Method for detecting or quantifying a target substance in a sample, and reagent for detecting or quantifying a target substance in a sample

Abstract

To provide a technique for detecting or quantifying a detection object of a trace quantity in a sample.SOLUTION: A method for detecting or quantifying a detection object in a sample includes the steps of: mixing fluorescent particles which contain first carrier particles, and first affinity substances carried by the first carrier particles and having affinity to the detection object, and which emit fluorescent light according to radiation of excitation light, quenching particles which contain second carrier particles, and second affinity substances carried by the second carrier particles and having affinity to the detection object, and which absorb fluorescent light emitted by the fluorescent particles, and the sample; and radiating the excitation light on a reaction mixture liquid obtained by the mixing, measuring an intensity of the fluorescent light emitted by the fluorescent particles, in order to detect or quantify the detection object in the sample on the basis of the intensity of the fluorescent light measured.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The embodiments disclosed in the present specification and drawings relate to methods for detecting or quantifying an analyte in a sample, and to reagents for detecting or quantifying an analyte in a sample. [Background technology]

[0002] Latex agglutination has traditionally been used as a method for detecting a target substance in a specimen. In the latex agglutination method, for example, when detecting an antigen in a specimen such as a biological sample, the specimen is mixed with latex particles carrying an antibody or a fragment thereof that specifically binds to the antigen, and the degree of agglutination of the latex particles is measured to detect or quantify the antigen.

[0003] In this latex agglutination method, antigens contained in a sample crosslink multiple latex particle-bound antibodies, promoting agglutination of the latex particles. However, when the amount of antigen is small, this crosslinking is difficult to occur, so the latex particles do not agglutinate sufficiently, and even if they do agglutinate, it is below the detection sensitivity and cannot be detected. This makes it difficult to detect small amounts of antigen. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 58-11575 Summary of the Invention [Problem to be solved by the invention]

[0005] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to provide a technology capable of detecting or quantifying a trace amount of a target substance in a sample. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described below can also be positioned as other problems. [Means for solving the problem]

[0006] According to an embodiment, there is provided a method for detecting or quantifying a target substance in a sample, comprising: fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; quenching particles that include second carrier particles and a second affinity substance that is carried by the second carrier particles and has affinity for the target substance, and that absorb the fluorescence emitted by the fluorescent particles; The specimen and and mixing the irradiating the reaction mixture obtained by the mixing with the excitation light, measuring the intensity of the fluorescence emitted by the fluorescent particles, and detecting or quantifying the target substance in the specimen based on the measured intensity of the fluorescence; A method is provided which includes: [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 10 is a diagram schematically illustrating an example of the state of a reaction mixture when the sample does not contain a target substance to be detected. [Figure 2] FIG. 1 is a diagram schematically illustrating an example of the state of a reaction mixture when a sample contains a low concentration of a detection target. [Figure 3] FIG. 1 is a diagram schematically illustrating an example of the state of a reaction mixture when a sample contains a high concentration of a detection target. [Figure 4] Graph showing the results of the fluorescence quenching method and the latex agglutination method. DETAILED DESCRIPTION OF THE INVENTION

[0008] The embodiments will be described in detail below.

[0009] 1. Method for detecting or quantifying a target substance in a sample The method according to the embodiment is a method for detecting or quantifying a target substance in a sample, fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; quenching particles that include second carrier particles and a second affinity substance that is carried by the second carrier particles and has affinity for the target substance, and that absorb the fluorescence emitted by the fluorescent particles; The specimen and and mixing the irradiating the reaction mixture obtained by the mixing with the excitation light, measuring the intensity of the fluorescence emitted by the fluorescent particles, and detecting or quantifying the target substance in the specimen based on the measured intensity of the fluorescence; Includes.

[0010] In the above method, when a sample contains a target substance, the fluorescent particles and the quenching particles bind to each other via the target substance, and the quenching particles can efficiently absorb the fluorescence emitted by the fluorescent particles in the vicinity of the fluorescent particles. Therefore, the above method can test whether the target substance is contained in the sample or determine the amount of the target substance in the sample by measuring the fluorescence intensity.

