metal thin films 10A and 10B according to the first embodiment and the test substance 70.

[0032] In the composite nanoparticle 70B, the first nanoparticle 10A and the second nanoparticle 10B are bound together via the test substance 70. The first nanoparticle 10A comprises a first metal nanoparticle 12A, a first polymer film 13A covering the surface of the first metal nanoparticle 12A, a first specific binding substance 14A bound to the first polymer film 13A and specifically binding to the test substance 70 in the sample, and a first electrochemiluminescent substance 16A bound to the first polymer film 13A. The second nanoparticle 10B comprises a second metal nanoparticle 12B, a second polymer film 13B covering the surface of the second metal nanoparticle 12B, a second specific binding substance 14B bound to the second polymer film 13B and specifically binding to the test substance 70 in the sample, and a second electrochemiluminescent substance 16B bound to the second polymer film 13B.

[0033] In a preferred embodiment, from the viewpoint of further increasing the intensity of electrochemiluminescence, the separation distance L1' between the metal nanoparticles of the nanoparticle body (first metal nanoparticle 12A of the first nanoparticle body 10A in Figure 5) and the metal thin film 25 is small, in a range in which the excited first electrochemiluminescent material 16A is less likely to be quenched. In this specification, the separation distance L1' between the first metal nanoparticle 12A and the metal thin film 25 refers to the shortest distance between the surface of the first metal nanoparticle 12A and the surface of the electrode portion 251. When the separation distance L1' is small, the first metal nanoparticle 12A and the metal thin film 25 are arranged in close proximity in the metal thin film 70C with composite nanoparticle body. Such close proximity effectively forms a near field and effectively contributes to the intensity of electrochemiluminescence. Therefore, in the metal thin film 70C with composite nanoparticle body, the intensity of electrochemiluminescence can be further increased when the separation distance L1' is small. The separation distance L1' is, for example, 1 nm to 10 nm. The separation distance L1' can be determined in the same way as the separation distance L1.

[0034] In a preferred embodiment, from the viewpoint of further increasing the intensity of electrochemiluminescence, the separation distance L2 between the metal nanoparticles of the nanoparticles bonded to each other via the test substance 70 in the composite nanoparticle-attached metal thin film 70C (in Figure 5, between the first metal nanoparticle 12A of the first nanoparticle 10A and the second metal nanoparticle 12B of the second nanoparticle 10B) is small, in a range in which the excited first and second electrochemiluminescent materials 16A and 16B are less likely to be quenched. In this specification, the separation distance L2 refers to the shortest distance between the surface of the first metal nanoparticle 12A and the surface of the second metal nanoparticle 12B. When the separation distance L2 is small, the first metal nanoparticle 12A and the second metal nanoparticle 12B are arranged in close proximity in the composite nanoparticle-attached metal thin film 70C. Such close proximity arrangement effectively forms a near field and effectively contributes to the intensity of electrochemiluminescence. Therefore, in the composite nanoparticle-attached metal thin film 70C, the intensity of electrochemiluminescence can be further increased when the separation distance L2 is small. The spacing distance L2 is, for example, 1 nm to 10 nm. The spacing distance L2 can be determined in the same way as the spacing distance L1.

[0035] In a preferred embodiment, from the viewpoint of further increasing the intensity of electrochemiluminescence, the first electrochemiluminescent material 16A is arranged at least between the first metal nanoparticles 12A and the metal thin film 25 in the composite nanoparticle-attached metal thin film 70C, as shown in Figure 5. The space between the first metal nanoparticles 12A and the metal thin film 25 in the composite nanoparticle-attached metal thin film 70C is a space where a near field is efficiently generated. When the first electrochemiluminescent material 16A is arranged at least between the first metal nanoparticles 12A and the metal thin film 25 in the composite nanoparticle-attached metal thin film 70C, the intensity of electrochemiluminescence can be further increased.

[0036] The nanoparticle body 10 according to the first embodiment may be a nanoparticle body used in an electrochemiluminescence immunoassay utilizing a plasmon-enhanced field, as described above. The nanoparticle body 10 can be used in an electrochemiluminescence immunoassay that does not utilize external light irradiation (i.e., does not use a light source). Because electrochemiluminescence immunoassay does not use a light source, it has a smaller background signal and can detect the detected light (electrochemiluminescence) with high sensitivity compared to fluorescence spectroscopy immunoassay. Furthermore, because electrochemiluminescence immunoassay does not use a light source, it is easier to construct a simpler measurement system compared to fluorescence spectroscopy immunoassay, and consequently, a simpler apparatus configuration can be adopted.

[0037] The nanoparticles 10 may also have their nonspecific binding sites blocked by a blocking agent. Blocked nanoparticles 10 suppress the formation of nonspecific binding of the specific binding agent 14 to substances other than the target substance (i.e., substances other than the test substance), thereby reducing background and false positive signals and improving the signal-to-noise ratio (S / N ratio). In such cases, detection sensitivity can be further improved. Examples of blocking agents include bovine serum albumin (BSA), proteins such as skim milk and casein, and chemically synthesized polymers.

[0038] When the nanoparticles 10 are present in a solvent, the dispersion of the nanoparticles 10 may further contain a dispersant to improve the dispersibility of the nanoparticles 10. Examples of such dispersants include sodium heparin.

[0039] [Composition of nanoparticles] The composition of the nanoparticle body 10 will now be described with reference to Figure 1. The nanoparticle body 10 comprises metal nanoparticles 12, a polymer film 13 covering the surface of the metal nanoparticles 12, a specific binding substance 14 bonded to the polymer film 13 and specifically binding to the test substance 70 in the sample, and an electrochemiluminescent substance 16 bonded to the polymer film 13. The nanoparticle body 10 may further include a hydrophilic substance that is bonded to the surface of the metal nanoparticles 12 via sulfur atoms.

[0040] (Metal nanoparticles) The metal nanoparticles 12 are coated with a polymer film 13 on their surface. Depending on the type of metal, the metal nanoparticles 12 interact with light of a specific wavelength, causing plasmon resonance. Silver nanoparticles have plasmon resonance peaks between 400 nm and 530 nm, while gold nanoparticles have peaks between 510 nm and 580 nm. This varies depending on the particle size. For example, silver nanoparticles with a particle size of 20 nm resonate with light of a wavelength of 405 nm. Gold nanoparticles with a particle size of 20 nm resonate with light of a wavelength of 524 nm. The particle size (average primary particle size) of the metal nanoparticles 12 is, for example, 5 nm to 100 nm. The particle size of the metal nanoparticles 12 can be obtained by taking an image of the metal nanoparticles 12 using a scanning electron microscope (SEM) or transmission electron microscope (TEM), measuring the particle size of the metal nanoparticles 12 in the image, and calculating the average value of multiple particle sizes (number of measurements: for example, at least 10 or more). The metal nanoparticles 12 preferably consist of gold or silver, and more preferably consist of silver. Although metal nanoparticles 12 have been described, the first and second metal nanoparticles 12A and 12B are synonymous with metal nanoparticles 12.

[0041] Individual metal nanoparticles may also be multilayered metal nanoparticles from the viewpoint of particle stability and optical properties. Examples of multilayered metal nanoparticles include metal nanoparticles containing multiple (two or more) metal layers. The multiple metal layers may be composed of different metals, and more specifically, particles having a double-shell structure such as gold nanoparticles with a gold layer and a silver layer (Au@Ag@Au nanoparticles) (Applied Physics Letters, 99 073107 (2011)).

[0042] (polymer membrane) The polymer film 13 covers the surface of the metal nanoparticles 12. The polymer film 13 functions as a film (quenching suppression film) that suppresses the quenching of the electrochemiluminescent material 16 in the excited state by the metal nanoparticles 12. In the metal thin film 70A with nanoparticles, the polymer film 13 can position the electrochemiluminescent material 16 at least by the thickness of the polymer film 13 away from the surface of the metal nanoparticles 12. Therefore, it is possible to suppress the quenching of the excited electrochemiluminescent material 16 by contact with the surface of the metal nanoparticles 12, thereby suppressing a decrease in detection sensitivity. The presence of the polymer film 13 can be confirmed by taking an image of the nanoparticles 10 using SEM or TEM and observing the nanoparticles 10 in the image.

