Electrochemiluminescence sensor, cell for mounting the electrochemiluminescence sensor, and measurement kit comprising these.

Abstract

To provide a small, easy-to-operate electrochemiluminescence sensor. [Solution] An electrochemiluminescence sensor for detecting a test substance in a sample using nanoparticles with an electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, comprising: a first substrate; an electrode provided on the surface of the first substrate, wherein a first specific binding substance that specifically binds to the test substance is bound to the surface of the electrode; the nanoparticles comprising: metal nanoparticles; a polymer film covering the surface of the metal nanoparticles; a second specific binding substance bound to at least one of the polymer film and the metal nanoparticles and specifically binding to the test substance; and an electrochemiluminescent substance bound to at least one of the polymer film and the second specific binding substance and capable of contacting a co-reactant, wherein the electrochemiluminescence sensor comprises: a first substrate; an electrode provided on the surface of the first substrate, wherein a first specific binding substance that specifically binds to the test substance is bound to the surface of the electrode; and an electrochemiluminescent substance that is bound to at least one of the polymer film and the second specific binding substance and capable of contacting a co-reactant.

Description

[Technical Field]

[0001] This disclosure relates to an electrochemiluminescence sensor, a cell for mounting the electrochemiluminescence sensor, and a measurement kit comprising the same. [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 the local plasmon-enhanced fluorescence sensor disclosed in Japanese Patent Publication No. 2008-216046 (Patent Document 1), excitation light is irradiated onto a fluorescently labeled composite, and fluorescence from the excited phosphor is detected. Furthermore, in the electrochemiluminescence immunoassay method disclosed in Japanese Patent Publication No. 2013-152215 (Patent Document 2), a primary antibody bound to magnetic particles and a secondary antibody labeled with an electrochemiluminescent substance form a complex in a flow system, sandwiching the test substance to be detected. The formed complex is captured by a magnetic field, and the co-reactants are further supplied to the flow system, and a voltage is applied. The resulting electrochemiluminescence is detected. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2008-216046 [Patent Document 2] Japanese Patent Publication No. 2013-152215 [Overview of the project] [Problems that the invention aims to solve]

[0005] By the way, after diligent investigation by the inventors into the measurement method described above, the following concerns were identified. Specifically, the local plasmon-enhanced fluorescence sensor disclosed in Patent Document 1 requires an excitation light source to excite the labeled phosphor, thus requiring a large measuring device and taking more time to obtain measurement results. Patent Document 2 requires a complex measuring device having a flow cell, a magnetic field generator, and a co-reactant supply unit, as well as a system for controlling these.

[0006] Based on the above, the inventors of the present invention have determined that there is room for improvement in the prior art, specifically in the large size and complexity of the measuring device, the time required for measurement (i.e., the need for a high level of skill from the operator), the length of the measurement time, and the large installation space occupied by the measuring device. Therefore, there is a desire for a measuring device that is smaller in size and easier to operate based on a simple structure.

[0007] Therefore, the primary object of this disclosure is to provide a small, easy-to-operate electrochemiluminescence sensor. Another object of this disclosure is to provide a cell for mounting such an electrochemiluminescence sensor. Yet another object of this disclosure is to provide a measurement kit comprising such an electrochemiluminescence sensor and a cell for the electrochemiluminescence sensor.

[0008] In light of these challenges, this disclosure has been diligently considered and, focusing on the fact that it does not require an excitation light source, has conceived of an electrochemiluminescence sensor for detecting electrochemiluminescence using a plasmon-enhanced field. That is, this disclosure includes the following embodiments. [Means for solving the problem]

[0009] An electrochemiluminescence sensor according to one embodiment of the present disclosure is An electrochemiluminescence sensor for detecting a test substance in a sample using nanoparticles, employing an electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, First substrate and An electrode provided on the surface of the first substrate, wherein a first specific binding substance that specifically binds to the test substance is bonded to the surface of the electrode and arranged thereon. comprising The nanoparticle body includes metal nanoparticles, a polymer film coating the surface of the metal nanoparticles, a second specific binding substance that is bound to at least one of the polymer film and the metal nanoparticles and specifically binds to the test substance, and an electrochemiluminescent substance that is bound to at least one of the polymer film and the second specific binding substance and is capable of contacting a co-reactant.

[0010] An electrochemiluminescent sensor cell according to another embodiment of the present disclosure is a cell for mounting the above-described electrochemiluminescent sensor, including a sensor holding part for detachably holding the electrochemiluminescent sensor, and a container housing capable of holding a second solution containing a co-reactant. comprising

[0011] A measurement kit according to another embodiment of the present disclosure comprises the above-described electrochemiluminescent sensor and the above-described electrochemiluminescent sensor cell.

Advantages of the Invention

[0012] The electrochemiluminescent sensor according to one embodiment of the present disclosure is small and easy to operate. Further, the electrochemiluminescent sensor cell according to another embodiment of the present disclosure can mount such an electrochemiluminescent sensor. Further, the measurement kit according to another embodiment of the present disclosure can comprise such an electrochemiluminescent sensor and an electrochemiluminescent sensor cell.

Brief Description of the Drawings

[0013] [Figure 1] FIG. 1 is a plan view showing an electrochemiluminescent sensor according to a first embodiment. [Figure 2] FIG. 2 is a cross-sectional view (cross-section I-I in FIG. 1) showing an electrochemiluminescent sensor according to a first embodiment. [Figure 3] FIG. 3 is a cross-sectional view schematically showing a nanoparticle body. [Figure 4]Figure 4 is a schematic cross-sectional view showing the working electrode (electrode with nanoparticles) of the electrochemiluminescence sensor according to the first embodiment in which nanoparticles are captured. [Figure 5] Figure 5 schematically illustrates the plasmon enhancement mechanism of electrochemiluminescence. [Figure 6] Figure 6 is a schematic cross-sectional view showing a composite nanoparticle body formed from nanoparticles. [Figure 7] Figure 7 is a schematic cross-sectional view showing the working electrode (electrode with composite nanoparticles) of an electrochemiluminescence sensor according to the first embodiment in which composite nanoparticles are captured. [Figure 8A] Figure 8A is a diagram illustrating the manufacturing method of the electrochemiluminescence sensor according to the first embodiment. [Figure 8B] Figure 8B is a diagram illustrating the manufacturing method of the electrochemiluminescence sensor according to the first embodiment. [Figure 8C] Figure 8C is a diagram illustrating the manufacturing method of the electrochemiluminescence sensor according to the first embodiment. [Figure 8D] Figure 8D is a diagram illustrating the manufacturing method of the electrochemiluminescence sensor according to the first embodiment. [Figure 8E] Figure 8E is a diagram illustrating the manufacturing method of the electrochemiluminescence sensor according to the first embodiment. [Figure 9] Figure 9 is an exploded perspective view showing an electrochemiluminescence sensor according to the second embodiment. [Figure 10] Figure 10 is a cross-sectional view (section II-II in Figure 9) showing an electrochemiluminescence sensor according to the second embodiment. [Figure 11] Figure 11 is an exploded perspective view showing an electrochemiluminescence sensor cell according to the third embodiment. [Figure 12] Figure 12 is a perspective view showing a cell for an electrochemiluminescence sensor according to the third embodiment. [Figure 13] Figure 13 is a perspective view showing an electrochemiluminescence sensor cell according to the third embodiment, mounted on an electrochemiluminescence immunoassay device. [Figure 14]Figure 14 is a cross-sectional view (section III-III in Figure 13) showing an electrochemiluminescence sensor cell according to the third embodiment, mounted on an electrochemiluminescence immunoassay device. [Figure 15] Figure 15 shows a reaction scheme illustrating an example of a method for producing nanoparticles. [Figure 16] Figure 16 is a schematic diagram illustrating the coating morphology of polymers by nanoparticles. [Figure 17] Figure 17 is a schematic diagram showing the nanoparticles of Example 1. [Modes for carrying out the invention]

[0014] Hereinafter, an electrochemiluminescence sensor, an electrochemiluminescence sensor cell, and a measurement kit comprising them, which are embodiments of the present disclosure, will be described in detail with reference to the illustrated embodiments. Note that the drawings include schematic representations and may not reflect actual dimensions or proportions.

[0015] The various numerical ranges referred to in this disclosure are intended to include the lower and upper numerical values ​​themselves, unless otherwise specified, such as "less than," "smaller than," and "greater than." In other words, for example, a numerical range of 1 nm to 10 nm should be interpreted as including both the lower limit of 1 nm and the upper limit of 10 nm.

[0016] The presence of polymer films in nanoparticles can be confirmed by imaging the nanoparticles using a scanning electron microscope (SEM) or transmission electron microscope (TEM), and observing the nanoparticles in the images.

[0017] Furthermore, the film thickness of the polymer film and the particle size of the metal nanoparticles can be obtained by using SEM or TEM to capture images of the nanoparticles, measuring the film thickness of the polymer film and the particle size of the metal nanoparticles in the images, and calculating the average value of multiple particle sizes (number of measurements: for example, at least 10).

[0018] In this specification, "above" broadly refers to a position in one direction from the surface of the member in question toward the outside of the member (for example, on an 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).

[0019] <First Embodiment: Electrochemiluminescence Sensor> The first embodiment relates to an electrochemiluminescence sensor. The electrochemiluminescence sensor according to the first embodiment is An electrochemiluminescence sensor for detecting a test substance in a sample using nanoparticles, employing an electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, First substrate and An electrode provided on the surface of a first substrate, wherein a first specific binding substance that specifically binds to the test substance is bonded to the surface of the electrode and arranged thereon. Equipped with, The nanoparticle body comprises metal nanoparticles, a polymer film coating the surface of the metal nanoparticles, a second specific binding substance bonded to the polymer film and specifically binding to the test substance, and an electrochemiluminescent substance bonded to the polymer film and capable of contacting the co-reactants.

[0020] [Mechanism of Action] The electrochemiluminescence sensor according to the first embodiment is small and easy to operate. The reason for this is not bound by any particular theory, but can be inferred as follows. The electrochemiluminescence sensor according to the first embodiment detects a test substance in a sample using nanoparticles containing an electrochemiluminescent material. For example, when a first solution containing the test substance and nanoparticles is applied to the electrochemiluminescence sensor, the first solution comes into contact with the nanoparticles and the electrode (more specifically, the working electrode). As a result, the nanoparticles are captured on the electrode side, forming an electrode with nanoparticles attached that are bound to the electrode via the test substance. Consequently, a space is formed in which the metal nanoparticles and the electrode are in close proximity, and a state is created in which the electrochemiluminescent material is present in the vicinity of this space. When a voltage is applied to this state under conditions in which a co-reactant is present, electrogenerated chemiluminescence (ECL) occurs, and this electrochemiluminescence is enhanced by localized surface plasmon resonance (LSPR, hereinafter also simply referred to as "plasmon resonance") generated by this. The electrochemiluminescence sensor can detect the electrochemiluminescence enhanced by plasmon resonance. In other words, the electrochemiluminescence sensor uses an electrochemiluminescence immunoassay method that utilizes a plasmon-enhancing field.