[0011] In the above method, typically, a plurality of fluorescent particles and a plurality of quenching particles are used. That is, in the above method, typically, a plurality of fluorescent particles, each of which contains a first carrier particle and a first affinity substance carried by the first carrier particle and having affinity for the target substance, and each of which emits fluorescent light when irradiated with excitation light; a plurality of quenching particles, each of which includes second carrier particles and a second affinity substance carried by the second carrier particles and having affinity for the target substance, and each of which absorbs the fluorescence emitted by the fluorescent particles; is used.

[0012] The above method includes the steps of "mixing" and "detection or quantification." The steps will be explained below in this order.

[0013] 1-1.Mixture In the mixing step, the "analyte," "fluorescent particles," and "quenching particles" are mixed. In this step, if the sample contains the target substance, a binding reaction occurs between the fluorescent particles and the quenching particles via the target substance (see Figures 2 and 3).

[0014] First, the "analyte," "fluorescent particle," and "quenching particle" will be explained in order below.

[0015] "Specimen" The specimen is any biological sample, for example, an extract of a body fluid or excrement, and specific examples include extracts of blood, serum, plasma, urine, lymph, sputum, and feces.

[0016] Detection targets contained in specimens include substances used in clinical diagnosis, and specific examples include human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen (fibrin and its degradation products), alpha-fetoprotein (AFP), C-reactive protein (CRP), myoglobin, ferritin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin (hCG), human placental lactogen (HPL), HIV virus antigen, allergens, bacterial toxins, bacterial antigens, enzymes, hormones (e.g., human thyroid-stimulating hormone (TSH), insulin, etc.), nucleic acids, nucleic acids amplified by PCR or the like, cytokines, drugs, etc., contained in body fluids, urine, sputum, feces, etc.

[0017] "Fluorescent particles" The fluorescent particles include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and emit fluorescence when irradiated with excitation light.

[0018] The first support particles can be support particles commonly used in agglomeration methods, such as cellulose particles, porous glass particles, silica particles, low-crosslinked and high-crosslinked polystyrene particles optionally crosslinked with divinylbenzene, grafted copolymer particles, polyacrylamide particles, latex particles, dimethylacrylamide particles optionally crosslinked with N,N-bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer. Alternatively, the support particles can be particles containing alkanethiolate-derivatized gold, polyamide, acrylic copolymer, nylon, dextran, polyacrolein, etc.

[0019] The first carrier particles have an average particle size of, for example, 20 to 800 nm, preferably 100 to 400 nm, and more preferably 150 to 200 nm.

[0020] The first affinity substance is preferably a substance that specifically binds to the target substance, more preferably a protein that specifically binds to the target substance, and even more preferably an antibody that specifically binds to the target substance. The antibody may be any type of immunoglobulin molecule, or may be an immunoglobulin molecule fragment having an antigen-binding site, such as Fab. The antibody may be a monoclonal or polyclonal antibody, but is preferably a monoclonal antibody that recognizes a different antigenic determinant of the antigen. Alternatively, the first affinity substance may be a protein other than an antibody, or may be a nucleic acid, lipid, sugar, etc.

[0021] The fluorescent particles have at least one of the first carrier particle and the first affinity substance labeled with a fluorescent substance.

[0022] According to one example, the first carrier particles are carrier particles labeled with a fluorescent substance. The first carrier particles may contain the fluorescent substance inside the particle, or may carry the fluorescent substance on the particle surface. Typical examples of fluorescently labeled carrier particles include fluorescent latex particles containing a fluorescent dye inside the particle, and fluorescent silica particles containing a fluorescent dye inside the particle. These are commercially available, and an example of the latter is Quartz Dot (Furukawa Electric Advanced Engineering Co., Ltd.).

