Measurement method

Abstract

Labels (2a, 2b) in a first label group and labels (2c, 2d) in a second label group have different labeling properties from one another. Each particle group includes a plurality of particle subgroups (a to d) that differ from each other in particle size. Furthermore, this measurement method comprises: a step for mixing a sample, particles (1a to 1d), and the labels (2a to 2d); a step for specifically binding the labels (2a to 2d) and the labels (2a to 2d) to a biomolecule; a step for separating each of the particles on the basis of the particle size; a step for detecting labeling properties of the labels; and a step for determining the type of biomolecules bound to the particles on the basis of the particle size and the labeling properties.

Description

Measurement method

[0001] The present invention relates to a measurement method, and more particularly to a method for measuring biomolecules.

[0002] Conventionally, objective evaluation methods for diagnosing disease and / or assessing therapeutic effects include measuring biomolecules called biomarkers in vivo. Among these, methods that consider disease as the sum of the effects of multiple biomolecules and diagnose and / or assess therapeutic effects based on the measurement results of multiple types of biomarkers are considered promising.

[0003] A method known as xMAP (registered trademark) is known as a method for simultaneously detecting multiple types of biomarkers. JP 2012-533052 A (Patent Document 1) discloses Luminex (registered trademark), a type of xMAP technology, as a method for detecting biomarkers. xMAP is a technology that uses beads that exhibit a predetermined fluorescence spectrum, in which two fluorescent dyes of different colors are combined and encapsulated at predetermined concentrations, as an identification library for types of biomarkers. In xMAP, beads with different fluorescence spectra are configured to bind different types of biomarkers.

[0004] Special Publication No. 2012-533052

[0005] However, with xMAP, beads must be distinguished solely by their fluorescence spectra, making it difficult to distinguish between beads that exhibit similar fluorescence spectra. Therefore, there is a theoretical limit to how many types of biomarkers can be detected at once. Therefore, there has been a demand for a technology different from xMAP that can detect multiple types of biomarkers at once.

[0006] The present disclosure has been made to solve such problems, and its purpose is to provide a technique for measuring multiple types of biomarkers at once.

[0007] A first aspect of the present disclosure is a method for measuring biomolecules contained in a sample derived from a biological specimen, comprising the steps of preparing particles belonging to a first particle group and particles belonging to a second particle group, and preparing labels belonging to a first labeling group corresponding to the first particle group and labels belonging to a second labeling group corresponding to the second particle group. The labels of the first labeling group and the labels of the second labeling group have labeling properties different from each other. The particles of the first particle group and the labels of the first labeling group are bound via a biomolecule. The particles of the second particle group and the labels of the second labeling group are bound via a biomolecule. Each particle group includes a plurality of particle subgroups. In each particle group, the plurality of particle subgroups have different particle sizes. The particles of the plurality of particle subgroups of the first particle group and the particles of the plurality of particle subgroups of the second particle group have first binding moieties that specifically bind to different types of biomolecules from each other. The measurement method further includes the steps of mixing the sample with particles of the first particle group and particles of the second particle group, and labels of the first labeling group and labels of the second labeling group; specifically binding biomolecules to the particles of the first particle group and particles of the second particle group and the labels of the first labeling group and labels of the second labeling group; separating each of the particles of the first particle group and the particles of the second particle group based on particle size; detecting label characteristics of the labels bound via biomolecules to each of the particles of the first particle group and particles of the second particle group separated based on particle size; and determining the types of biomolecules bound to the particles of the first particle group and particles of the second particle group based on the particle size and label characteristics.

[0008] A second aspect of the present disclosure is a method for measuring biomolecules contained in a sample derived from a biological specimen, comprising the steps of preparing particles belonging to a third particle group and particles belonging to a fourth particle group, and preparing a label belonging to a third label group corresponding to the third particle group and a label belonging to a fourth label group corresponding to the fourth particle group. The particles belonging to the third particle group and the particles of the fourth particle group have particle properties different from each other. The particles of the third particle group and the label of the third label group are bound via a biomolecule. The particles of the fourth particle group and the label of the fourth label group are bound via a biomolecule. Each particle group includes multiple particle subgroups. In each particle group, the multiple particle subgroups have different particle sizes. The particles of the multiple particle subgroups of the third particle group and the particles of the multiple particle subgroups of the fourth particle group have third binding moieties that specifically bind to different types of biomolecules from each other. The measurement method further includes the steps of mixing a sample with particles of the third particle group and particles of the fourth particle group, and labels of the third labeling group and labels of the fourth labeling group; specifically binding biomolecules to the particles of the third particle group and particles of the fourth particle group and the labels of the third labeling group and the labels of the fourth labeling group; separating each of the particles of the third particle group and the particles of the fourth particle group based on particle size; detecting particle characteristics and label characteristics of the labels bound to the particles via the biomolecules for each of the particles of the third particle group and particles of the fourth particle group separated based on particle size; and determining the types of biomolecules bound to the particles of the third particle group and particles of the fourth particle group based on the particle size, particle characteristics, and label characteristics.

[0009] A third aspect of the present disclosure is a method for measuring biomolecules contained in a sample derived from a biological sample, comprising the steps of preparing particles belonging to a first particle group and preparing a label belonging to a first label group corresponding to the first particle group. The particles of the first particle group and the label of the first label group are bound via biomolecules. The first particle group includes a plurality of particle subgroups. In the first particle group, the plurality of particle subgroups have different particle sizes. The particles of the plurality of particle subgroups of the first particle group have first binding moieties that specifically bind to different types of biomolecules. The measurement method further comprises the steps of mixing a sample with the particles of the first particle group and the labels of the first label group to specifically bind the particles of the first particle group and the labels of the first label group to the biomolecules, separating each of the particles of the first particle group based on particle size, detecting the label characteristic of the label bound via the biomolecules to each of the particles of the first particle group separated based on particle size, and determining the type of biomolecules bound to the particles of the first particle group based on the particle size and the label characteristic.

[0010] The control device according to the present disclosure can provide a technique for measuring multiple types of biomolecules at once.

[0011] FIG. 1 is a diagram illustrating the overall configuration of a measurement system according to the present embodiment; FIG. 2 is a diagram illustrating the structure of particles and labels; FIG. 3 is a diagram illustrating particle groups and label groups; FIG. 4 is a diagram illustrating a method of combining particles, biomarkers, and labels; FIG. 5 is a diagram illustrating separation results based on particle size; FIG. 6 is a diagram illustrating detection results of label characteristics; FIG. 7 is a flowchart illustrating a measurement process according to the present embodiment; FIG. 8 is a flowchart illustrating a measurement process according to Modification 1; FIG. 9 is a diagram illustrating a specific example of correction using first correction particles; FIG. 10 is a diagram illustrating a specific example of correction using second correction particles; FIG. 11 is a diagram illustrating the structure of particles and labels according to Modification 2; FIG. 12 is a diagram illustrating particle groups and label groups according to Modification 2; and FIG. 13 is a flowchart illustrating a measurement process according to Modification 2.

[0012] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals and description thereof will not be repeated.

[0013] 1. Configuration of Measurement System FIG. 1 is a diagram illustrating an overview of a measurement system 100 according to an embodiment of the present invention.

[0014] 1 , measurement system 100 includes a control device 4 and a measurement device 5. Measurement device 5 is a device for measuring biomarkers. Measurement device 5 includes a liquid delivery unit 6, a pretreatment unit 71, an injection unit 72, a separation unit 8, flow paths 51 and 52, and a detection unit 9.

[0015] In the measuring device 5, a flow path 51 is connected downstream of a liquid delivery unit 6. The liquid delivery unit 6 delivers a carrier (mobile phase) to the flow path 51. The liquid delivery unit 6 includes a container 61 that contains the carrier, and a liquid delivery pump 62 that sucks the carrier in the container 61.

[0016] The flow channel 51 is a flow channel that connects the liquid delivery section 6 and the separation section 8. An injection section 72 is arranged in the flow channel 51.

[0017] The injection unit 72 is a unit for injecting the mixed solution into the carrier of the flow channel 51. The mixed solution is a solution generated from the sample in the pretreatment unit 71. A method for generating the mixed solution by the pretreatment unit 71 will be described later. The mixed solution contains particles for measuring biomarkers. The particles include particles bound to biomarkers and particles not bound to biomarkers, but both are referred to as "particles" in the description of FIG. 1. A label having a predetermined labeling property (e.g., fluorescence) is bound to the biomarker. The injection unit 72 may be, for example, an autosampler or an injection port through which the user manually injects the mixed solution into the flow channel 51.

[0018] The separation unit 8 separates particles mixed in the carrier (hereinafter also referred to as "particles contained in the carrier") according to particle size. Generally, separating particles according to their size is called "classification." In one embodiment, the separation unit 8 is a classification device that uses, for example, a centrifugal FFF method, which is a type of FFF (Field Flow Fraction) method, or an AF4 (Asymmetrical Flow Field Flow Fraction) method.

[0019] The centrifugal FFF method uses centrifugal force to sediment large particles, thereby classifying particles based on differences in centrifugal force and diffusion coefficient. Using the centrifugal FFF method, particles can be classified according to their mass and size. For roughly spherical particles, particle size can be expressed, for example, by their diameter or volume. The centrifugal FFF method has the advantage of being able to distinguish a large number of biomarker types due to its relatively high size resolution. Furthermore, compared to the AF4 method, the centrifugal FFF method has smaller classification errors and can obtain classification results with high reproducibility. Therefore, there is no need to consider the influence of classification errors on the measurement results of the label characteristics in the detection unit 9. This improves the measurement accuracy of the label characteristics and improves the quantification accuracy of the biomarkers.

[0020] The AF4 method is a method for classifying particles based on the difference in moving speed in a laminar flow caused by generating a force field perpendicular to the moving direction. The centrifugal FFF method allows particles to be classified according to their size. When particles are approximately spherical, particle size can be expressed, for example, by the particle diameter or volume. On the other hand, the AF4 method has the advantage of being able to classify small and light particles. This allows the particle size of particles that can be used for biomarker detection to be set small, thereby increasing the number of types of biomarkers that can be distinguished.

[0021] The separation unit 8 may be a classification device using size exclusion chromatography. In size exclusion chromatography, a solution containing particles is passed through a column with many pores. The particles are then classified by utilizing the fact that smaller particles enter the pores and have a slower elution time. Size exclusion chromatography can classify particles according to their size. When the particles are approximately spherical, particle size can be expressed, for example, by the particle diameter or volume. A classification device using size exclusion chromatography can be configured more inexpensively than a classification device using the FFF (Field Flow Fraction) method.

[0022] The flow channel 52 is a flow channel that connects the separation unit 8 and the detection unit 9. The carrier containing the particles discharged from the separation unit 8 is introduced into the detection unit 9 via the flow channel 52.

[0023] The detection unit 9 detects the label characteristics of the labels bound to the particles separated according to particle size via the biomarkers. In this embodiment, the label characteristics are not particularly limited as long as they are capable of distinguishing the types of labels 2 corresponding to the types of biomarkers 3. In one example, the label characteristics are fluorescence. In this case, the detection unit 9 includes, for example, multiple fluorescence detectors or a multi-wavelength fluorescence detector. A multi-wavelength fluorescence detector is a detector capable of simultaneous measurement at multiple excitation wavelengths and fluorescence wavelengths. Using multiple fluorescence detectors as the detection unit 9 enables fluorescence detection with high sensitivity, thereby improving the measurement accuracy of the label characteristics and the quantification accuracy of the biomarkers. Using a multi-wavelength fluorescence detector as the detection unit 9 allows the number of detectors to be reduced, thereby reducing the cost required for the detection unit 9. In another example, the detection unit 9 is a radiation measurement device or an absorbance meter.