[0043] The polymer film 13 will be described with further reference to Figure 6. Figure 6 is an enlarged view of part A in Figure 1, and is an enlarged schematic diagram of the vicinity of the interface between the polymer film 13 of the nanoparticle body 10 and the surface of the metal nanoparticles 12. The polymer film 13 (polymer 13X constituting the polymer film 13) may include (for example, in its side chains) sulfur atom-mediated bonding sites 13a between it and the surface of the metal nanoparticles 12. The polymer film 13 (polymer 13X constituting the polymer film 13) may also include (for example, in its side chains) at least one selected from the group consisting of positively charged groups 13b and hydrophobic groups 13c. More specifically, in addition to the sulfur atom-mediated bonding sites 13a between it and the surface of the metal nanoparticles 12, the polymer film 13 (polymer 13X constituting the polymer film 13) may include a primary ammonium group (-NH3) as the positively charged group 13b. +The polymer may further contain a sulfur atom and a hydrophobic group 13c. The bonding site 13a bonds the surface of the metal nanoparticle 12 to the polymer film 13 (polymer 13X constituting the polymer film 13) via a sulfur atom. The positively charged group 13b forms an electrostatic bond (ionic bond) b with the negatively charged surface of the metal nanoparticle 12. The hydrophobic group 13c forms a hydrophobic bond c with the surface of the metal nanoparticle 12.

[0044] All three of the above bonds are relatively strong bonds to the surface of the metal nanoparticles 12. The polymer film 13 is stably fixed to the surface of the metal nanoparticles 12 by the bonding sites 13a. Furthermore, the polymer film 13 can be even more stably fixed to the surface of the metal nanoparticles 12 by hydrophobic bonds c and electrostatic bonds b formed by positively charged groups 13b. In this way, the polymer film 13 is stably fixed to the surface of the metal nanoparticles 12, and a predetermined distance can be stably separated between the electrochemiluminescent material 16 and the surface of the metal nanoparticles 12. Therefore, in the first embodiment, the quenching of the excited electrochemiluminescent material 16 is suppressed, and the decrease in detection sensitivity can be suppressed.

[0045] Furthermore, since the polymer film 13 may be composed of polymer 13X, it is easier to chemically modify than the silica layer, and the need for surface modification is reduced. As a result, the film thickness can be reduced compared to the silica layer, and the separation distance L1 in the nanoparticle-attached metal thin film 70A can be appropriately reduced. Moreover, the polymer film 13 can appropriately reduce the separation distances L1' and L2 in the composite nanoparticle-attached metal thin film 70C.

[0046] The presence of the sulfur atom-mediated bonding site 13a, the positively charged group 13b, and the hydrophobic group 13c can be confirmed by measuring the signals originating from them using infrared spectroscopy and nuclear magnetic resonance spectroscopy.

[0047] -Bonding site via sulfur atom- The sulfur atom-mediated bonding site 13a is formed, for example, by mixing a polymer having a site containing a disulfide bond as a side chain with metal nanoparticles 12.

[0048] -Positively charged group- The positively charged group 13b can form a relatively strong electrostatic bond b with the surface of the metal nanoparticles 12. In this specification, the positively charged group 13b is a group having a valency of 1 or more and being completely positively ionized. Considering multiple positively charged groups 13b contained in the polymer constituting the polymer film 13, the positively charged group 13b is expressed by the following formula (1):

number

[0049] The positively charged group 13b is preferably a primary ammonium group, a secondary ammonium group, a tertiary ammonium group, a quaternary ammonium group, and a guanidyl group (-NHC(=NH2) + It is at least one selected from the group consisting of )NH2).

[0050] -Hydrophobic group- The hydrophobic group 13c can form a hydrophobic bond c with the surface of the metal nanoparticle 12. The hydrophobic group 13c is, for example, at least one selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups.

[0051] Examples of aromatic cyclic groups include aromatic carbocyclic groups and aromatic heterocyclic groups. Aromatic carbocyclic groups are groups that do not contain aromatic heterocyclic groups but contain aromatic rings in which all member atoms are carbon atoms. Examples of aromatic carbocyclic groups include aryl groups (more specifically, phenyl groups, etc.) and arylalkyl groups (more specifically, benzyl groups, etc.). Aromatic heterocyclic groups are groups that contain aromatic rings in which at least one of the member atoms is a heteroatom (more specifically, oxygen atoms, sulfur atoms, and nitrogen atoms, etc.). Examples of aromatic heterocyclic groups include nitrogen-containing aromatic heterocyclic groups (more specifically, pyridyl groups (pyridinyl groups, etc.), sulfur-containing aromatic heterocyclic groups, and oxygen-containing aromatic heterocyclic groups.

[0052] Aliphatic cyclic groups are groups that contain a cyclic group consisting of a non-aromatic ring, without an aromatic ring. Examples of aliphatic cyclic groups include aliphatic carbocyclic groups and aliphatic heterocyclic groups. Aliphatic carbocyclic groups are groups that contain a non-aromatic ring in which all ring member atoms are carbon atoms, and examples include cycloalkyl groups. Aliphatic heterocyclic groups are groups that contain a non-aromatic ring in which at least one of the ring member atoms is a heteroatom.

[0053] Aliphatic chain groups are chain-like (more specifically, linear and branched) groups that do not contain aromatic or non-aromatic rings. Examples of aliphatic chain groups include aliphatic carbon chain groups (more specifically, alkyl and alkylene groups, etc.) and aliphatic heterochain groups. An example of an alkyl group is the butyl group. An example of an alkylene group is the n-butylene group.

[0054] The thickness of the polymer film 13 is preferably 1 nm to 50 nm, and more preferably 1 nm to 10 nm. When the thickness of the polymer film 13 is 50 nm or less, a near-field is more easily and efficiently formed in the space between the metal thin film 25 and the metal nanoparticles 12 in the metal thin film 70A with nanoparticles. Therefore, in this case, the detection sensitivity is further improved. Also, when the thickness of the polymer film 13 is 1 nm or more, the metal nanoparticles 12 and the electrochemiluminescent material 16 are arranged at a predetermined distance from each other, so the quenching of the electrochemiluminescent material 16 excited during measurement is suppressed, and the detection sensitivity is further improved. Although the numerical range for the film thickness of the polymer film 13 was explained using the nanoparticle-attached metal thin film 70A as an example, the same applies to the composite nanoparticle-attached metal thin film 70C. Furthermore, while polymer film 13 was explained, the first and second polymer films 13A and 13B are synonymous with polymer film 13.

[0055] (Electrochemiluminescent material) The electrochemiluminescent material 16 is labeled on the surface of the metal nanoparticles 12. The labeling site for the electrochemiluminescent material 16 is the surface of the polymer film 13.

[0056] When a voltage is applied to the electrochemiluminescent material 16, it undergoes an oxidation reaction, releasing electrons to become an oxide (oxide of electrochemiluminescent material 16). The generated oxide of electrochemiluminescent material 16 undergoes an electron exchange reaction with the co-reactant radicals generated by oxidation and putron elimination, producing an excited state of electrochemiluminescent material 16. The excited state of electrochemiluminescent material 16 (excited species) relaxes and emits light (electrochemiluminescence).

[0057] The emitted electrochemiluminescence induces plasmon resonance on the surfaces of the metal thin film 25 and metal nanoparticles 12 in the metal thin film 70A with nanoparticles. Furthermore, plasmon resonance may be induced on the surfaces of the metal thin film 25 and the first and second metal nanoparticles 12A and 12B in the metal thin film 70C with composite nanoparticles. The electrochemiluminescent material 16 in the excited state undergoes relaxation due to plasmon resonance, improving the emission quantum efficiency of the electrochemiluminescence.

[0058] As described above, the electrochemiluminescent material 16 is a material that undergoes an oxidation reaction, and the resulting oxide undergoes an electron exchange reaction with a co-reactant radical, emitting electrochemiluminescence upon relaxation of the excited species, and capable of inducing plasmon resonance in the metal thin film 25 and metal nanoparticles 12 with the emitted electrochemiluminescence. Examples of such electrochemiluminescent material 16 include at least one selected from the group consisting of metal complexes such as ruthenium (ruthenium complex), iridium complex, osmium complex, and rhenium complex. An example of a ruthenium complex is tris(bipyridine)ruthenium(II) which may have a counteranion. Although electrochemiluminescent material 16 has been described, the first and second electrochemiluminescent materials 16A and 16B are synonymous with electrochemiluminescent material 16.