[0023] In another example, the first affinity substance is an affinity substance labeled with a fluorescent substance. The fluorescently labeled affinity substance can be obtained by labeling the affinity substance with a fluorescent substance using a commercially available fluorescent labeling kit. When the affinity substance is an antibody, the fluorescently labeled affinity substance can be obtained by labeling the antibody with a fluorescent substance using a commercially available antibody fluorescent labeling kit.

[0024] Fluorescent particles usually contain many molecules of a fluorescent substance, and the fluorescent intensity emitted by fluorescent particles is high. Therefore, when fluorescent particles are used in the above-mentioned method (i.e., a method for detecting or quantifying a target substance in a sample), highly sensitive detection or quantification can be achieved.

[0025] Fluorescent particles can be produced by binding a first affinity substance to a first carrier particle. For example, when the first affinity substance is an antibody, the first affinity substance can be directly bound to the carrier particle using standard methods such as physical adsorption or chemical binding. Alternatively, the first affinity substance can be indirectly bound to the first carrier particle via substances that have affinity for each other (e.g., avidin and biotin, or glutathione and glutathione S-transferase).

[0026] "Quenching particles" The quenching particles include second carrier particles and a second affinity substance that is carried by the second carrier particles and has affinity for the target substance, and absorbs the fluorescence emitted by the fluorescent particles.

[0027] When the sample contains a target substance, the quenching particles bind to the fluorescent particles via the target substance and can efficiently absorb the fluorescence emitted by the fluorescent particles in the vicinity of the fluorescent particles (see Figure 3). On the other hand, when the sample does not contain a target substance, the quenching particles float in the reaction mixture without binding to the fluorescent particles via the target substance, and most of them are not present in the vicinity of the fluorescent particles, so they cannot efficiently absorb the fluorescence emitted by the fluorescent particles (see Figure 1). Therefore, by using the above-mentioned fluorescent particles and quenching particles in combination, the target substance in the sample can be detected and quantified.

[0028] The second carrier particles contained in the quenching particles can absorb the fluorescence emitted by the fluorescent particles. The second carrier particles are, for example, metal nanoparticles, preferably gold nanoparticles, silver nanoparticles, or platinum nanoparticles. It is known that the wavelength of light absorbed by metal nanoparticles varies depending on the type of metal, particle size, particle shape, etc. For example, it is known that spherical gold nanoparticles with a diameter of 20 nm exhibit an absorption peak at approximately 520 nm, and spherical gold nanoparticles with a diameter of 100 nm exhibit an absorption peak at approximately 570 nm. Therefore, it is preferable to select the second carrier particles appropriately according to the wavelength of the fluorescence emitted by the fluorescent particles so that they can efficiently absorb the fluorescence emitted by the fluorescent particles.

[0029] The second carrier particles have an average particle size of, for example, 10 to 100 nm, preferably 10 to 20 nm. The shape of the second carrier particles is not particularly limited and may be any shape, such as a sphere, an ellipsoid, or a rod, and is preferably a sphere.

[0030] The second affinity substance contained in the quenching particle is preferably a substance that specifically binds to the target substance, more preferably a protein that specifically binds to the target substance, and even more preferably an antibody that specifically binds to the target substance. The antibody may be any type of immunoglobulin molecule or an immunoglobulin molecule fragment having an antigen-binding site, such as Fab. The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody that recognizes a different antigenic determinant of the antigen. Alternatively, the first affinity substance may be a protein other than an antibody, or may be a nucleic acid, lipid, sugar, etc.

[0031] The second affinity substance contained in the quenching particle may be the same as or different from the first affinity substance contained in the fluorescent particle. In either case, the second affinity substance can bind to a site on the target substance that is different from the site on the target substance to which the first affinity substance binds. This allows both the first affinity substance and the second affinity substance to bind to a single target substance.

[0032] Preferably, the first affinity substance and the second affinity substance are each an antibody. In this case, the first antibody, which is the first affinity substance, and the second antibody, which is the second affinity substance, can be a combination of a capture antibody (solid-phase antibody) and a detection antibody used in the sandwich method. By using such a combination, the second antibody can bind to a site (epitope) on the target substance that is different from the site (epitope) on the target substance to which the first antibody binds.

