Analysis method and analysis device using polarization anisotropy-based measurements

Abstract

The purpose of the present invention is to provide an analysis method and an analysis device capable of analyzing the concentration of a target substance over a wide concentration range and with high sensitivity on the basis of polarization anisotropy. Provided is an analysis method for calculating the concentration of a target substance by measuring a value (R) related to polarization anisotropy using a light-emitting reagent bound to the target substance, the analysis method comprising: a mixing step of mixing a sample containing the target substance with the light-emitting reagent to provide a mixed solution; and a measurement step for measuring R of the mixed solution after the elapse of time T from the mixing, characterized in that, in the measurement step, when the time at which R reaches R1 is represented by T1, the concentration of the target substance is calculated on the basis of T1.

Description

Technical Field

[0001] The invention relates to an analysis method and an analysis device based on polarization anisotropy. Background Art

[0002] In the fields of medicine and clinical trials, there is a need for highly sensitive detection or quantification of trace amounts of biological components from collected parts, such as blood or organs, in order to study, for example, the cause and presence of a disease.

[0003] In the test technology for biological components, immunoassay is widely used. In many immunoassays, a washing step called bound / free (B / F) separation is required. As an immunoassay that does not require B / F separation, the latex agglutination method using antigen-antibody reaction is known. In the latex agglutination method, latex particles on which each load, for example, antibodies specifically bound to a target substance are mixed with a liquid that may contain the target substance, and the degree of agglutination of the latex particles is measured.

[0004] In the latex agglutination method, the target substance is captured by antibodies that are bound to latex particles and specific to the target substance, and a plurality of latex particles are cross-linked via the captured target substance, and as a result, agglutination of the latex particles occurs. That is, the amount of the target substance in a liquid sample such as a biological sample can be quantified by evaluating the degree of agglutination of the latex particles. The degree of agglutination can be quantified by measuring and evaluating the change in the amount of light transmitted through the liquid sample or scattered by the liquid sample.

[0005] The latex agglutination method can detect and quantitatively evaluate an antigen serving as a target substance in a simple and rapid manner, but involves a problem of a detection limit in which the antigen cannot be detected when the amount of the antigen in a liquid sample such as a biological sample is small.

[0006] In order to improve the detection sensitivity of the target substance, it is necessary to measure the degree of agglutination with higher sensitivity. That is, it is conceivable to replace the system for measuring the change in the amount of light transmitted through or scattered by the liquid sample with a detection / quantification method utilizing luminescence characteristics having higher sensitivity. Specifically, for example, a sample testing method utilizing a fluorescence depolarization method has been proposed (Patent Documents 1 and 2).

[0007] In Patent Document 1, an improved apparatus for a fluorescence depolarization method is proposed for clinical use.

[0008] In the fluorescence depolarization method, B / F separation required in general fluorescence measurement methods is not required.

[0009] Therefore, the fluorescence depolarization method enables simple sample testing like the latex agglutination method. In addition, it is conceivable that the fluorescence depolarization method can be used to perform measurement by the same test system as the latex agglutination method by mixing only the luminescent substance that specifically reacts with the target substance during the measurement. Meanwhile, in Patent Document 1, there is a proposal to use a single molecule such as fluorescein as a luminescent material, which is only applicable to drugs, low molecular weight antigens, etc. in principle.

[0010] Patent document 2 has solved the problem of patent document 1, that is, the fluorescence depolarization method is only applied to drugs, and low molecular weight antigens, etc. That is, in patent document 2, in order to apply the fluorescence depolarization method to macromolecules such as proteins, it is proposed to use a material obtained by adsorbing a dye having a long-life luminescence characteristic on latex particles as a luminescent material. In patent document 2, it is proposed to quantify high molecular weight substances by balancing the reduction of the rotational Brownian motion of the substance in the liquid due to the increase in particle size and the length of the luminescence life span based on the principle of the fluorescence depolarization method. However, in patent document 2, the fluorescent substance is loaded on the latex particles after the particle synthesis, so the interaction between the fluorescent substances adsorbed near the surface of the particles, etc. makes it difficult to stably determine the polarization anisotropy properties of the test particles. In addition, in patent document 2, bovine serum albumin (BSA) as a biomolecule is loaded on the surface of the particles to suppress nonspecific adsorption, so batch-to-batch variation may occur due to the wide particle size distribution and BSA as a protein.

[0011] Therefore, measurement is performed when the concentration of the target substance is on the order of μg / mL, which is not much different from the latex method in terms of measurement sensitivity.

[0012] Furthermore, in Patent Document 2, a method of measuring the difference between polarization anisotropy properties in aggregation between particles before and after a specific reaction time and a method of evaluating the aggregation degree based on the value of the polarization anisotropy property at the end of the lapse of the reaction time are proposed.

[0013] In the measurement method of Patent Document 2, the upper limit of the polarization anisotropy property is determined by the luminescent substance used as a probe. Therefore, when measuring in a wide range, it is necessary to adjust the amount of the reagent according to the concentration of the target substance. In other words, the concentration range that can be measured with one reagent is limited.

[0014] Therefore, even in Patent Document 2, it is difficult to say that detection of a target substance with high sensitivity and in a wide concentration range can be achieved.

[0015] Prior art literature

[0016] Patent Literature

[0017] PTL 1: Japanese Patent Publication No. H03-52575

[0018] PTL 2: Japanese Patent No.2893772 Summary of the invention

[0019] Problem that the invention aims to solve

[0020] The present invention has been made in view of such related art, and an object of the present invention is to provide an analysis method and an analysis apparatus each capable of analyzing the concentration of a target substance in a wide concentration range and with high sensitivity based on polarization anisotropy.

[0021] Solutions for solving problems

[0022] According to one embodiment of the present invention, there is provided an analytical method, which comprises calculating the concentration of a target substance by measuring a value of polarization anisotropy (R) using a luminescent agent bound to the target substance,

[0023] The analysis method comprises:

[0024] a mixing step of mixing a sample containing a target substance with a luminescent reagent to provide a mixed solution; and

[0025] The step of measuring R of the mixed liquid after a time T has passed since the mixing,

[0026] wherein in the measuring step, the time when R reaches R1 is represented by T1, and the concentration of the target substance is calculated based on T1, and

[0027] Wherein when R measured on the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

[0028] In addition, according to one embodiment of the present invention, there is provided an analysis apparatus configured to calculate the concentration of a target substance by measuring a value (R) of polarization anisotropy using a luminescent reagent bound to the target substance,

[0029] The analysis equipment comprises:

[0030] a mixing section configured to mix a sample containing a target substance with a luminescent reagent to provide a mixed solution;

[0031] a measuring unit configured to measure R of the mixed liquid after a time T has elapsed since the mixing; and

[0032] Control Department,

[0033] wherein when the time when R reaches R1 in the measuring section is represented by T1, the control section is configured to calculate the concentration of the target substance based on T1,

[0034] The condition is that when R measured for the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

[0035] Effects of the Invention

[0036] According to the analytical method of an embodiment of the present invention, the change of polarization anisotropy corresponding to the aggregation / dispersion behavior of particles can be detected with high sensitivity, and thus the amount of target substance can be measured in a wide concentration range. According to the analytical device of an embodiment of the present invention, the target substance can be detected and quantified with high sensitivity and in a wide concentration range based on polarization anisotropy.

[0037] Further features of the present invention will become apparent from the following description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] [ Figure 1 ] A schematic diagram for illustrating an analytical method according to an embodiment of the present invention.

[0039] [ Figure 2 ] A schematic diagram for illustrating an analytical method according to an embodiment of the present invention.

[0040] [ Figure 3 ]A schematic diagram for illustrating an apparatus according to an embodiment of the present invention.

[0041] [ Figure 4 ] is a schematic diagram for illustrating the luminescent reagent used in an embodiment of the present invention.

