JP2023168266A - Analysis method and analyzer by measurement based on polarization anisotropy - Google Patents

Abstract

To provide an analysis method and an analyzer that can analyze the concentration of a target substance within a wide concentration range and with high sensitivity based on polarization anisotropy.SOLUTION: An analysis method is to measure a value (R) related to polarization anisotropy by using a luminescent reagent bound to a target substance, thereby calculating the concentration of the target substance, and the method includes: a mixing step of mixing a sample including the target substance with the luminescent reagent to obtain a mixed solution; and a measuring step of measuring the R of the mixed solution after the lapse of time T from the time of mixing. In the measuring step, the time when the R reaches R1 is defined as T1, and the concentration of the target substance is calculated based on the T1.SELECTED DRAWING: Figure 1

Description

The present invention relates to an analysis method and an analysis apparatus based on polarization anisotropy.

In the fields of medicine and clinical testing, it is necessary to detect or quantify minute amounts of biological components from blood or a sampled organ part with high sensitivity in order to investigate the cause, presence or absence, etc. of a disease.
Among the testing methods for biological components, immunoassay is widely used. Many immunoassays require a washing step called B/F (Bound/Free) separation. One of the immunoassays that does not require B/F separation is a latex agglutination method that utilizes antigen-antibody reactions. In the latex agglutination method, latex particles carrying an antibody or the like that specifically binds to a target substance are mixed with a liquid that can contain the target substance, and the degree of aggregation of the latex particles is measured.

In the latex aggregation method, a target substance is captured by an antibody specific to the target substance bound to latex particles, and multiple latex particles are crosslinked via the captured target substance, resulting in aggregation of the latex particles. That is, the amount of a target substance in a liquid sample such as a biological sample can be quantified by evaluating the degree of aggregation of latex particles. The degree of aggregation can be quantified by measuring and evaluating changes in the amount of light transmitted or scattered through the liquid sample.

Although the latex agglutination method can easily and quickly detect and quantitatively evaluate target substances, antigens, it has the problem of a detection limit in that it cannot be detected if the amount of antigen in a liquid sample such as a biological sample is small.

In order to improve the detection sensitivity of target substances, it is required to measure the degree of aggregation with higher sensitivity. In other words, it is conceivable to replace the system that measures changes in the amount of light transmitted or scattered through a liquid sample with a detection and quantification method that utilizes luminescence characteristics with higher sensitivity. Specifically, specimen testing methods using fluorescence depolarization methods have been proposed (Patent Documents 1 and 2).

Patent Document 1 proposes to improve a device for fluorescence depolarization method for clinical use.
Fluorescence depolarization does not require the B/F separation required by common fluorescence measurements.
Therefore, using the fluorescence depolarization method, it is possible to easily test specimens in the same way as the latex agglutination method. Furthermore, when using the fluorescence depolarization method, it is considered possible to perform measurements using the same testing system as the latex agglutination method, by simply mixing a luminescent substance that specifically reacts with the target substance in the measurement process. On the other hand, Patent Document 1 proposes using a single molecule such as fluorescein as a luminescent material, but in principle it could only be applied to drugs, low-molecular antigens, and the like.

Patent Document 2 solves the problem of Patent Document 1, that the fluorescence depolarization method is only applicable to drugs, low-molecular antigens, and the like. That is, Patent Document 2 aims to apply fluorescence depolarization to polymers such as proteins, and proposes the use of a material in which a dye having long-life luminescent properties is adsorbed to latex particles as a luminescent material. In Patent Document 2, based on the principle of fluorescence depolarization, the reduction of the rotational Brownian motion of a substance in a liquid due to an increase in particle size, and the balance between the length of the luminescence lifetime, and the reduction of the polymer substance. Quantification is proposed. However, in Patent Document 2, the polarization anisotropy of the inspection particle can be stably determined by interactions between the fluorescent substances adsorbed near the particle surface, etc., because the fluorescent substance is supported on the particles after synthesis of the latex particles. is difficult. Furthermore, in Patent Document 2, in order to suppress non-specific adsorption, particles carry bovine serum albumin (BSA), a biomolecule, on their surfaces, so the particle size distribution is wide, and BSA, a protein, causes differences between lots. There was a possibility of variation.
Therefore, the concentration of the target substance is measured on the order of μg/mL, and there is no significant difference in measurement sensitivity from the latex method.

Furthermore, Patent Document 2 proposes a method of measuring the difference in polarization anisotropy in aggregation between particles before and after a specific reaction time, and a method of evaluating based on the value of polarization anisotropy at the end point of the reaction time. ing.

In the measurement method of Patent Document 2, the upper limit of polarization anisotropy is determined by the luminescent substance used as a probe, so when performing measurements over a wide range, the amount of reagent must be adjusted according to the concentration of the target substance. There is a need to. In other words, the concentration range that can be measured using one type of reagent is limited.

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 has been achieved.