[0059] (specific binding substance) The specific binding substance 14 is a nano-sized substance (with a maximum size of 3-15 nm) that specifically binds to the test substance in the sample.

[0060] The specific binding substance 14 is, for example, at least one selected from the group consisting of antibodies (hereinafter referred to as nanoantibodies), ligands, enzymes, and nucleic acid chains (more specifically, DNA chains and RNA chains). Among these, the specific binding substance 14 is preferably a nanoantibody. In the first embodiment, the nanoparticle body 10 to which such a specific binding substance 14 is bound exhibits superior detection sensitivity. For example, a nanoantibody as the specific binding substance 14 specifically binds to the antigen as the test substance at its tip (antigen binding site) through an antigen-antibody reaction to form a complex. A ligand as the specific binding substance 14 forms a complex with the protein as the test substance through a specific protein-ligand binding reaction. A nucleic acid chain as the specific binding substance 14 forms a pair (double helix) of complementary nucleic acid chains based on base pair complementarity. An enzyme as the specific binding substance 14 forms an enzyme-substrate complex with the substrate as the test substance at its active site (active site) based on substrate specificity (stereospecificity). These specific bonds are non-covalent bonds, such as hydrogen bonds, as well as bonds resulting from intermolecular forces, hydrophobic interactions, and charge interactions.

[0061] The nanoantibody is at least one selected from the group consisting of, for example, VHH (variable domain of heavy chain antibody) antibodies, fragmentation antibodies (more specifically, Fab (Fragment Antigen Binding) antibodies, etc.) and their variants. VHH antibodies are single-domain antibodies. Variants are antibodies in which a portion of the amino acid sequence has been rearranged or a substituent has been introduced, within a range that maintains specific binding to the antigen. Because the nanoantibody is at least one selected from the group consisting of VHH antibodies, fragmentation antibodies, and their variants, these nanoantibodies have a relatively small volume, which can narrow the separation distance L1 in the metal thin film 70A with nanoparticles attached (and further narrow the separation distances L1',L2 in the metal thin film 70C with composite nanoparticles attached), more efficiently form a near field, and further increase the intensity of electrochemiluminescence.

[0062] The molecular mass of the nanoantibody is preferably 60,000 Da or less, more preferably 30,000 Da or less, and even more preferably 20,000 Da or less. When the molecular mass is 60,000 Da or less (particularly 30,000 Da or less, or 20,000 Da or less), the volume of the nanoantibody is relatively small, which narrows the separation distance in the complex, allows for more efficient formation of the near field, and further increases the fluorescence intensity. Methods for measuring molecular mass include electrophoresis (SDS-PAGE), gel filtration chromatography, and static light scattering.

[0063] The specific binding substance 14 may be directly bound to the polymer film 13, or it may be a linker portion derived from the crosslinking agent (more specifically, SM(PEG) n The crosslinking agent may be indirectly bonded to the polymer film 13 via (where n is 4, 6, and 8, etc.). Examples of such crosslinking agents include amino group-sulfhydryl group crosslinking agents (more specifically, NHS-maleimide group crosslinking agents, etc.).

[0064] The specific binding substance 14 is bound to the polymer film 13, but may also be bound to the metal nanoparticles 12 (or their surface). Such a binding configuration to the metal nanoparticles 12 can be prepared, for example, as follows: Poly-L-lysine is reacted with a biotinylating reagent (N-hydroxysulfosuccinimide ester of biotin; Sulfo-NHS-SS-Biotin) to obtain a polymer as a raw material. In this reaction, the amino group at the end of the side chain of poly-L-lysine reacts with the ester bond of the biotinylating reagent to form an amide bond. The polymer as a raw material has a biotin moiety bound to the side chain via a disulfide bond. Next, by adding the polymer as a raw material to a dispersion of metal nanoparticles 12 and mixing it, a polymer film 13 is formed that coats the metal nanoparticles 12, as well as biotin that bonds to the surface of the metal nanoparticles 12 via sulfur atoms. After adding streptavidin to form a complex with biotin bound to the metal nanoparticles 12, biotinylated specific binding material 14 is added to form a complex with the streptavidin in the complex bound to the metal nanoparticles 12. This results in the formation of a specific binding material 14 that binds to the surface of the metal nanoparticles 12 via sulfur atom-biotin-streptavidin-biotin. Although the specific bonding substance 14 has been described, the first and second specific bonding substances 14A and 14B are synonymous with specific bonding substance 14.

[0065] The types of specific binding substances 14A, 14B, and 254 may be the same or different, as long as they can adequately capture the test substance 70 that is the target of detection. Taking the nanoparticle-attached working electrode 70A in Figure 2 as an example, if the test substance 70 is a monomeric antigen, it may be necessary that the first specific binding substance 14A, which specifically binds to the same test substance 70, and the third specific binding substance 254 of the working electrode 25 are of different types. This is because if there is one site (epitope or antigenic determinant) on the surface of the antigen that specifically binds to the first and third specific binding substances 14A and 254, and the first and third specific binding substances 14A and 254 are of the same type, only one of the first and third specific binding substances 14A and 254 will specifically bind to the antigen, and there is a risk that the nanoparticle-attached working electrode 70A will not be properly formed. If the test substance 70 is a homomultimeric antigen, multiple epitopes with the same structure may exist on the surface of the antigen. Therefore, even if the first and third specific binding substances 14A and 254 are of the same type, the working electrode 70A with nanoparticles can be properly formed. The same applies to the types of the first, second, and third specific binding materials in the working electrode 70C with composite nanoparticles.

[0066] (hydrophilic substance) The nanoparticle body 10 further comprises a hydrophilic substance that bonds to the surface of the metal nanoparticle 12 via sulfur atoms. When the nanoparticle body 10 contains a hydrophilic substance, the hydrophilic substance suppresses nonspecific adsorption to the nanoparticle body 10, thereby suppressing a decrease in the signal-to-noise ratio in the detection of the test substance and improving detection sensitivity.

[0067] Furthermore, if the nanoparticles 10 include a hydrophilic substance, the hydrophilic substance does not need to have a non-specific adsorption suppression function on the polymer film 13, thus allowing the film thickness of the polymer film 13 to be reduced. This makes it possible to reduce the distance between the metal nanoparticles 12 and the metal film 25 in the nanoparticle-attached metal film 70A formed during the detection of the test substance. In a preferred embodiment, the distance between the metal nanoparticles 12 and the distance between the metal nanoparticles 12 and the metal film 25 can be further reduced in the composite nanoparticle-attached metal film 70C formed during the detection of the test substance.

[0068] The hydrophilic material includes a sulfur atom-mediated bonding site between itself and the surface of the metal nanoparticle 12, and a polar group and / or an electrostatic group. The polar group includes, for example, at least one selected from the group consisting of a carboxyl group, a hydroxyl group, an amino group, a sulfonyl group, a phosphate group, and an alkylene oxide group. The electrostatic group includes, for example, at least one selected from the group consisting of a group that is charged by the ionization of a polar group 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.

[0069] Examples of hydrophilic substances include those with chemical formulas (a) to (d): [ka] [In chemical formulas (a) to (d), * indicates the bonding site with the surface of the metal nanoparticle 12.] Examples of compounds represented by [the formula shown] are given.

[0070] A hydrophilic substance that bonds to the surface of the metal nanoparticles 12 via sulfur atoms can be prepared, for example, as follows: A polymer is prepared as a raw material. The polymer as a raw material is the raw material for the polymer film 13 and has polar groups and / or charged groups (synonymous with the polar groups and charged groups in the hydrophilic substance described above) that are bonded to the side chains via disulfide bonds. The polymer as a raw material is mixed with a dispersion containing the metal nanoparticles 12. This produces a hydrophilic substance that bonds to the surface of the metal nanoparticles 12 via sulfur atoms.