[0042] [ Figure 5 ] A diagram showing the evaluation results, that is, showing the results of quantifying the CRP antigen concentration by the method according to an embodiment of the present invention. DETAILED DESCRIPTION

[0043] Preferred embodiments of the present invention are described in detail below. However, these embodiments are not intended to limit the scope of the present invention.

[0044] According to one embodiment of the present invention, the following analysis method is provided: an analysis method, which includes measuring the value (R) of polarization anisotropy by using a luminescent reagent combined with a target substance, thereby calculating the concentration of the target substance, the analysis method including: a mixing step of mixing a sample containing the target substance with the luminescent reagent to provide a mixed solution; and a measuring step of measuring R of the mixed solution after a time T has passed since the mixing, wherein in the measuring step, the time when R reaches R1 is represented by T1, and the concentration of the target substance is calculated based on T1, and wherein when the R measured for the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

[0045] (Value of polarization anisotropy)

[0046] In the present embodiment, the value of polarization anisotropy (sometimes referred to as R) is defined as follows. That is, R is a value indicating the relationship between the luminous intensity of the polarized light component in the direction parallel to the radiated polarized light and the luminous intensity of the polarized light component in the direction perpendicular to the radiated polarized light, with respect to the luminescence generated by exciting the luminescent substance by irradiating with polarized light. More specifically, R is a value calculated from the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of a given polarized light and the luminous intensity of the luminous component whose vibration direction is orthogonal (perpendicular) to the vibration direction of the polarized light, which luminous intensity is measured when the luminescent substance is excited by polarized light. Further, R is a value representing the ratio of the difference between the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam and the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the first polarized light beam when excited by the first polarized light beam, relative to the sum of the luminous intensities. R can be corrected by: the ratio between the luminous intensity of the luminous component having a vibration direction orthogonal to the vibration direction of the first polarized beam when excited by the second polarized beam having a vibration direction orthogonal to the vibration direction of the second polarized beam and the luminous intensity of the luminous component having a vibration direction parallel to the vibration direction of the second polarized beam when excited by the second polarized beam having a vibration direction orthogonal to the vibration direction of the first polarized beam; and other constants. The value of polarization anisotropy includes values ​​called "polarization anisotropy property" and "polarization degree" and the like.

[0047] More specifically, for example, R may be "r" in the following formula (1).

[0048] [Mathematical formula 1]

[0049]

[0050] (In formula (1),

[0051] I VV represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam,

[0052] I VH represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the first polarized light beam when excited by the first polarized light beam,

[0053] I HV represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam,

[0054] I HHrepresents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam, and

[0055] G indicates the correction value. )

[0056] In addition, R may be r' in the following formula (2).

[0057] [Mathematical formula 2]

[0058]

[0059] All symbols are the same as those in formula (1).

[0060] Regarding the measurement conditions of R, for example, it is preferably measured in a liquid at a temperature of 0° C. to 50° C., and the viscosity of the liquid is 0.5 mPa·s to 50 mPa·s. When the luminescent reagent contains particles containing a europium complex, it is preferably measured at a concentration of 0.001 mg / ml to 0.1 mg / ml, and the excitation wavelength is preferably 500 nm to 700 nm.

[0061] (About R1 and R0)

[0062] Depending on the purpose of the measurement and the properties of the target substance, the analyst can appropriately define R1.

[0063] For example, when the purpose is to analyze the concentration of the target substance with high sensitivity (in a low concentration range), it is preferred to satisfy R1-R0≥0.0001. For example, in order to analyze the target substance in the measurement sample within a measurement range of 1 pM to 10 nM, it is preferred to satisfy R1-R0≥0.0001. The maximum value of R1-R0 is 0.4, which is a theoretical value determined by the composition of the analysis method.

[0064] Meanwhile, when the purpose is to analyze the concentration of the target substance with low sensitivity (in a high concentration range), R1-R0 is preferably set low. For example, in order to analyze the target substance in the measurement sample within a measurement range of 1 nM to 1 M, R1-R0<0.0001 may be satisfied.

[0065] R1 can be determined as follows: a measurer measures the value of polarization anisotropy by using various standard samples having previously known concentrations to produce a calibration curve; and determines R1 from the curve.

[0066] In addition, regarding R0 which is R measured for the luminescent reagent not mixed with the target substance, it is preferable to satisfy R0 ≥ 0.001.

[0067] (About the mixing step)

[0068] For each step, please refer to Figure 1 . In the mixing step, a sample containing a target substance is mixed with a luminescent reagent to provide a mixed solution. The mixed solution is a liquid containing a luminescent reagent and a target substance, and may further contain, for example, additives other than the aforementioned. The mixing is preferably performed within a pH range of 3.0 to 11.0. In addition, the mixing temperature falls within a range of 20° C. to 50° C. The target substance and the luminescent reagent are described later.

[0069] (About measurement steps)

[0070] In the measuring step, R of the mixed solution after a time T has passed since the mixing is measured. The term "mixing" as used herein is not limited to the moment when the sample and the luminescent reagent are in contact with each other, and can be a moment after a predetermined time period has passed since the moment when the sample and the reagent are in contact with each other, within the extent that the measurement is appropriately performed. For example, mixing can be any one of the following: a moment after a predetermined time period (e.g., 10 seconds) has passed since the moment when the sample and the luminescent reagent have been in contact with each other; a moment after a stirring step performed after the sample and the luminescent reagent have been in contact with each other is completed; and a moment after a predetermined time period has passed since the moment when the stirring step is completed.

[0071] Regarding the measurement conditions, for example, it is preferred to measure in a liquid having a temperature of 0°C or higher and 50°C or lower, and a viscosity of the liquid of 0.5 mPa·s or higher and 50 mPa·s or lower. It is preferred to measure at a concentration of the luminescent reagent of 0.0001 mg / ml or higher and 0.1 mg / ml or lower, and the excitation wavelength is preferably 500 nm or higher and 700 nm or lower.

[0072] When a certain amount of the target substance exists in the target substance, the R of the mixed solution increases with the reaction time.

[0073] The rate of increase of the numerical value of R depends on the concentration of the target substance in the target substance. Specifically, when the concentration of the target substance is high, the rate of increase of the R value is large, and when the concentration is low, the rate of increase decreases.

[0074] That is, when the concentration of the target substance is high, the reaction time when the value of the polarization anisotropy property R reaches R1 is short, and when the concentration of the target substance is low, the reaction time is prolonged.

[0075] The time when R just changes to R1 is not necessarily the time during the measurement. Therefore, T1 can be determined by estimation from the time when the value close to R1 is measured.

[0076] T1 can be determined by calculation from the measurement results. For example, when the mixed solution is measured at certain time intervals, the rate of increase of the polarization anisotropy property R can be calculated relative to the reaction time. In other words, the time when the R value of the measured mixed solution reaches R1 can be determined by calculation from the gradient of the change of R relative to the reaction time.

[0077] When the value of R1 can be predicted, the measurement time can be shortened. Therefore, when the concentration of the target substance is low, the analysis method can be used appropriately.

[0078] In addition, the value of the polarization anisotropy property R1 can be determined later. Quantitative evaluation can be performed by using the following steps: after measuring data obtained by plotting the change of R with respect to T, the numerical value of R1 is determined and compared with the results of a similarly measured standard substance.

[0079] The analytical method using the present embodiment enables to determine the concentration of the target substance in a wide range without preparing many test reagents according to the concentration of the target substance. Usually, many reagents for the target substance of the same kind are usually problematic in analytical equipment, because the preparation is not only complicated but also affects the setting space, etc. In addition, when the concentration exceeds the measurement concentration range and the quantitative failure of the material, it is necessary to dilute and prepare the sample again, so the cost of a measurement increases. Therefore, the fact that a small amount of reagent can cover a wide measurement range has important value in the performance of sample testing.

[0080] The measuring steps are preferably repeated. Figure 1 As shown, when R does not reach R1, further measurement steps can be performed.