Special Publication No. 3-52575 Patent No. 2893772

The present invention has been made in view of such background technology, and provides an analysis method and an analysis device that enable high-sensitivity analysis of the concentration of a target substance over a wide concentration range based on polarization anisotropy. The purpose is to

In one embodiment, the present invention provides an analysis method for calculating the concentration of a target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance, comprising:
a mixing step of mixing the sample containing the target substance and the luminescent reagent to form a mixed solution; and a measuring step of measuring the R of the mixed solution after a time T has elapsed from the time of mixing.
has
In the measurement step, the time when the R reaches R1 is defined as T1, and the concentration of the target substance is calculated based on T1, and further, the luminescence reagent that is not mixed with the target substance is measured. When R is R0, R1>R0 is satisfied.
Provide an analysis method.

Further, as an embodiment of the present invention, there is provided an analysis device that calculates the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance,
a mixing unit that mixes the sample containing the target substance and the luminescent reagent to form a mixed solution;
a measuring unit that measures the R of the mixed liquid after a time T has elapsed from the time of the mixing, and a control unit;
The control section provides an analysis device characterized in that, in the measurement section, the time when the R reaches R1 is set as T1, and the concentration of the target substance is calculated based on the T1.
However, when the R measured for the luminescent reagent not mixed with the target substance is R0,
R1>R0 is satisfied.

According to the analysis method according to the embodiment of the present invention, changes in polarization anisotropy can be detected with high sensitivity in response to particle aggregation/dispersion behavior, and measurement can be performed over a wide concentration range. According to the analysis device according to the embodiment of the present invention, it is possible to detect and quantify a target substance with high sensitivity and over a wide concentration range based on polarization anisotropy.
Further features of the invention will become apparent from the following description of the embodiments and the drawings.

1 is a schematic diagram illustrating an analysis method according to an embodiment of the present invention. 1 is a schematic diagram illustrating an analysis method according to an embodiment of the present invention. 1 is a schematic diagram illustrating an apparatus according to an embodiment of the present invention. FIG. 1 is a schematic diagram illustrating a luminescent reagent used in an embodiment of the present invention. FIG. 3 is a diagram showing the evaluation results, that is, explaining the results of quantifying CRP antigen concentration using the method according to the embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail, but the scope of the present invention is not limited.
The present invention provides the following analysis method as one embodiment.
An analysis method for calculating the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance, the method comprising:
a mixing step of mixing the sample containing the target substance and the luminescent reagent to form a mixed solution; and a measuring step of measuring the R of the mixed solution after a time T has elapsed from the time of mixing.
has
In the measurement step, the time when the R reaches R1 is defined as T1, and the concentration of the target substance is calculated based on T1, and further, the luminescence reagent that is not mixed with the target substance is measured. When R is R0, R1>R0 is satisfied.
analysis method.

(value related to polarization anisotropy)
In this embodiment, the value related to polarization anisotropy (sometimes referred to as R) is determined as follows. In other words, it is a value indicating the relationship between the emission intensity of a polarized light component in a direction parallel to the irradiated polarized light and the emission intensity of a polarized light component in a direction perpendicular to the irradiated polarized light, for light emission generated by exciting a luminescent substance by irradiating it with polarized light. More specifically, R is the luminescence intensity of a luminescent component whose vibration direction is parallel to the polarization when a luminescent substance is excited with a certain polarized light, and the luminescence intensity of a luminescent component whose polarization and vibration direction are orthogonal (perpendicular). This value is calculated based on the luminescence intensity of . Furthermore, when excited with the first polarized light, the emission intensity of the emitted light component whose vibration direction is parallel to the first polarized light, and when excited with the first polarized light, the first polarized light and the vibration direction are orthogonal to each other. This is a value indicating the ratio between the difference in luminescence intensity of luminescent components and the sum of these. However, R is the emission intensity of the luminescent component whose vibration direction is orthogonal to the second polarized light when excited with the second polarized light whose vibration direction is orthogonal to the first polarized light, and whose vibration direction is orthogonal to the first polarized light. It may be corrected by the ratio of the emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited by the second polarization, and other constants. Values related to polarization anisotropy include values called polarization anisotropy, polarization degree, and the like.

（式（１）中、
Ｉ_ＶＶ・・・第一の偏光で励起したときの、第一の偏光と振動方向が平行な発光成分の発光強度
Ｉ_ＶＨ・・・第一の偏光で励起したときの、第一の偏光と振動方向が直交する発光成分の発光強度
Ｉ_ＨＶ・・・第一の偏光と振動方向が直交する第二の偏光で励起したときの、第二の偏光と振動方向が直交する発光成分の発光強度
Ｉ_ＨＨ・・・第一の偏光と振動方向が直交する第二の偏光で励起したときの、第二の偏光と振動方向が平行な発光成分の発光強度
Ｇ・・・補正値
である。） More specifically, for example, R can be r in the following formula (1).

(In formula (1),
I _VV ... Emission intensity of the luminescent component whose vibration direction is parallel to the first polarized light when excited with the first polarized light I _VH ... The first polarized light when excited with the first polarized light Emission intensity of a luminescence component whose vibration direction is orthogonal I _HV ...Emission intensity of a luminescence component whose vibration direction is orthogonal to the second polarized light when excited with a second polarized light whose vibration direction is orthogonal to the first polarized light _IHH ... Emission intensity of a light emitting component whose vibration direction is parallel to the second polarized light when excited with the second polarized light whose vibration direction is orthogonal to the first polarized light G... Correction value. )

各記号については式（１）に同じである。 Further, R can be r' in the following formula (2).