[0071] [Important points to consider when describing nanoparticles] The following describes important points that do not directly constitute the nanoparticle body 10, but are necessary for explaining the nanoparticle body 10.

[0072] (Test substance) The test substance 70 is a substance to be detected contained in the sample. Examples of the test substance 70 include antigens, proteins, substrates, and nucleic acid chains. The test substance 70 specifically binds to the specific binding substance 14. For example, an antigen has at least two antigenic determinants, which form a specific bond with the specific binding substance 14. Antigens are proteins such as c-reactive proteins, myoglobin, troponin T, troponin I, and BNP, as well as antigenic proteins of viruses such as influenza virus and RSV. The test substance 70 is a test substance derived from a sample such as blood, plasma, urine, or saliva. That is, examples of samples containing the test substance 70 are blood, plasma, serum, urine, and saliva. The sample may further contain a solvent and a buffer (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, and MES buffer).

[0073] (Co-reactants) The co-reactant excites the electrochemiluminescent material 16 in the nanoparticle-attached metal thin film 70A (and further, the first and second electrochemiluminescent materials 16A and 16B in the composite nanoparticle-attached metal thin film 70C) by an electrochemical reaction. The co-reactant is, for example, at least one selected from the group consisting of tripropylamine, triethylamine, and peroxosulfate ions.

[0074] (Metal thin film (working electrode)) The metal thin film (working electrode) 25 has an electrode portion 251 on its surface and a third specific binding substance 254 bonded to the surface of the electrode portion 251. The metal thin film 25 is, for example, the working electrode of an electrochemiluminescence sensor provided in an electrochemical immunoassay device.

[0075] The metal thin film (working electrode) 25 captures the nanoparticles 10 to form a nanoparticle-attached metal thin film (working electrode with nanoparticles) 70A. The metal thin film 25 can apply a voltage to the captured nanoparticles 10. The metal thin film 25 can also capture composite nanoparticles 70B to form a composite nanoparticle-attached metal thin film (working electrode with composite nanoparticles) 70C. The metal thin film 25 can apply a voltage to the captured composite nanoparticles 70B.

[0076] The metal thin film 25 comprises, for example, gold, silver, platinum, palladium, or indium tin oxide, and preferably substantially composed of gold, silver, platinum, palladium, or indium tin oxide. Here, substantially composed means that the component in question contains 95% or more by mass, 97% or more by mass, 99% or more by mass, or 100% by mass of the specific material.

[0077] The metal thin film 25 is a thin film with a thickness on the order of nanometers (e.g., a few nm to 100 nm). The metal thin film 25 induces plasmon resonance through interaction with light (e.g., visible light and near-infrared light).

[0078] -Third specific binding substance- The third specific bonding material 254 is bonded to the surface of the metal thin film 25. The third specific bonding material 254 is synonymous with the specific bonding material 14 except for its location.

[0079] <Second Embodiment: Electrochemiluminescence Immunoassay Method> The second embodiment relates to an electrochemiluminescence immunoassay method. In the second embodiment, reference numerals identical to those in the first embodiment have the same configuration as in the first embodiment, and therefore, their descriptions are generally omitted.

[0080] The electrochemiluminescence immunoassay method according to the second embodiment (hereinafter also simply referred to as the "measurement method") is as follows: An electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, A contact step involves bringing a first solution containing nanoparticles 10 and a test substance 70 according to the first embodiment into contact with a working electrode (metal thin film) 25 having a specific bonding substance 254, thereby bonding the nanoparticles 10 and the working electrode 25 via the test substance 70. A co-reactant supply step involves supplying a second solution containing co-reactants onto the working electrode 25, A voltage application step in which a voltage is applied to the working electrode to generate electrochemiluminescence, and the electrochemiluminescence is plasmon-enhanced, A detection step for detecting plasmon-enhanced electrochemiluminescence and It consists of including.

[0081] The measurement method according to the second embodiment can generate electrochemiluminescence without irradiating the sample with light from an external source. In other words, the measurement method according to the second embodiment does not have a light source for generating electrochemiluminescence. For this reason, the measurement method according to the second embodiment has a smaller background signal and can detect the detected light (electrochemiluminescence) with high sensitivity compared to measurement methods that use a light source (for example, fluorescence spectroscopy immunoassay). Furthermore, because the measurement method according to the second embodiment does not use a light source, it is easier to construct a simpler measurement system and, consequently, a simpler apparatus configuration can be adopted compared to measurement methods that use a light source.

[0082] Hereinafter, an example of an electrochemiluminescence immunoassay method according to the second embodiment will be described with reference to Figures 2 and 7. Figure 7 is a flowchart of an example of an electrochemiluminescence immunoassay method according to the second embodiment. As shown in Figure 7, the electrochemiluminescence immunoassay method comprises a contact step (step S102), a co-reactant supply step (step S104), a voltage application step (step S106), and a detection step (step S108).

[0083] Furthermore, the electrochemiluminescence sensor (hereinafter also simply referred to as "sensor") used in the electrochemiluminescence immunoassay method will be explained. Figures 11 and 12 are a plan view and a cross-sectional view of the electrochemiluminescence sensor, respectively. First, before explaining the electrochemiluminescence immunoassay method, the electrochemiluminescence sensor will be described. As shown in Figures 11 to 12, the electrochemiluminescence sensor 20 comprises a substrate 21, electrodes 25, 26, and 27 provided on the surface of the substrate 21, and a resist 23 covering parts of electrodes 25 to 27. The electrochemiluminescence sensor 20 also has hooks provided on the substantially rectangular substrate 21 so as to protrude in both directions in the X direction. The electrochemiluminescence sensor 20 is fixed to the electrochemiluminescence sensor cell by the hooks.

[0084] Electrodes 25-27 are the working electrode 25, the counter electrode 26, and the reference electrode 27. The working electrode 25 is the electrode that performs the electrochemical reaction (oxidation reaction) that contributes to electrochemiluminescence. The counter electrode 26 is the electrode opposite to the working electrode 25. The reference electrode 27 is the electrode that stabilizes the voltage applied to the working electrode 25, and is, for example, a silver / silver chloride electrode.

[0085] Electrodes 25-27 are electrically insulated from each other by a substantially recessed groove 22 and a resist 23 provided between them. Each electrode 25-27 has an electrode portion 251, 261, 271, a lead portion 252, 262, 272, and a terminal portion 253, 263, 273. The electrode portions 251, 261, 271 are exposed to the outside and can come into contact with the first and second solutions. The lead portions 252, 262, 272 are covered with resist 23. The terminal portions 253, 263, 273 are exposed to the outside and can be electrically connected to the voltage application section of the electrochemiluminescence immunoassay apparatus.

[0086] A third specific binding substance 254, which specifically binds to the test substance 70, is bonded to the surface of the electrode portion 251 of the working electrode 25. Therefore, the electrode portion 251 can capture the nanoparticles 10 to form a working electrode 70A with nanoparticles attached, and a voltage can be applied to the working electrode 70A with nanoparticles attached. Furthermore, the electrode portion 251 can capture the composite nanoparticles 70B to form a working electrode 70C with composite nanoparticles attached, and a voltage can be applied to the working electrode 70C with composite nanoparticles attached.

[0087] (Contact process: Step S102) In the contact step, a first solution containing the nanoparticles 10 and the test substance 70 according to the first embodiment is brought into contact with a working electrode 25 having a third specific binding substance 254, thereby bonding the nanoparticles 10 and the working electrode 25 via the test substance 70.

[0088] Specifically, first, a first solution containing nanoparticles 10 and the test substance 70 is prepared. The first solution is a solution in which nanoparticles 10 are further dissolved and / or dispersed in a test substance solution containing the test substance 70. In this specification, the term "test substance solution" refers to a solution containing the test substance prepared from a sample (which is directly collected from the individual being measured), in addition to the sample itself. Examples of solutions containing the test substance prepared from a sample include a solution in which a solvent has been added to the sample to adjust viscosity (sample diluent), and a solution containing the test substance 70 extracted from the sample (test substance extract). The first solution is prepared before being introduced into the electrochemiluminescence immunoassay apparatus (specifically, the electrochemiluminescence sensor (hereinafter also simply referred to as "sensor") 20 provided in the apparatus).