[0081] The repetitive measurement steps may be repeated periodically, and may be repeated in a cycle of, for example, (a+b) seconds, where "a" represents the time period required for the measurement, and "b" represents the measurement interval. That is, one measurement takes "a" seconds, and then, after an interval of "b" seconds has elapsed, the next measurement may be started. For example, "a" may be set to be greater than 1 millisecond and less than 10 seconds, and "b" may be set to be greater than 1 second and less than 1 minute.

[0082] For example, a+b can be set to preferably 1 second or more and 10 minutes or less, more preferably 1 second or more and 1 minute or less, still more preferably 15 seconds or more and 30 seconds or less.

[0083] In the repeated measurement steps, when R exceeds R1 for the first time in the nth measurement step, for example, any value between the end time of the (n-1)th measurement step and the start time of the (n+1)th measurement step can be used as T1.

[0084] (About R2)

[0085] The upper limit value (R2) of R can be predefined. That is, when R exceeds R2 even in the period when R exceeds R1 for the first time, it is preferred to adjust the period (R2) by shortening at least one of "a" or "b". Figure 2 ). For example, the case where the concentration of the target substance in the mixed liquid is high and therefore the value of the polarization anisotropy property R reaches R2 in a short period of time from the start of the reaction corresponds to the foregoing. In this case, the quantification of the high-concentration liquid is performed as follows: shorten "a" or "b", re-measure R, and then perform quantification based on a calibration curve matching R.

[0086] R2 can be set to, for example, R measured when the target substance is added to the extent that the luminescent reagent is saturated.

[0087] Similarly, when the concentration of the target substance in the mixed liquid is low and therefore the increase in the R value is slow, quantification of the low-concentration liquid can be performed by performing the following steps: extending "a" or "b", measuring the value of polarization anisotropy, and then comparing between the calibration curve matched to the value and the measurement result.

[0088] As described above, when the conditions of the step of quantifying a target substance by comparison with a calibration curve are classified according to the reaction time, a wide measurement range can be covered with a small number of kinds of reagents.

[0089] In addition, according to other embodiments of the present invention, the following measuring device is provided: a measuring device, which is configured to measure the concentration of the target substance by measuring the value of polarization anisotropy (R) using a luminescent reagent combined with the target substance, the measuring device comprising: a mixing section configured to mix a sample containing the target substance with the luminescent reagent to provide a mixed liquid; a measuring section configured to measure the R of the mixed liquid after a time T has passed since the mixing; and a control section, wherein when the time when R reaches R1 in the measuring section is represented by T1, the control section is configured to calculate the concentration of the target substance based on T1, provided that when the R measured for the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

[0090] (Target substance)

[0091] Examples of target substances include antigens, antibodies, low molecular weight compounds, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, transmitters and membrane proteins. Examples of antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, substances derived from blood, substances derived from food, substances derived from natural products and any low molecular weight compounds. Examples of nucleic acids include DNA, RNA or cDNA derived from bacteria, viruses or cells, parts or fragments thereof, synthetic nucleic acids, primers and probes. Examples of low molecular weight compounds include cytokines, hormones, neurotransmitters, transmitters and membrane proteins and their receptors. The analytical method according to the present embodiment can determine the presence or absence of the target substance by comparing the concentration of the target substance with a predetermined threshold. For example, when the concentration of the target substance is equal to or higher than a predetermined threshold, it can be determined that the target substance exists, and when the concentration is lower than a predetermined threshold, it can be determined that the target substance does not exist.

[0092] (Luminescent Reagent)

[0093] In the present embodiment, the luminescent agent is an agent that causes luminescence, in particular, an agent that is excited by light irradiation and emits light, and does not include agents such as luminol based on luminescence caused by chemical reactions. Luminescence includes phosphorescence and fluorescence, preferably phosphorescence. In the present embodiment, the luminescent agent more preferably comprises a europium complex. In addition, in the present embodiment, the luminescent agent more preferably comprises particles. In the present embodiment, the luminescent agent most preferably comprises particles containing a europium complex. In addition, the luminescent agent preferably has a ligand that is specific to the target substance. The presence of the ligand enables the particles of the present embodiment to detect and quantify the target substance based on polarization anisotropy. In the present embodiment, a ligand refers to a compound that specifically binds to a specific target substance.

[0094] Any compound that shows affinity for a specific substance can be used as a ligand. Examples of combinations of ligands and target substances, or target substances and ligands can include the following. That is, examples can include: antigens and antibodies; low molecular weight compounds and their receptors; enzymes and substrates; and nucleic acids that complement each other. In addition, examples can include antibodies and any of the following substances that are specific to them: allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, substances derived from blood, substances derived from food, substances derived from natural products, and any low molecular weight compounds. In addition, examples can include receptors and any of the following substances that are specific to them: low molecular weight compounds, cytokines, hormones, neurotransmitters, transmitters, and membrane proteins. In addition, examples can include DNA, RNA or cDNA, parts or fragments thereof, synthetic nucleic acids, primers or probes derived from bacteria, viruses or cells, and nucleic acids that are complementary thereto. In addition to the foregoing, any combination known to have affinity can be used as a combination of target substances and ligands. A typical example of a ligand in this embodiment is any one of an antibody, an antigen, and a nucleic acid.

[0095] Figure 4 Schematic diagram for explaining an example of the luminescent reagent used in the present embodiment. The luminescent reagent 4 used in the present embodiment preferably comprises: a particle substrate 1 containing a europium complex 3; and a hydrophilic layer 2 coated on the surface thereof. Figure 4 The diameter of the luminescent reagent in is greater than or equal to 25 nm and less than or equal to 500 nm.

[0096] The diameter of each particle can be determined by dynamic light scattering. When particles dispersed in a solution are illuminated with laser light and the resulting scattered light is observed with a photon detector, the intensity distribution caused by the interference of the scattered light constantly fluctuates because the particles constantly move their positions by Brownian motion.

[0097] Dynamic light scattering is a measurement method for observing the state of Brownian motion as fluctuations in the intensity of scattered light. The fluctuations of the scattered light relative to time are expressed as an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter is determined, and the size of each particle dispersed in the solution can be derived.

[0098] From the viewpoint of maintaining the uniformity and monodispersity of the particles, it is desirable that the luminescent reagent has nothing disposed on the surface of its particles. However, in order to be used in the analytical method according to the present embodiment, it is necessary to prevent nonspecific adsorption of substances other than the target on the particles, and therefore the luminescent reagent preferably includes a hydrophilic layer for maintaining its surface hydrophilicity.

[0099] A method including loading BSA on the surface of each particle is widely used as a technique for maintaining hydrophilicity, but this method may cause many changes. In view of the foregoing, the luminescent reagent preferably includes a hydrophilic layer containing a hydrophilic polymer. The concentration of the luminescent reagent in the mixed solution is preferably 0.000001 mass % or more and 1 mass % or less, more preferably 0.00001 mass % or more and 0.001 mass % or less.

[0100] The luminescent reagent used in the present embodiment comprises a europium complex, and therefore can emit phosphorescence with a long life. In the luminescent reagent used in the present embodiment, the average particle diameter as the average value of the diameter of the particles is preferably more than 25nm and less than 500nm, and the average particle diameter is more preferably more than 50nm and less than 300nm. When the average particle diameter is greater than 500nm, the polarization anisotropy property before agglutination becomes high, resulting in a small difference in the polarization anisotropy property after the agglutination reaction. In addition, when the average particle diameter is less than 25nm, the change between the sizes before and after agglutination becomes small, making it difficult to grasp the change of R by phosphorescent luminescence depolarization.

[0101] By reducing the particle size distribution of the luminescent reagent and introducing a europium complex as a luminescent molecule, even when the dispersion state of the particles in the liquid undergoes a slight change, the change in the polarized luminescence characteristics can be grasped. Specifically, even when the concentration of the target substance in the solution is from about nanograms to about picograms per milliliter (e.g., more than 1 picogram and less than 100 picograms), when the molecules of the luminescent reagent agglomerate via the target substance, the change in the rotational Brownian motion of the luminescent reagent can be grasped as a change in polarization anisotropy.