[0024] In the detection unit 9, detection of the labeling characteristics may be performed for each particle, or the labeling characteristics of multiple particles may be detected collectively. For example, when the detection unit 9 is configured so that the number of particles at the position where the labeled substance is measured is always one or less, the labeling characteristics of each particle are detected. For example, this applies when the flow path at the position where the labeled substance is measured is configured to be wide enough to allow only one particle to pass through, or when the concentration of particles in the carrier is sufficiently low so that only one molecule can be present at the measurement position.

[0025] On the other hand, when the system is configured so that there can be multiple particles at the position where the labeled substance is measured, a detection value corresponding to the sum of the labeled characteristics of the multiple particles is detected. In this case, by calculating the number of particles having the specific labeled characteristics from the detection value corresponding to the sum, the type and number of the corresponding biomarkers can be determined.

[0026] The control device 4 controls the measuring device 5 and analyzes the detection results of the detection unit 9. The control device 4 is typically a computer, and can be realized by a dedicated computer or a general-purpose personal computer.

[0027] The control device 4 includes a processor 40 , a memory 41 , an input unit 42 , and a display unit 43 .

[0028] The processor 40 includes, for example, a CPU (Central Processing Unit). The processor 40 loads a program stored in the memory 41 into a RAM or the like and executes the program.

[0029] The memory 41 includes, for example, a read-only memory (ROM), a random access memory (RAM), and a non-volatile memory. The program stored in the ROM is a program that describes the processing procedures of the measurement system 100. The non-volatile memory stores the detection results sent from the detection unit 9 as a data file. Note that the memory 41 may include a hard disk drive (HHD) and / or a solid state drive (SSD) instead of or in addition to the non-volatile memory.

[0030] The input unit 42 is a unit for inputting user instructions to the measurement system 100. For example, the input unit 42 includes a keyboard and a pointing device such as a mouse.

[0031] The display unit 43 includes a liquid crystal display, etc. The display unit 43 displays the detection results of the detection unit 9 and the analysis results thereof.

[0032] The control device 4 may be configured with multiple computers. Furthermore, some or all of the functions of the control device 4 described above may be located in an electronic computer, server, or the like that is physically separate from the measurement device 5. For example, the control device 4 may include a system controller, which is a dedicated computer, and a general-purpose personal computer that is connected to the system controller via a network.

[0033] [2. Conventional Biomarker Measurement Methods] In diagnosing a disease and / or assessing the effectiveness of treatment, it is necessary to objectively evaluate the presence or absence of the disease, the rate of progression, the severity of symptoms, etc. For such objective evaluation of disease, a method of measuring in vivo biomarkers and using them as indicators is useful. In particular, a method that regards a disease as the sum of interactions between many biomolecules and diagnoses it based on a set of measured values ​​of many types of biomarkers is considered promising.

[0034] Conventional methods such as ELISA (Enzyme-Linked Immuno-Sorbent Assay), in which biomarkers are specifically captured one by one, each captured biomarker is labeled, and then the label corresponding to each biomarker is detected, make it difficult to measure many types of biomarkers in terms of testing time and cost.

[0035] On the other hand, xMAP (registered trademark) is a method for measuring multiple types of biomarkers at once, and systems such as Luminex (registered trademark), which is a form of xMAP, are commercially available. In this specification, "measuring multiple types of biomarkers at once" means "labeling multiple types of biomarkers at once and detecting them at once." "Once" refers to, for example, a single process. In xMAP, microbeads are stained with two fluorescent dyes combined at various concentrations. Then, a fluorescent spectrum reflecting the combination pattern of the fluorescent dye concentrations in the beads is used as an identification code.

[0036] In biomarker measurement experiments using xMAP, first, a fluorescent labeling substance is bound to the biomarkers that have specifically bound to the beads, and then multiple types of biomarkers are measured by measuring the fluorescence spectrum of the beads.

[0037] However, when distinguishing beads based solely on their fluorescence spectra, it is difficult to distinguish between beads exhibiting similar fluorescence spectra. Due to this constraint, the number of beads that can be detected at one time, more specifically, beads exhibiting fluorescence spectra that are sufficiently different to be distinguishable from one another, is relatively small. "Fluorescence spectra that are sufficiently different to be distinguishable from one another" refers, for example, to a state in which the positions (wavelengths) on the horizontal axis corresponding to the peaks of each fluorescence spectrum are sufficiently far apart to be distinguishable from one another. For example, when distinguishing beads based on fluorescence spectra corresponding to the blending ratio of two color dyes, as in xMAP, the practical limit for the number of types of beads is thought to be around 30. This makes it theoretically difficult to further increase the number of types of biomarkers that can be detected at one time.

[0038] On the other hand, from the viewpoint that a large number of biomolecules may be involved in diseases, it is desirable to obtain measurements of many types of biomarkers (e.g., 100 types) for diagnosing diseases and / or assessing therapeutic effects, and therefore, in clinical and research settings, there is a demand for techniques that can measure many types of biomarkers simultaneously.

[0039] In view of this situation, the biomarker measurement method according to this embodiment uses particles of different particle sizes in addition to labeling characteristics such as fluorescence to distinguish between different types of biomarkers, thereby increasing the number of types of biomarkers that can be identified. This makes it possible to measure many types of biomarkers at once.

[0040] [3. Measurement Method According to the Embodiment] (3-1. Structure of Particles and Labels) First, the structures of the particles and labels used in the measurement method according to the present embodiment will be described.

[0041] 2 is a diagram illustrating the structure of the particle and the label, showing a biomarker 3, and a particle 1 and label 2 that bind to the biomarker 3.

[0042] In this specification, biomarker 3 refers to a biomolecule that serves as an indicator for quantitatively understanding biological changes in a living body, such as the presence or absence of a disease, the progression of the disease, or the effectiveness of a drug, which is the measurement target of the measurement method according to this embodiment. In one example, the biomarker is a protein. The biomarker may be at least one of a nucleic acid and a metabolite. The nucleic acid may include deoxyribonucleic acid (DNA), messenger RNA (ribonucleic acid), long non-coding RNA, or microRNA.

[0043] The particle 1 includes a particle body 11 and a first binding portion 12 that specifically binds to the biomarker 3 .

[0044] The particle body 11 typically has a spherical shape with a predetermined diameter, but may include shape variations within a predetermined range due to factors such as manufacturing processes. The predetermined range refers to, for example, a range within which classification by the separation unit 8 can be performed without problems. Spherical objects used in measuring biological samples, such as the particle body 11 or particle 1, or spherical objects modified with a predetermined modification, are also referred to as "beads" by those skilled in the art. The material forming the particle body 11 includes, for example, at least one of inorganic materials such as gold or silica (silicon dioxide), and resin materials such as polystyrene. When the particle body 11 is a gold particle, the diameter of the particle body 11 is preferably a predetermined value between 5 and 500 nm. When the particle body 11 is a silica particle, the diameter of the particle body 11 is preferably a predetermined value between 10 and 1,000 nm.

[0045] The gold particles and silica particles having nanoscale sizes as described above will hereinafter also be referred to as "gold nanoparticles" and "silica particles," respectively. The advantages of using gold nanoparticles and silica nanoparticles for the particle body 11 will be described below.

[0046] Gold nanoparticles are stable and resistant to degradation. This means that degradation over time and degradation due to chemicals or impacts are unlikely to occur during storage or measurement. This also makes it extremely unlikely that particle size will change during storage or measurement, improving the reliability of analysis. Furthermore, gold nanoparticles are characterized by easy size control during production. This makes it possible to produce particles with a small variation in size distribution. This allows for a large number of sizes that can be simultaneously separated. This means that when gold nanoparticles are used as the particle body 11, a large number of types of biomarkers 3 can be simultaneously detected.

[0047] As for silica particles, the refractive index of silica is closer to that of water than that of gold, so the scattered light from the particle surface, which can affect detection, is less than that of gold nanoparticles. Furthermore, unlike gold, silica particles do not exhibit high absorbance at specific wavelengths. As a result, when silica nanoparticles are used as the particle body 11, the measurement accuracy of fluorescence intensity and absorbance is improved. This increases the quantitative accuracy of the biomarker 3.

[0048] The advantage of using nanoparticles made of resin material as the particle body 11 is that nanoparticles made of highly reliable resin material with traceability are commercially available, and measurements can be performed relatively easily and accurately.

[0049] In one embodiment, the particle body 11 is a spherical gold particle with a silica coating on its surface. By using such a particle body 11, many types of biomarkers 3 can be detected at once, and the amount of the biomarkers can be measured with high accuracy.

[0050] It is preferable that the difference between the refractive index of the particle 1 and the refractive index of the carrier (hereinafter also referred to as "refractive index difference") is equal to or less than a predetermined value. More specifically, it is preferable that the difference between the refractive index of the particle body 11 and the carrier is equal to or less than a predetermined value. The refractive index difference is not limited thereto, but is preferably equal to or less than 0.1, and more preferably equal to or less than 0.05. The reason why the refractive index difference is preferably equal to or less than a predetermined value will be explained in detail below.

[0051] If there is a refractive index difference between particle 1 and the carrier, scattered light will be generated by the particle when the fluorescence detector irradiates it with excitation light. In this case, even when label 2 is not present, the scattered light may enter the fluorescence detector, causing the fluorescence detector to detect a signal. In order to accurately detect the fluorescence emitted from label 2, it is preferable to set the lower detection limit of the fluorescence detector so that it does not include a detection signal due to scattered light. It is also preferable to set the lower detection limit low enough to ensure the sensitivity required for diagnosis. Therefore, it is preferable to keep the amount of scattered light from particle 1 below a predetermined amount by designing the refractive index difference between the particle and the carrier to be below a predetermined value. This allows the lower detection limit of fluorescence to be set low, thereby improving detection sensitivity.

[0052] Conversely, if the difference in refractive index between the particle 1 and the carrier is greater than a predetermined value, the amount of scattered light from the particle 1 generated upon irradiation with excitation light also increases. Therefore, if the detection limit is raised according to the amount of scattered light, it may become impossible to detect the label 2 with the sensitivity required for diagnosis.

[0053] The first method for reducing the refractive index difference between the particle 1 and the carrier is to use a carrier with a refractive index close to that of the particle 1. The second method is to use a particle 1 with a refractive index close to that of the carrier. The third method is to make the refractive index of the particle 1 and the refractive index of the carrier close to each other.

[0054] In one embodiment, the refractive index of the particles 1 is approximately 1.5, and the refractive index of the carrier is 1.33. The particles 1 are, for example, silica particles (refractive index 1.46), and the carrier is, for example, water (refractive index 1.33). In this embodiment, an example of a first method is to replace the carrier with a refractive index of 1.33 with a carrier with a refractive index of 1.5. An example of a second method is to replace the particles 1 with a refractive index of 1.5 with particles 1 with a refractive index of 1.33. An example of a third method is to replace the carrier with a refractive index of 1.33 with a carrier with a refractive index of 1.4, and to replace the particles 1 with a refractive index of 1.5 with particles 1 with a refractive index of 1.4. The replacement of the particles 1 and carrier includes replacing the original particles 1 and carrier with new particles 1 and carrier, and modifying the original particles 1 and carrier to change the refractive index. Such modifications include, for example, adding a coating to the original particles 1, adding a new component to the carrier, and reformulating the carrier.

[0055] In addition to the refractive index difference, the degree of false detection of scattered light from particle 1 described above is also affected by factors such as the excitation wavelength, fluorescence wavelength, excitation wavelength bandwidth, and fluorescence wavelength bandwidth of the fluorescence detector. For example, the closer the excitation wavelength and fluorescence wavelength are, the higher the likelihood of false detection. Therefore, it is preferable that particle 1 and label 2 are designed and the fluorescence detector is configured so that the excitation wavelength, fluorescence wavelength, excitation wavelength bandwidth, fluorescence wavelength bandwidth, and the like can also reduce the probability of false detection and ensure the detection sensitivity required for diagnosis.