[0021] Thus, the electrochemiluminescence immunoassay method using the electrochemiluminescence sensor according to the first embodiment, which utilizes a plasmon-enhanced field, does not use external light irradiation (i.e., does not use a light source). Therefore, compared to fluorescence spectroscopy immunoassay, the electrochemiluminescence immunoassay method allows for the construction of a simpler measurement system and, consequently, a simpler apparatus configuration.

[0022] In addition, the electrochemiluminescence sensor comprises a first substrate and an electrode provided on the first substrate. Because the sensor configuration for measurement is so simple, it is compact and easy to operate. The electrochemiluminescence sensor does not require a flow cell, magnetic field generator, or co-reactant supply unit as disclosed in Patent Document 2.

[0023] The electrochemiluminescence sensor according to the first embodiment is small and easy to operate, making it portable. Furthermore, it does not require a highly knowledgeable and skilled operator (more specifically, a highly trained medical professional), and the measurement does not take much time. Therefore, the electrochemiluminescence sensor according to the first embodiment is suitable for use in medical settings where easy operation and rapid diagnosis are required, and is particularly suitable for immediate clinical testing (or simple rapid testing; Point of Care Testing (POCT)). In addition, the electrochemiluminescence sensor according to the first embodiment is disposable after use.

[0024] Furthermore, the electrochemiluminescence immunoassay method using the electrochemiluminescence sensor according to the first embodiment, which utilizes a plasmon-enhanced field, does not use external light irradiation, resulting in a low background signal and enabling high-sensitivity detection of the detected light (electrochemiluminescence).

[0025] The electrochemiluminescence sensor according to the first embodiment will be described with reference to Figures 1 and 2. Figure 1 is a plan view showing the electrochemiluminescence sensor according to the first embodiment. Figure 2 is a cross-sectional view (cross-sectional view II in Figure 1) showing the electrochemiluminescence sensor according to the first embodiment. In Figures 1 and 2, the direction perpendicular to the main surface of the electrochemiluminescence sensor is defined as the Z direction. The longitudinal direction of the main surface is defined as the Y direction, and the short direction of the main surface is defined as the X direction. The X, Y, and Z directions are orthogonal to each other.

[0026] The electrochemiluminescence sensor 1 according to the first embodiment is, for example, attached to an electrochemiluminescence immunoassay device. The electrochemiluminescence sensor 1 detects the test substance in the sample using an electrochemiluminescence immunoassay method that utilizes a plasmon-enhanced field.

[0027] The electrochemiluminescence sensor 1 according to the first embodiment comprises a first substrate 20 and electrodes (working electrode 31, counter electrode 32, reference electrode 33) provided on the surface of the first substrate 20. The electrochemiluminescence sensor 1 has a substantially rectangular shape when viewed from a direction perpendicular to its main surface (the surface of the first substrate 20) (in an XY plane view).

[0028] (1st base material) The first substrate 20 includes a first insulating substrate 21, grooves 22 disposed between electrodes 31, 32, and 33 placed on the first insulating substrate 21, a resist 23 covering the electrodes 31, 32, and 33 and a portion of the grooves 22, and a hook portion 24.

[0029] -groove- The groove 22 is positioned between electrodes 31, 32, and 33 (working electrode 31, counter electrode 32, and reference electrode 33), has a substantially concave shape in cross-sectional view (for example, ZX cross-sectional view), and electrically insulates the electrodes 31, 32, and 33.

[0030] -Register- The resist 23 covers the lead portions 312, 322, and 332 of the electrodes 31, 32, and 33 (working electrode 31, counter electrode 32, and reference electrode 33), allowing only the electrode portions 311, 321, and 331 to be exposed to the test substance solution, the first solution (containing the test substance and nanoparticles), and the second solution (containing co-reactants). The resist 23 also fills a portion of the groove 22 between the electrodes 31, 32, and 33, electrically insulating the electrodes 31, 32, and 33 (working electrode 31, counter electrode 32, and reference electrode 33). The resist 23 is mainly composed of resin. The resist 23 may further contain fillers. Examples of fillers include silica particles and / or magnetite oxide particles.

[0031] -Hook part- The hook portion 24 is provided on the substantially rectangular first base material 20 so as to protrude in both directions in the X direction. In the second embodiment described later, the electrochemiluminescence sensor 1 is hooked onto and fixed to the electrochemiluminescence sensor cell.

[0032] (electrode) Electrodes 31, 32, and 33 are thin films composed substantially of metal, with a thickness on the order of nanometers (e.g., 1 nm to 100 nm). Here, "substantially composed" means that the component in question is composed of a specific material in proportions of 97% or more, 99% or more, or 100% by mass.

[0033] The electrodes 31, 32, and 33 are provided on the surface of the first substrate 20 (specifically, the first insulating substrate 21). The electrodes 31, 32, and 33 have electrode portions 311, 321, and 331 located at one end of the electrochemiluminescence sensor 1, which is substantially rectangular in plan view, and terminal portions 313, 323, and 333 located at the other end, electrically connected to the electrode portions 311, 321, and 331 via lead portions 312, 322, and 332. The electrodes 31, 32, and 33 include a working electrode 31, a counter electrode 32, and a reference electrode (or reference electrode, comparison electrode, or reference electrode) 33. A first specific binding substance is bound to the surface of the working electrode 31. The first specific binding substance specifically binds to the test substance. The first specific binding substance is synonymous with the second specific binding substance 14 described later, except that the binding site is the surface of the electrode portion 311. Preferably, at least one of the first specific binding substance and the second specific binding substance 14 is a nano-antibody described later.

[0034] -Working electrode- The working electrode 31 has an electrode portion 311 and a terminal portion 313 that is electrically connected to the electrode portion 311 via a lead portion 312. The working electrode 31 is an electrode that performs the electrochemical reaction covered by this disclosure, and more specifically, an oxidation reaction is performed in the electrode portion 311.

[0035] The working electrode 31 is made of, for example, gold, silver, platinum, palladium, or indium tin oxide. The terminal portion 313 is electrically connected to the outside of the electrochemiluminescence sensor 1 (for example, the voltage application portion of an electrochemiluminescence immunoassay device). A first specific binding substance is bonded to and arranged on the surface of the electrode portion 311.

[0036] The working electrode 31 is a thin metal film with a thickness on the order of nanometers (e.g., a few nm to 100 nm). The working electrode 31 (or its electrode portion 311) induces plasmon resonance through interaction with light (e.g., visible light and near-infrared light).

[0037] The working electrode 31 is positioned apart from the counter electrode 32 and the reference electrode 33 by grooves 22, and is electrically insulated by the grooves 22 and the resist 23 filling the grooves 22. The counter electrode 32 and the reference electrode 33 are similarly electrically insulated.

[0038] - Opposite electrode - The counter electrode 32 has an electrode portion 321 and a terminal portion 323 that is electrically connected to the electrode portion 321 via a lead portion 322. The counter electrode 32 is the paired electrode of the working electrode 31, and the reduction reaction takes place at the electrode portion 321. The area of ​​the electrode portion 321 of the counter electrode 32 is larger than the area of ​​the electrode portion 311 of the working electrode 31. This relative size of the areas of the electrode portions 311 and 321 is set to allow the desired electrochemical reaction to proceed sufficiently at the working electrode 31.

[0039] -Reference electrode- The reference electrode 33 has an electrode portion 331 and a terminal portion 333 that is electrically connected to the electrode portion 331 via a lead portion 332. The presence of the reference electrode 33 in the sensor 1 creates a triode system (an electrode system composed of the working electrode 31, the counter electrode 32, and the reference electrode 33), which stabilizes the voltage applied to the working electrode 31 compared to a biode system (an electrode system composed of the working electrode 31 and the counter electrode 32). The reference electrode 33 is, for example, a silver / silver chloride electrode.

[0040] (Nanoparticle material) The nanoparticles 10 are used in the electrochemiluminescence sensor 1 to detect the test substance in the sample.

[0041] The nanoparticle body 10 will be described with reference to Figures 3 and 4. Figure 3 is a schematic cross-sectional view of the nanoparticle body 10. As shown in Figure 3, the nanoparticle body 10 comprises metal nanoparticles 12, a polymer film 13 covering the surface of the metal nanoparticles 12, a second specific binding substance 14 bonded to the polymer film 13 and specifically binding to the test substance 70, and an electrochemiluminescent substance 16 bonded to the polymer film 13 and in contact with the co-reactant.

[0042] Figure 4 is a schematic cross-sectional view showing the working electrode 31 (electrode with nanoparticles) of the electrochemiluminescence sensor 1 that has captured the nanoparticles 10. As shown in Figure 4, the nanoparticles 10 are bonded to the electrode portion 311 of the working electrode 31 via the (same) test substance 70 to form the electrode with nanoparticles 70A.

[0043] -Metal nanoparticles- The metal nanoparticles 12 are coated on their surface with a polymer film 13. Depending on the type of metal, the metal nanoparticles 12 interact with light having a specific wavelength, causing plasmon resonance. Silver nanoparticles have a plasmon resonance peak between 400 nm and 530 nm, while gold nanoparticles have a plasmon resonance peak 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 at a wavelength of 405 nm. Gold nanoparticles with a particle size of 20 nm resonate with light at 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 metal nanoparticles 12 preferably consist of gold or silver, and more preferably consist of silver. Although metal nanoparticles 12 have been described, metal nanoparticles 12A and 12B described later are synonymous with metal nanoparticles 12.

[0044] -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 nanoparticle body 10, 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 contacting the surface of the metal nanoparticles 12, and thus suppress the decrease in detection sensitivity.

[0045] The polymer film 13 (the polymer constituting the polymer film 13) may also include at least one selected from the group consisting of sulfur atom-mediated bonding sites, positively charged groups, and hydrophobic groups (for example, in its side chains) between it and the surface of the metal nanoparticles 12. The bonding sites bond between the surface of the metal nanoparticles 12 and the polymer film 13 (the polymer constituting the polymer film 13) via sulfur atoms.

[0046] The positively charged groups form electrostatic bonds (ionic bonds) with the surface of the negatively charged metal nanoparticles 12. The positively charged groups are preferably primary ammonium groups, secondary ammonium groups, tertiary ammonium groups, quaternary ammonium groups, and guanidyl groups (-NHC(=NH2 + It is at least one selected from the group consisting of )NH2).