Each symbol is the same as in equation (1).

The conditions for measuring R are, for example, preferably in a liquid at a temperature of 0° C. or more and 50° C. or less, and a viscosity of the liquid of 0.5 Pa·s or more and 50 mPa·s or less. When the luminescent reagent is a particle containing a europium complex, the concentration of the luminescent reagent is preferably measured at 0.001 mg/ml or more and 0.1 mg/ml or less, and the excitation wavelength is preferably 500 nm or more and 700 nm or less. .

(About R1 and R0)
R1 can be appropriately determined by the analyst depending on the purpose of measurement and the properties of the target substance.
For example, when the purpose is to analyze the concentration of a target substance with high sensitivity (in a low concentration range), it is preferable that R1-R0≧0.0001. For example, in order to analyze a target substance in a measurement sample within a measurement range of 1 pM or more and 10 nM or less, it is preferable that R1-R0≧0.0001. Note that the maximum value of R1-R0 is 0.4, which is a theoretical value determined from the configuration of the analysis method.

On the other hand, when the purpose is to analyze the concentration of a target substance with low sensitivity (in a high concentration range), it is preferable to lower R1-R0. For example, in order to analyze a target substance in a measurement sample within a measurement range of 1 nM or more and 1M or less, R1-R0<0.0001 can be set.

R1 can be determined by the measurer in advance by measuring values related to polarization anisotropy using standard samples with various known concentrations and creating a calibration curve.

Further, R0, which is the R measured for the luminescent reagent not mixed with the target substance, preferably satisfies R0≧0.001.

(About the mixing process)
FIG. 1 can be referred to for each step. In the mixing step, a sample containing a target substance and a luminescent reagent are mixed to form a mixed solution. The mixed liquid is a liquid containing a luminescent reagent and a target substance, and may also contain other additives. It is preferable that the mixing is carried out in a range of pH 3.0 or more and pH 11.0 or less. Further, the mixing temperature is in the range of 20°C or more and 50°C or less. The target substance and luminescent reagent will be described later.

(About the measurement process)
In the measurement step, R of the mixed liquid after time T has elapsed from the time of mixing is measured. Here, the time of mixing is not limited to the time when the sample and the luminescent reagent touch, but may be the time after a certain period of time has elapsed from the time of contact, as long as the measurement can be performed appropriately. For example, the time after a certain period of time (for example, 10 seconds) has elapsed since the time when the sample and the luminescent reagent came into contact, the time when the stirring process that is performed after the sample and the luminescent reagent have come into contact, and the time when the stirring process has been completed. It may be any time after a certain period of time has elapsed. As for the measurement conditions, for example, it is preferable that the temperature of the liquid be 0° C. or more and 50° C. or less, and the viscosity of the liquid be 0.5 mPa·s or more and 50 mPa·s or less. The concentration of the luminescent reagent is preferably measured at 0.0001 mg/ml or more and 0.1 mg/ml or less, and the excitation wavelength is preferably 500 nm or more and 700 nm or less.

When a certain target substance is present in the target substance, R of the mixture increases with reaction time.
The rate of increase in the 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 in the R value is fast, and when the concentration is low, the rate of increase is slow.

That is, the reaction time for the polarization anisotropy R to reach R1 is short when the target substance is at a high concentration, and becomes long when the target substance is at a low concentration.

Since the time when R1 is exactly reached does not necessarily mean that the measurement is in progress, T1 can be estimated and determined from the time when a value close to R1 is measured.

T1 can also be calculated and determined from the measurement results. For example, if a mixed liquid is measured at regular time intervals, the rate of increase in polarization anisotropy R relative to the reaction time can be calculated. That is, it is possible to calculate and determine the time for the R value of the liquid mixture being measured to reach R1 from the slope of the change in R with respect to the reaction time.

Since the R1 value can be predicted and the measurement time can be shortened, this is an analysis method that can be suitably used when the target substance is at a low concentration.

Further, the value of polarization anisotropy R1 may be determined later. Quantitative evaluation is also possible by using a process in which the value of R1 is determined after measuring data plotting the change in R against T, and compared with the results of a standard substance measured in the same manner.

By using the analysis method of this embodiment, the concentration can be determined over a wide range without adjusting many test reagents depending on the concentration of the target substance. Generally, preparing a large number of reagents for the same type of target substance in an analysis device is not only complicated, but also often poses a problem because it affects the installation space. Furthermore, if quantification fails because the concentration exceeds the measurement concentration range, it is necessary to dilute and adjust the sample again, which increases the cost for one measurement. Therefore, being able to cover a wide measurement range with a small number of reagents has important value in performing sample tests.

It is preferable that the measurement process is performed repeatedly. As shown in FIG. 1, further measurement steps can be performed when R does not reach R1.

The repeatedly performed measurement step can be repeated periodically, for example, with a period of (a+b) seconds. However, a is the time required for measurement, and b is the measurement interval. That is, a measurement takes a second, and then the next measurement can be started after an interval of b seconds. For example, a can be 1 millisecond or more and 10 seconds or less, and b can be 1 second or more and 1 minute or less.
For example, a+b can be 1 second or more and 10 minutes or less, more preferably 1 second or more and 1 minute or less, furthermore, 15 seconds or more and 30 seconds or less.