[0033] Quenching particles can be prepared by binding a second affinity substance to a second carrier particle. For example, when the second affinity substance is an antibody, the second affinity substance can be directly bound to the carrier particle using conventional methods such as physical adsorption or chemical binding. Alternatively, the second affinity substance can be indirectly bound to the second carrier particle via substances that have affinity for each other (e.g., avidin and biotin, glutathione and glutathione S-transferase).

[0034] "mixture" In the method according to the embodiment, a sample, fluorescent particles, and quenching particles are mixed, and when the sample contains a target substance, a binding reaction between the fluorescent particles and the quenching particles occurs via the target substance. In this specification, the mixture obtained by mixing the sample, fluorescent particles, and quenching particles is referred to as a "reaction mixture."

[0035] The mixing may be performed by simultaneously adding fluorescent particles and quenching particles to the specimen, causing a binding reaction between the fluorescent particles and the quenching particles via the target substance. Alternatively, the mixing may be performed by adding fluorescent particles to the specimen, causing a binding reaction between the target substance and the fluorescent particles, and then adding quenching particles to the resulting mixture, causing a binding reaction between the complex of the target substance and the fluorescent particles and the quenching particles. Alternatively, the mixing may be performed by adding quenching particles to the specimen, causing a binding reaction between the target substance and the quenching particles, and then adding fluorescent particles to the resulting mixture, causing a binding reaction between the complex of the target substance and the quenching particles and the fluorescent particles.

[0036] The reaction mixture may be stirred to facilitate the binding reaction, or may be left to stand for the time required for the binding reaction (e.g., 5 minutes), or may be heated to a temperature suitable for the binding reaction (e.g., 37°C).

[0037] The total volume of the reaction mixture is not particularly limited, but can be, for example, 50 to 3000 L, and preferably 100 to 400 L. The reaction mixture may further contain a buffer solution.

[0038] The concentration of fluorescent particles in the reaction mixture can be, for example, 0.001 to 0.02% by mass, preferably 0.001 to 0.01% by mass. The concentration of quenching particles in the reaction mixture can be, for example, 0.001 to 0.02% by mass, preferably 0.001 to 0.01% by mass. In the reaction mixture, the ratio of the concentration of fluorescent particles to the concentration of quenching particles is, for example, 1:3 to 3:1, preferably 1:2 to 2:1.

[0039] 1-2. Detection or quantification In the method according to the embodiment, the reaction mixture is irradiated with excitation light, the intensity of the fluorescence emitted by the fluorescent particles is measured, and the target substance in the sample is detected or quantified based on the measured fluorescence intensity.

[0040] The excitation light can be a laser beam that excites the fluorescent particles. The wavelength of the excitation light can be determined based on the excitation spectrum and fluorescence spectrum of the fluorescent particles. For example, when Quartz Dot (Furukawa Electric Advanced Engineering Co., Ltd.) is used as the fluorescent particle, laser light with a wavelength of 500 nm can be irradiated and light with a wavelength of 550 nm can be detected.

[0041] The fluorescence intensity can be measured after a predetermined time has elapsed since the specimen, fluorescent particles, and quenching particles were mixed (i.e., after the binding reaction between the fluorescent particles and quenching particles via the target substance has finished). In this case, the fluorescence intensity serving as a reference value is measured immediately after the specimen, fluorescent particles, and quenching particles are mixed, and if the fluorescence intensity measured after the binding reaction has finished is lower than the reference value, it can be determined that the specimen contains the target substance.