[0047] The hydrophobic group forms a hydrophobic bond with the surface of the metal nanoparticle 12. The hydrophobic group is, for example, at least one selected from the group consisting of aromatic cyclic groups (more specifically, aromatic carbocyclic groups and aromatic heterocyclic groups), aliphatic cyclic groups, and aliphatic chain groups. An aromatic carbocyclic group is a group that does not contain an aromatic heterocyclic group and contains an aromatic ring in which all ring member atoms are carbon atoms, and examples include aryl groups (more specifically, phenyl groups, etc.) and arylalkyl groups (more specifically, benzyl groups, etc.). An aromatic heterocyclic group is a group that contains an aromatic ring in which at least one of the ring 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.

[0048] 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.

[0049] 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.

[0050] 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, for example, a near-field is more easily formed efficiently in the space between the electrode portion 311 and the metal nanoparticles 12 in the nanoparticle-attached electrode 70A. 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 polymer film 13 has been described, polymer films 13A and 13B described later are synonymous with polymer film 13.

[0051] -Electrochemiluminescent materials- The electrochemiluminescent material 16 is labeled on the polymer film 13. When a voltage is applied to the electrochemiluminescent material 16, it releases electrons through an oxidation reaction 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 to produce an excited state of electrochemiluminescent material 16. The excited state of electrochemiluminescent material 16 (excited species) relaxes and emits light (electrochemiluminescence).

[0052] The emitted electrochemiluminescence induces plasmon resonance on the surface of the electrode portion 311 and the metal nanoparticles 12 in the nanoparticle-attached electrode 70A. Furthermore, plasmon resonance may be induced on the surface of the electrode portion 311 and the metal nanoparticles 12A and 12B in the composite nanoparticle-attached electrode described later. The electrochemiluminescent material 16 in the excited state undergoes relaxation due to plasmon resonance and emits electrochemiluminescence.

[0053] 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. It emits electrochemiluminescence upon relaxation of the excited species, and the emitted electrochemiluminescence can induce plasmon resonance in the electrode portion 311 and the metal nanoparticles 12. 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, electrochemiluminescent materials 16A and 16B described later are synonymous with electrochemiluminescent material 16.

[0054] -Second specific binding substance- The second specific binding substance 14 is a nano-sized substance (with a maximum size of 3-15 nm) that specifically binds to the test substance 70 in the sample.

[0055] The second 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 second specific binding substance 14 is preferably a nanoantibody. In the first embodiment, the nanoparticle body 10 to which such a second specific binding substance 14 is bound exhibits superior detection sensitivity. For example, a nanoantibody as the second specific binding substance 14 specifically binds to an antigen, such as the test substance 70, at its tip (antigen binding site) through an antigen-antibody reaction, forming a complex. A ligand as the second specific binding substance 14 specifically binds to a protein, such as the test substance 70, through a ligand-receptor reaction, forming a complex. A nucleic acid chain as the second specific binding substance 14 forms a pair of nucleic acid chains (double helix) with complementary nucleic acid chains based on base pair complementarity. The enzyme, as the second specific binding substance 14, forms an enzyme-substrate complex with the substrate, the test substance 70, at its active site (active site) based on substrate specificity (stereospecificity). These specific bindings are non-covalent bonds, such as hydrogen bonds, as well as bonds resulting from intermolecular forces, hydrophobic interactions, and charge interactions.

[0056] 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 (and further the separation distances L1', L2 in the composite nanoparticle electrode described later) at the nanoparticle-attached electrode 70A, more efficiently form a near field, and further increase the intensity of electrochemiluminescence.

[0057] The second specific binding substance 14 may be directly bound to the polymer film 13, and may also 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.). Although the second specific binding substance 14 has been described, the second specific binding substances 14A and 14B described later are synonymous with the second specific binding substance 14.

[0058] The types of specific binding substances 14A, 14B, and 314 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 4 as an example, if the test substance 70 is a monomeric antigen, it may be necessary that the second specific binding substance 14A, which specifically binds to the same test substance 70, and the first specific binding substance 314 of the working electrode 31 be 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 second specific binding substances 314 and 14A, and the first and second specific binding substances 314 and 14A are of the same type, only one of the first or second specific binding substances 314 and 14A 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 second specific binding substances 314 and 14A are of the same type, the working electrode 70A with nanoparticles can be properly formed. The same applies to the types of the first and second specific binding materials 314, 14A, and 14B in the working electrode 70C with composite nanoparticles.

[0059] [Detection of test substance: Measurement method for electrochemiluminescence] In addition to Figures 1-4, an example of a method for detecting a test substance 70 using the electrochemiluminescence sensor 1 (a method for measuring electrochemiluminescence) will be described with reference to Figure 5 and Scheme 1. Figure 5 and Scheme 1 show the plasmon enhancement mechanism of electrochemiluminescence (co-reaction type electrochemiluminescence) in the electrochemiluminescence measurement method using the electrochemiluminescence sensor 1 according to the first embodiment.

[0060] A method for measuring electrochemiluminescence using the electrochemiluminescence sensor 1 includes, for example, a step of applying a solution of the substance to be tested, a contact step, a co-reactant supply step, a voltage application step, and a detection step. This measurement method may further include a cleaning step after the contact step.

[0061] (Test substance application process) The test substance application step involves applying a first solution containing the test substance 70 and nanoparticles 10 to the electrochemiluminescence sensor 1. Specifically, in the test substance application step, first, the nanoparticles 10 are mixed with the test substance solution containing the test substance to prepare the first solution. Next, the first solution is dropped onto the electrode portion 311 of the working electrode 31 of the electrochemiluminescence sensor 1.

[0062] In this specification, a test substance solution refers to a solution containing the test substance 70 to be detected, and includes not only the sample (taken directly from the individual being measured) itself, but also solutions prepared from the sample. Examples of solutions prepared from a sample include solutions (sample diluents) obtained by adding a solvent and buffer (more specifically, phosphate-buffered saline (PBS), Tris buffer, HEPES buffer, MOPS buffer, and MES buffer, etc.) to the sample for viscosity adjustment, etc., and solutions (test substance extracts) obtained by extracting the test substance 70 from the sample. The test substance 70 is the substance to be detected contained in the sample. Examples of the test substance 70 include antigens, proteins, substrates, and nucleic acid chains. Examples of samples containing the test substance 70 include blood, plasma, serum, urine, and saliva.

[0063] (contact process) In the contact step, a first solution containing nanoparticles 10 and the test substance 70 is brought into contact with the working electrode 31 (the electrode portion 311) having a first specific binding substance 314, thereby bonding the nanoparticles 10 and the working electrode 31 (the electrode portion 311) via the test substance 70.

[0064] Upon contact of the first solution with the electrode portion 311, as shown in Figure 4, the test substance 70 in the first solution is captured by the first specific binding substance 314 of the electrode portion 311. The nanoparticles 10 specifically bind to the test substance 70 captured by the first specific binding substance 314 of the electrode portion 311. As a result, the nanoparticles 10 are captured by the electrode portion 311, and an electrode 70A with nanoparticles is formed. In other words, upon contact of the first solution containing the test substance 70 and nanoparticles 10 with the working electrode 31, the nanoparticles 10 are captured on the working electrode 31 side via the test substance 70, thereby forming an electrode 70A with nanoparticles.

[0065] (Washing process) This measurement method may further include a cleaning step after the contact step, in which the working electrode 31 (the electrode portion 311) is cleaned. This removes unreacted substances (e.g., nanoparticles 10, test substance 70, and composite nanoparticles 70B) from the electrode portion 311, thereby improving the signal-to-noise ratio (S / N ratio). The cleaning of the electrode portion 311 can be performed, for example, by immersing the electrode portion 311 of the electrochemiluminescence sensor 1 in a cleaning solution (e.g., pure water and buffer solution). The cleaning can be performed multiple times. Alternatively, the cleaning of the electrode portion 311 can also be performed by pouring the cleaning solution onto the electrode portion 311 of the electrochemiluminescence sensor 1 to remove unreacted substances. After cleaning the electrode section 311, the electrode section 311 is dried. For example, drying after cleaning is carried out in the dark at room temperature for 30 minutes to 1 hour.

[0066] (Co-reactant supply process) In the co-reactant supply step, a second solution containing the co-reactant is supplied onto the working electrode 31. Here, the co-reactant is a substance that excites the electrochemiluminescent material 16 in the nanoparticle-attached electrode 70A by an electrochemical reaction. The co-reactant is, for example, at least one selected from the group consisting of tripropylamine, triethylamine, and peroxosulfate ions.

[0067] (Voltage application process) In the voltage application step, a voltage is applied to the working electrode 31 to generate electrochemiluminescence, and the electrochemiluminescence is plasmon-enhanced. After the co-reactant supply step, an electrode 70A with nanoparticles is present, and the metal nanoparticles 12 and the electrode portion 311 are arranged in close proximity to each other on the electrode 70A with nanoparticles. Furthermore, co-reactants are present around the electrochemiluminescent material 16 of the electrode 70A with nanoparticles, and the electrochemiluminescent material 16 is able to physically come into contact with the co-reactants.

[0068] When a voltage is applied to the working electrode 31, see Figure 5 and Scheme 1: [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 nanoparticle 12 and electrode portion 311 in the nanoparticle-attached electrode 70A. * M represents the plasmon resonance-excited metal nanoparticles 12 and the plasmon resonance-excited electrode portion 311 in the nanoparticle-attached electrode 70A. Note that the symbols in Figure 5 also represent the same chemical species as the symbols in Scheme 1. As shown in the elementary processes (1) and (3), the electrochemiluminescent substance (ECLM) 16 and the coreactant (CR) in the electrode 70A with the nanoparticle body are oxidized by voltage application, and the oxide of the electrochemiluminescent substance 16 (ECLM + ) and the oxide of the coreactant (the radical cation CR of the coreactant ·+ ) are respectively generated. Here, as shown in the elementary process (2), the oxide of the coreactant dissociates protons to generate the radical of the coreactant (CR · ).

[0069] As shown in the elementary process (4), the oxide of the electrochemiluminescent substance 16 (ECLM + ) reacts (electron exchange reaction) with the radical of the coreactant (CR · ) to generate the electrochemiluminescent substance in the excited state ( * ECLM) 16 and the product of the coreactant (CR product).

[0070] As shown in the elementary process (5), a part of the electrochemiluminescent substance 16 in the excited state relaxes to emit electrochemiluminescence. As shown in the elementary process (6), the emitted electrochemiluminescence is irradiated onto the metal nanoparticles 12 and the electrode portion 311 in the electrode 70A with the nanoparticle body. Thereby, surface plasmon resonance is induced on the surfaces of the metal nanoparticles 12 and the electrode portion 311 (particularly, the space between the metal nanoparticles 12 and the electrode portion 311), and the metal nanoparticles 12 and the electrode portion 311 ([[]] * M) in the plasmon resonance excitation state are generated. The relaxation of other electrochemiluminescent substances 16 in the excited state is induced by the surface plasmon resonance (induced relaxation), and the luminescence quantum efficiency (luminescence quantum yield) of the electrochemiluminescence is improved (that is, the electrochemiluminescence is plasmon-enhanced).