[0056] The first binding portion 12 is bound to the particle body 11. The first binding portion 12 is a binding site on the particle 1 that specifically binds to a predetermined type of biomarker 3. This gives the particle 1 binding specificity for the biomarker 3. In one embodiment, when the biomarker 3 is a predetermined type of protein, the first binding portion 12 typically includes an antibody that binds to the predetermined type of protein. In this case, the antibody and the predetermined type of protein have a structure that specifically binds to each other through forces such as hydrogen bonds, Coulomb forces, or van der Waals forces. In another embodiment, when the biomarker 3 is a nucleic acid having a predetermined sequence, the first binding portion 12 includes a nucleic acid having a sequence complementary to a first sequence that is a part of the predetermined sequence. The nucleic acid includes microRNA. In the complementary sequence, for example, adenine-uracil and guanine-cytosine hydrogen bonds are formed between the first sequence and the nucleic acid having the complementary sequence. In yet another embodiment, when the biomarker 3 is a predetermined type of metabolite, the first binding portion 12 typically includes a molecule that specifically binds to a part of the predetermined type of metabolite.

[0057] The label 2 includes a labeling portion 21 and a second binding portion 22 that specifically binds to the biomarker 3 .

[0058] The labeling portion 21 is a portion containing a substance (hereinafter also referred to as "labeling substance") for labeling the particle 1 to which the biomarker 3 is bound. The labeling substance exhibits predetermined labeling characteristics. The labeling characteristics of the labeling portion 21 in the label 2 for different types of biomarkers 3 are configured to be distinguishable from each other. This allows the type of biomarker 3 to be identified based on the difference in the labeling characteristics of the labeling substance.

[0059] In one embodiment, the labeling substance is a fluorescent substance, and the labeling characteristic is fluorescence. The fluorescence may be detected as a fluorescence spectrum showing the relationship between wavelength and fluorescence intensity, or as a pattern (graph shape) thereof, or as the intensity at a predetermined wavelength only. The fluorescent substance is typically a single fluorescent dye, but a mixture of two or more fluorescent dyes may also be used. For example, the fluorescent dye may be a dye typically used in FACS (Fluorescence-Activated Cell Sorting). More specifically, dyes commonly used for labeling, such as dyes with different wavelengths from the Alexa Fluor (registered trademark) family, rhodamine, or PI (Propidium Iodide), may be used.

[0060] The fluorescence spectrum of each type of label 21 corresponding to the type of biomarker 3 has, for example, a fluorescence wavelength that characterizes the respective fluorescence spectrum. The fluorescence wavelength that characterizes the fluorescence spectrum of a predetermined type of label 21 is, for example, a fluorescence wavelength at which the fluorescence intensity of the predetermined type of label 21 is high but the fluorescence intensity of other types of label 21 is very low. In this case, by referring to the fluorescence intensity for that wavelength, it is possible to determine whether or not the predetermined type of label 21 is present.

[0061] More specifically, it is preferable that the fluorescence spectra of the different types of labeling portions 21 corresponding to the different types of biomarkers 3 are configured so that their peaks do not overlap with each other (see FIG. 6). Specifically, the labeling portions 21 are fabricated so that the wavelengths at which the peaks of the respective types of labeling portions 21 are sufficiently separated. Furthermore, at the wavelength at which the peak of a given type of labeling portion 21 is at its peak, the fluorescence intensity of other types of labeling portions 21 is hardly detected. In this case, the types of labeling portions 21 can be distinguished by referring to the fluorescence intensity at the wavelength at which the peak of each type of labeling portion 21 is at its peak.

[0062] In another embodiment, the labeling substance is a radioisotope and the labeling characteristic is the amount of radiation, which may be detected as a radiation spectrum or its pattern (shape of a graph) showing the relationship between wavelength and amount of radiation, or simply the intensity of radiation for a given wavelength.

[0063] In yet another embodiment, the marker substance is a substance exhibiting a predetermined absorbance, and the marker characteristic is absorbance. The absorbance may be detected as an absorbance spectrum showing the relationship between wavelength and absorbance amount, or as its pattern (graph shape), or only the absorbance at a predetermined wavelength may be detected. When a substance exhibiting a predetermined absorbance is used as the marker substance, since gold has a high absorbance of purple and blue light, it is preferable that the surface of the particle body 11 be composed of a material other than gold (e.g., silica) so that the particle body 11 does not affect the detection of the marker substance. The marker substance may be a substance exhibiting a color at a wavelength recognizable by the naked eye, and the marker characteristic may be that color. However, examples of the marker substance and marker characteristic are not limited to those described above, as long as the marker portions 21 corresponding to each type of biomarker 3 can be distinguished from one another.

[0064] The second binding portion 22 is bound to the label portion 21. The second binding portion 22 is a binding site in the label 2 that specifically binds to a predetermined type of biomarker 3. This gives the label 2 binding specificity for the biomarker 3. The second binding portion 22 binds to a site in the biomarker 3 that is different from the binding site of the first binding portion 12. This allows the label 2 to bind to the biomarker 3 while the particle 1 is bound to the biomarker 3. In one embodiment, if the biomarker 3 is a predetermined type of protein, the second binding portion 22 typically includes an antibody that binds to the predetermined type of protein. In another embodiment, if the biomarker 3 is a nucleic acid having a predetermined sequence, the second binding portion 22 typically includes a nucleic acid having a sequence complementary to a second sequence that is a part of the predetermined sequence. The second sequence is a sequence different from the first sequence to which the first binding portion 12 binds. In still another example, when the biomarker 3 is a predetermined type of metabolite, the second binding part 22 typically specifically binds to the predetermined type of metabolite at a site other than the site to which the first binding part 12 binds. As described above, the measurement method according to this embodiment can measure biomarkers 3 belonging to any of the categories of proteins, nucleic acids, and metabolites.

[0065] In one embodiment, the second binding portion 22 specifically binds to the biomarker 3 in a state where it is previously bound to the labeling portion 21. However, the second binding portion 22 may also be configured to specifically bind to the biomarker 3 before being bound to the labeling portion 21, and then specifically bind to the labeling portion 21.

[0066] The mode of binding between the second binding portion 22 and the labeling portion 21 is not particularly limited, but may be, for example, amide bond formation using a carbodiimide reaction, avidin-biotin interaction, or click chemistry using cyclooctyne-azide.

[0067] (3-2. Sizes of Particle, Biomarker, and Label) Here, the relationship in size between the particle body 11, the first binding portion 12, the biomarker 3, and the label 2 will be further explained.

[0068] In the measurement method according to this embodiment, particle bodies 11 of different sizes are used, as detailed in FIG. 3 . More specifically, a plurality of particle bodies 11 having a plurality of sizes that can be classified by the separation unit 8 are used. In this specification, the "size" of an object refers to a value indicating the size and / or mass of the object. The size of an object can be expressed, for example, by the maximum outer diameter (diameter in the case of a spherical object) and / or volume. In one embodiment, the composition of the particle bodies 11 is approximately the same regardless of the size of the particle bodies 11, and the specific gravity of the particle bodies 11 is also approximately the same. In this case, the diameter, volume, and / or mass of the particle bodies 11 are correlated with each other.

[0069] Furthermore, it is preferable that the difference in size between the plurality of particle bodies 11 having different sizes is sufficiently larger than the sizes of the first binding portions 12, the biomarkers 3, and the labels 2. Here, size refers to, for example, a physical quantity (e.g., maximum outer dimension, volume, and / or mass) that indicates the size that affects classification in the separation unit 8. With this configuration, classification can be performed in the separation unit 8 based on the size of the particle bodies 11, regardless of the types of the first binding portions 12, biomarkers 3, and labels 2 that are bound to the particle bodies 11.

[0070] Similarly, among the plurality of particle bodies 11 having different sizes, it is preferable that the size of the particle body 11 having the smallest size is also configured to be sufficiently larger than the sizes of the first binding portion 12, the biomarker 3, and the label 2. With this configuration, the particle bodies 11 can be classified in the separation unit 8 based on the size thereof, regardless of the types of the first binding portion 12, the biomarker 3, and the label 2 bound to the particle body 11. Furthermore, the free label 2 contained in the mixed solution can be accurately classified from the particle body 11 having the smallest size. This eliminates the risk of the free label 2 being erroneously detected as the labeled particle body 11 having the smallest size.

[0071] As a result, the size of particle 1 formed by binding first binding portion 12 to particle body 11 of a predetermined size is approximately equal to the size of the original particle body 11. Furthermore, the sizes of the complex formed by binding biomarker 3 to particle 1 (hereinafter also referred to as "biomarker complex") and the complex formed by binding label 2 to this complex (hereinafter also referred to as "labeled complex") are also approximately unchanged from the size of the original particle body 11. That is, the sizes of the original particle body 11, particle 1, biomarker complex, and labeled complex are approximately equal. In view of the above circumstances, in this specification, the sizes of particle body 11 and particle 1 are collectively referred to as "particle size." Furthermore, when biomarker 3 binds to particle 1 to form a biomarker complex, the size of the biomarker complex is also referred to as particle size. Furthermore, when biomarker 3 and label 2 bind to particle 1 to form a labeled complex, the size of the labeled complex is also referred to as particle size.

[0072] In this specification, "particle sizes are different from each other" means, for example, that the particle size distributions of different particle groups (e.g., different particle subgroups) do not overlap. Alternatively, when a certain degree of overlap is allowed, the average particle diameters may be different. Alternatively, particle sizes may be selected appropriately as long as particles belonging to the average particle diameter of one particle group can be separated from those of another particle group by particle size separation means such as FFF.

[0073] According to the above configuration, it is possible to prepare a labeled complex containing the particle 1 via the first binding part 12 and the second binding part 22. Furthermore, it is possible to distinguish the type of the labeled complex based on the particle size mainly attributable to the particle body 11 and the labeling characteristics of the labeling part 21. This makes it possible to identify the type of biomarker 3 corresponding to the labeled complex.

[0074] (3-3. Particle Groups and Labeled Groups) In the measurement method according to this embodiment, the labeled complexes are separated based on the particle size and then distinguished based on the label characteristics. Next, the groups of particles 1 defined by the particle size and label characteristics will be described.

[0075] Fig. 3 is a diagram illustrating particle groups and label groups, showing groups including particles 1a to 1d and labels 2a to 2d that specifically bind to four types of biomarkers 3a to 3d, respectively.

[0076] In this embodiment, a "particle group" is a group of particles 1 that are bound to a label 2 having a particular labeling property. In Figure 3, a first particle group and a second particle group are shown.

[0077] Furthermore, a "label group" is a group of labels 2 corresponding to a specific particle group. In FIG. 3, a first label group corresponding to a first particle group and a second label group corresponding to a second particle group are shown. Particles 1 of the first particle group and labels 2 of the first label group are bonded via biomarkers 3. Particles 1 of the second particle group and labels 2 of the second label group are bonded via biomarkers 3.

[0078] The markers 2 (2a, 2b) of the first marker group and the markers 2 (2c, 2d) of the second marker group have different marker characteristics. Referring to Figure 3, the marker 2 of the first marker group includes a marker portion 21a having a predetermined marker characteristic. The marker 2 of the second marker group includes a marker portion 21c having a marker characteristic different from that of the first marker group.

[0079] Each of the first particle group and the second particle group (hereinafter also referred to as "each particle group") includes a plurality of particle subgroups.

[0080] Each particle subgroup includes particles 1 that have the same particle size and are labeled with labels 2 that have the same labeling properties. With reference to FIG. 3 , for example, particle subgroup a is made up of particles 1a that have the same particle size and are labeled with labels 2a. Each labeled group includes multiple labeled subgroups that correspond to the multiple particle subgroups in each particle group. A group of labels 2a that corresponds to particle subgroup a is referred to as "label subgroup" a. Furthermore, particle subgroup a and label subgroup a are collectively referred to as "particle-label subgroup" a. The number of types of particle-label subgroups corresponds to the number of types of distinguishable biomarkers 3.