In repeated measurement steps, if R exceeds R1 for the first time in the n-th measurement step, for example, sometime between the end time of the (n-1)th measurement step and the start time of the (n+1)-th measurement step. The numerical value of can be set as T1.

(About R2)
The upper limit value (R2) of R can be determined in advance. That is, if R also exceeds R2 in a period in which R exceeds R1 for the first time, it is preferable to adjust the period by shortening at least one of a and b (FIG. 2). For example, this is the case when the concentration of the target substance in the mixed solution is high and the R value of polarization anisotropy reaches R2 in a short time from the start of the reaction. In such a case, a or b is shortened, R is newly measured, and the high concentration liquid is quantified based on a calibration curve that fits the measurement.

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

Similarly, if the concentration of the target substance in the mixture is low and the R value rises slowly, measure the value related to polarization anisotropy by lengthening a or b, and then use the calibration curve and measurement results that match it. By performing the comparison step, low concentration liquids can be quantified.

In this way, by dividing the conditions of the step of quantifying by comparison with a calibration curve based on the reaction time, it becomes possible to cover a wide measurement range with a small number of types of reagents.

Moreover, the present invention provides the following measuring device as a further embodiment.
A measuring device that measures the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance,
a mixing unit that mixes the sample containing the target substance and the luminescent reagent to form a mixed solution;
a measuring unit that measures the R of the mixed liquid after a time T has elapsed from the time of the mixing, and a control unit;
A measuring device, wherein the control unit calculates the concentration of the target substance based on T1 when the R reaches R1 in the measuring unit;
However, when the R measured for the luminescent reagent not mixed with the target substance is R0,
R1>R0 is satisfied.

(Target substance)
Examples of target substances include antigens, antibodies, low molecular weight compounds, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, information transmitters, membrane proteins, and the like. Examples of antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of low-molecular-weight compounds. Nucleic acids include DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc. Examples of low-molecular compounds include cytokines, hormones, neurotransmitters, information transmitters, membrane proteins, and their receptors. The analysis method according to the present embodiment can also determine the presence or absence of a target substance by comparing the concentration of the target substance with a predetermined threshold value. For example, it can be determined that the target substance is present when the concentration of the target substance is greater than or equal to a predetermined threshold, and that the target substance is absent when the concentration of the target substance is less than the predetermined threshold.

(Luminescence reagent)
In the present embodiment, the luminescent reagent is a reagent that generates luminescence, particularly a reagent that emits luminescence when excited by irradiation with light, excluding those that emit luminescence caused by a chemical reaction, such as luminol. Luminescence includes phosphorescence and fluorescence, preferably phosphorescence. More preferably, the luminescent reagent in this embodiment includes a europium complex. Also, more preferably, in this embodiment, the luminescent reagent includes particles. Most preferably, in this embodiment, the luminescent reagent comprises particles comprising a europium complex. Moreover, it is preferable that the luminescent reagent has a ligand specific to the target substance. By having the ligand, the particles of this embodiment can detect and quantify a target substance based on polarization anisotropy. In this embodiment, a ligand is a compound that specifically binds to a specific target substance.

Any ligand can be used as long as it shows affinity for a specific substance. Examples of combinations of a ligand and a target substance or a combination of a target substance and a ligand include the following. That is, examples include antigens and antibodies, low-molecular compounds and their receptors, enzymes and substrates, and complementary nucleic acids. Furthermore, antibodies and their specific allergens, bacteria, viruses, cells, cell membrane constituents, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, all kinds of low-molecular compounds, etc. can be mentioned. Further examples include receptors and specific low molecular weight compounds, cytokines, hormones, neurotransmitters, information transmitters, membrane proteins, and the like. Further examples include DNA, RNA, cDNA derived from bacteria, viruses, cells, etc., parts or fragments thereof, synthetic nucleic acids, primers, probes, etc., and nucleic acids complementary thereto. In addition to the above, any combination known to have affinity can be used as a combination of a target substance and a ligand. The ligand in this embodiment typically includes any one of an antibody, an antigen, and a nucleic acid.

FIG. 4 is a schematic diagram showing an example of a luminescent reagent used in this embodiment. The luminescent reagent 4 used in this embodiment preferably comprises a particle substrate 1 containing a europium complex 3, and a hydrophilic layer 2 covering the surface thereof. The diameter of the luminescent reagent in FIG. 4 is 25 nm or more and 500 nm or less.

The diameter of the particles can be determined by dynamic light scattering. When particles dispersed in a solution are irradiated with a laser beam and the scattered light is observed with a photon detector, the intensity distribution due to the interference of the scattered light is constantly changing because the particles are constantly moving their positions due to Brownian motion. It's shaking.
Dynamic light scattering is a measurement method that observes this Brownian motion as fluctuations in the intensity of scattered light. The fluctuation of scattered light with respect to time is expressed by an autocorrelation function, and the translational diffusion coefficient is determined. By determining the Stokes diameter from the determined diffusion coefficient, the size of the particles dispersed in the solution can be derived.

From the viewpoint of maintaining particle uniformity and monodispersity, it is desirable that no luminescent reagent be applied to the surface of the particles. However, in order to use the analysis method according to the present embodiment, it is necessary to prevent non-specific adsorption of substances other than the target onto the particles, so it is necessary to have a hydrophilic layer to keep the surface of the luminescent reagent hydrophilic. is preferred.