[0042] Alternatively, the measurement of fluorescence intensity may be performed by mixing a sample with fluorescent particles to induce a binding reaction between the target substance and the fluorescent particles, then adding quenching particles to the resulting mixture, and measuring the fluorescence intensity for a predetermined period of time starting immediately after the addition of the quenching particles. In this case, the measurement of fluorescence intensity may be performed intermittently at regular intervals or continuously over time. By measuring the fluorescence intensity over a predetermined period of time in this manner, the increase in the number of quenching particles bound to the fluorescent particles via the target substance can be detected as a decrease in fluorescence intensity over time, thereby enabling highly accurate detection of the target substance.

[0043] Alternatively, fluorescence intensity may be measured by mixing the sample with quenching particles to induce a binding reaction between the target substance and the quenching particles, then adding fluorescent particles to the resulting mixture, and measuring the fluorescence intensity for a predetermined period of time starting immediately after adding the fluorescent particles. In this case, fluorescence intensity may also be measured intermittently at regular intervals or continuously over time. By measuring fluorescence intensity over a predetermined period of time in this manner, the increase over time of fluorescent particles bound to the quenching particles via the target substance can be detected as a decrease over time in fluorescence intensity, allowing for highly accurate detection of the target substance. Adding the fluorescent particles last in this manner also has the advantage of suppressing the fluorescence quenching phenomenon (i.e., the phenomenon of a decrease in fluorescence intensity) of the fluorescent particles.

[0044] The quantity of the target substance can be determined by calculating the quantity of the target substance from the measured value of the fluorescence intensity based on a correlation equation between the quantity of the target substance and the fluorescence intensity.

[0045] It is preferable to prepare a correlation equation between the amount of the target substance and the fluorescence intensity in advance. The more data there is on the measurement of the amount of the target substance and the fluorescence intensity that constitutes this correlation equation, the more reliable the correlation equation will be. Therefore, the data may relate to the amounts of two or more target substances, and preferably relate to the amounts of three or more target substances.

[0046] By substituting the measured fluorescence intensity value into the created correlation equation, the amount of the target substance in the sample can be calculated.

[0047] 1-3.Specific examples Examples of the appearance of the reaction mixture when the first affinity substance and the second affinity substance are each an antibody are shown schematically in Figures 1 to 3. Figure 1 shows an example of the appearance of the reaction mixture when the sample does not contain the target substance, Figure 2 shows an example of the appearance of the reaction mixture when the sample contains a low concentration of the target substance, and Figure 3 shows an example of the appearance of the reaction mixture when the sample contains a high concentration of the target substance.

[0048] 1 to 3, fluorescent particles 10 are composed of first carrier particles 11 labeled with a fluorescent substance and first antibodies 12 carried by the first carrier particles 11 and specifically binding to the analyte 30. Also, in FIGS. 1 to 3, quenching particles 20 are composed of second carrier particles 21 that absorb the fluorescence emitted by the fluorescent particles and second antibodies 22 carried by the second carrier particles 21 and specifically binding to the analyte 30. As described above, the second antibodies 22 bind to a site (epitope) on the analyte 30 that is different from the site (epitope) on the analyte 30 to which the first antibodies 12 bind. For example, when the analyte 30 is ferritin, each of the first antibodies 12 and the second antibodies 22 can be an anti-ferritin monoclonal antibody.

[0049] In Figure 1, the sample does not contain the target substance, so the quenching particles 20 are unable to bind to the fluorescent particles 10 via the target substance and are suspended in the reaction mixture. Most of the quenching particles 20 suspended in the reaction mixture are not present in the vicinity of the fluorescent particles 10 and are therefore unable to efficiently absorb the fluorescence L2 emitted by the fluorescent particles 10. For this reason, when the reaction mixture is irradiated with excitation light L1, the fluorescence L2 emitted by the fluorescent particles 10 is detected with high fluorescence intensity.

[0050] In FIG. 2, the sample contains a low concentration of the target substance 30, so the quenching particles 20 bind to the fluorescent particles 10 via the low concentration of the target substance 30. Therefore, a small number of quenching particles are bound to the fluorescent particles 10 via the target substance 30. The small number of quenching particles 20 bound to the fluorescent particles 10 can efficiently absorb part of the fluorescence L2 emitted by the fluorescent particles 10 in the vicinity of the fluorescent particles 10. Therefore, the fluorescence intensity detected here is lower than the fluorescence intensity detected in the case of FIG. 1.