[0081] Since the particles 1 of each particle subgroup bind to different types of biomarkers 3, the first binding portions 12 are also different. Since the labeled subgroups bind to different types of biomarkers 3, the second binding portions 22 are also different. In other words, the particles 1 of each particle subgroup have first binding portions 12 structured to specifically bind to different types of biomarkers 3. Furthermore, the labels 2 of each labeled subgroup have second binding portions 22 structured to specifically bind to biomarkers 3 to which the particles 1 of multiple particle subgroups of the corresponding particle group specifically bind. Referring to FIG. 3, particles 1a to 1d include first binding portions 12a to 12d, respectively. Referring to FIG. 3, labels 2a to 2d include second binding portions 22a to 22d, respectively.

[0082] In each particle group, the particle subgroups differ from one another in particle size. Referring to Figure 3, in the first particle group, the diameter of the particle body 11a of particle 1a in particle subgroup a is larger than the diameter of the particle body 11b of particle 1b in particle subgroup b. In the second particle group, the diameter of the particle body 11a of particle 1c in particle subgroup c is larger than the diameter of the particle body 11b of particle 1d in particle subgroup d. This allows the particle subgroups in each particle group to be distinguished by particle size.

[0083] Next, the second particle group and the second labeled group will be further described. In the particle-label subgroup c of the second particle group, the particle size is the same as that of the particle-label subgroup a, but the labeling characteristics of the labeled portion 21 are different. With reference to FIG. 3 , the particle-label subgroup c and the particle-label subgroup a can be distinguished by the labeling characteristics. Furthermore, in the particle-label subgroup d of the second particle group, the particle size is the same as that of the particle-label subgroup b, but the labeling characteristics of the labeled portion 21 are different. As a result, the particle-label subgroup d and the particle-label subgroup b can be distinguished by the labeling characteristics.

[0084] As described above, labeled complexes corresponding to a particle-label subgroup corresponding to a given type of biomarker 3 can be distinguished from labeled complexes corresponding to other particle-label subgroups based on particle size and label characteristics. More specifically, if the given particle-label subgroup and the other particle-label subgroups correspond to the same particle group, they can be separated by particle size. Furthermore, if the given particle-label subgroup and the other particle-label subgroups correspond to different particle groups, they can be distinguished by label characteristics. This allows the types of biomarkers 3 to be distinguished based on particle size and label characteristics.

[0085] Naturally, the number of particle subgroups and the number of label groups included in each particle group may each be three or more, and similarly, the number of label subgroups included in each particle group and each label group may each be three or more. For example, if the number of particle subgroups in each particle group is 10 to 20 and the number of label groups is 4 to 5, it is possible to measure 40 to 100 types of biomarkers at once. Furthermore, if the number of particle subgroups in each particle group is 10 to 20 and the number of label groups is approximately 20, it is possible to measure approximately 200 to 400 types of biomarkers at once. In this way, by combining variations in label characteristics and variations in particle size, it is possible to increase the number of types of biomarkers that can be measured at once.

[0086] Furthermore, in the example of Figure 3, two particle size patterns and two label characteristic patterns are combined in a matrix to form four particle-label subgroup patterns. However, it goes without saying that the particle sizes and label characteristics do not necessarily have to be combined in a matrix. For example, the particle size pattern of the particle subgroups of the first particle group and the particle size combination of the subgroups of the second particle group may be different. However, when particle size patterns and label characteristic patterns are combined in a matrix to form particle-label subgroups, there is the advantage that the number of particle size patterns and label characteristic patterns required to form all particle-label subgroups can be minimized. This allows the maximum number of particle-label subgroups to be formed by maximizing the particle size resolution of the separation unit 8 and the resolution of the detected values ​​of the label characteristics of the detection unit 9. In other words, the number of distinguishable biomarker types can be maximized.

[0087] (3-4. Method for binding particles, biomarkers, and labels) Next, a method for binding particles 1, biomarkers 3, and labels 2 in the pretreatment unit 71 will be described. In one embodiment, this method is performed manually by a user using general molecular biology laboratory equipment, but it may also be performed by an automatic pretreatment device. In other words, the pretreatment unit 71 may be an automatic pretreatment device or may be laboratory equipment handled by the user.

[0088] FIG. 4 is a diagram illustrating a method for binding particles, biomarkers, and labels. First, a user prepares a sample containing a biomolecule called a biomarker to be measured. The sample may be, for example, a biological specimen such as urine or blood from a subject. The sample may be appropriately prepared and / or purified in advance. In one embodiment, the sample is contained in a container typically used for preparing biological samples, such as a microtube.

[0089] Next, the user mixes particles 1 into the sample. For example, the user adds a solution containing a predetermined number of particles 1 belonging to each particle subgroup to a container containing the sample, mixes them, and creates a mixed solution. The particles 1 are made to be capable of binding to the biomarkers 3 to be measured. As a result, the biomarkers 3 to be measured contained in the sample bind to the corresponding particles 1. In one embodiment, the number of particles 1 is mixed so that it is sufficiently greater than the number of biomarkers 3 to be measured, so that almost all of the biomarkers 3 to be measured can bind to the particles 1. As a result, the biomarkers 3 to be measured exist in the mixed solution in the form of biomarker complexes.

[0090] Next, the user further mixes labels 2 into the mixed solution. As a result, the labels 2 specifically bind to the biomarkers 3, forming biomarker complexes. In one embodiment, the number of labels 2 is mixed so that it is sufficiently greater than the number of biomarkers 3, so that almost all of the biomarkers 3 to be measured are labeled. As a result, the biomarkers 3 to be measured exist in the mixed solution in the form of labeled complexes.

[0091] As described above, a labeling method in which a substance to be measured (here, biomarker 3) is sandwiched between substances that specifically bind to the substance (here, first binding portion 12 of particle 1 and second binding portion 22 of label 2) is generally called the sandwich method. By using the sandwich method, multiple types of biomarkers 3 can be specifically labeled simultaneously in a single step simply by adding multiple types of labels 2 corresponding to the multiple types of biomarkers 3.

[0092] The mixed solution prepared as described above is injected into the measuring device 5, whereby the labeled complexes are separated by particle size in the separation unit 8, and the label characteristics can be detected in the detection unit 9.

[0093] The mixed solution also contains free biomarkers that are not the measurement target, but because they are smaller in size than the labeled complex with the smallest particle size, they are separated from the labeled complex that is the measurement target in separation unit 8. This does not affect the detection results of the labeled complex. The mixed solution also contains particles 1 that are not bound to biomarker 3 and label 2 that are the measurement target, but because they are not labeled, they are not detected in detection unit 9. This does not affect the detection results of the labeled complex.

[0094] Next, the results of separation based on actual particle size and the results of detection of label characteristics in the measuring device 5 will be described.

[0095] (3-5. Separation Results by Particle Size and Detection Results of Labeling Characteristics) Figure 5 shows the separation results by particle size. The horizontal axis of Figure 5 represents elution time. The elution time is the time from when the mixed solution is injected into the sample injection section 72 until a predetermined component is detected. The vertical axis represents the baseline-corrected absorbance. Referring to Figure 5, particles 1 with diameters of 7 nm, 10 nm, 15 nm, 45 nm, 75 nm, and 110 nm can be detected with elution times that do not overlap. In other words, the labeled complex can be separated based on particle size.

[0096] Figure 6 shows the results of detecting the labeling characteristics. In Figure 6, the horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. Referring to Figure 6, three fluorescence spectra are detected in a manner that allows them to be distinguished from one another. In other words, the labeled complexes can be separated based on the fluorescence spectra, which are the labeling characteristics.

[0097] 5 and 6, it is possible to distinguish particle-label subgroups based on particle size and label characteristics, which allows us to determine which type of biomarker is detected.

[0098] (3-6. Measurement Process) Next, a process for carrying out the measurement method according to the embodiment will be described.

[0099] 7 is a flowchart showing the measurement process according to this embodiment. The steps shown in FIG.

[0100] In the figure, "S" is used as an abbreviation for "STEP." In S1, the user prepares particles 1 belonging to a first particle group and particles 1 belonging to a second particle group. The user also prepares labels 2 belonging to a first label group corresponding to the first particle group and labels 2 belonging to a second label group corresponding to the second particle group.

[0101] In S2, the user uses the pretreatment unit 71 to mix the sample with particles 1 of the first particle group and particles 1 of the second particle group, thereby specifically binding biomarker 3 to particles 1 of the first particle group and particles 1 of the second particle group.

[0102] In S3, the user uses the pre-treatment unit 71 to mix the mixed solution prepared in S2 with label 2 of the first labeling group and label 2 of the second labeling group, thereby specifically binding label 2 of the first labeling group and label 2 of the second labeling group to biomarker 3.

[0103] In S4, the processor 40 separates each of the particles 1 of the first particle group and the particles 1 of the second particle group based on particle size. In one embodiment, the processor 40 introduces the mixed solution injected from the injection unit 72 into the separation unit 8, and separates the labeled complex and the particle 1 alone in the mixed solution based on particle size in the separation unit 8. For example, if the separation unit 8 is a centrifugal FFF, molecules with smaller particle sizes will flow out first. As a result, after the free label 2 in the mixed solution flows out, it is fractionated according to particle size, such as particle 1 alone or labeled complex containing the particle 1 with the smallest particle size, particle 1 alone or labeled complex containing the particle 1 with the second smallest particle size, particle 1 alone or labeled complex containing the particle 1 with the third smallest particle size, etc.

[0104] The processes of S2 and S3 may be performed by the processor 40 controlling a device (e.g., an automatic pretreatment device) that performs the same processes as the user's operations. The order of S2 and S3 may be reversed, or they may be performed simultaneously. Specifically, the sample and label 2 may be mixed first to bind the biomarker 3 and label 2, and then the particle 1 may be mixed to bind the particle 1 to the biomarker 3. Alternatively, the sample, particle 1, and label 2 may be mixed simultaneously to bind the particle 1 to the biomarker 3 in a single step.

[0105] In S5, the processor 40 detects the label characteristics of the labels 2 bound to each of the particles 1 of the first particle group and the particles 1 of the second particle group separated based on particle size via the biomarkers 3. For example, if the detection unit 9 is a plurality of fluorescence detectors or a multi-wavelength fluorescence detector, the processor 40 irradiates the particles 1 with excitation light and detects the intensity of the generated fluorescence.

[0106] At S6, the processor 40 determines the type of biomarker 3 bound to each of the particles 1 of the first particle group and the particles 1 of the second particle group based on the particle size and label characteristics.

[0107] In S7, the processor 40 measures the amount of each type of biomarker 3 based on the result of determining the type of biomarker 3. The amount of each type of biomarker 3 is, for example, the concentration or number of each biomarker 3 in the sample.

[0108] In one embodiment of S6 and S7, the processor 40 determines the type of biomarker 3 corresponding to the label 2 that has emitted fluorescence based on the fluorescence intensity, and measures the amount of each type of biomarker 3.

[0109] More specifically, when detecting fluorescence for each particle (i.e., for each particle 1 alone or labeled complex), the processor 40 identifies the label 2 corresponding to the fluorescence intensity and identifies the type of biomarker 3 corresponding to the label 2. Then, each time a type of biomarker 3 is identified, the number of identified biomarkers 3 is added up. This determines the "number of biomarkers 3 contained in the labeled complexes in the mixed solution." In one embodiment, the number of particles 1 and the number of labels 2 are adjusted so that they are sufficient to convert all biomarkers 3 in the mixed solution into labeled complexes. This means that the "number of biomarkers 3 contained in the labeled complexes in the mixed solution" is considered to correspond to the "number of biomarkers 3 contained in the sample." The processor 40 can determine the "concentration of biomarkers 3 contained in the sample" based on the "number of biomarkers 3 contained in the sample" and the "total amount of sample before the mixed solution was prepared."