As a method of maintaining hydrophilicity, a method of supporting BSA on the surface of particles is commonly used, but this method may cause lot variations. Therefore, the luminescent reagent preferably includes a hydrophilic layer containing a hydrophilic polymer. The concentration of the luminescent reagent in the mixed liquid is preferably 0.000001% by mass or more and 1% by mass or less, more preferably 0.00001% by mass or more and 0.001% by mass or less.

The luminescent reagent used in this embodiment can emit long-lived phosphorescence by containing a europium complex. The luminescent reagent used in this embodiment preferably has an average particle diameter of 25 nm or more and 500 nm or less, more preferably 50 nm or more and 300 nm or less. When the average particle diameter exceeds 500 nm, the polarization anisotropy before aggregation becomes high, and the difference from the polarization anisotropy after the aggregation reaction becomes small. Further, if the average particle diameter is less than 25 nm, the change in size before and after aggregation becomes small, and it becomes difficult to capture the change in R by depolarizing the phosphorescent emission.

By reducing the particle size distribution of the luminescent reagent and introducing a europium complex as a luminescent molecule, it is possible to detect changes in polarized luminescence characteristics even if there is a slight change in the dispersion state of particles in the liquid. . Specifically, even if the concentration of the target substance in the solution is on the order of nanograms to picograms per mL, for example from 1 picogram to 100 picograms, when the luminescent reagent aggregates via the target substance, the rotation of the luminescent reagent Changes in Brownian motion can be understood as changes in polarization anisotropy.

Polarized light emission refers to the fact that in a light emitting material whose transition moment (transition dipole moment) is anisotropic, if excitation light is polarized along the transition moment, the emitted light will also be polarized along the transition moment. say. Europium complexes exhibit fluorescence emission based on energy transfer from the ligand to the central metal ion, so the transition moment is complicated, but red emission around 610 nm is derived from electronic transition from the lowest excited state 5D0 to 7F2. emits polarized light.

The principle of polarization anisotropy is to measure the deviation of the transition moment due to the rotational movement of the luminescent material during the time when polarized light emission is occurring. The rotational motion of the luminescent material can be expressed by equation (3).
Q=3Vη/kT...(3)
here,
Q: rotational relaxation time of material V: volume of material η: viscosity of solvent k: Boltzmann constant T: absolute temperature.

The rotational relaxation time of a material is the time required for molecules to rotate through an angle θ (68.5°) where cos θ=1/e.

From equation (3), it can be seen that the rotational relaxation time of the luminescent material is proportional to the volume of the material, that is, when the luminescent material is in the form of particles, to the cube of the particle size. On the other hand, the relationship between the luminescence lifetime of a luminescent material and the degree of polarization, which is a value related to polarization anisotropy, can be expressed by equation (4).
p0/p=1+A(τ/Q)...(4)
here,
p0: degree of polarization when the material is at rest (Q=∞) p: degree of polarization A: constant τ: luminescence lifetime of the material Q: rotational relaxation time.

From equations (3) and (4), the degree of polarization is affected by the luminescent material's luminescent lifetime and rotational relaxation time, that is, the volume (particle size) of the luminescent material, that is, the balance between the luminescent material's particle size and luminescent lifetime. It can be seen that this has an influence.

When determining the degree of polarization of a luminescent material expressed by equation (4) through experiments, polarized light may be incident on the luminescent material, and luminescence may be detected in a direction 90 degrees from the traveling direction and vibration direction of the excitation light. At this time, the detection light may be detected separately into polarization components parallel to and perpendicular to the polarization of the incident light, and the polarization anisotropy shown in equation (5) may be taken as a value related to polarization anisotropy, for example.
R(t)=(I∥(t)-GI⊥(t))/(I∥(t)+2GI⊥(t))...(5)
here,
R(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, which is the ratio of I⊥/I∥ measured using excitation light whose vibration direction is 90 degrees different from that used for sample measurement.

In other words, if the particle size and luminescence lifetime are within appropriate ranges, changes in the size of the luminescent material due to reactions with target substances, etc. can be sensitively read as changes in polarization anisotropy. That is, the R(t) of non-agglomerated luminescent materials is observed to be low, and the R(t) of aggregated luminescent materials to be observed to be high. This is the principle of polarization anisotropy.

Note that the value related to polarization anisotropy may be corrected using G and 2G, or may be a value excluding G and 2G.

(Particle matrix 1)
FIG. 4 shows an example of a spherical luminescent reagent 4 , which comprises a particle matrix 1 . Although FIG. 4 shows an example in which both the luminescent reagent 4 and the particle substrate 1 are spherical, the shapes of the luminescent reagent 4 and the particle substrate 1 used in this embodiment are not limited. The particle substrate 1 is not particularly specified as long as it can stably incorporate the europium complex, but it is preferably a polymer containing a styrene unit and an organic silane unit, particularly a polymer containing styrene as a main component and a radically polymerizable organic silane. Polymers obtained by polymerizing compositions containing the above-mentioned components are preferably used. By containing styrene as a main component in the composition, it is possible to produce particles with a very uniform particle size distribution by the emulsion polymerization method described below, and by using a polymer containing an organic silane unit, Silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, and siloxane bonds (Si-O-Si) are formed with each other on the surface of the particle substrate, through which a hydrophilic layer and a ligand, which will be described later, can be provided. can. The particles according to this embodiment preferably have a ligand-binding functional group capable of binding a ligand on the outside of the particle matrix.