[0102] "Polarized luminescence" refers to the phenomenon that when a luminescent material having anisotropic properties in the transition moment (transition dipole moment) uses polarized light along its transition moment as excitation light, its luminescence is also polarized light along the transition moment. Europium complexes show fluorescent luminescence based on energy transfer from ligands to central metal ions, so the transition moment is complex, but red luminescence around 610nm generated by the electron transition from the lowest excited state 5D0 to 7F2 is emitted as polarized light.

[0103] The principle of polarization anisotropy is to measure the shift of the transition distance caused by the rotational motion of the luminescent material during polarized luminescence. The rotational motion of the luminescent material can be expressed by equation (3):

[0104] Q=3Vη / kT … (3)

[0105] Where Q represents the rotational relaxation time of the material, V represents the volume of the material, η represents the viscosity of the solvent, k represents the Boltzmann constant, and T represents the absolute temperature.

[0106] The rotational relaxation time of a material is the time required for a molecule to rotate through an angle θ (68.5°) (where cosθ = 1 / e).

[0107] It is found from formula (3) that the rotational relaxation time of the luminescent material is proportional to the volume of the material, that is, when the luminescent material has a particle shape, it is proportional to the cube of the particle size. At the same time, the relationship between the luminescence lifetime of the luminescent material and the polarization degree used as the value of polarization anisotropy can be expressed by formula (4):

[0108] p0 / p=1+A(τ / Q) … (4)

[0109] Where p0 represents the degree of polarization when the material is stationary (Q=∞), "p" represents the degree of polarization, A is a constant, τ represents the luminescence lifetime of the material, and Q represents the rotational relaxation time.

[0110] It is found from equations (3) and (4) that the polarization degree is affected by the luminescence lifetime and rotational relaxation time of the luminescent material, that is, the volume (particle size) of the luminescent material, that is, by the balance between the particle size and luminescence lifetime of the luminescent material.

[0111] When the polarization degree of the light emitting material represented by the formula (4) is experimentally determined, it is appropriate to allow polarized light to enter the light emitting material and detect the light emission in a direction of 90° with respect to the traveling direction and the vibration direction of the excitation light. In this case, it is appropriate that the detected light is detected by dividing it into polarized light components in a parallel direction and a perpendicular direction with respect to the polarized light as the incident light, and, for example, the polarization anisotropy property represented by the formula (5) is adopted as the value of the polarization anisotropy:

[0112] R(t)=(I‖(t)-GI⊥(t)) / (I‖(t)+2GI⊥(t)) … (5)

[0113] Wherein R(t) represents the polarization anisotropy property at time "t", I‖(t) represents the luminous intensity of the luminous component parallel to the excitation light at time "t", I⊥(t) represents the luminous intensity of the luminous component perpendicular to the excitation light at time "t", and G represents the correction value, which is the ratio of I⊥ / I‖ measured using excitation light whose vibration direction is 90° different from the excitation light used for sample measurement.

[0114] That is, when the particle size and the luminescence lifetime fall within an appropriate range, the change in the size of the luminescent material caused by, for example, a reaction with a target substance can be sensitively read as a change in polarization anisotropy properties. That is, the R(t) of the unagglomerated luminescent material is observed to be low, and the R(t) of the agglomerated luminescent material is observed to be high. This is the principle of polarization anisotropy.

[0115] The value of polarization anisotropy may be corrected with G and 2G, or may be a value excluding G and 2G.

[0116] (Particle Matrix 1)

[0117] Figure 4 FIG. 4 is a diagram showing an example of a luminescent agent 4 having a spherical shape, and the luminescent agent 4 includes a particle matrix 1. Figure 4 In the embodiment, the luminescent agent 4 and the particle matrix 1 are both spherical in shape, but the shapes of the luminescent agent 4 and the particle matrix 1 used in the present embodiment are not limited. There is no particular provision for the particle matrix 1, as long as the particle matrix 1 is a material capable of stably introducing a europium complex, but it is preferably a polymer comprising a styrene unit and an organosilane unit. In particular, for example, a polymer obtained by polymerizing a composition comprising styrene as a main component and a free radical polymerizable organosilane is suitable for use. When the composition comprises styrene as a main component, particles having an extremely uniform particle size distribution can be produced by the emulsion polymerization method described later. In addition, when a polymer containing an organosilane unit is used, a silanol group (Si-OH) is generated in the polymer in an aqueous solvent, and a siloxane bond (Si-O-Si) is formed on the surface of the particle matrix to each other, and a hydrophilic layer or ligand described later can be provided via the bond. The particles according to the present embodiment each preferably have a ligand binding functional group capable of binding a ligand to the outside of the particle matrix.

[0118] (Hydrophilic layer 2)

[0119] The hydrophilic layer 2 can be formed by introducing a hydrophilic polymer or a hydrophilic molecule on the outside of the particle matrix 1. The hydrophilic polymer or the hydrophilic molecule is a polymer or a molecule containing a hydrophilic group, and specific examples of the hydrophilic group include molecules or polymers each having a hydroxyl, ether, pyrrolidone or betaine structure. Specific examples of the hydrophilic polymer include polyethylene glycol, polyvinyl pyrrolidone, a polymer of sulfobetaine, a polymer of phosphate betaine, and a polymethacrylate glycidyl ester having a molecule having an end modified by a hydroxyl group by ring-opening a glycidyl group, and these hydrophilic polymers can each be used as a main component of the hydrophilic layer 2. Alternatively, the hydrophilic layer 2 can be formed by directly arranging a single molecule having a hydrophilic group on the surface of the particle matrix 1 by using a silane coupling agent or the like. There is no limitation on the thickness of the hydrophilic layer 2, but it does not need to be set to be so large as to exceed the thickness that can exhibit hydrophilicity. When the hydrophilic layer 2 is too thick, the hydrophilic layer may become hydrogel-like and hydrated by the influence of ions in the solvent, thereby making its thickness unstable. The thickness of the hydrophilic layer 2 is appropriately 1 nm or more and 15 nm or less.

[0120] (Europium complex 3)

[0121] The luminescent reagent used in the present embodiment may include europium complex 3 as a luminescent dye. Europium complex 3 has the characteristics that the wavelength and intensity of its luminescence are hardly affected by the surrounding environment and therefore the luminescence has a long life. Europium complex 3 is composed of europium element and ligand. Taking into account the luminescence lifetime and visible luminescence wavelength region, etc., the luminescent dye is preferably a europium complex. The luminescence lifetime of europium is generally more than 0.1ms and less than 1.0ms. The luminescence lifetime and the rotational relaxation time obtained by formula (1) need to be appropriately adjusted. In the case of europium in an aqueous dispersion, when the diameter of the luminescent reagent is more than 50nm and less than 300nm, R changes significantly before and after aggregation.

[0122] At least one constituent ligand of the europium complex 3 is a ligand having a light-collecting function. "Light-collecting function" refers to the action of being excited at a specific wavelength to excite the central metal of the complex by energy transfer. In addition, it is preferred that the constituent ligands of the europium complex 3 include ligands such as β-diketone to prevent the coordination of water molecules. Ligands such as β-diketone coordinated to europium ions suppress the deactivation process due to energy transfer to solvent molecules, etc., thereby providing strong luminescence.

[0123] The europium complex 3 may be a polynuclear complex.

[0124] In addition, specific examples of the europium complex include [tris(2-thenoyltrifluoroacetone)(bis(triphenylphosphine oxide))europium(III)], [tris(2-thenoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzyl sulfoxide)europium(III)] and [tris(2-thenoyltrifluoroacetone)phenanthroline)europium(III)].

[0125] When the Brownian rotation of the europium complex 3 is considered to be stationary in the medium, the polarization anisotropy property represented by formula (3) is expected to be 0.08 or more. The state where the Brownian rotation can be considered to be stationary refers to a state where the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of the europium complex 3.