[0110] On the other hand, when the detection unit 9 simultaneously detects the fluorescence of multiple particles, the processor 40 calculates, from the collection of the fluorescence spectra of the multiple particles, how many labels 2 each having which label characteristic (for example, fluorescence intensity at a predetermined wavelength) are included. As a more specific example, the processor 40 determines the fluorescence intensity or peak area at a predetermined wavelength in the fluorescence spectra of the multiple particles, and then determines the type and number of biomarkers 3 corresponding to the labels 2.

[0111] Even when the detection unit 9 detects fluorescence particle by particle, it may output an aggregate of fluorescence spectra of multiple particles by adding together the detection results within a predetermined range. In this case, the processor 40 performs processing in the same manner as when detecting fluorescence from multiple particles simultaneously. The predetermined range is, for example, a range in which particle sizes are equivalent. The range in which particle sizes are equivalent is, for example, defined in advance based on the time since the separation process began in the separation unit 8.

[0112] In S8, the user diagnoses a disease and / or determines the effectiveness of a treatment based on the amounts of each type of biomarker 3. For example, based on a combination of the amounts of multiple types of biomarkers 3 that are thought to be related to a specific disease and the effectiveness of the treatment, the presence or absence of the disease, the severity of the disease, the rate of progression of the disease, the effectiveness of the treatment, etc. may be determined. Note that by storing the combinations of the amounts of multiple types of biomarkers 3 in memory 41, the processor 40 may be automated to perform processing equivalent to the above-mentioned diagnosis by the user.

[0113] As described above, the measurement method according to this embodiment can distinguish and detect many types of biomarkers 3 by using a combination of particles 1 with different particle sizes and labels 2 with different labeling properties. This allows many types of biomarkers 3 to be measured at once. Furthermore, it is possible to diagnose a disease or determine the effectiveness of a treatment based on the amount of each type of biomarker measured at once. In other words, diagnosis based on multiple types of biomarkers can be easily performed.

[0114] While the above describes a configuration for identifying the type of biomarker 3 using particle size and labeling characteristics, it is of course also possible to identify the biomarker 3 using only particle size. For example, by using only the first particle group and first labeling group in FIG. 3, it is possible to identify the type of biomarker 3 corresponding to each subgroup of the first particle group. In this case, the number of types of biomarker 3 corresponding to the number of particle sizes can be identified. In this way, a technology can be provided for simultaneously measuring multiple types of biomolecules based on differences in particle size.

[0115] [4. Measurement Method According to Modification 1] The majority of the detected value (e.g., peak area) of the labeling characteristic (e.g., fluorescence intensity) detected in S5 of FIG. 7 is attributable to the labeled portion 21. However, there is a possibility that components from other parts of the labeled complex may also be included. In this way, detection value components due to influences other than those of the part being measured are generally referred to as "background." In the measurement method according to this embodiment, it may be preferable to correct the background, particularly the influence on the detection value due to the particle body 11, which accounts for the majority of the labeled complex. In Modification 1 of this embodiment, a configuration for removing the background and correcting the detection value will be described.

[0116] (4-1. Measurement Process Including Correction Process) Next, a measurement method according to Modification 1 of this embodiment will be described. The measurement method according to Modification 1 includes a process of correcting the detection value obtained by the process of detecting the marker characteristics. In Modification 1, the detection value before correction and the detection value after correction are referred to as the detection value before correction and the detection value after correction, respectively.

[0117] FIG. 8 is a flowchart showing a measurement process according to Modification 1. The steps shown in FIG. 8 are performed using measurement system 100. In FIG. 8, steps S51 and S52 are performed instead of S5 in FIG. 7. Steps S1 to S4 and S6 to S8 in FIG. 8 correspond to steps S1 to S4 and S6 to S8 in FIG. 7, respectively. Descriptions of steps in FIG. 8 that overlap with those in FIG. 7 will not be repeated.

[0118] In S51 of FIG. 8, the processor 40 detects the label characteristics of the label 2 bound to the particle 1 to obtain an uncorrected detection value.

[0119] In S52, the processor 40 corrects the pre-correction detection value to obtain a corrected detection value. Next, as specific aspects of the correction in S52, a correction method using first correction particles and a correction method using second correction particles will be described.

[0120] (4-2. Correction Method Using First Correction Particles) As the first step in the correction, first correction particles are prepared, each having a size corresponding to the particle size of each of the plurality of particle subgroups of each particle group. The first correction particles do not have the labeling portion 21 bonded thereto.

[0121] In one embodiment, the first correction particle is a particle that affects the detection value in the same manner as particle 1, and is typically an object equivalent to particle 1. An object equivalent to particle 1 is, for example, an object having the same size, composition, or structure. The first correction particle includes a particle body 11, which is a portion that is expected to have a relatively large effect on the detection value of the label characteristic, next to the label portion 21. The first correction particle may further include a first binding portion 12 that is expected to have a relatively small effect on the detection value of the label characteristic. This allows for more accurate correction.

[0122] In one embodiment, the label characteristic is fluorescence intensity, and in this case, scattered light on the surface of the particle body 11 may affect the detected value of the label characteristic. More specifically, the effect of scattered light appears as a peak area caused by scattered light.

[0123] As a next step, the labeling characteristics of the first correction particle in a "state in which the labeling portion 21 is not bound" are detected. Note that for the first correction particle in this state, the biomarker 3 and the second binding portion 22, which are portions that are thought to have a relatively small effect on the detected value of the labeling characteristics, may each be bound. This allows for more accurate correction that takes into account the influence of the biomarker 3 and the second binding portion 22.

[0124] In one embodiment, the first correction particles are measured in the same manner as the measurement of the mixed sample containing the labeled complex to be measured. Specifically, the first correction particles are introduced through the injection unit 72, separated according to their size in the separation unit 8, and then the label characteristics are detected in the detection unit 9.

[0125] As the final step, for each particle size, the pre-correction detection value of the labeling characteristics of the labeling portion 21 bound to particle 1 of the first particle group and particle 1 of the second particle group is corrected based on the detection value of the labeling characteristics of the first correction particle to obtain the corrected detection value.

[0126] FIG. 9 is a diagram illustrating a specific example of correction using first correction particles. In the example of FIG. 9, the label characteristic is fluorescence intensity. The graph in FIG. 9 shows the detection intensity of a predetermined fluorescence wavelength as a function of elution time. The horizontal axis of the graph in FIG. 9 indicates the elapsed time after injection from the injection section 72. In other words, the horizontal axis correlates with particle size. The vertical axis is fluorescence intensity at a predetermined fluorescence wavelength λa. More specifically, it is fluorescence intensity at a fluorescence wavelength that characterizes the fluorescence spectrum of the labeling portion 21a of the particles 1a and 1b to be measured. The fluorescence wavelength that characterizes the fluorescence spectrum of the labeling portion 21a is, for example, a fluorescence wavelength that corresponds to a peak in the fluorescence spectrum of the labeling portion 21a and is hardly detected in the fluorescence spectra of the labeling portions 21 of other particles.

[0127] 9, a peak with a peak area Sar is detected corresponding to the first correction particle 1ar in a state in which the labeling moiety 21 is not bound, and a peak with a peak area Sbr is detected corresponding to the first correction particle 1br in a state in which the labeling moiety 21 is not bound. Note that the first correction particle 1ar is an object equivalent to the particle 1a. The first correction particle 1br is an object equivalent to the particle 1b. The peak areas Sar and Sbr correspond to the background.

[0128] Furthermore, the detection results of the labeling characteristics for the labeled complex containing the labeled portion 21 a and the particle 1 a, and the labeled complex containing the labeled portion 21 a and the particle 1 b are shown as a peak with a peak area Sa and a peak with a peak area Sb. The peak areas Sa and Sb correspond to examples of "pre-correction detected values."

[0129] A correction is performed by subtracting the detection value of the first correction particle 1ar and the detection value of the first correction particle 1br from such pre-correction detection values. As a result, the corrected peak area Sax for the labeled complex containing particle 1a becomes Sax = Sa - Sar. As a result, the corrected peak area Sbx for the labeled complex containing particle 1b becomes Sbx = Sb - Sbr. The peak areas Sax and Sbx correspond to examples of "corrected detection values."

[0130] 9, the influence of scattered light on the surface of the particle body 11 can be eliminated. As a result, a component that correlates with the number of detected labeled moieties 21a remains in the corrected peak area. This improves the accuracy of quantifying the number of detected labeled moieties 21a based on the corrected peak area.

[0131] As described above, by using the first correction particles to remove background data contained in the pre-correction detection values, the post-correction detection values ​​are mainly attributable to the labeling characteristics of the labeling portion 21. This allows the number of each type of biomarker 3 to be determined more accurately based on the post-correction detection values. This improves the quantitative accuracy of the biomarkers 3 in the measurement method according to this embodiment.

[0132] The first correction particles are of the same particle size as the measurement target particles 1, so the number of particle sizes to be measured is not reduced. This allows background correction while maximizing the number of distinguishable biomarker types.

[0133] (4-3. Other Forms of Correction Method) In the correction using the above-mentioned first correction particles, it was necessary to measure the first correction particles separately from the measurement of the labeled complex to be measured. Next, a method of performing correction by a single measurement by adding correction particles as an internal standard to the labeled complex to be measured will be described.

[0134] As a first step in the correction, first, second correction particles having particle sizes different from the particle sizes of each of the plurality of particle subgroups of each particle group are prepared.

[0135] The second correction particles do not have any labeling portion 21. In one embodiment, the second correction particles include particle bodies 11 having sizes different from the sizes of the particle bodies 11 of each of the plurality of particle subgroups of each particle group.

[0136] As a next step, the labeling characteristics of the second correction particles are detected in a state where the labeling moiety 21 is not bound to them. In one embodiment, the second correction particles are mixed with a mixed sample containing a labeled complex containing the particle 1 to be measured, and then introduced from the injection unit 72. The second correction particles and the labeled complex to be measured are then classified by the separation unit 8, and the labeling characteristics of each are detected in the detection unit 9. This makes it possible to simultaneously obtain the detection value of the second correction particles and the pre-correction detection value of the labeled complex to be measured.

[0137] As the final step, for each particle 1 of the first particle group and particle 1 of the second particle group, the pre-correction detection value of the labeling characteristics of the bound labeling portion 21 is corrected based on the detection value of the labeling characteristics of the second correction particle to obtain a corrected detection value.

[0138] FIG. 10 is a diagram illustrating a specific example of correction using second correction particles. In the example of FIG. 10, the label characteristic is fluorescence intensity. The horizontal axis of the graph in FIG. 10 indicates the elapsed time after injection from the injection portion 72. As a result, the horizontal axis correlates with particle size. The vertical axis is fluorescence intensity at a predetermined wavelength. More specifically, the vertical axis of the upper graph in FIG. 10 is fluorescence intensity at a fluorescence wavelength λa that characterizes the fluorescence spectrum of the labeling portion 21a of particles 1a and 1b. The vertical axis of the lower graph in FIG. 10 is fluorescence intensity at a fluorescence wavelength λc that characterizes the fluorescence spectrum of the labeling portion 21c of particles 1c and 1d.

[0139] FIG. 10 shows the results of measurement after the second correction particles 1r are simultaneously injected into the measuring device 5 in addition to the labeled complexes containing the measurement target particles 1a to 1d, respectively.

[0140] Specifically, in the "graph of fluorescence intensity versus wavelength λa" in the upper part of Fig. 10, a peak with a peak area Sr1 is detected corresponding to the second correction particle 1r. Furthermore, the detection results of the labeling characteristics for the labeled complex containing the labeled moiety 21a and the particle 1a, and the labeled complex containing the labeled moiety 21a and the particle 1b are shown as a peak with a peak area Sa and a peak with a peak area Sb.