(Hydrophilic layer 2)
The hydrophilic layer 2 can be comprised of a hydrophilic polymer or hydrophilic molecules on the outside of the particle matrix 1 . A hydrophilic polymer or a hydrophilic molecule is a polymer or molecule containing a hydrophilic group, and specific examples of the hydrophilic group include molecules and polymers having a hydroxyl group, ether, pyrrolidone, betaine structure, etc. Specific examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid with ring-opened glycidyl groups and modified hydroxyl groups at the end of the molecule. It can be the main component of the hydrophilic layer 2. Alternatively, the hydrophilic layer 2 may be formed by directly applying a monomolecule having a hydrophilic group to the surface of the particle substrate 1 using a silane coupling agent or the like. Although there is no limitation on the thickness of the hydrophilic layer 2, it is not necessary to make it thicker than the thickness that can exhibit hydrophilicity. If the hydrophilic layer 2 is too thick, it will look like a hydrogel, and the thickness of the hydrophilic layer may become unstable due to hydration due to the influence of ions in the solvent. The thickness of the hydrophilic layer 2 is preferably 1 nm or more and 15 nm or less.

(Europium complex 3)
The luminescent reagent used in this embodiment can contain europium complex 3 as a luminescent dye. The europium complex 3 has the characteristics that the wavelength and intensity of the emitted light are not easily influenced by the surroundings, and the emitted light has a long life. Europium complex 3 is composed of europium element and a ligand. Considering the luminescence lifetime, visible emission wavelength range, etc., the luminescent dye is preferably a europium complex. Europium generally has a luminescence lifetime of 0.1 ms or more and 1.0 ms or less. It is necessary to appropriately adjust this emission lifetime and the rotational relaxation time obtained from equation (1). In the case of europium in an aqueous dispersion, if the diameter of the luminescent reagent is from 50 nm or more to 300 nm or less, R changes significantly before and after aggregation.

At least one of the ligands constituting the europium complex 3 is a ligand having a light condensing function. The light condensing function is an action that excites the central metal of the complex through energy transfer by excitation at a specific wavelength. Further, it is preferable that a ligand such as β-diketone exists in the ligand constituting the europium complex 3 to prevent coordination of water molecules. Ligands such as β-diketones coordinated to europium ions suppress the deactivation process due to energy transfer to solvent molecules, etc., resulting in strong luminescence.

The europium complex 3 may be a polynuclear complex.
Further, as a specific example of the europium complex, [Tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III)]([Tris(2-thenoyltrifluoroacetone)(Bis(triphenylphosphineoxide))] III)] ), [Tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide) europium(III)]([Tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfox ide) europium (III)]), Examples include [Tris(2-thenoyltrifluoroacetone)phenanthroline europium(III)].

When the Brownian rotational motion of the europium complex 3 in the medium is considered to have stopped, it is desirable that the polarization anisotropy expressed by formula (3) is 0.08 or more. The state in which the Brownian rotational motion can be considered to have stopped refers to a state in which the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of the europium complex 3.

It is preferable that a large amount of europium complex 3 is incorporated into the particle matrix 1 because the luminescence intensity per particle becomes stronger. On the other hand, when the europium complex 3 aggregates in the particle matrix 1, the interaction between the ligands affects the excitation efficiency, etc. of the europium complex 3, making it difficult to measure polarization anisotropy while maintaining reproducibility. It becomes difficult. Whether the europium complex 3 exhibits non-aggregating luminescence behavior in the particle matrix 1 can be determined from the excitation spectrum of the sample.

Particles with strong luminescence not only enable highly sensitive measurements, but also maintain luminescence even when the particle size is reduced, making it possible to accelerate biochemical reaction rates. The smaller the particle size, the larger the diffusion coefficient of Brownian motion in the liquid, making it possible to detect reactions in a shorter time.

When a liquid in which such particles are dispersed is used in the analysis method according to the present embodiment, changes in the anisotropy of polarized light emission can be detected with high sensitivity in response to particle aggregation/dispersion behavior. . A dispersion in which such particles are dispersed in an aqueous solvent can be used as a highly sensitive test reagent using polarization anisotropy. A buffer solution may be used as the water solvent. Further, in order to increase the stability of the liquid in which the particles are dispersed, a surfactant, a preservative, a sensitizer, etc. may be added to the aqueous solvent.

(Method for producing luminescent reagent)
Next, an example of a method for manufacturing the luminescent reagent used in this embodiment will be described.
A method for producing a luminescent reagent includes preparing an emulsion by mixing at least a radically polymerizable monomer containing styrene and a radically polymerizable organic silane, a radical polymerization initiator, a polarized luminescent europium complex, and a hydrophilic polymer with an aqueous medium. (first step).

The method for producing a luminescent reagent includes a step (second step) of heating an emulsion and polymerizing a radically polymerizable monomer.