[0051] In FIG. 3, the sample contains a high concentration of the target substance 30, so the quenching particles 20 bind to the fluorescent particles 10 via the high concentration of the target substance 30. As a result, a large number of quenching particles are bound to the fluorescent particles 10 via the target substance 30. The large number of quenching particles 20 bound to the fluorescent particles 10 can efficiently absorb much of the fluorescence L2 emitted by the fluorescent particles 10 in the vicinity of the fluorescent particles 10. As a result, the fluorescence intensity detected here is lower than the fluorescence intensity detected in the case of FIG. 2.

[0052] 1 to 3, the detected fluorescence intensity varies depending on the amount of the target substance in the sample. That is, the greater the amount of target substance 30 contained in the sample, the more quenching particles 20 bind to the fluorescent particles 10 via the target substance 30, and the more efficiently the fluorescence emitted by the fluorescent particles 10 can be absorbed, resulting in a lower fluorescence intensity. On the other hand, if the sample does not contain the target substance 30, the quenching particles 20 cannot bind to the fluorescent particles 10 via the target substance 30, but instead float in the reaction mixture and cannot efficiently absorb the fluorescence emitted by the fluorescent particles 10, resulting in a high fluorescence intensity.

[0053] 1-4.Effects As described above, the method according to the embodiment can detect or quantify a target substance in a sample by correlating the presence of the target substance in the sample with a decrease in fluorescence intensity. On the other hand, the conventional latex agglutination method detects aggregates obtained by a crosslinking reaction between a large number of target substances (antigens) and a large number of latex particle-bound antibodies, and therefore, as described in the Background Art section, cannot detect a target substance (antigen) in a small amount. Compared to the conventional latex agglutination method, the method according to the embodiment can detect or quantify a small amount of target substance.

[0054] 2. Reagents According to an embodiment, there is provided a reagent for detecting or quantifying a target substance in a sample, comprising: fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; second carrier particles; and quenching particles that are carried on the second carrier particles and contain a second affinity substance having affinity for the target substance, and that absorb the fluorescence emitted by the fluorescent particles. A reagent comprising:

[0055] The reagent includes the fluorescent particles described above in the "Fluorescent Particles" section and the quenching particles described above in the "Quenching Particles" section. The reagent can be mixed with a sample to prepare a reaction mixture. The reagent may further include a buffer solution.

[0056] The reagent typically comprises a plurality of fluorescent particles and a plurality of quenching particles. a plurality of fluorescent particles, each of which contains a first carrier particle and a first affinity substance carried by the first carrier particle and having affinity for the target substance, and each of which emits fluorescent light when irradiated with excitation light; a plurality of quenching particles, each of which includes second carrier particles and a second affinity substance carried by the second carrier particles and having affinity for the target substance, and each of which absorbs the fluorescence emitted by the fluorescent particles; Includes.

[0057] The reagent may contain the fluorescent particles and the quenching particles in separate packages, or may contain the fluorescent particles and the quenching particles in the same package.

[0058] In the former case, the reagent according to a preferred embodiment comprises: a first package including the above-mentioned plurality of fluorescent particles and a first dispersion medium in which the plurality of fluorescent particles are dispersed; a second package including the plurality of quenching particles and a second dispersion medium in which the plurality of quenching particles are dispersed; Here, each of the first dispersion medium and the second dispersion medium can be a buffer solution that constitutes the reaction mixture.

[0059] In the latter case, the reagent according to a preferred embodiment comprises: a plurality of fluorescent particles as described above; a plurality of quenching particles as described above; a dispersion medium in which the plurality of fluorescent particles and the plurality of quenching particles are dispersed; Here, the dispersion medium may be a buffer solution that constitutes the reaction mixture.