[0071] (Detection step) In the detection step, the plasmon-enhanced electrochemiluminescence is detected. Specifically, in the detection step, the plasmon-enhanced electrochemiluminescence in the voltage application step travels outside the electrochemiluminescence sensor 1 and is detected by the optical detection unit of the electrochemiluminescence immunoassay device.

[0072] 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 working electrode 31 in the nanoparticle-attached electrode 70A is small, in a range in which the excited electrochemiluminescent material 16 is less likely to be quenched. Separation distance L1 refers to the shortest distance between the surface of the metal nanoparticles 12 and the surface of the electrode portion 311 in the nanoparticle-attached electrode 70A. When the separation distance L1 is small, the metal nanoparticles 12 and the electrode portion 311 are positioned closer together in the nanoparticle-attached electrode 70A. Such close proximity effectively forms a near field and contributes to increasing the intensity of electrochemiluminescence. Therefore, in the nanoparticle-attached electrode 70A, the intensity of electrochemiluminescence can be further increased when the separation distance L1 is small. The separation distance L1 between the captured nanoparticles 10 and the working electrode 31 (electrode portion 311) in the nanoparticle-attached electrode 70A is 1 nm to 10 nm.

[0073] 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 working electrode 31 in the nanoparticle-attached electrode 70A, as shown in Figure 4. The space between the metal nanoparticles 12 and the working electrode 31 in the nanoparticle-attached electrode 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 working electrode 31 in the nanoparticle-attached electrode 70A, the intensity of electrochemiluminescence can be further increased.

[0074] (Plasmon enhancement of electrochemiluminescence by electrodes with composite nanoparticles) As described above, electrochemiluminescence is plasmon-enhanced by an electrode 70A with nanoparticles 10 trapped on the working electrode 31 of the electrochemiluminescence sensor 1 according to the first embodiment (Figures 1-5 and Scheme 1). In addition, electrochemiluminescence can also be plasmon-enhanced by an electrode with composite nanoparticles trapped on the working electrode 31 of the electrochemiluminescence sensor 1. Refer to Figures 6-7 in addition to Figures 1-5 and Scheme 1 to explain the plasmon enhancement of electrochemiluminescence by an electrode with composite nanoparticles. Figure 6 is a schematic cross-sectional view showing a composite nanoparticle formed from nanoparticles 10. Figure 7 is a schematic cross-sectional view showing the working electrode 31 (electrode with composite nanoparticles) of the electrochemiluminescence sensor 1 according to the first embodiment with composite nanoparticles trapped.

[0075] In the test substance application step of the electrochemiluminescence measurement method, a first solution containing the test substance and nanoparticles 10 is prepared. During the preparation of the first solution, the second specific binding substances 14A and 14B of the nanoparticles 10A and 10B can specifically bind to the (same) test substance 70. As a result, as shown in Figure 6, a composite nanoparticle 70B can be formed in which nanoparticles 10A and 10B are bound via the (same) test substance 70. In this case, the first solution contains the test substance 70 and nanoparticles 10, as well as the composite nanoparticle 70B. The composite nanoparticle 70B is formed in which nanoparticles 10A and 10B are bound via the (same) test substance 70.

[0076] In the contact step, the first solution comes into contact with the electrode portion 311 of the working electrode 31. As shown in Figure 7, the contact of the first solution with the electrode portion 311 causes the composite nanoparticles 70B to be captured on the working electrode 31, thereby forming the electrode 70C with the composite nanoparticles. Specifically, the second specific binding substance 14A in the composite nanoparticles 70B specifically binds to the test substance 70 captured by the first specific binding substance 314 of the electrode portion 311 of the working electrode 31 (similarly, in other embodiments, the second specific binding substance 14B in the composite nanoparticles 70B can also specifically bind to the test substance 70 captured by the first specific binding substance 314 of the electrode portion 311). As a result, the composite nanoparticles 70B are captured by the working electrode 31, and the electrode 70C with the composite nanoparticles is formed.

[0077] In the co-reactant supply step, the second solution is supplied onto the working electrode 31 (electrode portion 311). Due to the contact caused by the movement of the second solution onto the working electrode 31, the electrochemiluminescent materials 16A and 16B of the composite nanoparticle electrode 70C can come into (physical) contact with the co-reactant. In this way, the electrochemiluminescent material 16A between the composite nanoparticle 70B and the working electrode 31 (electrode portion 311), and the electrochemiluminescent materials 16A and 16B between the nanoparticles 10A and 10B of the composite nanoparticle electrode 70C come into contact with the co-reactant.

[0078] In the charge application step, electrochemiluminescence is plasmon-enhanced by the electrode 70C with composite nanoparticles, as shown below. The electrode 70C with composite nanoparticles has a sandwich structure in which the composite nanoparticles 70B and the working electrode 31 sandwich the test substance 70. In the electrode 70C with composite nanoparticles, the metal nanoparticles 12A and the electrode portion 311 are arranged in close proximity. Therefore, when the generated electrochemiluminescence is irradiated onto the metal nanoparticles 12A and the electrode portion 311, which are in close proximity to each other, plasmon resonance occurs, and a near-field is efficiently formed near the surfaces of the metal nanoparticles 12A and the electrode portion 311 (particularly near the space between the metal nanoparticles 12A and the electrode portion 311).

[0079] Furthermore, the electrode 70C with composite nanoparticles has a sandwich structure in which nanoparticles 10A and 10B sandwich the test substance 70 within the composite nanoparticles 70B. In the electrode 70C with composite nanoparticles, metal nanoparticles 12A and 12B are arranged in close proximity. Therefore, when the generated electrochemiluminescence is irradiated onto the metal nanoparticles 12A and 12B which are in close proximity to each other, plasmon resonance occurs, and a near-field is efficiently formed near the surfaces of the metal nanoparticles 12A and 12B (particularly near the space between the metal nanoparticles 12A and 12B).

[0080] The plasmon enhancement mechanism of electrochemiluminescence by the composite nanoparticle electrode 70C is explained in the same manner as in Figure 5 and Scheme 1, which show the plasmon enhancement mechanism of electrochemiluminescence by the nanoparticle electrode 70A, except that ECLM is changed from the electrochemiluminescent material 16 in the nanoparticle electrode 70A to the electrochemiluminescent materials 16A and 16B in the composite nanoparticle electrode 70C, and M is changed from the metal nanoparticles 12 and electrode portion 311 in the nanoparticle electrode 70A to the metal nanoparticles 12A and 12B and electrode portion 311 in the composite nanoparticle electrode 70C.

[0081] 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 (metal nanoparticles 12A of nanoparticle body 10A in Figure 7) and the working electrode 31 in the electrode 70C with composite nanoparticle body is small, in a range in which the excited electrochemiluminescent material 16A is less likely to be quenched. In this specification, the separation distance L1' between the metal nanoparticles 12A and the working electrode 31 refers to the shortest distance between the surface of the metal nanoparticles 12A and the surface of the electrode portion 311. When the separation distance L1' is small, the metal nanoparticles 12A and the electrode portion 311 are positioned closer together in the electrode 70C with composite nanoparticle body. Such close proximity effectively forms a near field and effectively contributes to the intensity of electrochemiluminescence. Therefore, in the electrode 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.

[0082] 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 electrode 70C with the composite nanoparticles (in Figure 7, between the metal nanoparticle 12A of nanoparticle 10A and the metal nanoparticle 12B of nanoparticle 10B) is small, in a range in which the excited electrochemiluminescent materials 16A and 16B are less likely to be quenched. The separation distance L2 refers to the shortest distance between the surface of the metal nanoparticle 12A and the surface of the metal nanoparticle 12B in this specification. When the separation distance L2 is small, the metal nanoparticles 12A and 12B are positioned closer together in the electrode 70C with the composite nanoparticles. Such close proximity effectively forms a near field and effectively contributes to the intensity of electrochemiluminescence. Therefore, in the electrode 70C with the composite nanoparticles, the intensity of electrochemiluminescence can be further increased when the separation distance L2 is small. The separation distance L2 is, for example, 1 nm to 10 nm.

[0083] In a preferred embodiment, from the viewpoint of further increasing the intensity of electrochemiluminescence, the electrochemiluminescent material 16A is arranged at least between the metal nanoparticles 12A and the electrode portion 311 in the composite nanoparticle electrode 70C, as shown in Figure 7. The space between the metal nanoparticles 12A and the electrode portion 311 in the composite nanoparticle electrode 70C is a space where a near field is efficiently generated. When the electrochemiluminescent material 16A is arranged at least between the metal nanoparticles 12A and the electrode portion 311 in the composite nanoparticle electrode 70C, the intensity of electrochemiluminescence can be further increased.

[0084] [Manufacturing method for electrochemiluminescence sensors] An example of a method for manufacturing the electrochemiluminescence sensor 1 according to the first embodiment will be described with reference to Figures 8A to 8E. Figures 8A to 8E are diagrams illustrating the method for manufacturing the electrochemiluminescence sensor 1. The method for manufacturing the electrochemiluminescence sensor 1 includes a conductive layer formation step, an electrode pair formation step, a reference electrode formation step, a resist formation step, a specific bonding substance immobilization step, and a dicing step. In this example, a mother assembly containing the electrochemiluminescence sensors 1 is fabricated, and then the electrochemiluminescence sensors 1 are obtained by dicing them into individual pieces.

[0085] (Conductive layer formation process) In the conductive layer formation process, a conductive layer is formed on the first insulating substrate 21. Specifically, as shown in Figure 8A, in the conductive layer formation process, a conductive layer (for example, a metal layer or metal oxide layer composed of gold, silver, platinum, palladium, or indium tin oxide) 30' is formed on the entire surface of the first insulating substrate 21 using a sputtering method. Furthermore, the first insulating substrate 21 may be pre-annealed to suppress expansion and contraction in subsequent processes. The reference holes 25 are located near both ends in the longitudinal direction of the first insulating substrate 21 and serve as reference holes for processing in subsequent processes.

[0086] (Electrode pair formation process) In the electrode pair formation process, the conductive layer 30' is patterned to form an electrode pair (working electrode 31 and counter electrode 32). Specifically, as shown in Figure 8B, a laser ablation method is used to draw the electrode (working electrode 31, counter electrode 32, reference electrode 33) pattern and the outline pattern of the first substrate 20 on the conductive layer 30' on the first insulating substrate 21 using laser light. This removes a portion of the conductive layer 30' to form a roughly concave groove 22 (in the ZX cross-section) that electrically insulates the electrodes, thereby forming the working electrode 31 and counter electrode 32.