[0126] It is preferable to introduce the europium complex 3 into the particle matrix 1 in a larger amount because the luminescence intensity of each particle becomes stronger. Meanwhile, when the europium complex 3 is agglomerated in the particle matrix 1, the interaction between the ligands affects the excitation efficiency of the europium complex 3 and the like, making it difficult to measure the polarization anisotropy property while maintaining reproducibility. Whether the europium complex 3 exhibits non-agglomerated luminescence behavior in the particle matrix 1 can be judged from the excitation spectrum of the sample.

[0127] Particles with strong luminescence not only enable highly sensitive measurements, but also increase biochemical reaction rates because they maintain luminescence even when their particle size decreases. As the particle size becomes smaller, the diffusion coefficient of Brownian motion in the liquid becomes larger, so the reaction can be detected in a shorter time.

[0128] When a liquid in which such particles are dispersed is used in the analytical method according to the present embodiment, a change in the anisotropic property of polarized luminescence corresponding to the aggregation / dispersion behavior of the particles can be detected with high sensitivity. The dispersion obtained by dispersing such particles in an aqueous solvent can be used as a highly sensitive test reagent using polarization anisotropy. A buffer solution can be used in an aqueous solvent. In addition, a surfactant, a preservative or a sensitizer, etc. can be added to an aqueous solvent to improve the stability of the liquid in which the particles are dispersed.

[0129] (Production method of luminescent reagent)

[0130] Next, an example of a production method of the luminescent reagent used in the present embodiment is described.

[0131] The method for producing a luminescent agent includes the step of mixing a radical polymerizable monomer including at least styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarized luminescent europium complex and a hydrophilic polymer with an aqueous medium to prepare an emulsion (first step).

[0132] The method for producing a luminescent agent includes a step of heating the emulsion to polymerize the radical polymerizable monomer (second step).

[0133] In addition, the production method of the luminescent reagent can include a step (third step) of arranging a ligand binding functional group described later on the surface of the luminescent reagent. Here, the ligand binding functional group refers to a functional group capable of binding a ligand. Specifically, any one of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimide group or an alkoxysilyl group (silicon alkoxide structure) can be used.

[0134] (Free radical polymerizable monomer)

[0135] The production of the luminescent agent is carried out by polymerizing free radical polymerizable monomers, and the free radical polymerizable monomers include at least styrene and free radical polymerizable organosilanes. The free radical polymerizable monomers may also include monomers selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of monomers may include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, in addition to styrene and free radical polymerizable organosilanes, one or more of these monomers may also be used. In addition, monomers having two or more double bonds per molecule, such as divinylbenzene, may be used as crosslinking agents.

[0136] Including a radical polymerizable organosilane in the radical polymerizable monomer provides a siloxane bond to the particle matrix 1. Examples of the radical polymerizable organosilane may include vinyl trimethoxysilane, vinyl triethoxysilane, p-phenylene trimethoxysilane, 3-methacryloxypropyl methyl dimethoxysilane, 3-methacryloxypropyl trimethoxysilane, 3-methacryloxypropyl methyl diethoxysilane, 3-methacryloxypropyl triethoxysilane, 3-acryloxypropyl trimethoxysilane, and combinations thereof. The use of the radical polymerizable organosilane is used to form a skeleton of an inorganic oxide in the particle matrix 1 to improve the physical and chemical stability of the luminescent agent. In addition, the use of the radical polymerizable organosilane enhances the affinity between the particle matrix 1 and each of the hydrophilic layer 2 and the ligand binding functional group.

[0137] In addition, the radical polymerizable organosilane included in the radical polymerizable monomer provides silanol groups to the surface of the particle substrate 1. The silanol groups form hydrogen bonds with the hydrophilic polymer such as PVP. Therefore, the hydrophilic polymer such as PVP is more strongly adsorbed to the surface of the particle substrate 1.

[0138] (Free Radical Polymerization Initiator)

[0139] A wide range of compounds selected from, for example, azo compounds and organic peroxides can each be used as a free radical polymerization initiator. Specific examples thereof may include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid dimethyl ester), tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), and potassium persulfate (KPS).

[0140] (Hydrophilic polymer)

[0141] The luminescent agent may include a hydrophilic polymer as a hydrophilic layer. The hydrophilic polymer preferably inhibits nonspecific adsorption. Examples of hydrophilic polymers include hydrophilic polymers each containing a unit having an ether, betaine or pyrrolidone ring. Preferably, the hydrophilic layer is contained in the synthesized luminescent agent and is mainly present on the particle surface on the outside of the particle matrix. Here, the polymer with a pyrrolidone ring is sometimes abbreviated as "PVP". When PVP is supplied when synthesizing the luminescent agent, nonspecific adsorption inhibition ability and ligand binding ability can be provided to the luminescent agent at the same time. The PVP to be supplied during synthesis has a higher hydrophilicity than the hydrophilicity of the free radical polymerizable monomer, and is therefore present at the interface between the solvent and the particle matrix being polymerized during synthesis. The particle matrix adsorbs PVP on its outside by including a portion of PVP during polymerization, or by physical / chemical adsorption such as the interaction between the pyrrolidone ring and styrene (free radical polymerizable monomer).

[0142] The molecular weight of PVP is preferably 10,000 or more and 100,000 or less, more preferably 40,000 or more and 70,000 or less. When the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent reagent is weak, and therefore nonspecific adsorption is easily caused. When the molecular weight is greater than 100,000, the hydrophilic layer becomes so thick that it gels, thereby becoming difficult to handle.

[0143] In addition to PVP, other hydrophilic polymers can be added as protective colloids when synthesizing the particle matrix.

[0144] Furthermore, the luminescent reagent preferably satisfies A2-A1≤0.1.

[0145] A1 and A2 are defined as follows. That is, for a mixture obtained by adding 30 μL of a 0.1 wt% luminescent reagent dispersion to 60 μL of a buffer solution mixed with 16 μL of 15-fold diluted human serum, the absorbance of the mixture immediately after the addition is represented by A1, and the absorbance of the mixture after standing at 37°C for 5 minutes after the addition is represented by A2. The absorbance is measured at an optical path of 10 mm and a wavelength of 572 nm.

[0146] Particles showing an A2-A1 of 0.1 or less are preferred because they hardly nonspecifically adsorb impurities in serum.

[0147] (Water-based media)

[0148] The aqueous medium (aqueous solution) used in the production method of the above-mentioned luminescent reagent preferably contains more than 80wt% and less than 100wt% of water in the medium. The aqueous solvent is preferably water or a water-soluble organic solvent, examples of which include solutions obtained by mixing water with methanol, ethanol, isopropanol or acetone. When an organic solvent other than water is introduced in an amount exceeding 20wt%, dissolution of the polymerizable monomer may occur when producing particles.

[0149] Furthermore, it is preferred that the pH of the aqueous medium is preliminarily adjusted to be not less than 6 and not more than 9. When the pH value is less than 6 or greater than 9, the alkoxide group or silanol group of the radical polymerizable organosilane may undergo polycondensation or reaction with other functional groups before forming a polymer, resulting in agglomeration of the particles to be obtained. In the present embodiment, the alkoxide is not intentionally subjected to polycondensation before polymerization.

[0150] The above pH is preferably adjusted with a pH buffer, but may also be adjusted with an acid or a base.

[0151] In addition to the foregoing, a surfactant, a defoaming agent, a salt, a thickener, and the like may be used by adding them in a ratio of 10% or less relative to the aqueous medium.

[0152] In the production of the luminescent reagent, it is preferred that, first, PVP is dissolved in an aqueous medium adjusted to a pH of 6 to 9. The content of PVP is preferably 0.01 wt% or more and 10 wt% or less, more preferably 0.03 wt% or more and 5 wt% or less relative to the aqueous medium. When the content is less than 0.01 wt%, the amount adsorbed to the particle matrix is ​​small, and the effect cannot be exhibited. In addition, when the content is greater than 10 wt%, the viscosity of the aqueous medium may increase, thereby preventing sufficient stirring.