[0060] When the above-mentioned reagent is used to detect or quantify a target substance in a sample, the presence of the target substance in the sample can be correlated with a decrease in fluorescence intensity, making it possible to detect or quantify minute amounts of the target substance, compared to conventional latex agglutination methods (methods for detecting aggregates obtained by crosslinking a large number of target substances (antigens) with a large number of latex particle-bound antibodies). [Example]

[0061] 1. Method <Example: Fluorescence quenching method> Test solution containing the target substance: avidin solution Fluorescent particle dispersion: Dispersion of biotin-loaded Quartz Dots (particle size: 200 nm) (Furukawa Electric Advanced Engineering Co., Ltd.) Quenching particle dispersion: Dispersion of biotin-loaded gold nanoparticles (particle size: 100 nm) Reaction vessel: Fluorescence cell

[0062] Seven avidin solutions were prepared as test solutions, with the avidin concentrations in the reaction mixtures being 0.1 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.3 μg / mL, 2.5 μg / mL, 5 μg / mL, and 50 μg / mL. A fluorescent particle dispersion was prepared with a fluorescent particle concentration of 0.005% by mass in the reaction mixture. Two quenching particle dispersions were prepared, with a quenching particle concentration of 0.006% by mass or 0.012% by mass in the reaction mixture.

[0063] 10 μL of the test solution was dispensed into a reaction vessel, followed by 180 μL of the fluorescent particle dispersion, and the resulting intermediate mixture was stirred. After stirring, 10 μL of the quenching particle dispersion was dispensed into the reaction vessel containing the intermediate mixture to obtain a reaction mixture.

[0064] Immediately after dispensing the quenching particle dispersion, the sample was irradiated with excitation light at 500 nm and the fluorescence intensity at 550 nm was measured. The fluorescence intensity measured here is the baseline fluorescence intensity.

[0065] The reaction mixture was stirred and then allowed to stand at 37°C for 5 minutes. It was then irradiated with 500 nm excitation light and the fluorescence intensity at 550 nm was measured. The fluorescence intensity measured here was subtracted from the baseline fluorescence intensity value to calculate the rate of decrease in fluorescence intensity. The results are shown in Figure 4.

[0066] <Comparative Example: Latex Agglutination Method> Test solution containing the target substance: avidin solution Latex particle dispersion: Dispersion of biotin-loaded latex particles (particle size: 150 nm) Reaction vessel: Spectrophotometer cell

[0067] Seven different concentrations of avidin solutions were prepared as test solutions, with the concentrations of avidin in the reaction mixture being 0.1 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.3 μg / mL, 2.5 μg / mL, 5 μg / mL, and 50 μg / mL. The latex particle dispersion was prepared so that the concentration of latex particles in the reaction mixture was 0.005% by mass.

[0068] 10 μL of the test solution was dispensed into the reaction vessel, and then 180 μL of the latex particle dispersion was dispensed to obtain a reaction mixture.

[0069] Immediately after dispensing the latex particle dispersion, the reaction vessel was irradiated with light and the intensity of the light transmitted through the reaction vessel was measured. The absorbance was calculated based on the measured transmitted light intensity. The calculated absorbance here is the reference absorbance.

[0070] The reaction mixture was stirred and then allowed to stand at 37°C for 5 minutes. Light was then irradiated, and the intensity of the light transmitted through the reaction vessel was measured. The absorbance was calculated based on the measured transmitted light intensity. The absorbance increase rate was calculated by subtracting the reference absorbance value from the calculated absorbance value. The results are shown in Figure 4.

[0071] 2.Results In the graph of Figure 4, the horizontal axis represents the avidin concentration, and the vertical axis represents the rate of decrease in fluorescence intensity on the left scale and the rate of increase in absorbance (Δabs) on the right scale. In Figure 4, ◯ represents the rate of decrease in fluorescence intensity when the concentration of quenching particles (gold nanoparticles) is 0.006% by mass in the fluorescence quenching method, △ represents the rate of decrease in fluorescence intensity when the concentration of quenching particles (gold nanoparticles) is 0.012% by mass in the fluorescence quenching method, and □ represents the rate of increase in absorbance (Δabs) in the latex agglutination method.