[0087] (Reference electrode formation process) In the reference electrode formation process, the reference electrode 33 is formed. Specifically, as shown in Figure 8C, an Ag / AgCl layer is laminated on the conductive layer 30' to form the electrode portion 331 of the reference electrode 33. This forms the reference electrode 33. In the formation of the Ag / AgCl layer, a mixed dispersion containing Ag particles and AgCl particles is screen printed to form a coating film, and then the coating film is subjected to heat treatment. This forms the silver / silver chloride electrode that serves as the reference electrode 33.

[0088] (Resist formation process) In the resist formation process, a resist 23 is formed. Specifically, as shown in Figure 8D, in the resist formation process, a liquid containing an insulating material is applied to the lead portions 312, 322, 332 of the electrodes 31, 32, 33 (working electrode 31, counter electrode 32, and reference electrode 33) and to a portion of the groove 22 between the electrodes 31, 32, 33 to form a coating film, and the coating film is subjected to a heat treatment to form the resist 23.

[0089] (Specific binding substance immobilization process) In the specific bonding substance immobilization step, the first specific bonding substance 314 is fixed to the surface of the electrode portion 311 of the working electrode 31. Specifically, as shown in Figure 8E, for example, the first specific bonding substance 314 having a disulfide bond is brought into contact with the surface of the electrode portion 311 to form a bonding site in which the first specific bonding substance 314 bonds to the surface of the electrode portion 311 via sulfur atoms. This fixes the first specific bonding substance 314 to the surface of the electrode portion 311 of the working electrode 31.

[0090] (Dicing process) In the dicing process, the electrochemiluminescence sensor 1 is divided into individual pieces by dicing. Specifically, the electrochemiluminescence sensor 1 is divided into individual pieces by dicing (for example, using a pinnacle blade®) according to the outer shape of the sensor to obtain the electrochemiluminescence sensor 1 shown in Figures 1 and 2.

[0091] <Second Embodiment: Electrochemiluminescence Sensor> The second embodiment relates to an electrochemiluminescence sensor. The second embodiment differs from the first embodiment mainly in that it further comprises a nanoparticle body supported on a first substrate and a second substrate positioned opposite the first substrate via a spacer. The second embodiment will mainly describe this difference. Reference numerals that are the same as those in the first embodiment will generally not be described, as they have the same configuration as in the first embodiment.

[0092] The electrochemiluminescence sensor according to the second embodiment will be described with reference to Figures 9 and 10. Figure 9 is an exploded perspective view showing the electrochemiluminescence sensor according to the second embodiment. Figure 10 is a cross-sectional view (section II-II in Figure 9) showing the electrochemiluminescence sensor according to the second embodiment. As shown in Figures 9 and 10, the electrochemiluminescence sensor 1A according to the second embodiment further comprises a nanoparticle body 10 supported on a first substrate 20 and a second substrate 40 positioned opposite the first substrate 20 via a spacer 50. The electrochemiluminescence sensor 1A is provided with a flow channel 60 having an open space by the first substrate 20, the second substrate 40 and the spacer 50.

[0093] (Second base material) The second substrate 40 is positioned opposite the first substrate 20 via a spacer 50. The second substrate 40 has a second insulating substrate 41 and a first opening 42 provided in the second insulating substrate 41.

[0094] The second substrate 40 (the second insulating substrate 41) is transparent. That is, the second substrate 40 can transmit visible light. As a result, the resulting electrochemiluminescence can be received by the optical detection unit of the electrochemiluminescence immunoassay device located outside the electrochemiluminescence sensor 1A.

[0095] The first opening 42 is positioned opposite the working electrode 31. Since the first opening 42 is in communication with the flow path 60, the test substance solution can be easily introduced from the first opening end 611 into the flow path 60 and moved to the electrode 311.

[0096] (Spacer) The spacer 50 is laminated with the first base material 20 and the second base material 40, and the first base material 20 and the second base material 40 are separated by the thickness of the spacer 50. The spacer 50 has a second opening 52. The second opening 52 constitutes part of the flow path 60. The second opening 52 has a linear shape parallel to the Y direction.

[0097] (Flow channel) The flow path 60 comprises a first flow path 61 and a second flow path 62 communicating with the first flow path 61. The first flow path 61 occupies region R1 in the flow path 60 and extends from a first open end 611 to the working electrode 31 (electrode portion 311). The second flow path 62 occupies region R2 in the flow path 60 and extends from a second open end 621 to the working electrode 31 (electrode portion 311). At least a portion of the electrode portions 311, 321, and 331 of the electrodes 31, 32, and 33 (working electrode 31, counter electrode 32, and reference electrode 33) are exposed on the inner wall of the flow path 60.

[0098] The channel 60 also communicates from the first open end 611 to the second open end 621. The first channel 61 can introduce the test substance solution containing the test substance from outside the electrochemiluminescence sensor 1 into the inside (channel 60) and move it towards the electrode portion 311 of the working electrode 31. The first open end 611 of the first channel 61 is the inlet for the test substance solution. Nanoparticles 10 are supported between the first open end 611 and the working electrode 31 (electrode portion 311) in the first channel 61. As a result, the test substance solution can move along the first channel 61 in direction D1, come into contact with the nanoparticles 10, dissolve and / or disperse, and prepare a first solution (containing the test substance and nanoparticles) in the first channel 61, which can then reach the electrode portion 311. In other words, the first channel 61 can move the first solution from the first open end 611 side to the working electrode 31 (electrode portion 311) side.

[0099] The working electrode 31 (the electrode portion 311) and the first solution are in contact with each other via a channel 60 (first channel 61). Upon contact of the first solution with the working electrode 31 (the electrode portion 311), the nanoparticles 10 are captured on the working electrode 31 (the electrode portion 311) side via the test substance 70, thereby forming an electrode 70A with nanoparticles. Furthermore, through the same contact, composite nanoparticles 70B may be captured on the working electrode 31 (the electrode portion 311) side via the test substance 70, thereby forming an electrode 70C with composite nanoparticles. The channel 60 has a substantially rectangular parallelepiped shape parallel to the Y direction.

[0100] Similarly, the second solution containing the co-reactants can also reach the electrode portion 311 by passing through the first channel 61 from outside the electrochemiluminescence sensor 1. In other words, the first channel 61 can move the second solution from the first open end 611 side to the working electrode 31 (the electrode portion 311). In the first channel 61, the second solution (containing the co-reactants) can move from the first open end 611 side to the working electrode 31 side. As a result of the contact that occurs when the second solution moves to the working electrode 31 (the electrode portion 311), the electrochemiluminescent material 16 of the nanoparticle-attached electrode 70A comes into contact with the co-reactants. Furthermore, through this contact, the electrochemiluminescent materials 16A and 16B of the composite nanoparticle-attached electrode 70C can come into contact with the co-reactants.

[0101] The channel 60 is a capillary channel, and the test substance solution and the second solution can move along direction D1 from the first open end 611, which is the inlet, to the electrode section 311 by capillary action. Therefore, no special components are required for injecting the test substance solution and the second solution into the electrochemiluminescence sensor 1A or for their movement to the electrode section 311 after injection. Thus, the electrochemiluminescence sensor 1A is small and easy to operate. Regarding the thickness of the channel 60, as shown in Figure 10, the length T1 in the Z direction from the bottom surface of the second substrate 40 to the top surface of the electrode sections 311, 321, 331 (working electrode 31, counter electrode 32, reference electrode 33) of the first substrate 20 is 0.05 to 0.5 mm, and the length T2 in the Z direction from the bottom surface of the second substrate 40 to the top surface of the resist 23 is 0.05 to 0.5 mm. Note that in Figure 10, T1 and T2 are shown as having different thicknesses for the sake of explanation, but the thickness of the resist 23 (length in the Z direction) is much smaller than T1 and T2. Therefore, please note that the numerical ranges of T1 and T2 are equivalent.

[0102] (Nanoparticle material) The nanoparticles 10 are supported (in a dry state) on the first substrate 20 (more specifically, between the first open end 611 in the flow channel 60 and the electrode portion 311 of the working electrode 31).

[0103] [Detection of test substance: Measurement method for electrochemiluminescence] A method for measuring electrochemiluminescence using the electrochemiluminescence sensor 1A includes, for example, a step of applying a solution of the substance to be tested, a contact step, a co-reactant supply step, a voltage application step, and a detection step.

[0104] (Test substance application process) The test substance application step involves applying a test substance solution containing the test substance 70 to the electrochemiluminescence sensor 1A. Specifically, in the test substance application step, the test substance solution is dropped onto the first open end 611 of the electrochemiluminescence sensor 1A.

[0105] (contact process) In the contact process, the test substance solution dropped onto the first open end 611 is moved into the flow channel 60 from the first open end 611, which is the inlet, by capillary action, and then to the electrode section 311. During this movement of the test substance solution from the first open end 611 to the electrode section 311, the test substance solution first comes into contact with the nanoparticles 10 arranged in the flow channel 60, and a first solution containing the test substance 70 and the nanoparticles 10 is prepared.

[0106] Next, the prepared first solution comes into contact with the electrode portion 311 of the working electrode 31. In this way, in the electrochemiluminescence sensor 1A, the working electrode 31 and the first solution containing the test substance 70 and the nanoparticles 10 are able to come into contact with each other via the flow path 60. Upon contact of the first solution with the working electrode 31, the nanoparticles 10 are captured on the working electrode 31 side via the test substance 70, thereby forming an electrode 70A with nanoparticles. Furthermore, through the above contact, composite nanoparticles 70B may be captured on the working electrode 31 side via the test substance 70, thereby forming an electrode 70C with composite nanoparticles.

[0107] (Co-reactant supply process) In the co-reactant supply process, the second solution is introduced from the first open end 611 of the flow channel 60 and moved onto the electrode section 311. The second solution can move within the flow channel 60 by capillary action. In this way, the second solution can move from the first open end 611 side to the working electrode 31 side in the first flow channel 61 of the electrochemiluminescence sensor 1. Contact arising from the movement of the second solution to the working electrode 31 allows the electrochemiluminescent material 16 of the nanoparticle-attached electrode 70A to come into (physical) contact with the co-reactant. Furthermore, this contact may allow the electrochemiluminescent materials 16A and 16B of the composite nanoparticle-attached electrode 70C to come into (physical) contact with the co-reactant.

[0108] (Detection process) In the detection process, plasmon-enhanced electrochemiluminescence proceeds outside the electrochemiluminescence sensor 1 via the second substrate 40 and is detected by the optical detection unit of the electrochemiluminescence immunoassay device.