Furthermore, the method for producing a luminescent reagent can include a step (third step) of providing the surface of the luminescent reagent with a ligand-binding functional group, which will be described later. Here, the ligand-binding functional group is a functional group that can bind a ligand, and specifically includes a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or an alkoxysilyl group (silicon alkoxide structure) can be used.

(radical polymerizable monomer)
The luminescent reagent is produced by polymerizing radically polymerizable monomers, and the radically polymerizable monomers include at least styrene and radically polymerizable organic silane. The radically polymerizable monomer can further include a monomer selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of the monomer include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and radically polymerizable organic silane. Furthermore, a monomer having two or more double bonds in one molecule, such as divinylbenzene, may be used as a crosslinking agent.

Since the radically polymerizable monomer contains a radically polymerizable organic silane, the particle substrate 1 is provided with siloxane bonds. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyl Mention may be made of diethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. By using a radically polymerizable organic silane, an inorganic oxide skeleton is formed within the particle substrate 1, which plays a role in improving the physical and chemical stability of the luminescent reagent. Furthermore, by using a radically polymerizable organic silane, the affinity between the particle substrate 1 and the hydrophilic layer 2 or the ligand-binding functional group increases.

Furthermore, since the radically polymerizable monomer contains a radically polymerizable organic silane, silanol groups are provided on the surface of the particle substrate 1. Silanol groups and hydrophilic polymers, such as PVP, form hydrogen bonds. As a result, the hydrophilic polymer such as PVP is more firmly adsorbed onto the surface of the particle matrix 1.

(radical polymerization initiator)
As the radical polymerization initiator, a wide variety of azo compounds, organic peroxides, etc. can be used. Specifically, 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, dimethyl 2,2'-azobis(2-methylpropionate), teRt-butylhydroperoxide , benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), and the like.

(hydrophilic polymer)
The luminescent reagent can include a hydrophilic polymer as a hydrophilic layer. The hydrophilic polymer preferably suppresses non-specific adsorption. Examples of hydrophilic polymers include hydrophilic polymers containing units having ether, betaine, pyrrolidone rings, and the like. The hydrophilic layer is included in the synthesized luminescent reagent and is preferably present mainly on the particle surface outside the particle matrix. In this specification, a polymer having a pyrrolidone ring may be abbreviated as "PVP". By adding PVP during the synthesis of the luminescent reagent, it becomes possible to impart the luminescent reagent with the ability to suppress nonspecific adsorption and the ability to bind a ligand at the same time. Since PVP introduced during synthesis has higher hydrophilicity than the radically polymerizable monomer, it is present at the interface between the solvent and the particle substrate during polymerization during synthesis. The particle matrix adsorbs PVP on the outside by partially involving PVP during polymerization or by physical/chemical adsorption such as interaction between the pyrrolidone ring and styrene (a radically polymerizable monomer).

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 nonspecific adsorption is likely to occur. When the molecular weight is greater than 100,000, the hydrophilic layer becomes too thick, gels, and becomes difficult to handle.

In addition to PVP, a hydrophilic polymer may be added as a protective colloid during particle matrix synthesis.

Further, the luminescent reagent preferably satisfies A2-A1≦0.1.
A1 and A2 are defined as follows. That is, the absorbance of a mixture obtained by adding 30 μL of a 0.1% by weight luminescence reagent dispersion to 60 μL of a buffer containing 16 μL of 15-fold diluted human serum is defined as A1, and the absorbance immediately after the addition is 37° C. The absorbance after being left for 5 minutes is defined as A2. The absorbance is measured with an optical path of 10 mm and a wavelength of 572 nm.
Particles with A2-A1 of 0.1 or less are preferable because non-specific adsorption of impurities in serum is small.

(aqueous medium)
The aqueous medium (aqueous solution) used in the above-described method for producing a luminescent reagent preferably contains water in an amount of 80% by weight or more and 100% by weight or less. The aqueous solvent is preferably water or an organic solvent soluble in water, and examples include a solution of methanol, ethanol, isopropyl alcohol, or acetone mixed in water. If the organic solvent other than water is contained in an amount exceeding 20% by weight, there is a risk that the polymerizable monomer will be dissolved during particle production.

Moreover, it is preferable that the pH of the aqueous medium is adjusted in advance to 6 or more and 9 or less. If the pH is less than 6 or greater than 9, the alkoxide groups and silanol groups of the radically polymerizable organosilane will undergo polycondensation or react with other functional groups before forming a polymer, resulting in agglomeration of the resulting particles. There is a risk. In this embodiment, the alkoxide is not intentionally subjected to condensation polymerization before polymerization.

It is preferable to adjust the pH using a pH buffer, but it may also be adjusted using an acid or a base.

In addition, surfactants, antifoaming agents, salts, thickeners, etc. may be added to the aqueous medium at a rate of 10% or less.

When producing a luminescent reagent, it is preferable to first dissolve PVP in an aqueous medium whose pH is adjusted to 6 to 9. The content of PVP is preferably 0.01% by weight or more and 10% by weight or less, more preferably 0.03% by weight or more and 5% by weight or less based on the aqueous medium. If it is less than 0.01% by weight, the amount of adsorption to the particle matrix will be small and the effect will not be exhibited. Moreover, if the amount is more than 10% by weight, the viscosity of the aqueous medium increases, and there is a possibility that sufficient stirring cannot be performed.