[0089] Next, the first solution is brought into contact with the working electrode 25 having the third specific binding substance 254. Specifically, the prepared first solution is dropped onto the electrode portion 251 of the sensor 20 using a pipette. When contact is made in this way, as shown in Figure 2, the specific binding substance 14 of the nanoparticles 10 specifically binds to the test substance 70 captured by the third specific binding substance 254 of the working electrode 25, thereby capturing the nanoparticles 10 on the working electrode 25 and forming a working electrode 70A with nanoparticles.

[0090] In the contact step, after bringing the first solution into contact with the working electrode 25 (the electrode portion 251), the working electrode 25 (the electrode portion 251) may be washed. This removes unreacted substances (e.g., nanoparticles 10, test substance 70, and composite nanoparticles 70B) from the electrode portion 251, thereby improving the signal-to-noise ratio. The washing of the electrode portion 251 can be performed, for example, by immersing the electrode portion 251 of the sensor 20 in a washing solution (e.g., pure water and buffer solution). Washing can be performed multiple times. Alternatively, the washing of the electrode portion 251 can also be performed by pouring the washing solution onto the electrode portion 251 of the sensor 20 to remove unreacted substances. After cleaning the electrode section 251, the electrode section 251 is dried. For example, drying after cleaning is carried out in the dark at room temperature for 30 minutes to 1 hour.

[0091] (Co-reactant supply process: Step S104) In the co-reactant supply step, a second solution containing the co-reactant is supplied onto the working electrode 25. First, the sensor 20 from the contact step is placed in an electrochemiluminescence sensor cell (hereinafter also simply referred to as "cell"). Before explaining the co-reactant supply step, we will explain using a cell used in the electrochemiluminescence immunoassay method with reference to Figure 13. Figure 13 is a perspective view showing a cell with the sensor 20 attached.

[0092] As shown in Figure 13, the cell 30 is a cell for mounting the sensor 20 and comprises a sensor holding portion 31 for detachably holding the sensor 20 and a container housing 32 capable of holding a second solution 323 containing a co-reactant. The sensor holding portion 31 is a male member and is fitted and connected to the female member of the container housing 32. The sensor holding portion 31 has a sensor insertion opening 311 for inserting and holding the sensor 20 and a second solution injection opening 312 for injecting the second solution. The container housing 32 has a transparent first wall surface 321.

[0093] The second solution 323 is filled into the container housing 32 of the cell 30. The sensor 20 is inserted into the sensor holder 31 and fixed so that the working electrode 25 is positioned on the bottom side (bottom surface 322 side) of the container housing 32. This immerses the sensor 20 in the second solution 323 inside the container housing 32, supplying the second solution containing the co-reactants onto the working electrode 25. As a result of this contact process, the working electrode 25 now has a working electrode 70A with nanoparticles attached. When the second solution is supplied onto the working electrode 25, the co-reactants are placed around the electrochemiluminescent substance 16 on the working electrode 70A with nanoparticles attached. In other words, the co-reactants can physically contact the electrochemiluminescent substance 16 on the working electrode 70A with nanoparticles attached. The sensor 20 is fixed to the cell 30 by a portion of its electrode portion 261 being sandwiched on the bottom surface 322 side of the container housing 32 (see Figure 15 below).

[0094] (Voltage application process: Step S106) In the voltage application step, a voltage is applied to the working electrode 25 to generate electrochemiluminescence, and this electrochemiluminescence is plasmon-enhanced. First, the sensor 20 held in the cell 30 is placed in the electrochemiluminescence immunoassay apparatus (hereinafter also simply referred to as "apparatus"). Before explaining the voltage application step, we will explain using the apparatus with reference to Figures 14 and 15. Figure 14 is a perspective view showing the apparatus. Figure 15 is a cross-sectional view showing the apparatus (cross-sectional view taken along line II-II in Figure 14).

[0095] The device 100 includes a cell mounting section 110 for mounting the cell 30, an optical detection section 120 for receiving electrochemiluminescence, a connector 140 for applying voltage to the sensor 20, and a photomultiplier tube (PMT) 150 for amplifying the received electrochemiluminescence. The cell 30 holding the sensor 20 is mounted in the cell mounting section 110 of the device 100. Furthermore, the connector 140 is electrically connected to the terminal sections 253, 263, and 273 exposed to the outside of the cell 30.

[0096] When the sensor 20 is mounted on the device 100, the transparent region of the first wall surface 321 faces the working electrode 25 of the sensor 20. In the Z direction from the electrode portion 251 of the working electrode 25, the transparent region of the first wall surface 321 of the cell 30 is positioned, and beyond that, the optical detection unit 120 and the photomultiplier tube 150 are positioned. Furthermore, when the sensor 20 is mounted on the device 100, the working electrode 25 has a working electrode 70A with nanoparticles attached in the presence of co-reactants.

[0097] When a voltage is applied to the working electrode 25, an electrochemical reaction occurs between the co-reactant (CR) and the electrochemiluminescent material (ECLM) 16, as shown in Scheme 1 above, and the co-reactant radical (CR) generated therefrom · ) and oxidized electrochemiluminescent substance 16 (ECLM + ) undergoes an electron exchange reaction, and as a result, the excited state of the electrochemiluminescent substance 16 (ECLM) undergoes an electron exchange reaction, and as a result, the electrochemiluminescent substance 16 (ECLM) * ) is generated. Electrochemiluminescent material 16 (ECLM) in the excited state is produced. * ) is relaxed and electrochemiluminescence is emitted.

[0098] Next, when the resulting electrochemiluminescence irradiates the metal nanoparticles 12 and the working electrode 25 in the working electrode 70A with the nanoparticle body, a near-field is efficiently formed between the metal nanoparticles 12 and the working electrode 25, which are positioned in close proximity in the working electrode 70A with the nanoparticle body (particularly near the space between the metal nanoparticles 12 and the metal thin film 25). This near-field enhances the electrochemiluminescence (plasmon enhancement).

[0099] (Detection process: Step S108) In the detection step, plasmon-enhanced electrochemiluminescence is detected. In the detection step, the plasmon-enhanced electrochemiluminescence from the voltage application step is received by the optical detection unit 120 and amplified by the photomultiplier tube 150. In this way, the electrochemiluminescence is detected.

[0100] In the contact step, a composite nanoparticle body 70B is formed by two or more nanoparticles bonding via the test substance 70, and the composite nanoparticle body 70B can be captured on the working electrode 25 side. In other words, in the contact step, a working electrode 70A with nanoparticles attached is formed, but a working electrode 70C with composite nanoparticles attached can also be formed.

[0101] Further details will be provided with reference to Figures 4-5. The composite nanoparticle 70B can be formed in the preparation of the first solution containing the nanoparticle 10 and the test substance 70. For example, the composite nanoparticle 70B shown in Figure 4 can be formed by the bonding of two nanoparticles 10A and 10B via the same test substance 70. Therefore, the first solution may contain the composite nanoparticle 70B.

[0102] As shown in Figure 4, the working electrode 70C with composite nanoparticles can be formed by contacting a working electrode 25 having a third specific binding substance 254 with a first solution containing composite nanoparticles 70B. In other words, the composite nanoparticles 70B in the first solution specifically bind to the third specific binding substance 254 present on the surface of the working electrode 25 and are captured by the working electrode 25, thereby forming the working electrode 70C with composite nanoparticles.

[0103] In the working electrode 70C with composite nanoparticles, the first and second metal nanoparticles 12A and 12B of the two first and second nanoparticles 10A and 10B are arranged in close proximity to each other, and the first metal nanoparticle 12A of one of the first nanoparticles 10A and the working electrode 25 are also arranged in close proximity to each other. Therefore, in the subsequent voltage application step, a near-field is efficiently formed, particularly in the vicinity of the space between the first and second metal nanoparticles 12A and 12B in the close proximity arrangement, and between the first metal nanoparticle 12A and the working electrode 25.

[0104] <Other Embodiments> This disclosure is not limited to the embodiments described above, and design modifications are possible without departing from the gist of this disclosure.