[0109] [Manufacturing method for electrochemiluminescence sensors] An example of a method for manufacturing the electrochemiluminescence sensor 1A according to the second embodiment will be described. The method for manufacturing the electrochemiluminescence sensor 1A further includes a nanoparticle loading step and a channel formation step between the specific binding substance immobilization step and the dicing step of the method for manufacturing the electrochemiluminescence sensor 1 according to the first embodiment.

[0110] (Nanoparticle loading process) In the nanoparticle loading process, the (dry) nanoparticles 10 are loaded onto the first substrate 20. Specifically, in the first channel 61 of the channel 60 to be formed in a subsequent process, the nanoparticles 10 are positioned on the opposing electrode 32 (electrode portion 321) of the first substrate 20 so as to be located in front of the electrode portion 311, moving from the first opening end 611 side to the electrode portion 311 side.

[0111] (Flow channel formation process) In the channel formation process, the spacer 50 and the second base material 40 are laminated onto the first base material 20. Specifically, the spacer 50 having a predetermined pattern is laminated onto the first base material 20, and the second base material 40 is laminated on top of the spacer 50. This forms the channel 60.

[0112] <Third Embodiment: Cell for Electrochemiluminescence Sensor> The third embodiment relates to a cell for an electrochemiluminescence sensor. In the third embodiment, the same reference numerals as in the first embodiment have the same configuration as in the first embodiment, and therefore, their description will be omitted in principle.

[0113] The cell for the electrochemiluminescence sensor according to the third embodiment is: A cell for mounting an electrochemiluminescence sensor 1 according to the first embodiment, A sensor holding part that detachably holds the electrochemiluminescence sensor 1, A container housing capable of holding the second solution containing the co-reactants and It is equipped with.

[0114] The electrochemiluminescence sensor cell according to the third embodiment will be described with reference to Figures 11 to 14. Figure 11 is an exploded perspective view showing the electrochemiluminescence sensor cell according to the third embodiment. Figure 12 is a perspective view showing the electrochemiluminescence sensor cell according to the third embodiment. Figure 13 is a perspective view showing the electrochemiluminescence sensor cell according to the third embodiment mounted on an electrochemiluminescence immunoassay device. Figure 14 is a cross-sectional view (section III-III in Figure 13) showing the electrochemiluminescence sensor cell according to the third embodiment mounted on an electrochemiluminescence immunoassay device.

[0115] The electrochemiluminescence sensor cell 2 is a cell for mounting the electrochemiluminescence sensor 1 according to the first embodiment. The electrochemiluminescence sensor cell 2 comprises a sensor holding portion 80 and a container housing 90. The sensor holding portion 80 is a male-type member and is fitted and connected to the female-type member of the container housing 90.

[0116] (Sensor holding part) The sensor holder 80 detachably holds the electrochemiluminescence sensor 1. The sensor holder 80 has a sensor insertion opening 81 and a second solution injection opening 82. The other end of the electrochemiluminescence sensor 1 (i.e., the terminal portion 313, 323, 333 side) is held by the sensor holder 80.

[0117] -Sensor insertion opening- The sensor insertion opening 81 is used to mount the electrochemiluminescence sensor 1 into the electrochemiluminescence sensor cell 2. The sensor insertion opening 81 is approximately rectangular in shape in a ZX cross-section. The length of the sensor insertion opening 81 in the Z direction is slightly greater than the length of the electrochemiluminescence sensor 1 in the Z direction. The length of the sensor insertion opening 81 in the X direction is slightly greater than the length of the electrochemiluminescence sensor 1 in the X direction excluding the hook portion 24. The electrochemiluminescence sensor 1 is also fixed to the sensor holding portion 80 by the hook portion 24 catching on the sensor insertion opening 81.

[0118] -Second solution injection opening- The second solution injection port 82 allows the second solution 93 to be injected into the container housing 90 after the electrochemiluminescence sensor 1 has been mounted in the electrochemiluminescence sensor cell 2. For example, the second solution 93 is injected with a pipette 130 fitted into the second solution injection port 82. Injection of the second solution 93 into the container housing 90 is possible even when the sensor holding part 80 is engaged with the container housing 90. Of course, injection of the second solution 93 into the container housing 90 is also possible even when the electrochemiluminescence sensor 1 is not mounted in the electrochemiluminescence sensor cell 2.

[0119] (Container housing) The container housing 90 is capable of holding a second solution 93 containing co-reactants. The container housing 90 has a transparent first wall surface 91. As shown in Figure 14, the bottom surface 92 of the container housing 90 has a narrower thickness in the Z direction of the inner wall of the container housing 90, allowing the electrochemiluminescence sensor 1 to be sandwiched. As a result, one end of the electrochemiluminescence sensor 1 is held inside the container housing 90. By holding the electrochemiluminescence sensor 1 not only with the sensor holding part 80 but also with the container housing 90 in this way, misalignment of the sensor when mounted on the electrochemiluminescence sensor cell 2 is prevented, thereby suppressing a decrease in the amount of light emitted by the detected electrochemiluminescence.

[0120] The narrowing of the bottom side (bottom surface 92 side) of the container housing 90 reduces the internal volume of the container housing 90, thereby reducing the volume of the second solution used in the measurement of electrochemiluminescence. Furthermore, while the electrochemiluminescence sensor 1 is mounted on the electrochemiluminescence sensor cell 2, at least a portion of the electrode portions 311, 321, and 331 of the electrodes 31, 32, and 33 may be immersed in the second solution 93.

[0121] The fact that one end of the electrochemiluminescence sensor 1 (i.e., the electrode portion 311 side) is held inside the container housing 90 indicates that one end of the electrochemiluminescence sensor 1 is positioned on the bottom side (bottom surface 92 side) of the container housing 90. When the electrochemiluminescence sensor 1 is mounted in the electrochemiluminescence sensor cell 2, the electrode portions 311, 321, and 331 of the electrochemiluminescence sensor 1 are positioned in the opposite Y direction to the liquid surface of the second solution 93 held in the container housing 90 (i.e., the electrode portions 311, 321, and 331 are immersed in the second solution 93), so that the second solution can be supplied to the electrode portion 311 of the working electrode 31.

[0122] [Method for measuring electrochemiluminescence] An example of a method for measuring electrochemiluminescence using the electrochemiluminescence sensor cell 2 according to the second embodiment will be described.

[0123] The electrochemiluminescence sensor cell 2 is mounted in the cell mounting section 110 of the electrochemiluminescence immunoassay analyzer 100. The second solution 93 containing the co-reactant is poured into the electrochemiluminescence sensor cell 2.

[0124] A first solution containing the test substance 70 and the nanoparticles 10 is applied to the electrode portion 311 of the working electrode 31 of the electrochemiluminescence sensor 1. As a result, as described in the first embodiment, an electrode 70A with nanoparticles is formed on the electrode portion 311 of the working electrode 31, and a composite nanoparticle electrode 70C may be formed.

[0125] The electrochemiluminescence sensor 1 is mounted in the electrochemiluminescence sensor cell 2. When the electrochemiluminescence sensor 1 is mounted in the electrochemiluminescence sensor cell 2, the terminals 313, 323, and 333 of the electrochemiluminescence sensor 1 are exposed to the outside of the electrochemiluminescence sensor cell 2. For example, a connector 140 is connected to the terminals 313, 323, and 333, and the terminals 313, 323, and 333 are electrically connected to the voltage application unit of the electrochemiluminescence immunoassay analyzer 100 via the connector 140.

[0126] Furthermore, when the electrochemiluminescence sensor 1 is mounted in the electrochemiluminescence sensor cell 2, one end of the electrochemiluminescence sensor 1 is positioned on the bottom side (bottom surface 92 side) of the container housing 90. In other words, the electrode portions 311, 321, and 331 of the electrodes 31, 32, and 33 (working electrode 31, counter electrode 32, and reference electrode 33) of the electrochemiluminescence sensor 1 are immersed in the second solution. As a result, as described in the first embodiment, the co-reactants are supplied to the electrode portion 311.

[0127] Furthermore, when the electrochemiluminescence sensor 1 is mounted in the electrochemiluminescence sensor cell 2, the transparent area of ​​the first wall surface 91 faces the working electrode 31 of the electrochemiluminescence sensor 1. In the Z direction from the electrode portion 311 of the working electrode 31, the transparent areas of the first wall surface 91 of the electrochemiluminescence sensor cell 2 are arranged in order, and the optical detection unit 120 is positioned beyond them.

[0128] A voltage is applied to the electrochemiluminescence sensor 1 (the electrode portion 311) by the voltage application unit, and the emitted electrochemiluminescence is received by the optical detection unit 120. The received electrochemiluminescence can be amplified by the photomultiplier tube (PMT) 150.

[0129] <Fourth Embodiment: Cell for Electrochemiluminescence Sensor> The fourth embodiment relates to a cell for an electrochemiluminescence sensor. The fourth embodiment differs from the third embodiment mainly in that it is an electrochemiluminescence sensor cell for which the electrochemiluminescence sensor 1A according to the second embodiment is provided. The fourth embodiment will mainly describe this difference. In the fourth embodiment, reference numerals that are the same as those in the first to third embodiments have the same configuration as in the first embodiment, so their description will be omitted in principle.

[0130] The cell for the electrochemiluminescence sensor according to the fourth embodiment is a cell for mounting the electrochemiluminescence sensor 1A according to the second embodiment. When the electrochemiluminescence sensor 1A is mounted in the electrochemiluminescence sensor cell, one end of the electrochemiluminescence sensor 1A (i.e., the first open end 611 side of the flow path 60) is held inside the container housing 90 and is positioned on the bottom side (bottom surface 92 side) of the container housing 90.

[0131] The electrochemiluminescence sensor 1A is held by the container housing 90 with a gap between one end of the electrochemiluminescence sensor 1A (i.e., the first open end 611 of the first channel 61) and the bottom of the container housing 90 (the bottom surface of the inner wall). Since the first open end 611 is in contact with the second solution 93 held in the container housing 90, the second solution 93 can be introduced from the first open end 611 into the channel 60 and supplied to the electrode portion 311 of the working electrode 31 via the first channel 61. As described in the second embodiment, since the second solution 93 can move within the channel 60 by capillary action, the first open end 611 only needs to be positioned in the opposite Y direction to the liquid surface of the second solution 93 in the container housing 90.

[0132] When the electrochemiluminescence sensor 1A is mounted in the electrochemiluminescence sensor cell, the second opening 52 of the spacer 50 and the transparent second substrate 40 of the electrochemiluminescence sensor 1A, and the transparent region of the first wall surface 91 of the electrochemiluminescence sensor cell 2 are arranged in the Z direction from the electrode portion 311 of the working electrode 31, and the optical detection unit 120 is positioned beyond them.