[0153] Subsequently, a radical polymerizable monomer including styrene (A) and a radical polymerizable organosilane (B) is added to the above-mentioned aqueous medium to prepare an emulsion. The weight ratio between styrene (A) and the radical polymerizable organosilane (B) is 6:4 to 100:1. In addition, the prepared emulsion is mixed with a europium complex. At this time, when the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The weight ratio between the europium complex and the radical polymerizable monomer is 1:1,000 to 1:10.

[0154] When the weight ratio between styrene (A) and free radical polymerizable organosilane (B) is less than 6:4, the specific gravity of the particles as a whole may increase, resulting in significant sedimentation of the particles. In addition, in order to increase the adhesion between PVP and the luminescent particles, it is desirable that the weight ratio between styrene (A) and free radical polymerizable organosilane (B) is set to 100:1 or more.

[0155] The weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is preferably 5:5 to 9.5:0.5. When the weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is less than 5:5, significant agglomeration of the particles to be produced may occur. In addition, when the weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is greater than 9.5:0.5, the production amount of the particles may decrease, although there is no problem in the production of the particles.

[0156] The radical polymerization initiator is used by being dissolved in water, a buffer solution, etc. The radical polymerization initiator can be used in an amount of 0.5 mass % to 10 mass % in the emulsion based on the total weight of styrene (A) and the radical polymerizable organosilane (B).

[0157] In the step of heating the emulsion, it is only necessary to uniformly heat the entire emulsion. The heating temperature can be arbitrarily set at 50° C. to 80° C., and the heating time can be arbitrarily set at 2 hours to 24 hours. By heating the emulsion, the free radical polymerizable monomer is polymerized.

[0158] The luminescent agent may have a ligand binding functional group on its surface. There is no particular limitation on the ligand binding functional group, as long as the functional group can bind to an antibody, an antigen or an enzyme, etc., but it may be, for example, a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimide group or a silicon alkoxide group, etc., or contain any of these functional groups. For example, a silane coupling agent having a ligand binding functional group and a synthetic particle may be mixed to provide a functional group to the particle surface. Specifically, an aqueous solution of a silane coupling agent having a carboxyl group may be prepared and mixed with a dispersion of synthetic particles to provide a carboxyl group to the particle surface. At this point, a dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature may be arbitrarily set at 0°C or more and 80°C or less, and the reaction time may be arbitrarily set at 1 hour or more and 24 hours or less. In order to suppress the sudden condensation reaction of the silane coupling agent, it is appropriate to set the temperature to be equal to or less than room temperature of about 25°C, and to set the reaction time to be 3 hours or more and 14 hours or less. Depending on the ligand binding functional group, the reaction with the particle surface can be promoted by adding acid or base catalysts.

[0159] The luminescent reagent can be used as a particle for sample testing by binding a ligand such as any of various antibodies. It is only necessary to select the best technique for binding the antibody of interest or the like by utilizing the functional groups present on the hydrophilic layer 2.

[0160] (Introduction of ligand)

[0161] In the range of the degree that can achieve the purpose of the present invention, the method known so far can be applied to the chemical reaction for making the ligand binding functional group and the ligand chemically bond. In addition, when the part is an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used.

[0162] The luminescent reagent used in the present embodiment can be preferably applied to the latex immunoagglutination measurement method widely used in the fields of clinical trials and biochemical research.

[0163] (Analytical Equipment)

[0164] According to one embodiment of the present invention, the following analysis method is provided: an analysis device configured to calculate the concentration of a target substance by measuring a value (R) of polarization anisotropy using a luminescent reagent combined with a target substance, the analysis device comprising: a mixing section configured to mix a sample containing the target substance with the luminescent reagent to provide a mixed liquid; a measuring section configured to measure R of the mixed liquid after a time T has passed since mixing; and a control section, wherein when the time when R reaches R1 in the measuring section is represented by T1, the control section is configured to calculate the concentration of the target substance based on T1, provided that when the R measured for the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

[0165] Figure 3 An overview of the analysis apparatus according to the present embodiment is described in FIG.

[0166] The mixing section is a section that performs a mixing step. The measuring section is a section that performs a measuring step. The control section controls the mixing section and the measuring section.

[0167] Mixing part and measuring part can each include, for example, a nozzle that can inhale and discharge liquid, a container for storing various samples, an orifice plate and a stand for moving these components. In addition, the measuring part can include an optical system, which includes, for example, a light source, a polarizing filter or a spectrophotometer. The control unit has a computer function. For example, the control unit can be formed integrally with a desktop personal computer (PC), a laptop PC, a tablet PC or a smart phone, etc. In order to realize the function of a computer that performs operations and stores, the control unit includes a CPU, a RAM, a ROM and a HDD. In addition, the control unit may include a communication interface (I / F), a display device and an input device.

[0168] (Reagents)

[0169] The analytical method according to the present embodiment can be used for sample testing or in vitro diagnosis. The reagent for such test or diagnosis may include: the luminescent reagent used in the present embodiment; and a dispersion medium in which the luminescent reagent is dispersed. The amount of the luminescent reagent to be mixed into the reagent is preferably 0.000001% by mass or more and 20% by mass or less, more preferably 0.0001% by mass or more and 1% by mass or less. In the range of the degree to which the purpose of the present invention can be achieved, in addition to the luminescent reagent, the reagent may include a third substance, such as an additive or a blocking agent. The reagent may include a combination of two or more third substances such as additives and blocking agents. The example of the dispersion medium used in the present embodiment includes various buffer solutions, such as phosphate buffer solution, glycine buffer solution, Good buffer solution, Tris buffer solution and ammonia buffer solution, but the dispersion medium including the test reagent in the present embodiment is not limited thereto. When the reagent is used to detect an antigen or antibody in a sample, the antibody or antigen may be used as a ligand.

[0170] Example

[0171] The present invention will be specifically described below by way of examples. However, the present invention is not limited to these examples.

[0172] (1) Production of luminescent particles

[0173] Polyvinyl pyrrolidone (PVP-K 30: manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in a 2-morpholineethanesulfonic acid (MES) buffer solution (manufactured by Kishida Chemical Co., Ltd.) having a pH of 7 to prepare a solvent A.

[0174] [Tris(2-thenoyltrifluoroacetone)(bis(triphenylphosphine oxide))europium(III)] (manufactured by Central Techno Corporation, hereinafter referred to as "Eu(TTA)3(TPPO)2"), styrene monomer (manufactured by Kishida Chemical Co., Ltd.), and 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter referred to as "MPS") used as a europium complex are mixed to prepare a reaction liquid B. The reaction liquid B is added to a four-necked flask containing the solvent A, and the mixture is stirred with a mechanical stirrer set at 300 rpm. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath is set to 70°C, and nitrogen flow is performed for an additional 15 minutes. After the mixed solution is heated and stirred, an aqueous solution in which potassium persulfate (hereinafter referred to as "KPS") (manufactured by Sigma-Aldrich) is dissolved is added to the reaction solution, and emulsion polymerization is performed for 20 hours. After the polymerization reaction, the resulting suspension was subjected to ultrafiltration with about 4 L of ion-exchanged water by using an ultrafiltration membrane having a molecular weight cutoff of 100K to wash the product, thereby providing a dispersion of light-emitting particles.

[0175] Take a sample (aliquot) of the dispersion of the luminescent particles obtained by emulsion polymerization, and add it to an aqueous solution in which 1% by mass of Tween 20 (manufactured by Kishida Chemical Co., Ltd.) is dissolved. After stirring for 10 minutes, add silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.), and stir the mixture overnight. After stirring, centrifuge the dispersion, remove the supernatant, and redisperse the precipitate with pure water. The operation of centrifugation and redispersion is performed more than 3 times to wash the product. The washed precipitate is redispersed in pure water. Therefore, the ligand binding functional group is introduced into particles 1 to 8. The mass ratio between the loaded particles, pure water and X12-1135 is set to 1:300:2.