[0141] In the "graph of fluorescence intensity versus wavelength λc" graph in the lower part of Fig. 10, a peak with a peak area Sr2 is detected corresponding to the second correction particle 1r. Furthermore, the detection results of the labeling characteristics for the labeled complex containing the labeled portion 21c and particle 1c, and the labeled complex containing the labeled portion 21c and particle 1d are shown as a peak with a peak area Sc and a peak with a peak area Sd. The peak areas Sa to Sd correspond to examples of "pre-correction detected values."

[0142] These uncorrected detection values ​​are corrected using correction coefficients Ka to Kd calculated based on the peak areas Sr1 and Sr2 of the second correction particle 1r. The correction coefficients Ka and Kb are numbers indicating how much the peak area of ​​the particle bodies 11a and 11b changes relative to the peak area Sr1 of the second correction particle. The correction coefficients Kc and Kd are numbers indicating how much the peak area of ​​the particle bodies 11a and 11b changes relative to the peak area Sr2 of the second correction particle. The correction coefficients Ka to Kd are calculated using a Mie scattering model or a Rayleigh scattering model. The correction coefficients Ka to Kd may be determined based on the detection values ​​obtained by performing a measurement on an unlabeled particle 1 in advance. This allows the peak area of ​​the second correction particle 1r to be used to calculate the influence on the peak area caused by measurement particles having a particle size different from that of the second correction particle 1r.

[0143] Using the correction coefficients calculated as above, the corrected peak area Sax for the labeled complex containing particle 1a is Sax = Sa - Ka x Sr1. The corrected peak area Sbx for the labeled complex containing particle 1b is Sbx = Sb - Kb x Sr1. The corrected peak area Scx for the labeled complex containing particle 1c is Scx = Sc - Kc x Sr2. The corrected peak area Sdx for the labeled complex containing particle 1d is Sdx = Sd - Kd x Sr2.

[0144] In the example of Figure 10, the effect of scattered light (background) on the surface of particle body 11 of a predetermined size is calculated based on the peak area of ​​second correction particles, which serve as internal standards. By excluding this background, the corrected detection value is primarily attributable to the labeling characteristics of the labeling moiety. This makes it possible to improve the quantitative accuracy of biomarker 3 without the cost and time required for separately measuring the background. As described above, the use of second correction particles makes it possible to easily improve the quantitative accuracy of biomarker 3.

[0145] The above correction can be performed whether the peaks in FIGS. 9 and 10 are the detection result of the sum of the labeling characteristics of a plurality of particles or the detection result of the labeling characteristics of a single particle.

[0146] [5. Modification 2] As described above, in the measurement method according to this embodiment, labeled complexes can be distinguished and biomarkers 3 can be identified based on particle size and the labeling characteristics of the label 2. On the other hand, by configuring the particles themselves to have characteristics (particle characteristics), it is also possible to distinguish labeled complexes and identify biomarkers 3 based on particle size and particle characteristics.

[0147] (5-1. Structure of particles and labels according to Modification 2) FIG. 11 is a diagram illustrating the structure of particles and labels according to Modification 2. The particles and labels according to Modification 2 have different characteristics from the particles 1 and labels 2 according to the embodiment and Modification 1, and therefore are referred to as particles 1z and labels 2z in Modification 2. In FIG. 11, only the parts that differ from FIG. 2, which illustrates the structure of particles and labels according to the embodiment, are described.

[0148] The particle 1z includes a particle body 11z and a first binding portion 12z that specifically binds to the biomarker 3.

[0149] The particle body 11z has particle characteristics. The particle characteristics are not particularly limited as long as they enable the type of particle 1z corresponding to the type of biomarker 3 to be distinguished from one another. In one embodiment, the particle characteristics are fluorescence spectrum patterns. The fluorescence spectrum pattern is a pattern of fluorescence intensity versus fluorescence wavelength, and more specifically, the shape of a graph of fluorescence intensity versus fluorescence wavelength. Having a common fluorescence spectrum pattern, which is a particle characteristic, means that the pattern of fluorescence intensity versus wavelength is common; the strength of the fluorescence intensity at a specific wavelength does not necessarily have to be the same. Note that when the particle characteristics are fluorescence spectrum patterns, the material forming the outer surface of the particle body 11z is preferably a colorable material (e.g., silica). However, the particle characteristics may be at least one of a radioactive spectrum pattern and an absorbance spectrum pattern. In the measurement method according to Variation 2, the type of biomarker 3 is identified based on the differences in these particle characteristics.

[0150] The first binding portion 12z is bound to the particle body 11z and is a binding site on the particle 11z for specifically binding to a predetermined type of biomarker 3.

[0151] The label 2z includes a label portion 21z and a second binding portion 22z that specifically binds to the biomarker 3.

[0152] The labeling portion 21z is a portion containing a labeling substance for labeling the particle 1z to which the biomarker 3 is bound. The labeling substance exhibits predetermined labeling characteristics. As will be described later, the labeling portion 21z of the label 2z only needs to indicate that the particle 1z is bound to the biomarker 3, and does not need to indicate the type of the biomarker 3. Thus, the labeling portion 21z may be equivalent regardless of the type of biomarker 3 to which the label 2z containing the labeling portion 21z is bound.

[0153] The second binding part 22z is bound to the label part 21z. The second binding part 22z is a binding site in the label 2z for specifically binding to a predetermined type of biomarker 3.

[0154] (5-2. Particle Groups and Label Groups According to Modification 2) Fig. 12 is a diagram illustrating particle groups and label groups according to Modification 2. Fig. 12 shows sets of particles 1az-1dz and labels 2az-2dz that specifically bind to each of four types of biomarkers 3a-3d. In Fig. 12, only the parts that are different from Fig. 3, which illustrates particle groups and label groups according to the embodiment, are described.

[0155] In Modification 2, a "particle group" is a group of particles 1z having specific particle characteristics. In FIG. 12, a third particle group and a fourth particle group are shown. Furthermore, a "label group" is a group of labels 2z corresponding to a specific particle group. In FIG. 12, a third label group corresponding to the third particle group and a fourth label group corresponding to the fourth particle group are shown. Particles 1z of the third particle group and labels 2z of the third label group are bonded via biomarkers 3. Particles 1z of the fourth particle group and labels 2z of the fourth label group are bonded via biomarkers 3.

[0156] The particles 1z of the third particle group and the particles 1z of the fourth particle group have different particle properties. Referring to Fig. 12, the particles 1az and 1bz of the third particle group include particle bodies 11az and 11bz having common particle properties. The particles 1cz and 1dz of the fourth particle group include particle bodies 11cz and 11dz having common particle properties.

[0157] Each of the third particle group and the fourth particle group (hereinafter also referred to as "each particle group") includes a plurality of particle subgroups.

[0158] Each particle subgroup includes particles 1z that share a common particle size and particle properties. Referring to FIG. 12 , for example, particle subgroup az is made up of particles 1az that have the same particle size and particle properties. Note that a group of labels 2az corresponding to particle subgroup az is referred to as a "label subgroup" az. Furthermore, particle subgroup az and label subgroup az are collectively referred to as a "particle-label subgroup" az. The number of particle-label subgroups corresponds to the number of types of distinguishable biomarkers 3.

[0159] Since the particles 1z in each particle subgroup bind to different types of biomarkers 3, the first binding portions 12z are also different. Since the labeled subgroups bind to different types of biomarkers 3, the second binding portions 22z are also different. In other words, the particles 1z in each particle subgroup have first binding portions 12z that bind to different types of biomarkers 3. Furthermore, the labels 2z in each labeled subgroup have second binding portions 22z that bind to different types of biomarkers 3. Referring to FIG. 3, the particles 1az to 1dz each include first binding portions 12az to 12dz. Referring to FIG. 3, the labels 2az to 2dz each include second binding portions 22az to 22dz. Note that the first binding portion 12z and the second binding portion 22z in Modification 2 may be made of the same material as the first binding portion 12 and the second binding portion 22 in the embodiment, respectively, if they bind to the same types of biomarkers. Specifically, the first binding portions 12az to 12dz and the first binding portions 12a to 12d of the particle 1 according to the embodiment may be made of equivalent materials (e.g., materials having the same molecular structure), and the second binding portions 22az to 22dz and the second binding portions 22a to 22d of the label 2 according to the embodiment may be made of equivalent materials (e.g., materials having the same molecular structure).

[0160] In each particle group, the particle sizes of the multiple particle subgroups are different from one another. Referring to FIG. 3, in the third particle group, the diameter of the particle body 11az of the particle 1az in the particle subgroup az is larger than the diameter of the particle body 11bz of the particle 1bz in the particle subgroup bz. In the fourth particle group, the diameter of the particle body 11cz of the particle 1cz in the particle subgroup cz is larger than the diameter of the particle body 11dz of the particle 1dz in the particle subgroup dz. This allows the particle subgroups in each particle group to be distinguished by particle size. In one embodiment, the diameter of the particle body 11az is equal to the diameter of the particle body 11cz. Furthermore, the diameter of the particle body 11bz is equal to the diameter of the particle body 11dz.

[0161] Next, the fourth particle group and the fourth label group will be further described. In the particle-label subgroup cz of the fourth particle group, the particle size is common to the particle-label subgroup az, but the particle characteristics are different from each other. As a result, the particle-label subgroup cz and the particle-label subgroup az can be distinguished by particle characteristics. Furthermore, in the particle-label subgroup dz of the fourth particle group, the particle size is common to the particle-label subgroup bz, but the particle characteristics are different from each other. As a result, the particle-label subgroup dz and the particle-label subgroup bz can be distinguished by particle characteristics.

[0162] As described above, particles 1z included in a predetermined particle subgroup can be distinguished from particles 1z included in other particle subgroups based on particle size and particle characteristics. More specifically, if the predetermined particle subgroup and the other particle subgroups belong to the same particle group, they can be separated by particle size. Furthermore, if the predetermined particle subgroup and the other particle subgroups belong to different particle groups, they can be distinguished by particle characteristics.

[0163] As described above, in Modification 2, particles 1z included in a predetermined particle subgroup can be identified based on particle size and particle characteristics. This allows the type of corresponding biomarker 3 to be identified based on particle size and particle characteristics. This eliminates the need to identify the type of corresponding biomarker 3 based on the label characteristics of label 2z. Specifically, label 2z only needs to indicate that biomarker 3 is bound to particle 1z, regardless of the type of biomarker 3.

[0164] As a result, in Modification 2, the marker characteristics of the markers 2z of each marker subgroup may be the same. More specifically, the detection value of the marker characteristic of the third marker group may be equivalent to the detection value of the marker characteristic of the fourth marker group. In the example of FIG. 9 , the marker unit 21az and the marker unit 21cz may be objects that exhibit the same detection value, or more specifically, may be the same object. In this way, configuring all marker units to have the same marker characteristics reduces the complexity of preparing the marker units.

[0165] However, for each of the particles of the third particle group and the particles of the fourth particle group, the detected value of the particle characteristic and the detected value of the label characteristic must be distinguishable. The particle characteristic and the label characteristic may be the same type of characteristic and configured to be distinguishable from each other. For example, the particle characteristic and the label characteristic may be fluorescent spectra that can be distinguished from each other. On the other hand, the particle characteristic and the label characteristic may be different types of characteristics (e.g., fluorescent spectrum and absorbance spectrum) and detected by different detection methods or detectors. This allows the presence or absence of binding of the biomarker 3 and the type of the biomarker 3 to be determined simultaneously based on the detection value of the detection unit 9. In one embodiment, the fluorescent spectrum of each of the particles of the third particle group and the particles of the fourth particle group is configured so as not to overlap with the fluorescent spectrum of the label characteristic of the labeling units 21az and 21cz.

[0166] (5-3. Measurement Process According to Modification 2) Fig. 13 is a flowchart showing the measurement process according to Modification 2. The steps shown in Fig. 13 are performed using measurement system 100.