[0105] In the first embodiment, as shown in Figure 1, the electrochemiluminescent substance 16 was bonded to the polymer film 13, but it is not limited to this. The electrochemiluminescent substance 16 may also be bonded to the specific bonding substance 14.

[0106] In the first embodiment, it was explained that the test substance 70 binds to the third specific binding substance 254 on the metal thin film 25, and as a result of the nanoparticles 10 binding to the captured test substance 70, a nanoparticle-attached metal thin film 70A is formed. However, the embodiment is not limited to this. The test substance 70 may bind to the nanoparticles 10, and as a result of the third specific binding substance 254 on the metal thin film 25 binding to the test substance 70 captured by the nanoparticles 10, a nanoparticle-attached metal thin film 70A may be formed. The same applies to the composite nanoparticle-attached metal thin film (composite nanoparticle-attached working electrode) 70C. Furthermore, the composite nanoparticle-attached metal thin film 70C may be formed by bonding the nanoparticles 10 to the nanoparticle-attached working electrode 70A via another test substance 70.

[0107] In the second embodiment, the first solution was prepared in the contact step before being introduced into the electrochemiluminescence immunoassay apparatus (and the electrochemiluminescence sensor 20 provided therein), but is not limited thereto. For example, the first solution may be mixed and prepared within the electrochemiluminescence immunoassay apparatus (and the electrochemiluminescence sensor 20 provided therein). [Examples]

[0108] The present disclosure will be described in more detail below with reference to examples. However, the present disclosure is not limited in any way by the following examples. Unless otherwise specified, parts and percentages in the examples are by mass. The examples and comparative examples were carried out under ambient air and room temperature (1 atmosphere, 25°C) unless otherwise specified.

[0109] Furthermore, in the examples and comparative examples, the concentration of metal nanoparticles in the dispersion is sometimes expressed in terms of absorbance. Absorbance was measured using a UV-Vis spectrophotometer (TECAN Japan Co., Ltd. "infinite M200 PRO"). Since the absorption wavelength differs for each sample, it is listed for each sample. The number appended to the absorbance notation OD indicates the absorption wavelength. For example, OD 455 =0.1 indicates that the absorbance OD at a wavelength of 455 nm is 0.1. The unit of concentration, M, represents mol / L.

[0110] <Example 1> [1. Formation of polymer films] The method for forming the polymer film will be explained with reference to Figures 8 and 9. Figure 8 is a schematic diagram showing the method for forming the polymer film in Example 1. Figure 9 is a schematic diagram illustrating the polymer coating morphology of the nanoparticles in Example 1. As shown in Figure 8, poly-L-lysine (Peptide Laboratories, Inc., "3075") and 3-(2-pyridyldithio)propionamide-PEG4-NHS (Thermo Fisher Scientific, Ltd., lot number "26128", "NHS-PEG4-SPDP") were mixed and stirred at room temperature for 4 hours using a small rotary incubator (Tytec Co., Ltd., "RT-30mini"). As a result, a polymer was obtained. This synthesis reaction is a nucleophilic substitution reaction in which the primary amino group of poly-L-lysine attacks the NHS ester group of 3-(2-pyridyldithio)propionamide-PEG4-NHS. The synthesized polymer had disulfide bonds in its side chains. More specifically, the synthesized polymer had a hydrophobic group (pyridyl group) 13c and a positively charged group (primary ammonium group) 13b linked via disulfide bonds.

[0111] The obtained polymer was brought into contact with metal nanoparticles 12 to form a polymer film 13. More specifically, the obtained polymer was used as the metal nanoparticle 12, which was silver nanoparticle (nanocomposix "AGCB80-1M", diameter 80 nm, OD 455 The mixture was added to 1 mL of a dispersion of (=0.1) and stirred and mixed at room temperature and overnight using a small rotary incubator (RT-30mini, manufactured by Taitec Co., Ltd.). As a result, a dispersion of silver nanoparticles coated with a polymer membrane was obtained.

[0112] As shown in Figure 9, the polymer film 13 contains a sulfur atom-mediated bonding site 13a on the surface of the silver nanoparticles 12, a hydrophobic group (pyridyl group) 13c that forms a hydrophobic bond with the surface of the silver nanoparticles 12, and a positively charged group (primary ammonium group) 13b that forms an electrostatic bond b with the surface of the silver nanoparticles 12. In other words, the polymer 13X constituting the polymer film 13 has a sulfur atom-mediated bonding site 13a on the surface of the silver nanoparticles 12, a hydrophobic group (pyridyl group) 13c that forms a hydrophobic bond c with the surface of the silver nanoparticles 12, and a positively charged group (primary ammonium group) 13b that forms an electrostatic bond with the surface of the silver nanoparticles 12. An SEM image (magnification 500,000x) of the obtained silver nanoparticles 12 was created, and it was confirmed that the surface of the silver nanoparticles 12 is continuously coated by the polymer film 13 (hereinafter, silver nanoparticles coated with the polymer film 13 will also be referred to as "polymer-coated silver nanoparticles").

[0113] [2. Fabrication of Nanoparticles] Figure 10 is a schematic diagram showing the structure of the nanoparticle 10 to which the antibody (hereinafter also referred to as the labeled antibody) labeled with the electrochemiluminescent substance 16 of Example 1 was bound. The nanoparticle 10 shown in Figure 10 was first prepared by binding a crosslinking agent to the surface of polymer-coated metal nanoparticles, separately binding the electrochemiluminescent substance 16 and the crosslinking agent to the nanoantibody, and then binding the crosslinking agent bound to the polymer-coated metal nanoparticles to the crosslinking agent bound to the nanoantibody. The details of the preparation of the nanoparticles to which the labeled antibody was bound will be described below.

[0114] (2-1. Binding of crosslinking agents to polymer-coated silver nanoparticles) To 1 mL of the dispersion of the prepared polymer-coated silver nanoparticles, the crosslinking agent SM(PEG)2 (PEGylated, long-chain SMCC crosslinker) (ThermoFisher SCIENTIFIC, "22105") and heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd., "081-00136") were added, and the mixture was stirred and mixed at room temperature for 1 hour using a small rotary incubator (Tytec Corporation, "RT-30mini"). As a result, a dispersion of silver nanoparticles in which the crosslinking agent SM(PEG)2 was bound to the polymer membrane 13 (hereinafter also referred to as polymer-coated silver nanoparticles bound with SM(PEG)2 linkers) was obtained. The SM(PEG)2 linkers bound to the polymer-coated silver nanoparticles had maleimide groups.

[0115] (2-2. Labeling of VHH antibodies with electrochemiluminescent substances) [Chemical Formula 5] is obtained for 100 μg of VHH antibody (RePHAGEN, molecular weight 18,000 Da): [ka] An NHS-labeled Ru complex derivative represented by ("Ruthenium(II)tris(Bipyridyl)-C5-NHS ester" manufactured by Tokyo Chemical Industry Co., Ltd.) was added and mixed by stirring at room temperature for 1 hour using a small rotary incubator ("RT-30mini" manufactured by Taitec Co., Ltd.). As a result, a VHH antibody conjugated with electrochemiluminescent substance 16 (hereinafter also referred to as labeled VHH antibody) was obtained. The NHS-labeled Ru complex derivative is an electrochemiluminescent substance with a maximum emission wavelength in the range of 500 to 700 nm.

[0116] (2-3. Binding of the crosslinking agent to the labeled VHH antibody) Next, 3-(2-pyridyldithio)propionamide-PEG4-NHS (Thermo Fisher SCIENTIFIC, product number "26128", "NHS-PEG4-SPDP"), used as an NHS-bipyridyl disulfide crosslinking agent, was added in an 8-fold molar equivalent volume to the substance-labeled VHH antibody. The mixture was then stirred and mixed at room temperature for 1 hour using a small rotary incubator (Tytec Co., Ltd., "RT-30mini"). As a result, a VHH antibody conjugated with electrochemiluminescent substance 16 and an SPDP linker (hereinafter also referred to as labeled VHH antibody conjugated with an SPDP linker) was obtained.