[0176] (Production of anti-CRP antibody modified luminescent reagent)

[0177] Take a sample of 0.25 mL of a particle dispersion corresponding to 1.2 wt% of the synthesized luminescent particles, and replace the solvent with 1.6 mL of a MES buffer solution with a pH of 6.0. To the particle MES buffer solution, add 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimidesodium, and react the mixture at 25 ° C for 1 hour. After the reaction, wash the dispersion with a MES buffer solution with a pH of 5.0, add anti-CRP antibodies at 100 μg / mL, and bind the anti-CRP antibodies to the particles at 25 ° C for 2 hours. After binding, wash the particles with a Tris buffer solution with a pH of 8. After the reaction, wash the particles with a phosphate buffer solution to provide a luminescent reagent (sometimes referred to as "affinity particles") modified with an anti-CRP antibody at a concentration of 0.3 wt%.

[0178] The binding of the antibody to the particles was identified by measuring the decrease in antibody concentration in the buffer solution to which the antibody was added by means of the BCA assay.

[0179] (Preparation of Luminescent Reagent Liquid)

[0180] The obtained luminescent reagent was diluted with a phosphate buffer solution (PBS) having a pH of 7.4 to a concentration of 0.1 mg / mL to prepare a luminescent reagent liquid.

[0181] (Preparation of diluent)

[0182] A 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution and a PBS buffer solution were mixed at a volume ratio of 1:1 to prepare a dilution solution.

[0183] Reaction between target substance and ligand (antigen-antibody reaction)

[0184] A liquid obtained by mixing a CRP antigen into a HEPES buffer solution was prepared and warmed to a temperature of 37°C. A luminescent reagent liquid was added to the prepared mixed solution, and the whole was quickly stirred, and then the polarization anisotropy of the mixed solution was observed. Polarization anisotropy was studied while fixing the concentration of the luminescent reagent at 0.01 mg / mL or 0.0025 mg / mL and setting the concentration of the CRP antigen to 0 pM to 1,000 pM. The observation was performed at a temperature of 37°C. Polarization anisotropy is described later.

[0185] (Example 1)

[0186] After setting the value of the specific polarization anisotropy property R1 to 0.062, the value of the polarization anisotropy property R was measured by performing an antigen-antibody reaction using the apparatus 1 described later. The time when the polarization anisotropy property R1 reached 0.062 was measured, and a time-CRP antigen concentration graph was prepared, and then the CRP antigen concentration was evaluated. The concentration of the luminescent reagent was set to 0.01 mg / mL.

[0187] (Example 2)

[0188] The CRP antigen concentration was evaluated by the same method as in Example 1, except that the value of the specific polarization anisotropy property R1 was set to 0.043; and an apparatus 2 described later was used.

[0189] (Example 3)

[0190] The measurement of the antigen-antibody reaction was performed by the same method as in Example 1. Then, the result of subtracting the value of the polarization anisotropy property R 5 minutes after the start of the reaction from the value of R 1 minute after the start of the reaction and the result of subtracting the value of the polarization anisotropy property R 40 minutes after the start of the reaction from the value of R 1 minute after the start of the reaction were evaluated together.

[0191] (Example 4)

[0192] After setting the value of the specific polarization anisotropy property R1 to 0.076, the value of the polarization anisotropy property R was measured by performing an antigen-antibody reaction using the apparatus 1 described later. The time when the polarization anisotropy property R1 reached 0.076 was measured, and a time-CRP antigen concentration graph was prepared, and then the CRP antigen concentration was evaluated. Sodium alginate was added to the diluent to thicken the liquid, thereby setting the concentration of the luminescent reagent to 0.0025 mg / mL.

[0193] (Comparative Example 1)

[0194] The measurement of the antigen-antibody reaction was performed by the same method as in Example 3, and only the result of the subtraction between the numerical value of the polarization anisotropy property R 5 minutes after the start of the reaction and the R value 1 minute after the start of the reaction was evaluated.

[0195] (Comparative Example 2)

[0196] The measurement of the antigen-antibody reaction was performed by the same method as in Example 3, and only the result of the subtraction between the numerical value of the polarization anisotropy property R 40 minutes after the start of the reaction and the R value 1 minute after the start of the reaction was evaluated.

[0197] (Product Evaluation)

[0198] The products of Examples and Comparative Examples were evaluated as described below.

[0199] The shape of the product was evaluated with an electron microscope (S5500 manufactured by Hitachi High-Technologies Corporation).

[0200] The average particle size of the product was evaluated by using dynamic light scattering (Zetasizer Nano S manufactured by Malvern).

[0201] The concentration of the suspension in which the product was dispersed was evaluated with a gravimetric analyzer (Thermo plus TG 8120 manufactured by Rigaku Corporation).

[0202] The measurement of R was performed using the apparatus 1 and the apparatus 2. The apparatus 1 is an apparatus having such a configuration as described below.

[0203] An LED light source with excitation light of a wavelength of 340 nm was prepared, and a polarization filter (manufactured by Sigmakoki Co., Ltd., NSPFU-30C) and a short-wave pass filter (manufactured by Edmund Optics, Inc., 84-706) were inserted into the light path to set up an optical system capable of irradiating a 1 cm quartz square cell with light. A polarization filter (manufactured by Thorlabs, Inc., PIVISC050) and a bandpass filter (manufactured by Thorlabs, Inc., FB610-10) were set in a direction of 90° relative to the incident light. In order to simultaneously measure the two directions as I VV and I VH For the light emission, two groups in which the structure of the polarizer changes the direction of 90° relative to the incident light are prepared. For the detection of polarized light, spectral measurement is performed using QEPRo manufactured by Ocean Optics, Inc. A temperature controller is set in the sample holder so that the polarization anisotropy property R can be measured at 37°C. The polarization anisotropy property R is measured while fixing the output of the LED light source at 12mW and setting the accumulation time to 3 seconds. The measurement interval is set to 15 seconds. Based on the luminescence spectrum of the polarized luminescence thus obtained, the polarization anisotropy property R is determined by substituting the luminescence intensity in the wavelength range from 600nm to 630nm into formula (4).

[0204] The apparatus 2 used in the evaluation of the polarization anisotropy property R of the product and the CRP antigen-antibody reaction is an apparatus having such a configuration as described below.

[0205] An LED light source with an excitation light having a wavelength of 340 nm was prepared, and a polarization filter (manufactured by Sigmakoki Co., Ltd., NSPFU-30C) and a short-wave pass filter (manufactured by Edmund Optics, Inc., 84-706) were inserted into the optical path to set an optical system capable of irradiating a quartz cell having an optical path length of 5 cm with light. The polarized luminescence generated from the sample was split into two directions by arranging an excitation light cutting filter (manufactured by Edmund Optics, Inc., 33-910), a polarization beam splitter (manufactured by Edmund Optics, Inc., 47-127) and a polarizer (manufactured by Sigmakoki Co., Ltd., SPF-30C-32) in a prescribed order in the optical path on the same straight line as the optical path of the excitation light passing through the sample. The polarized luminescence (two directions) of the split light is detected with an avalanche photodiode (APD, manufactured by Hamamatsu Photonics KK, C15522-3010SA). A temperature controller is set in the sample holder so that the polarization anisotropy property R can be measured at 37°C. The polarization anisotropy property R is measured when the output of the LED light source is fixed at 60mW and the accumulation time is set to 8 milliseconds. The measurement interval is set to 30 seconds. The signal of the polarized luminescence thus obtained is measured with an oscilloscope, and the polarization anisotropy property R is determined by substituting the signal into formula (1).

[0206] The nonspecific agglutination inhibition evaluation of the products was performed as follows.

[0207] 60 μl of a human serum solution diluted 15 times with a buffer solution was added to a luminescent reagent dispersion (3 mg / mL), and the mixture was kept at a temperature of 37°C for 5 minutes. The absorbance at 527 nm was measured before and after the temperature was kept, and the change in absorbance before and after the temperature was kept was measured 3 times. Table 1 shows the average value of the 3 times. Evaluation was performed as follows: When the change in the "absorbance × 10,000" value was less than 1,000, it was determined that nonspecific agglutination was inhibited, and when the amount was more than 1,000, it was determined that nonspecific agglutination occurred.