[0133] <Fifth Embodiment: Measurement Kit> The fifth embodiment relates to a measurement kit. In the fifth embodiment, the same reference numerals as those in the first to fourth embodiments are used to indicate the same configuration as in the first to fourth embodiments, and therefore, their descriptions are generally omitted.

[0134] The measurement kit according to the fifth embodiment comprises an electrochemiluminescence sensor 1 according to the first embodiment and an electrochemiluminescence sensor cell 2 according to the third embodiment, or an electrochemiluminescence sensor 1A according to the second embodiment and an electrochemiluminescence sensor cell according to the fourth embodiment.

[0135] <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.

[0136] In the first embodiment, the electrochemiluminescent substance 16 is bonded to the polymer film 13 in the nanoparticle body 10, but is not limited to this. The electrochemiluminescent substance 16 may also be bonded to the second specific bonding substance 14. Furthermore, in the first embodiment, the second specific binding substance 14 is bound to the polymer film 13 in the nanoparticle body 10, but the embodiment is not limited to this. The second specific binding substance 14 may also be bound to the metal nanoparticles 12.

[0137] In the first embodiment, the composite nanoparticle 70B consisted of two nanoparticles 10A and 10B bonded together via the (same) test substance 70, but is not limited to this. The composite nanoparticle 70B may be composed of three or more nanoparticles.

[0138] In the first embodiment, a silver / silver chloride electrode was used as the reference electrode 33, but the invention is not limited to this.

[0139] In the first embodiment, electrodes 31, 32, and 33 were arranged along the long axis of the substantially rectangular electrochemiluminescence sensor 1, but are not limited to this. Furthermore, the shape and arrangement of the electrode portions 311, 321, 331 and the terminal portions 313, 323, 333 are not limited to the shapes and arrangements shown in Figure 1. The same applies to the second embodiment.

[0140] In the second embodiment, the second opening 52 of the spacer 50 has a substantially rectangular shape parallel to the Y direction, but is not limited thereto. The second opening 52 can have other shapes in the range in which the flow path 60 it comprises exposes only the electrode portions 311, 321, and 331 of the electrodes 31, 32, and 33, has an inlet for introducing the test substance solution and the second solution from outside to inside the electrochemiluminescence sensor 1, and moves the introduced test substance solution and second solution 93 from the inlet side to the electrode portions 311, 321, and 331 side. Other shapes include, for example, a shape in which the flow path 60 has three or more open ends.

[0141] In the second embodiment, both the test substance solution and the second solution 93 were introduced into the flow path 60 from the first open end 611 of the flow path 60, but the embodiment is not limited to this. Both the test substance solution and the second solution 93 may be introduced from the second open end 621 of the flow path 60, or one of the test substance solution and the second solution may be introduced from the first open end 611 and the other from the second open end 621.

[0142] For example, when the test substance solution is introduced from the first open end 611 and the second solution is introduced from the second open end 621, the first channel 61 can move the first solution (containing the test substance 70 and nanoparticles 10) from the first open end 611 side to the working electrode 31 (electrode portion 311), and the second channel 62 can move the second solution (containing co-reactants) from the second open end 621 side to the working electrode 31 (electrode portion 311).

[0143] Alternatively, when the test substance solution is introduced from the second open end 621 and the second solution is introduced from the first open end 611, the nanoparticles 10 are supported between the second open end 621 and the electrode portion 311. In this case, the second channel 62 can move the first solution from the second open end 621 side to the working electrode 31 (electrode portion 311) side, and the first channel 61 can move the second solution from the first open end 611 side to the working electrode 31 (electrode portion 311) side.

[0144] In the second embodiment, the container housing 90 had a first wall surface 91 that was entirely transparent, but is not limited thereto. With the electrochemiluminescence sensor 1 mounted in the electrochemiluminescence sensor cell 2, the first wall surface 91 may have at least a portion of a transparent area in the area where the transparent area faces the working electrode 31 (electrode portion 311) of the electrochemiluminescence sensor 1.

[0145] In the second embodiment, the electrochemiluminescence sensor 1 was mounted in the electrochemiluminescence sensor cell 2, and then the second solution 93 containing the co-reactant was added to the electrochemiluminescence sensor cell 2. However, the embodiment is not limited to this. The second solution 93 may also be added to the electrochemiluminescence sensor cell 2 via the second solution injection opening 82 before mounting the electrochemiluminescence sensor 1 in the electrochemiluminescence sensor cell 2.

[0146] 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.

[0147] 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.

[0148] <Example 1> [1. Formation of polymer films] The method for forming the polymer film will be explained with reference to Figures 15-16. Figure 15 is a schematic diagram showing the method for forming the polymer film in Example 1. Figure 16 is a schematic diagram illustrating the polymer coating morphology of the nanoparticles in Example 1. As shown in Figure 15, 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.

[0149] 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 12 coated with a polymer membrane 13 was obtained.

[0150] As shown in Figure 16, 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").

[0151] [2. Fabrication of Nanoparticles] Figure 17 is a schematic diagram showing the structure of the nanoparticle body to which the (electrochemiluminescent substance 16) labeled antibody (hereinafter also referred to as the labeled antibody) of Example 1 was bound. The nanoparticle body shown in Figure 17 was prepared by first 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 nanoparticle body to which the labeled antibody was bound will be described below.

[0152] (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.

[0153] (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.

[0154] (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.

[0155] (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.

[0156] (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 to which the labeled VHH antibody was conjugated via the linker portion (see Figure 17).

[0157] [3. Electrochemiluminescence Immunoassay] (3-1. Contact process) To the phosphate buffer solution of the obtained nanoparticles, C Reactive Protein (ADVY CHEMICAL Sigma-Aldrich "00-AGN-AP-CRP-00") (hereinafter also referred to as CRP antigen) was added as test substance 70, and 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. -14 ~5.0×10 -10 The result was M. Additionally, a phosphate-buffered solution of nanoparticles without CRP antigen 70 was prepared separately as a blank.

[0158] Next, an electrochemiluminescence sensor 1 (with a length of 6.5 mm in the short axis direction (X direction) (the length in the X direction including the two hook portions 24 is 8.0 mm) × a length of 30 mm in the long axis direction (Y direction), and a thickness (length in the Z direction) of 0.2 mm) was prepared and placed so that the XY plane was horizontal and the electrode portion 311 was in the inverted vertical direction (horizontal state). The electrochemiluminescence sensor 1 had a first specific binding substance 314 that was bound to the electrode portion 311 of the working electrode 31 (approximately Φ3 mm in diameter in the XY plane view). Using a pipette, the prepared first solution (2.5 μL) was dropped onto the electrode portion 311 of the electrochemiluminescence sensor 1. After dropping, the electrochemiluminescence sensor 1 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).

[0159] The electrode portion 311 of the electrochemiluminescence sensor 1 was positioned so that its electrode portion 311 faced the bottom side (bottom surface 92 side) of the cylindrical cleaning container. The electrode portion 311 of the electrochemiluminescence sensor 1 was then immersed in the cleaning solution inside the cleaning container, and the operation of removing the electrode portion 311 from the cleaning solution was repeated multiple times to clean the electrode portion 311 of the working electrode 31. The cleaning solution was pure water prepared using an ultrapure water production system (Millipore's "Milli-Q®"). After cleaning, the electrochemiluminescence sensor 1 was left undisturbed in a dark, room-temperature environment for 1 hour in a horizontal position to dry the electrode portion 311 of the working electrode 31.

[0160] (3-2. Co-reactant supply process) The container housing 90 of the electrochemiluminescence sensor cell 2 was filled with the second solution 93. The electrochemiluminescence sensor 1 was inserted into the sensor holder 80 and fixed to the electrochemiluminescence sensor cell 2 so that the electrode portion 311 of the working electrode 31 was located on the bottom side (bottom surface 92 side) of the container housing 90. The electrode portion 311 of the working electrode 31 of the electrochemiluminescence sensor 1 was immersed in the second solution 93 held in the container housing 90, and the co-reactants were supplied onto the working electrode 31 (the electrode portion 311).

[0161] (3-3. Voltage Application Process) The electrochemiluminescence sensor cell 2 was mounted on the electrochemiluminescence immunoassay analyzer 100. Connectors 140 were connected to the terminals 313, 323, and 333 of the electrochemiluminescence sensor 1 that were exposed from the electrochemiluminescence sensor cell 2. As a result, the electrode portion 311 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 311 of the electrochemiluminescence sensor 1 under the conditions of an applied voltage of 0.5 to 1.2 V and an application time of 0.02 seconds.

[0162] (3-3. Detection Process) Using an optical detection unit 120 synchronized with the voltage application unit, the electrochemiluminescence emitted from the electrochemiluminescence sensor 1 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).

[0163] The blank was measured in the same manner. The enhanced electrochemiluminescence intensity for this disclosure was obtained by subtracting the electrochemiluminescence intensity of the blank from the obtained electrochemiluminescence intensity.