[0072] The results in Figure 4 show that the fluorescence quenching method is able to detect and quantify trace amounts of avidin compared to the latex agglutination method. The fluorescence quenching method was able to detect and quantify trace amounts of avidin compared to the latex agglutination method, both when the quenching particle concentration was 0.006% by mass and when the quenching particle concentration was 0.012% by mass.

[0073] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0074] 10... fluorescent particles, 11... first carrier particles, 12... first antibody, 20... quenching particles, 21... second carrier particles, 22... second antibody, 30...detection target, L1: Excitation light, L2: Fluorescence light

Claims

1. A method for detecting or quantifying a target substance in a sample, comprising: fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; quenching particles, which include second carrier particles and a second affinity substance carried by the second carrier particles and having affinity for the target substance, and which absorb the fluorescence emitted by the fluorescent particles; The specimen and and mixing the irradiating the reaction mixture obtained by the mixing with the excitation light, measuring the intensity of the fluorescence emitted by the fluorescent particles, and detecting or quantifying the target substance in the specimen based on the measured intensity of the fluorescence; Including, of the first carrier particles and the second carrier particles, only the first carrier particles are carrier particles labeled with a fluorescent substance, The method wherein the second support particles are metal nanoparticles.

2. The method of claim 1 , wherein the second carrier particles are gold nanoparticles, silver nanoparticles, or platinum nanoparticles.

3. The method according to claim 1 or 2, wherein each of the first affinity substance and the second affinity substance is an antibody.

4. A method for detecting or quantifying a target substance in a sample, comprising: fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; quenching particles, which include second carrier particles and a second affinity substance carried by the second carrier particles and having affinity for the target substance, and which absorb the fluorescence emitted by the fluorescent particles; The specimen and and mixing the irradiating the reaction mixture obtained by the mixing with the excitation light, measuring the intensity of the fluorescence emitted by the fluorescent particles, and detecting or quantifying the target substance in the specimen based on the measured intensity of the fluorescence; Including, the first affinity substance is an affinity substance labeled with a fluorescent substance, A method wherein each of the first affinity substance and the second affinity substance is an antibody.

5. The method of claim 4 wherein the second support particles are metal nanoparticles.

6. The method according to claim 4 or 5, wherein the second carrier particles are gold nanoparticles, silver nanoparticles, or platinum nanoparticles.

7. A reagent for detecting or quantifying a target substance in a sample, fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; second carrier particles; and quenching particles that are carried on the second carrier particles and contain a second affinity substance having affinity for the target substance, and that absorb the fluorescence emitted by the fluorescent particles. Including, of the first carrier particles and the second carrier particles, only the first carrier particles are carrier particles labeled with a fluorescent substance, The reagent, wherein the second support particles are metal nanoparticles.

8. The reagent according to claim 7 , wherein the second carrier particles are gold nanoparticles, silver nanoparticles, or platinum nanoparticles.

9. The reagent according to claim 7 or 8, wherein each of the first affinity substance and the second affinity substance is an antibody.

10. A reagent for detecting or quantifying a target substance in a sample, fluorescent particles that include first carrier particles and a first affinity substance that is carried by the first carrier particles and has affinity for the target substance, and that emit fluorescence when irradiated with excitation light; second carrier particles; and quenching particles that are carried on the second carrier particles and contain a second affinity substance having affinity for the target substance, and that absorb the fluorescence emitted by the fluorescent particles. Including, the first affinity substance is an affinity substance labeled with a fluorescent substance, A reagent, wherein each of the first affinity substance and the second affinity substance is an antibody.

11. The reagent according to claim 10, wherein the second carrier particles are metal nanoparticles.

12. The reagent according to claim 10 or 11, wherein the second carrier particles are gold nanoparticles, silver nanoparticles, or platinum nanoparticles.

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