[0167] In S11, the user prepares particles 1z belonging to a third particle group and particles 1z belonging to a fourth particle group. The user also prepares labels 2z belonging to a third label group corresponding to the third particle group and labels 2z belonging to a fourth label group corresponding to the fourth particle group. As described above, the particles belonging to the third particle group and the particles of the fourth particle group have different particle characteristics.

[0168] In S12, the user uses the pretreatment unit 71 to mix the sample with particles 1z of the third particle group and particles 1z of the fourth particle group, thereby specifically binding the biomarker 3 to the particles 1z of the third particle group and the particles 1z of the fourth particle group.

[0169] In S13, the user uses the pre-treatment unit 71 to mix the mixed solution prepared in S12 with the label 2z of the third labeling group and the label 2z of the fourth labeling group, thereby specifically binding the label 2z of the third labeling group and the label 2z of the fourth labeling group to the biomarker 3.

[0170] In S14, the processor 40 separates each of the particles 1z of the third particle group and the particles 1z of the fourth particle group based on particle size.

[0171] In S15, the processor 40 detects particle characteristics and label characteristics of the labels 2z bound to the particles 1z of the third particle group and the fourth particle group 1z separated based on particle size, for each of the particles 1z of the third particle group and the fourth particle group via the biomarkers 3. For example, when the detection unit 9 is a multiple fluorescence detector or a multi-wavelength fluorescence detector, the processor 40 irradiates excitation light and detects the pattern of the generated fluorescence spectrum.

[0172] In S16, the processor 40 determines the type of biomarker 3 bound to each of the particles 1z of the third particle group and the particles 1z of the fourth particle group based on the particle size, particle characteristics and label characteristics.

[0173] In one embodiment, the processor 40 determines the type of biomarker 3 bound to each of the particles 1z based on the pattern of the fluorescence spectrum. More specifically, the processor 40 first determines whether or not a biomarker 3 is bound to the particle 1z based on the peak corresponding to the label 2z in the pattern of the fluorescence spectrum, which is the detection value. Next, the processor 40 determines the type of the particle 1z based on the peaks corresponding to the particle characteristics of each of the particles 1z of the third particle group and the particles 1z of the fourth particle group, and determines the type of the corresponding biomarker 3.

[0174] 7, the description of S17 to S18 will not be repeated. According to the measurement method of Modification 2, particles 1z having different combinations of particle size and particle characteristics can be used to distinguish and detect multiple types of biomarkers 3. This makes it possible to measure multiple types of biomarkers 3 at once, similar to the measurement method of the embodiment.

[0175] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0176] (Section 1) A measurement method according to one aspect is a method for measuring biomolecules contained in a sample derived from a biological specimen, and includes the steps of preparing particles belonging to a first particle group and particles belonging to a second particle group, and preparing labels belonging to a first labeling group corresponding to the first particle group, and labels belonging to a second labeling group corresponding to the second particle group.

[0177] The labels of the first labeling group and the labels of the second labeling group have labeling properties different from each other. The particles of the first particle group and the labels of the first labeling group are bound via biomolecules. The particles of the second particle group and the labels of the second labeling group are bound via biomolecules. Each particle group includes multiple particle subgroups. In each particle group, the multiple particle subgroups have different particle sizes. The particles of the multiple particle subgroups of the first particle group and the particles of the multiple particle subgroups of the second particle group have first binding moieties that specifically bind to different types of biomolecules from each other.

[0178] The measurement method further includes the steps of mixing the sample with particles of the first particle group and particles of the second particle group, and labels of the first labeling group and labels of the second labeling group; specifically binding biomolecules to the particles of the first particle group and particles of the second particle group and the labels of the first labeling group and labels of the second labeling group; separating each of the particles of the first particle group and the particles of the second particle group based on particle size; detecting label characteristics of the labels bound via biomolecules to each of the particles of the first particle group and particles of the second particle group separated based on particle size; and determining the types of biomolecules bound to the particles of the first particle group and particles of the second particle group based on the particle size and label characteristics.

[0179] According to the measurement method described in paragraph 1, it is possible to provide a technique for measuring multiple types of biomolecules at once based on differences in particle size and differences in labeling characteristics.

[0180] (Item 2) The measurement method according to item 1 further includes a step of measuring the amount of each type of biomolecule based on the result of determining the type of biomolecule.

[0181] According to the measurement method described in paragraph 2, it is possible to provide a technique for measuring the amount of each type of biomolecule at once for multiple types of biomolecules.

[0182] (Item 3) The measurement method according to item 2 further comprises a step of diagnosing a disease or determining the effectiveness of a treatment based on the amount of each type of biomolecule.

[0183] According to the measurement method described in paragraph 3, a disease can be diagnosed or the effectiveness of a treatment can be determined based on the amount of each type of biomolecule obtained by a single measurement. In other words, diagnosis based on multiple types of biomolecules can be easily performed.

[0184] (Item 4) In the measurement method described in any one of Items 1 to 3, the particles of each particle group further include a particle body, and the labels of each labeled group include a labeling portion and a second binding portion that specifically binds to a biomolecule.

[0185] According to the measurement method described in paragraph 4, a labeled complex containing particles can be prepared via the first binding portion and the second binding portion. Furthermore, the type of labeled complex can be distinguished based on the particle size, which is mainly attributed to the particle body, and the labeling characteristics of the labeling portion. This allows the type of biomolecule corresponding to the labeled complex to be identified.

[0186] (Item 5) In the measurement method described in Item 4, the labeling portion includes at least one of a fluorescent substance, a radioisotope, and a substance that exhibits a predetermined absorbance.

[0187] According to the measurement method described in item 5, the type of biomolecule can be distinguished based on the difference in the labeling properties of the labeling substance.

[0188] (Item 6) In the measurement method described in Items 4 or 5, the biological molecule includes a protein, the first binding moiety includes an antibody that binds to the protein, and the second binding moiety includes an antibody that binds to a site on the protein that is different from the binding site of the first binding moiety.

[0189] According to the measurement method described in paragraph 6, each of the first binding part and the second binding part can bind to a predetermined type of protein, which is a biomolecule, by utilizing the specificity of the antibody. Furthermore, in a state where the particle is bound to the protein, the label can also be bound to the protein.

[0190] (7) In the measurement method according to any one of items 1 to 6, the biomolecule includes at least one of a nucleic acid and a metabolite.

[0191] According to the measurement method described in Section 7, not only proteins but also biomolecules belonging to nucleic acids and / or metabolites can be targeted by the measurement method according to this embodiment.

[0192] (Item 8) In the measurement method according to any one of items 1 to 7, the particles of each particle group contain at least one of an inorganic material and a resin material.

[0193] According to the measurement method described in paragraph 8, it is possible to increase the number of types of biomolecules that can be detected simultaneously, to increase the accuracy of quantifying biomolecules, and / or to perform measurements with high accuracy relatively easily.

[0194] (Item 9) In the measurement method described in Item 8, the inorganic material includes at least one of gold and silica.

[0195] According to the measurement method described in item 9, it is possible to increase the number of types of biomolecules that can be detected simultaneously, and / or to increase the accuracy of quantification of biomolecules.

[0196] (Item 10) In the measurement method described in item 8, the resin material includes polystyrene. According to the measurement method described in item 10, nanoparticles made of highly reliable resin materials with traceability are commercially available, and measurements can be performed relatively easily and accurately.

[0197] (Item 11) In the measurement method described in items 1 to 10, the separating step includes separating particles based on particle size using at least one of centrifugal FFF (Field Flow Fraction) method, AF4 (Asymmetrical Flow Field Flow Fraction) method, and size exclusion chromatography.

[0198] In the measurement method described in paragraph 11, if the centrifugal FFF method is used, a large number of types of biomolecules can be distinguished and the accuracy of quantification of biomolecules is high. If the AF4 method is used, small and light molecules can also be classified. If size exclusion chromatography is used, an inexpensive device configuration is possible.

[0199] (Item 12) In the measurement method according to any one of Items 1 to 11, the step of detecting the label characteristics uses a plurality of fluorescence detectors or a multi-wavelength fluorescence detector.

[0200] In the measurement method described in paragraph 12, if multiple fluorescence detectors are used, fluorescence can be detected with high sensitivity, improving the measurement accuracy of labeling characteristics and the quantitative accuracy of biomolecules. If a multi-wavelength fluorescence detector is used, the number of detectors can be reduced, thereby reducing the cost required for the detection unit.

[0201] (Item 13) The detecting step of the measurement method described in Item 12 includes a step of irradiating excitation light onto particles of each particle group mixed in a carrier. The particle preparing step includes a step of preparing particles of each particle group and a carrier. The step of preparing particles of each particle group and a carrier includes a step of preparing particles of each particle group and a carrier such that the difference between the refractive index of the particles of each particle group and the refractive index of the carrier is equal to or less than a predetermined value.

[0202] According to the measurement method described in paragraph 13, the amount of scattered light originating from particles can be kept below a predetermined amount, thereby enabling the lower detection limit of fluorescence to be set low and improving detection sensitivity.

[0203] (Item 14) In the measurement method described in any one of items 1 to 13, the step of detecting the label characteristics further includes a step of correcting the detection values ​​of the label characteristics of the labels bound to each of the particles of the first particle group and the particles of the second particle group.

[0204] According to the measurement method described in item 14, the accuracy of quantifying biomolecules in the measurement method is improved.

[0205] (Item 15) In the measurement method described in Item 14, the correction step includes the steps of preparing first correction particles of a size corresponding to each particle size of a plurality of particle subgroups of each particle group, detecting the label characteristics of the first correction particles in a state in which no labeling moiety is bound to them for each particle size, and correcting, for each particle size, the detected values ​​of the label characteristics of the labeling moieties bound to the particles of the first particle group and the particles of the second particle group based on the detected values ​​of the label characteristics of the first correction particles.

[0206] According to the measurement method described in item 15, background can be corrected while maximizing the number of distinguishable types of biomolecules.

[0207] (Item 16) In the measurement method described in Item 15, the step of detecting the labeling characteristics of the first correction particle includes a step of detecting the labeling characteristics for each particle size while the first correction particle including the first binding portion is bound to a biomolecule and a second binding portion.

[0208] According to the measurement method described in item 16, more accurate correction can be performed by taking into account the influence of the biomolecule and the second binding portion.

[0209] (Item 17) In the measurement method described in Item 14, the detecting step includes a step of detecting the label characteristics of second correction particles having particle sizes different from the particle sizes of each of the multiple particle subgroups of each particle group in a state where no label portion is bound to them, and the correcting step includes a step of correcting the detected values ​​of the label characteristics of the labels bound to the particles of the first particle group and the particles of the second particle group based on the detected values ​​of the label characteristics of the second correction particles.

[0210] According to the measurement method described in paragraph 17, the quantitative accuracy of biomolecules can be improved without the cost and time required for separate background measurement.

[0211] (Item 18) In the measurement method described in Item 17, the step of correcting based on the detection value of the labeling characteristic of the second correction particle includes a step of correcting the detection results of the labeling characteristic binding to the particles of the first particle group and the particles of the second particle group using a correction coefficient calculated based on the peak area of ​​the labeling characteristic of the second correction particle.

[0212] According to the measurement method described in item 18, the influence on the peak area caused by measurement particles having a particle size different from that of the second correction particles can be calculated using the peak area of ​​the second correction particles.

[0213] (Clause 19) A measurement method according to another aspect is a method for measuring biomolecules contained in a sample derived from a biological specimen, and includes the steps of preparing particles belonging to a third particle group and particles belonging to a fourth particle group, and preparing labels belonging to a third labeling group corresponding to the third particle group, and labels belonging to a fourth labeling group corresponding to the fourth particle group.