[0208] (Performance Evaluation)

[0209] The synthesized luminescent agent has a particle size of about 100 nm, and exhibits strong red luminescence under the excitation light having a wavelength of 340 nm.

[0210] According to the results of the nonspecific agglutination inhibition evaluation, the change in absorbance was equal to or less than a specific value (the amount of change in the value of "absorbance x 10,000" was 1,000 or less), and thus it was confirmed that the particles were able to inhibit nonspecific adsorption.

[0211] Figure 5 The measurement results of Example 1 are shown. The time required to reach R1 is plotted on the abscissa axis, and the measured CRP concentration is plotted on the ordinate axis. It can be seen from the figure that when R1 is set to 0.062, the time required for the R value to reach 0.062 is extended according to the CRP concentration. Figure 5 It was confirmed that the region corresponding to the CRP antigen measurement range of 1,000 pM to 5 pM could be measured within a measurement time of 400 seconds.

[0212] The results of Examples 1 to 4 are shown in Table 1. The initial polarization anisotropy property R0 measured using only the luminescent reagent in each of Example 1 and Example 3 studied with Device 1 was 0.0585, and the polarization anisotropy property R0 studied with Device 2 was 0.0388. This difference reflects the difference in light receiving sensitivity between the polarized light components of the device and does not affect the nature of the measurement. In Example 4, the polarization anisotropy property was studied with Device 1. As a result, the initial polarization anisotropy property R0 measured using only the luminescent reagent was 0.0740. This is because sodium alginate, which has a thickening effect, is added to the reagent, so that the R0 of Example 4 is greater than the R0 of Example 1. At the same time, the value R2 of the saturated polarization anisotropy property was determined from a sample left to stand overnight using a large excess of CRP antigen (100,000 pM). As a result, the initial polarization anisotropy property R2 measured using only the luminescent agent in each of Example 1, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 investigated with the apparatus 1 was 0.1150, and the polarization anisotropy property R2 investigated with the apparatus 2 was 0.0900. It was confirmed in each example that the preset value of the polarization anisotropy property R1 existed between R0 and R2.

[0213] In Example 1, measurement was performed with R1 set to 0.062. As a result, it was confirmed that the detection concentration range of the CRP antigen was 5 pM to 1,000 pM.

[0214] In Example 2, measurement was performed with R2 set to 0.043. As a result, it was confirmed that the detection concentration range of the CRP antigen was 0.16 pM to 200 pM.

[0215] The difference in this embodiment may be due to the difference in the sensitivity of the measuring instrument and the preset conditions for the value of R1.

[0216] In Example 3, for two measurement times, the antigen-antibody reaction is further evaluated separately. As can be seen from the results of Example 3, when the measurement time is as short as 5 minutes, the measurement sensitivity (from 50pM to 1,000pM) in the low concentration region (about 5pM) is not obtained, and in contrast, when the measurement time is as long as 40 minutes, the antigen-antibody reaction in the high concentration region (about 1,000pM) is saturated, and therefore the polarization anisotropy property R does not increase, thereby being equal to or greater than the value of R2 (from 5pM to 200pM). In other words, it is revealed that the measurement range in the results of quantitative evaluation within a time period similar to the comparative example is narrowed, and it is revealed that when the R value is measured and quantitatively evaluated for multiple measurement times, it is possible to measure in a wide measurement range (from 5pM to 1,000pM).

[0217] In Example 4, the CRP antigen concentration is evaluated in a state where the concentration of the luminescent reagent to be used is reduced, and sodium alginate used as a thickener is added. The value of R2 is set to 0.0760. As a result, it is confirmed that the detection concentration range of the CRP antigen is 0.05pM to 100pM in a measurement of up to 600 seconds. It is revealed that when the concentration of the luminescent reagent is reduced, even a slight antigen-antibody reaction causes a large change in the polarization anisotropy property, and the promotion of the agglutination reaction of the particles in the luminescent reagent by adding a thickener can not only be measured in a wide range, but also can achieve high sensitivity and short-time measurement.

[0218] Table 1

[0219]

[0220] From the foregoing, it is revealed that the analysis method according to the present embodiment is a method capable of measuring the concentration of a CRP antigen serving as a target substance with high sensitivity and in a wide measurement range by using one reagent composition.

[0221] Therefore, the use of the analytical method according to the present embodiment enables the concentration of the target substance to be measured in a wide measurement range without preparing many test reagents according to the concentration of the target substance. Usually, it is usually a problem to prepare many reagents for the target substance of the same kind in an analytical device, because the preparation is not only complicated but also affects the setting space, etc. In addition, when the concentration exceeds the measurement concentration range and the quantitative determination of the substance ends in failure, it is necessary to dilute and prepare the sample again, so the cost of a single measurement increases. Therefore, the analytical method according to the present embodiment that can cover a wide measurement range with a small amount of reagents is useful as a method for sample testing.

[0222] The present invention is not limited to the above-described embodiments, and various changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, the following claims are attached hereto to disclose the scope of the present invention.

[0223] This application claims priority based on Japanese Patent Application No. 2022-079524 filed on May 13, 2022 and Japanese Patent Application No. 2023-076794 filed on May 8, 2023, the entire contents of the foregoing patent applications are incorporated herein by reference.

Claims

1. An analytical method comprising measuring a value R of polarization anisotropy by using a luminescent reagent bound to a target substance, thereby calculating the concentration of the target substance, The analysis method comprises: A mixing step of mixing a sample containing the target substance with the luminescent reagent to provide a mixed solution; and The step of measuring R of the mixed solution after a period of time T has passed since the mixing time, wherein in the measuring step, the time when R reaches R1 is represented by T1, and the concentration of the target substance is calculated based on T1, and Wherein when R measured on the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

2. The analytical method according to claim 1, wherein R1-R0≥0.0001 is satisfied. The analytical method according to claim 1 , wherein R0 ≥ 0.001 is satisfied. The analytical method according to claim 1 , wherein the luminescent reagent comprises luminescent particles. The analysis method according to claim 4 , wherein the luminescent particle comprises a europium complex. The analytical method according to claim 1 , wherein the luminescent reagent comprises a ligand that binds to the target substance.

7. The analytical method according to claim 1, wherein the R is defined as "r" in the following formula (1): In formula (1), I VV represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam, I VH represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the first polarized light beam when excited by the first polarized light beam, I HV represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam, I HH represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam, and G represents the correction value. The analytical method according to claim 1 , wherein the measuring step is performed repeatedly.

9. The analysis method according to claim 8, wherein in the repeated measurement steps, when R exceeds R1 for the first time in the nth measurement step, any value between the end time of the n-1th measurement step and the start time of the n+1th measurement step is used as T1.

10. The analysis method according to claim 8, wherein the repeatedly performed measuring step is repeated with a period of a+b seconds, wherein "a" represents a time period required for measurement, and "b" represents a measurement interval.

11. The analysis method according to claim 8, wherein in the repeatedly performed measuring step, when R1 exceeds a predefined value R2 when R1 reaches R1 for the first time, at least one of "a" or "b" is set to be short.

12. An analysis device configured to calculate the concentration of a target substance by measuring a value R of polarization anisotropy using a luminescent reagent bound to the target substance, The analysis equipment comprises: a mixing unit configured to mix the sample containing the target substance with the luminescent reagent to provide a mixed solution; a measuring unit configured to measure R of the mixed liquid after a time T has passed since the mixing; and Control Department, wherein when the time when R reaches R1 in the measuring section is represented by T1, the control section is configured to calculate the concentration of the target substance based on T1, The condition is that when R measured for the luminescent reagent not mixed with the target substance is represented by R0, R1>R0 is satisfied.

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