[0117] (2-4. Thiolation of labeled VHH antibody bound to a crosslinking agent) Next, the labeled VHH antibody bound to the SPDP linker was mixed with a reducing agent TCEP (ThermoFisher SCIENTIFIC "77720") in a molar ratio of 2 equivalents, and stirred using a stirrer (BioSan "TS-100") at 37°C for 1 hour. As a result, an electrochemiluminescent substance and a VHH antibody bound to a reduced SPDP linker (hereinafter also referred to as the reduced SPDP linker) were obtained (hereinafter also referred to as the labeled VHH antibody bound to the reduced SPDP linker). The reduced SPDP linker had a thiol group (-SH group) generated by the reduction of the disulfide bond.

[0118] (2-5. Binding of labeled VHH antibody to silver nanoparticles) Next, a dispersion of polymer-coated silver nanoparticles to which a maleimide group-containing SM(PEG)6 linker is attached (OD 430 A labeled VHH antibody conjugated to a reduced SPDP linker was added to a solution of 0.1 (=0.1), and the mixture was stirred and mixed at room temperature and overnight using a small rotary incubator (RT-30mini, manufactured by Taitec Co., Ltd.). As a result, the maleimide group of the SM(PEG)6 linker reacted with the thiol group of the reduced SPDP linker to obtain nanoparticles 10 to which the labeled VHH antibody was conjugated via the linker portion (see Figure 10).

[0119] [3. Electrochemiluminescence Immunoassay] (3-1. Contact process) To the phosphate buffer solution of the obtained nanoparticles 10, C Reactive Protein (ADVY CHEMICAL Sigma-Aldrich "00-AGN-AP-CRP-00") (hereinafter also referred to as CRP antigen) as the test substance 70 was added, and the mixture was stirred using a small rotary incubator (Tytec Co., Ltd. "RT-30mini") at room temperature (25°C) for 15 minutes to obtain the first solution (OD 455 A solution (=0.4) was prepared. The first solution contained CRP antigen (test substance 70), nanoparticle 10, and composite nanoparticle 70B. The concentration of CRP antigen was 1.0 × 10⁻⁶. -12 ~5.0×10 -10 The result was M. Additionally, a phosphate-buffered solution of nanoparticle 10 without CRP antigen 70 was prepared separately as a blank.

[0120] Next, an electrochemiluminescence sensor 20 (length in the short axis direction (X direction) 6.5 mm (length in the X direction including the two hook parts is 8.0 mm) × length in the long axis direction (Y direction) 30 mm, thickness (length in the Z direction) 0.2 mm) was prepared and placed so that the XY plane was horizontal and the electrode part 251 was in the inverted vertical direction (horizontal state). The electrochemiluminescence sensor 20 had a third specific binding substance 254 that was bound to the electrode part 251 of the working electrode 25 (diameter Φ3 mm in the XY plane view). Using a pipette, the prepared first solution (2.5 μL) was dropped onto the electrode part 251 of the electrochemiluminescence sensor 20. After dropping, the electrochemiluminescence sensor 20 was kept in a horizontal state and left to stand for 1 hour in the dark, at room temperature and high humidity (temperature 25°C and humidity 85% RH).

[0121] The electrode portion 251 of the electrochemiluminescence sensor 20 was positioned so that its electrode portion 251 faced the bottom side (bottom surface 322 side) of the cylindrical cleaning container. The electrode portion 251 of the electrochemiluminescence sensor 20 was immersed in the cleaning solution in the cleaning container, and the operation of removing the electrode portion 251 from the cleaning solution was repeated multiple times to clean the electrode portion 251 of the working electrode 25. The cleaning solution was pure water prepared using an ultrapure water production system (Millipore's "Milli-Q®"). After cleaning, the electrochemiluminescence sensor 20 was left to stand in a horizontal position for 1 hour in the dark at room temperature to dry the electrode portion 251 of the working electrode 25.

[0122] (3-2. Co-reactant supply process) The container housing 32 of the electrochemiluminescence sensor cell 30 was filled with the second solution 323. The electrochemiluminescence sensor 20 was inserted into the sensor holder 31 and fixed to the electrochemiluminescence sensor cell 30 so that the electrode portion 251 of the working electrode 25 was located on the bottom side (bottom surface 322 side) of the container housing 32. The electrode portion 251 of the working electrode 25 of the electrochemiluminescence sensor 20 was immersed in the second solution 323 held in the container housing 32 so that the co-reactants were supplied onto the working electrode 25 (the electrode portion 251).

[0123] (3-3. Voltage Application Process) The electrochemiluminescence sensor cell 30 was mounted on the electrochemiluminescence immunoassay analyzer 100. Connectors 140 were connected to the terminals 253, 263, and 273 of the electrochemiluminescence sensor 20 exposed from the electrochemiluminescence sensor cell 30. As a result, the electrode portion 251 was electrically connected to the electrode application portion (not shown: Dual electrochemical analyzer (ALS700E, manufactured by BAS Corporation)) of the electrochemiluminescence immunoassay analyzer 100. A voltage was applied to the electrode portion 251 of the electrochemiluminescence sensor under the conditions of an applied voltage of 0.5 to 1.2 V and an application time of 0.02 seconds.

[0124] (3-3. Detection Process) Using an optical detection unit 120 synchronized with the voltage application unit, the electrochemiluminescence emitted from the electrochemiluminescence sensor 20 was received by the optical detection unit 120 for 25 to 45 seconds immediately after the voltage was applied (detection wavelength range 400 nm to 650 nm) to obtain the photon count. The optical detection unit 120 was a photon counting detector. The measured photon count (electrochemiluminescence intensity) was obtained by comparing the applied voltage (0.5 to 1.2 V) and the CRP concentration (1.0 × 10⁻¹⁶). -12 ~5.0×10 -10 Each showed linearity with respect to M).

[0125] The blank was measured in the same manner. The electrochemiluminescence intensity of the blank was subtracted from the obtained electrochemiluminescence intensity to obtain the enhanced electrochemiluminescence intensity described herein.