[0214] The particles belonging to the third particle group and the particles of the fourth particle group have particle properties different from each other. The particles of the third particle group and the labels of the third labeling group are bound via biomolecules. The particles of the fourth particle group and the labels of the fourth labeling group are bound via biomolecules. Each particle group includes multiple particle subgroups. In each particle group, the multiple particle subgroups have different particle sizes. The particles of the multiple particle subgroups of the third particle group and the particles of the multiple particle subgroups of the fourth particle group have third binding moieties that specifically bind to different types of biomolecules from each other.

[0215] The measurement method further includes the steps of mixing a sample with particles of the third particle group and particles of the fourth particle group, and labels of the third labeling group and labels of the fourth labeling group; specifically binding biomolecules to the particles of the third particle group and particles of the fourth particle group and the labels of the third labeling group and the labels of the fourth labeling group; separating each of the particles of the third particle group and the particles of the fourth particle group based on particle size; detecting particle characteristics and label characteristics of the labels bound to the particles via the biomolecules for each of the particles of the third particle group and particles of the fourth particle group separated based on particle size; and determining the types of biomolecules bound to the particles of the third particle group and particles of the fourth particle group based on the particle size, particle characteristics, and label characteristics.

[0216] According to the measurement method described in paragraph 19, it is possible to provide a technique for measuring multiple types of biomolecules at once based on differences in particle size and differences in particle properties.

[0217] (Item 20) In the measurement method described in Item 19, the particle characteristics include at least one of a fluorescence spectrum pattern, a radiation spectrum pattern, and an absorbance spectrum pattern.

[0218] According to the measurement method described in paragraph 20, the type of biomolecule can be identified based on the difference in these particle properties.

[0219] (Item 21) In the measurement method described in Item 19 or 20, in the detection step, the detection value of the particle characteristic and the detection value of the label characteristic are distinguishable for each of the particles of the third particle group and the particles of the fourth particle group.

[0220] According to the measurement method described in paragraph 21, the presence or absence of binding of a biomolecule and the type of the biomolecule can be determined at once based on the detection value of the detection unit.

[0221] (Item 22) In the measurement method described in any one of Items 19 to 21, the detected value of the marker characteristic of the third marker group is equivalent to the detected value of the marker characteristic of the fourth marker group.

[0222] According to the measurement method described in paragraph 22, the complexity of preparing the labeling portion can be reduced. (paragraph 23) A measurement method according to still another aspect is a method for measuring biomolecules contained in a sample derived from a biological sample, and includes the steps of preparing particles belonging to a first particle group and preparing labels belonging to a first labeling group corresponding to the first particle group.

[0223] The particles of the first particle group and the labels of the first label group are bound to each other via biomolecules. The first particle group includes a plurality of particle subgroups. In the first particle group, the plurality of particle subgroups have different particle sizes. The particles of the plurality of particle subgroups of the first particle group have first binding moieties that specifically bind to different types of biomolecules.

[0224] The measurement method further includes the steps of: mixing a sample with particles of the first particle group and labels of the first labeling group to specifically bind the particles of the first particle group and the labels of the first labeling group to biomolecules; separating each of the particles of the first particle group based on particle size; detecting the labeling characteristics of the labels bound to each of the particles of the first particle group separated based on particle size via biomolecules; and determining the type of biomolecules bound to the particles of the first particle group based on the particle size and the labeling characteristics.

[0225] According to the measurement method described in paragraph 23, particles of different particle sizes can be bound to each of multiple types of biomolecules contained in a sample, providing a technology for measuring multiple types of biomolecules at once based on differences in particle size.

[0226] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims.

[0227] 1, 1a to 1d, 1z, 1az to 1dz Particle, 1ar, 1br First correction particle, 1r Second correction particle, 2, 2a to 2d, 2z, 2az to 2dz Label, 3, 3a to 3d Biomarker, 4 Control device, 5 Measuring device, 6 Liquid section, 8 Separation section, 9 Detection section, 11, 11a, 11b, 11az to 11dz Particle body, 12, 12a to 12d, 12z, 12az to 1dz First binding section, 21, 21a, 21c, 21az, 21cz Label section, 22, 22a to 22d Second binding section, 40 Processor, 41 Memory, 42 Input section, 43 Display section, 51, 52 Analysis flow path, 61 Container, 62 Pump, 71 Pretreatment section, 72 Injection section, 100 Measurement system.

Claims

1. A method for measuring a biomolecule contained in a sample derived from a biological sample, comprising: providing particles belonging to a first particle group and particles belonging to a second particle group; preparing a marker belonging to a first marker group corresponding to the first particle group, and a marker belonging to a second marker group corresponding to the second particle group; the markers of the first marker group and the markers of the second marker group have marker characteristics different from each other; the particles of the first particle group and the labels of the first label group are bound via a biomolecule; the particles of the second particle group and the labels of the second label group are bound via a biomolecule; each of the particle groups includes a plurality of particle subgroups; In each of the particle groups, the particle subgroups have different particle sizes, the particles of the plurality of particle subgroups of the first particle group and the particles of the plurality of particle subgroups of the second particle group have first binding portions that specifically bind to different types of biomolecules; The measurement method further comprises: mixing the sample with the particles of the first particle group and the particles of the second particle group and with the labels of the first label group and the labels of the second label group; specifically binding biomolecules to the particles of the first particle group and the particles of the second particle group and to the labels of the first label group and the labels of the second label group; separating each of the first group of particles and the second group of particles based on particle size; detecting a label characteristic of a label bound via a biomolecule to each of the particles of the first particle group and the particles of the second particle group separated based on particle size; and determining the types of biomolecules bound to the particles of the first particle group and the particles of the second particle group based on particle size and label characteristics.

2. The measurement method according to claim 1 , further comprising the step of measuring the amount of each type of biomolecule based on a result of determining the type of the biomolecule.

3. The method according to claim 2 , further comprising a step of diagnosing a disease or assessing a treatment effect based on the amounts of each type of biomolecule.

4. The particles of each particle group further include a particle body, The measurement method according to any one of claims 1 to 3, wherein the labels of each of the labeling groups include a labeling portion and a second binding portion that specifically binds to a biomolecule.

5. The measurement method according to claim 4 , wherein the label includes at least one of a fluorescent substance, a radioisotope, and a substance exhibiting a predetermined absorbance.

6. Biomolecules include proteins, the first binding moiety comprises an antibody that binds to the protein; The method of claim 4 , wherein the second binding moiety comprises an antibody that binds to a site on the protein that is different from the binding site on the protein to which the first binding moiety binds.

7. The method according to any one of claims 1 to 3, wherein the biomolecule comprises at least one of a nucleic acid and a metabolite.

8. The measurement method according to any one of claims 1 to 3, wherein the particles of each of the particle groups include at least one of an inorganic material and a resin material.

9. The method of claim 8 , wherein the inorganic material includes at least one of gold and silica.

10. The method according to claim 8 , wherein the resin material includes polystyrene.

11. The method according to any one of claims 1 to 3, wherein the separating step includes a step of separating particles based on particle size using at least one of a centrifugal field flow fraction (FFF) method, an asymmetrical flow field flow fraction (AF4) method, and size exclusion chromatography.

12. The measurement method according to any one of claims 1 to 3, wherein the step of detecting the label characteristic uses a plurality of fluorescence detectors or a multi-wavelength fluorescence detector.

13. The step of detecting the label characteristic includes a step of irradiating the particles of each of the particle groups mixed in a carrier with an excitation light; The step of preparing the particles includes the step of preparing particles of each of the particle groups and the carrier; The step of preparing the particles of each particle group and the carrier includes a step of preparing the particles of each particle group and the carrier, the difference between the refractive index of the particles of each particle group and the refractive index of the carrier being a predetermined numerical value or less. The measurement method according to claim 12.

14. The measurement method according to any one of claims 1 to 3, wherein the step of detecting the label characteristic further includes a step of correcting the detection value of the label characteristic of the label bound to each of the particles of the first particle group and the particles of the second particle group.

15. The correcting step includes: providing first correction particles having a size corresponding to each particle size of a plurality of particle subgroups of each of the particle groups; detecting the label characteristic of the first correction particle without a label portion bound thereto for each particle size; The measurement method according to claim 14, further comprising a step of correcting, for each particle size, a detection value of the labeling characteristic of a labeling portion that binds to a particle of the first particle group and a particle of the second particle group based on a detection value of the labeling characteristic of the first correction particle.

16. The step of detecting the label characteristic of the first correction particle includes: The measurement method according to claim 15, further comprising: detecting labeling characteristics for each particle size while a biomolecule and a second binding portion are bound to the first correction particle including a first binding portion.

17. The detecting step includes: detecting a marker characteristic of second correction particles having a particle size different from each of the particle subgroups of each particle group in a state where no marker portion is bound thereto; The correcting step includes: The measurement method according to claim 14, further comprising a step of correcting detection values ​​of label characteristics of the labels bound to the particles of the first particle group and the particles of the second particle group based on detection values ​​of the label characteristics of the second correction particles.

18. The step of correcting based on the detection value of the label characteristic of the second correction particle includes: The measurement method according to claim 17, further comprising a step of correcting a detection result of the label characteristics of the labels bound to the particles of the first particle group and the particles of the second particle group using a correction coefficient calculated based on a peak area of ​​the label characteristics of the second correction particles.

19. A method for measuring a biomolecule contained in a sample derived from a biological sample, comprising: preparing particles belonging to a third particle group and particles belonging to a fourth particle group; preparing a marker belonging to a third marker group corresponding to the third particle group, and a marker belonging to a fourth marker group corresponding to the fourth particle group; the particles belonging to the third particle group and the particles of the fourth particle group have different particle properties; the particles of the third particle group and the labels of the third label group are bound via a biomolecule; the particles of the fourth particle group and the labels of the fourth label group are bound via a biomolecule; each of the particle groups includes a plurality of particle subgroups; In each of the particle groups, the particle subgroups have different particle sizes, the particles of the plurality of particle subgroups of the third particle group and the particles of the plurality of particle subgroups of the fourth particle group have third binding portions that specifically bind to different types of biomolecules; The measurement method further comprises: mixing the sample with particles of the third particle group and particles of the fourth particle group and labels of the third label group and labels of the fourth label group; specifically binding a biomolecule to the particles of the third particle group and the particles of the fourth particle group and to the labels of the third label group and the labels of the fourth label group; separating each of the third group of particles and the fourth group of particles based on particle size; Detecting a particle characteristic and a label characteristic of a label bound via a biomolecule for each of the particles of the third particle group and the particles of the fourth particle group separated based on particle size; and determining the types of biomolecules bound to the particles of the third particle group and the particles of the fourth particle group based on particle size, particle characteristics and label characteristics.

20. 20. The method of claim 19, wherein the particle characteristics include at least one of a fluorescence spectrum pattern, a radiation spectrum pattern, and an absorbance spectrum pattern.

21. The measurement method according to claim 19 or 20, wherein in the detecting step, the detection value of the particle characteristic and the detection value of the label characteristic are distinguishable for each of the particles of the third particle group and the particles of the fourth particle group.

22. The measurement method according to claim 19 or 20, wherein the detection value of the marker characteristic of the third marker group is equivalent to the detection value of the marker characteristic of the fourth marker group.

23. A method for measuring a biomolecule contained in a sample derived from a biological sample, comprising: Providing particles belonging to a first particle group; and preparing markers belonging to a first marker group corresponding to the first particle group; the particles of the first particle group and the labels of the first label group are bound via a biomolecule; the first particle group includes a plurality of particle subgroups; In the first particle group, the particle subgroups have different particle sizes, Particles of the plurality of particle subgroups of the first particle group have first binding portions that specifically bind to different types of biomolecules, The measurement method further comprises: mixing the sample with the particles of the first particle group and the labels of the first label group, thereby specifically binding the particles of the first particle group and the labels of the first label group to biomolecules; separating each of the particles in the first group of particles based on particle size; detecting a label characteristic of a label attached via a biomolecule to each of the particles of the first group of particles separated based on particle size; and determining the type of biomolecule bound to the particles of the first particle group based on particle size and label characteristics.