[0164] The embodiments of the electrochemiluminescence sensor, the cell equipped with the electrochemiluminescence sensor (cell for electrochemiluminescence sensor), and the measurement kit equipped therewith relating to this disclosure are as follows. <1> An electrochemiluminescence sensor for detecting a test substance in a sample using nanoparticles, employing an electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, First substrate and An electrode provided on the surface of the first substrate, wherein a first specific binding substance that specifically binds to the test substance is bonded to the surface of the electrode and arranged thereon. Equipped with, The electrochemiluminescent sensor comprises a nanoparticle body containing metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, a second specific binding substance bonded to at least one of the polymer film and the metal nanoparticles and specifically binding to the test substance, and an electrochemiluminescent substance bonded to at least one of the polymer film and the second specific binding substance and capable of contacting a co-reactant. <2> The system further comprises a nanoparticle body supported on the first substrate and a second substrate positioned opposite the first substrate via a spacer, The first substrate, the second substrate, and the spacer provide a flow channel having an open space, the flow channel extending from the open end to the electrode, The nanoparticles are supported between the open end and the electrode. <1> The electrochemiluminescence sensor described above. <3> The open end of the flow path is the inlet for the test substance solution containing the test substance. <2> The electrochemiluminescence sensor described above. <4> The electrode and the first solution containing the test substance and the nanoparticles are in contact with each other via the flow path. <2> or <3> The electrochemiluminescence sensor described above. <5> Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the nanoparticles are captured on the electrode side via the test substance, thereby forming an electrode with nanoparticles. <1> ~ <4> An electrochemiluminescence sensor as described in any one of the following. <6> Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the composite nanoparticles are captured on the electrode side, thereby forming an electrode with composite nanoparticles. The composite nanoparticle body is formed in which two or more nanoparticle bodies are bonded together via the test substance. <1> ~ <5> An electrochemiluminescence sensor as described in any one of the following. <7> In the aforementioned flow path, the second solution containing the co-reactant is capable of moving from the open end side to the electrode side. <2> The electrochemiluminescence sensor described above. <8> In the aforementioned flow path, the second solution containing the co-reactant is capable of moving from the open end side to the electrode side. Upon contact with the electrode as the second solution moves to it, the electrochemiluminescent material of the nanoparticle-attached electrode comes into contact with the coreactant. <2> To quote <5> The electrochemiluminescence sensor described above. <9> Upon contact of the second solution containing the co-reactant with the electrode attached to the composite nanoparticles, the electrochemiluminescent substance between the composite nanoparticles and the electrode, and between the nanoparticles of the composite nanoparticles, comes into contact with the co-reactant. <1> or <6> The electrochemiluminescence sensor described above. <10> The aforementioned channel is a capillary channel. <2> To quote <3> ~ <9> An electrochemiluminescence sensor as described in any one of the following. <11> The second substrate further has an opening that is positioned opposite the electrode and communicates with the flow path. <2> or <2> To quote <3> ~ <10> An electrochemiluminescence sensor as described in any one of the following. <12> The system further comprises a second substrate positioned opposite the first substrate via a spacer, The second substrate is transparent. <1> ~ <11> An electrochemiluminescence sensor as described in any one of the following. <13> The aforementioned flow path comprises a first flow path and a second flow path. The first channel moves the first solution containing the test substance and the nanoparticles from the open end side of the space towards the electrode side. The second channel moves the second solution containing the co-reactants from the open end side of the space towards the electrode side. <2> or <2> To quote <3> ~ <12> An electrochemiluminescence sensor as described in any one of the following. <14> At least one of the first specific binding substance and the second specific binding substance is a nanoantibody. <1> ~ <13> An electrochemiluminescence sensor as described in any one of the following. <15> Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the nanoparticles are captured on the electrode side via the test substance, thereby forming an electrode with nanoparticles. The separation distance between the nanoparticles and the electrode in the electrode with the nanoparticles is 1 nm to 10 nm. <1> ~ <14> An electrochemiluminescence sensor as described in any one of the following. <16> Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the composite nanoparticles are captured on the electrode side via the test substance, thereby forming an electrode with composite nanoparticles. The distance between the nanoparticles in the electrode with the composite nanoparticles is 1 nm to 10 nm. The composite nanoparticles consist of two or more nanoparticles bonded together via the test substance. <1> ~ <15> An electrochemiluminescence sensor as described in any one of the following. <17> <1> ~ <16> A cell for mounting an electrochemiluminescence sensor as described in any one of the following: A sensor holding part that detachably holds the electrochemiluminescence sensor, A container housing capable of holding the second solution containing the co-reactants and A cell for an electrochemiluminescence sensor, comprising the following features. <18> The sensor holding portion further has a sensor insertion opening for mounting the electrochemiluminescence sensor into the cell. <17> Cell for electrochemiluminescence sensors as described above. <19> The sensor holding portion further has a second solution injection opening for injecting the second solution into the container housing after the electrochemiluminescence sensor has been mounted on the cell. <17> or <18> Cell for electrochemiluminescence sensors as described above. <20> The container housing has a wall surface in which at least a portion is transparent, With the electrochemiluminescence sensor mounted on the cell, the transparent region faces the electrode of the electrochemiluminescence sensor. <17> ~ <19> An electrochemiluminescence sensor cell as described in any one of the following. <21> One end of the electrochemiluminescence sensor is held within the container housing, and the other end is held by the sensor holding portion. <17> ~ <20> An electrochemiluminescence sensor cell as described in any one of the following. <22> One end of the electrochemiluminescence sensor is positioned on the bottom side of the container housing. <17> ~ <21> An electrochemiluminescence sensor cell as described in any one of the following. <23> With a gap provided between one end of the electrochemiluminescence sensor and the bottom of the container housing, the container housing holds the electrochemiluminescence sensor. <17> ~ <22> An electrochemiluminescence sensor cell as described in any one of the following. <24> The electrochemiluminescence sensor and, <17> ~ <23> A measurement kit comprising an electrochemiluminescence sensor cell as described in one of the following. [Explanation of symbols]

[0165] 1.1A ···Electrochemical luminescence sensor 2 ···Cell for electrochemiluminescence sensor 3... Measuring kit 10, 10A, 10B ... Nanoparticles 12, 12A, 12B ··· Metal nanoparticles 13,13A,13B...Polymer membrane 14,14A,14B...Second specific binding substance 16, 16A, 16B... Electrochemiluminescent materials 20...1st base material 31...Working electrode 311 ···(The electrode part of the working electrode) 314...first specific binding substance 40...Second base material 42 ···First opening 50...spacer 60 ···flow channel 61 ···First channel 611...First opening end 62 ···Second channel 621...Second opening end 70 ···Test substance 70A ··· Electrode with nanoparticles 70B ···Composite Nanoparticles 70C ···Electrode with composite nanoparticles 80...Sensor holding part 81...Sensor insertion opening 82...Second solution injection opening 90 ···Container / Housing 91 ···First Wall 92 ···Bottom 93 ···Second Solution L1,L1',L2...Separation distance (separation distance)

Claims

1. An electrochemiluminescence sensor for detecting a test substance in a sample using nanoparticles, employing an electrochemiluminescence immunoassay method utilizing a plasmon-enhanced field, First substrate and An electrode provided on the surface of the first substrate, wherein a first specific binding substance that specifically binds to the test substance is bonded to the surface of the electrode and arranged thereon. Equipped with, The electrochemiluminescent sensor comprises a nanoparticle body containing metal nanoparticles, a polymer film covering the surface of the metal nanoparticles, a second specific binding substance bonded to at least one of the polymer film and the metal nanoparticles and specifically binding to the test substance, and an electrochemiluminescent substance bonded to at least one of the polymer film and the second specific binding substance and capable of contacting the co-reactant.

2. The system further comprises a nanoparticle body supported on the first substrate and a second substrate positioned opposite the first substrate via a spacer, The first substrate, the second substrate, and the spacer provide a flow channel having an open space, and the flow channel extends from the open end to the electrode. The electrochemiluminescence sensor according to claim 1, wherein the nanoparticles are supported between the open end and the electrode.

3. The electrochemiluminescence sensor according to claim 2, wherein the open end of the flow path is an inlet for the test substance solution containing the test substance.

4. The electrochemiluminescence sensor according to claim 2, wherein the electrode and the first solution containing the test substance and the nanoparticles are in contact with each other via the flow path.

5. The electrochemiluminescence sensor according to claim 1 or 4, wherein, upon contact of the electrode with the first solution containing the test substance and the nanoparticles, the nanoparticles are captured on the electrode side via the test substance, thereby forming an electrode with nanoparticles.

6. Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the composite nanoparticles are captured on the electrode side, thereby forming an electrode with composite nanoparticles. The electrochemiluminescence sensor according to claim 1 or 4, wherein the composite nanoparticles consist of two or more nanoparticles bonded together via the test substance.

7. The electrochemiluminescence sensor according to claim 2, wherein in the flow path, the second solution containing the co-reactant is movable from the open end side to the electrode side.

8. In the aforementioned flow path, the second solution containing the co-reactant is capable of moving from the open end side to the electrode side. The electrochemiluminescent sensor according to claim 5, wherein the electrochemiluminescent material of the nanoparticle-attached electrode comes into contact with the coreactant upon contact as the second solution moves to the electrode.

9. The electrochemiluminescent sensor according to claim 1 or 6, wherein the electrochemiluminescent substance between the composite nanoparticles and the electrode, and between the nanoparticles of the composite nanoparticles, comes into contact with the coreactant upon contact of the second solution containing the coreactant with the electrode with the composite nanoparticles.

10. The electrochemiluminescence sensor according to claim 2, wherein the aforementioned channel is a capillary channel.

11. The electrochemiluminescence sensor according to claim 2, wherein the second substrate is positioned opposite the electrode and further has an opening that communicates with the flow path.

12. The system further comprises a second substrate positioned opposite the first substrate via a spacer, The electrochemiluminescence sensor according to claim 1 or 2, wherein the second substrate is transparent.

13. The aforementioned flow path comprises a first flow path and a second flow path. The first channel moves the first solution containing the test substance and the nanoparticles from the open end side of the space towards the electrode side. The electrochemiluminescence sensor according to claim 2, wherein the second channel moves the second solution containing the co-reactants from the open end side of the space to the electrode side.

14. The electrochemiluminescence sensor according to claim 1 or 2, wherein at least one of the first specific binding substance and the second specific binding substance is a nanoantibody.

15. Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the nanoparticles are captured on the electrode side via the test substance, thereby forming an electrode with nanoparticles. The electrochemiluminescence sensor according to claim 1 or 2, wherein the separation distance between the nanoparticles and the electrode in the electrode with nanoparticles is 1 nm to 10 nm.

16. Upon contact of the first solution containing the test substance and the nanoparticles with the electrode, the composite nanoparticles are captured on the electrode side via the test substance, thereby forming an electrode with composite nanoparticles. The distance between the nanoparticles in the electrode with the composite nanoparticles is 1 nm to 10 nm. The electrochemiluminescence sensor according to claim 1 or 2, wherein the composite nanoparticles consist of two or more nanoparticles bonded together via the test substance.

17. A cell for mounting the electrochemiluminescence sensor according to claim 1 or 2, A sensor holding part that detachably holds the electrochemiluminescence sensor, A container housing capable of holding a second solution containing co-reactants and A cell for an electrochemiluminescence sensor, comprising the following features.

18. The electrochemiluminescence sensor cell according to claim 17, wherein the sensor holding portion further has a sensor insertion opening for mounting the electrochemiluminescence sensor into the cell.

19. The electrochemiluminescence sensor cell according to claim 17, wherein the sensor holding portion further has a second solution injection opening for injecting the second solution into the container housing after the electrochemiluminescence sensor has been mounted in the cell.

20. The container housing has a wall surface in which at least a portion is transparent, The electrochemiluminescence sensor cell according to claim 17, wherein, with the electrochemiluminescence sensor mounted in the cell, the transparent region faces the electrode of the electrochemiluminescence sensor.

21. The electrochemiluminescence sensor cell according to claim 17, wherein one end of the electrochemiluminescence sensor is held within the container housing and the other end is held by the sensor holding portion.

22. The electrochemiluminescence sensor cell according to claim 17, wherein one end of the electrochemiluminescence sensor is positioned on the bottom side of the container housing.

23. The electrochemiluminescence sensor cell according to claim 17, wherein the container housing holds the electrochemiluminescence sensor with a gap provided between one end of the electrochemiluminescence sensor and the bottom of the container housing.

24. A measurement kit comprising the electrochemiluminescence sensor and the electrochemiluminescence sensor cell according to claim 17.

Images