[0126] The embodiments of the nanoparticles and the electrochemiluminescence immunoassay method using the nanoparticles relating to this disclosure are as follows. <1> Metal nanoparticles and A polymer film covering the surface of the metal nanoparticles, A specific binding substance is bonded to at least one of the metal nanoparticles and the polymer film, and specifically binds to the test substance in the sample, An electrochemiluminescent material bonded to at least one of the polymer film and the specific bonding material. A nanoparticle body comprising the following. <2> These are nanoparticles used in electrochemiluminescence immunoassays utilizing plasmon-enhanced fields. <1> The nanoparticles described above. <3> The luminescence produced by the electrochemical reaction of the electrochemiluminescent material is enhanced by plasmons. <1> or <2> The nanoparticles described above. <4> Used in electrochemiluminescence immunoassays that do not utilize external light irradiation, <1> ~ <3> A nanoparticle material described in any one of the following. <5> The electrochemiluminescent material is at least one selected from the group consisting of ruthenium complexes, europium complexes, iridium complexes, osmium complexes, and rhenium complexes. <1> ~ <4> A nanoparticle material described in any one of the following. <6> The aforementioned specific binding substance is a nanoantibody. <1> ~ <5> A nanoparticle material described in any one of the following. <7> The aforementioned metal nanoparticles contain gold or silver. <1> ~ <6> A nanoparticle material described in any one of the following. <8> A metal thin film with nanoparticles bonded to it via a test substance is formed. The aforementioned metal thin film has a specific bonding substance bonded to its surface. <1> ~ <7> A nanoparticle material described in any one of the following. <9> The aforementioned nanoparticles include a first nanoparticle and a second nanoparticle. The first nanoparticle and the second nanoparticle are bonded together via the test substance to form a composite nanoparticle. <1> ~ <8> A nanoparticle material described in any one of the following. <10> The composite nanoparticles form a metal thin film with composite nanoparticles via a metal thin film and a test substance, The aforementioned metal thin film has a specific bonding substance bonded to its surface. <9> The nanoparticles described above. <11> The test substance is derived from a sample that is blood, plasma, urine, or saliva. <1> ~ <10> A nanoparticle material described in any one of the following. <12> The polymer constituting the polymer film includes a sulfur atom-mediated bonding site between it and the surface of the metal nanoparticles. <1> ~ <11> A nanoparticle material described in any one of the following. <13> The polymer forming the polymer film includes the binding site between the side chain of the polymer and the surface of the metal nanoparticles via the sulfur atom, <1> ~ <12> A nanoparticle material described in any one of the following. <14> The polymer constituting the polymer film further contains positively charged groups in the side chains of the polymer, The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH 2+ )NH 2 At least one selected from the group consisting of ) <1> ~ <13> A nanoparticle material described in any one of the following. <15> The polymer constituting the polymer film further contains hydrophobic groups in the side chains of the polymer, The hydrophobic group is selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. It is at least one of the following types: <1> ~ <14> A nanoparticle material described in any one of the following. <16> The thickness of the polymer film is 1 nm to 10 nm. <1> ~ <15> A nanoparticle material described in any one of the following. <17> An electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, <1> ~ <16> A contact step involves bringing a first solution containing nanoparticles and a test substance as described in any one of the above into contact with a working electrode having a specific binding substance, thereby bonding the nanoparticles and the working electrode via the test substance. A co-reactant supply step involves supplying a second solution containing co-reactants onto the working electrode, A voltage application step in which a voltage is applied to the working electrode to generate electrochemiluminescence, and the electrochemiluminescence is plasmon-enhanced, A detection step for detecting the plasmon-enhanced electrochemiluminescence, An electrochemiluminescence immunoassay method comprising the following: <18> In the contact step, a composite nanoparticle body is formed in which two or more nanoparticle bodies are bonded via the test substance, and the composite nanoparticle body is captured on the working electrode side. <17> The electrochemiluminescence immunoassay method described below. <19> The aforementioned co-reactant is at least one selected from the group consisting of tripropylamine, triethylamine, and peroxosulfate ions. <17> or <18> The electrochemiluminescence immunoassay method described below. <20> The aforementioned metal nanoparticles consist of gold or silver, The working electrode comprises gold, silver, platinum, palladium, or indium tin oxide. <17> ~ <19> An electrochemiluminescence immunoassay method described in any one of the following. <21> The separation distance between the captured nanoparticles and the working electrode is 1 nm to 10 nm. <17> ~ <20> An electrochemiluminescence immunoassay method described in any one of the following. <22> In the contact step, a composite nanoparticle body is formed in which two or more nanoparticle bodies are bonded via the test substance, and the composite nanoparticle body is captured on the working electrode side. The separation distance between the nanoparticles in the composite nanoparticle body is 1 nm to 10 nm. <17> ~ <21> The electrochemiluminescence immunoassay method described below. <23> To generate the electrochemiluminescence described above without irradiating the sample with light from the outside, <17> ~ <22> An electrochemiluminescence immunoassay method described in any one of the following. [Explanation of Symbols]

[0127] 10. Nanoparticles 12. Metal nanoparticles 13...Polymer membrane 13X... Polymers that constitute polymer films 13a ···Bonding site via sulfur atom 13b ···Positively charged group 13c ···Hydrophobic group 14...specific binding substance 16. Electrochemiluminescent substances 10A ···First Nanoparticle 12A ···First metal nanoparticle 13A...First polymer membrane 14A...First specific binding substance 16A ···First electrochemiluminescent material 10B ···Second Nanoparticle 12B ···Second metal nanoparticles 13B...Second polymer membrane 14B...Second specific binding substance 16B ···Second Electrochemiluminescent Material 25...Metal thin film (working electrode) 251...electrode section 254...Third specific binding substance 70 ···Test substance 70A ···Metal thin film with nanoparticles (working electrode with nanoparticles) 70B ···Composite Nanoparticles 70C ···Metal thin film with composite nanoparticles (working electrode with composite nanoparticles) L1,L1',L2...Separation distance (separation distance)

Claims

1. Metal nanoparticles and A polymer film covering the surface of the metal nanoparticles, A specific binding substance is bonded to at least one of the metal nanoparticles and the polymer film, and specifically binds to the test substance in the sample, An electrochemiluminescent material bonded to at least one of the polymer film and the specific bonding material. A nanoparticle body comprising the following.

2. The nanoparticle material according to claim 1, which is a nanoparticle material used in an electrochemiluminescence immunoassay using a plasmon-enhanced field.

3. The nanoparticle body according to claim 1 or 2, which enhances the luminescence produced by the electrochemical reaction of the electrochemiluminescent material by plasmons.

4. A nanoparticle body according to claim 1 or 2, for use in an electrochemiluminescence immunoassay that does not utilize external light irradiation.

5. The nanoparticle body according to claim 1 or 2, wherein the electrochemiluminescent material is at least one selected from the group consisting of ruthenium complex, europium complex, iridium complex, osmium complex, and rhenium complex.

6. The nanoparticle body according to claim 1 or 2, wherein the specific binding substance is a nanoantibody.

7. The nanoparticle body according to claim 1 or 2, wherein the metal nanoparticles comprise gold or silver.

8. A metal thin film with nanoparticles bonded to it via a test substance is formed. The nanoparticle body according to claim 1 or 2, wherein the metal thin film has a specific bonding substance bonded to its surface.

9. The aforementioned nanoparticle body includes a first nanoparticle body and a second nanoparticle body. The nanoparticle body according to claim 1 or 2, wherein the first nanoparticle body and the second nanoparticle body are bonded together via a test substance to form a composite nanoparticle body.

10. The composite nanoparticles form a metal thin film with composite nanoparticles via a metal thin film and a test substance, The nanoparticle body according to claim 9, wherein the metal thin film has a specific bonding substance bonded to its surface.

11. The test substance is derived from a sample that is blood, plasma, urine, or saliva. The nanoparticle body according to claim 1 or 2.

12. The nanoparticle body according to claim 1 or 2, wherein the polymer constituting the polymer film includes a sulfur atom-mediated bonding site between itself and the surface of the metal nanoparticle.

13. The nanoparticle body according to claim 1 or 2, wherein the polymer forming the polymer film includes a sulfur atom-mediated bonding site in the side chain of the polymer between it and the surface of the metal nanoparticle.

14. The polymer constituting the polymer film further contains positively charged groups in the side chains of the polymer, The positively charged groups include primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH ２＋ ) NH ２ The nanoparticle body according to claim 1 or 2, which is at least one selected from the group consisting of ).

15. The polymer constituting the polymer film further contains hydrophobic groups in the side chains of the polymer, The hydrophobic group is selected from the group consisting of aromatic cyclic groups, aliphatic cyclic groups, and aliphatic chain groups. The nanoparticle body according to claim 1 or 2, wherein at least one of the following types is present.

16. The nanoparticle body according to claim 1 or 2, wherein the thickness of the polymer film is 1 nm to 10 nm.

17. An electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, A contact step of bringing a first solution containing the nanoparticles and the test substance according to claim 1 or 2 into contact with a working electrode having a specific binding substance, thereby bonding the nanoparticles and the working electrode via the test substance, A co-reactant supply step involves supplying a second solution containing co-reactants onto the working electrode, A voltage application step in which a voltage is applied to the working electrode to generate electrochemiluminescence, and the electrochemiluminescence is plasmon-enhanced, A detection step for detecting plasmon-enhanced electrochemiluminescence and An electrochemiluminescence immunoassay method comprising the following:

18. The electrochemiluminescence immunoassay method according to claim 17, wherein in the contact step, two or more nanoparticles are bound together via the test substance to form a composite nanoparticle body, and the composite nanoparticle body is captured on the working electrode side.

19. The electrochemiluminescence immunoassay method according to claim 17, wherein the co-reactant is at least one selected from the group consisting of tripropylamine, triethylamine, and peroxosulfate ions.

20. The aforementioned metal nanoparticles consist of gold or silver, The electrochemiluminescence immunoassay method according to claim 17, wherein the working electrode comprises gold, silver, platinum, palladium, or indium tin oxide.

21. The electrochemiluminescence immunoassay method according to claim 17, wherein the separation distance between the captured nanoparticles and the working electrode is 1 nm to 10 nm.

22. The electrochemiluminescence immunoassay method according to claim 18, wherein the separation distance between the nanoparticles in the composite nanoparticles is 1 nm to 10 nm.

23. The electrochemiluminescence immunoassay method according to claim 17, wherein the electrochemiluminescence is generated without irradiating the sample to be measured with light from an external source.

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