Subsequently, a radically polymerizable monomer containing styrene (A) and radically polymerizable organic silane (B) is added to the aqueous medium to form an emulsion. The weight ratio of styrene (A) and radically polymerizable organic silane (B) is from 6:4 to 100:1. Furthermore, a europium complex is mixed into the prepared emulsion. At this time, if the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The weight ratio of the europium complex to the radically polymerizable monomer is 1:1000 to 1:10.

If the weight ratio of styrene (A) and radically polymerizable organic silane (B) is less than 6:4, the specific gravity of the entire particles may increase, and sedimentation of the particles may become significant. Furthermore, in order to improve the adhesion between PVP and the luminescent particles, it is desirable that the weight ratio of styrene (A) and radically polymerizable organic silane (B) be 100:1 or more.

The weight ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is preferably from 5:5 to 9.5:0.5. If the weight ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is less than 5:5, there is a possibility that the agglomeration of the produced particles will become significant. Moreover, if the weight ratio of the weight of the aqueous medium to the total amount of the radically polymerizable monomer is greater than 9.5:0.5, there will be no problem in the generation of particles, but there is a risk that the amount of generation will be reduced.

The radical polymerization initiator is used after being dissolved in water, a buffer, or the like. The amount of the radical polymerization initiator based on the total weight of styrene (A) and radically polymerizable organic silane (B) can be used in an amount of 0.5% by mass or more and 10% by mass or less in the emulsion.

In the step of heating the emulsion, the entire emulsion may be heated uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the radically polymerizable monomer is polymerized.

The luminescent reagent can have a ligand-binding functional group on its surface. The ligand-binding functional group is not particularly limited as long as it is a functional group capable of binding an antibody, antigen, enzyme, etc., but examples thereof include a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, a silicon alkoxide group, etc. or can contain these functional groups. For example, by mixing a silane coupling agent having a ligand-binding functional group with synthesized particles, it is possible to impart a functional group to the particle surface. Specifically, a carboxy group can be imparted to the particle surface by preparing an aqueous solution of a silane coupling agent having a carboxyl group and mixing it with the synthesized particle dispersion. At this time, a dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature can be set arbitrarily between 0° C. or higher and 80° C. or lower, and the reaction time can be set arbitrarily between 1 hour or more and 24 hours or less. In order to suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the reaction time at a temperature from about 25° C. to room temperature or lower and a reaction time of 3 hours or more and 14 hours or less. Depending on the ligand-binding functional group, an acid or alkali catalyst may be added to promote the reaction on the particle surface.

By binding a luminescent reagent with a ligand such as various antibodies, it can be used as a particle for specimen testing. What is necessary is to select the optimal method for binding the target antibody or the like using the functional groups present in the hydrophilic layer 2.

(Introduction of ligand)
For the chemical reaction for chemically bonding the ligand-binding functional group and the ligand, conventionally known methods can be applied to the extent that the object of the present invention can be achieved. Further, when a ligand is bonded to an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

The luminescent reagent used in this embodiment can be preferably applied to immunolatex agglutination assays that are widely used in fields such as clinical testing and biochemical research.

(Analysis device)
The present invention provides the following analysis method as one embodiment.
An analysis device that calculates the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance,
a mixing unit that mixes the sample containing the target substance and the luminescent reagent to form a mixed solution;
a measuring unit that measures the R of the mixed liquid after a time T has elapsed from the time of the mixing, and a control unit;
An analysis device characterized in that the control section calculates the concentration of the target substance based on T1 when the R reaches R1 in the measurement section;
However, when the R measured for the luminescent reagent not mixed with the target substance is R0,
R1>R0 is satisfied.

The analysis device according to this embodiment is schematically shown in FIG.
The mixing section is a section that performs a mixing process. The measurement section is a section that performs a measurement process. The control section controls the mixing section and the measuring section.
The mixing section and the measuring section can each include a nozzle capable of sucking and discharging a liquid, containers for storing various samples, a well plate, a stage for moving these, and the like. Further, the measurement unit can include an optical system including a light source, a polarizing filter, a spectrophotometer, and the like. The control unit has a computer function. For example, the control unit may be configured integrally with a desktop PC (Personal Computer), laptop PC, tablet PC, smartphone, or the like. The control unit includes a CPU, RAM, ROM, and HDD, and can also include a communication I/F (interface), a display device, and an input device in order to realize functions as a computer that performs calculations and storage.

(reagent)
The analysis method according to this embodiment can be used for sample testing and in vitro diagnosis. The reagent used for these can include the luminescent reagent used in this embodiment and a dispersion medium in which the luminescent reagent is dispersed. The amount of the luminescent reagent contained in 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 addition to the luminescent reagent, the reagent may also contain a third substance such as an additive or a blocking agent, as long as the object of the present invention can be achieved. A combination of two or more types of third substances such as additives and blocking agents may be included. Examples of the dispersion medium used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer. The dispersion medium included is not limited to these. When the reagent is used to detect an antigen or antibody in a specimen, the antibody or antigen can be used as the ligand.

Hereinafter, the present invention will be specifically explained with reference to Examples. However, the present invention is not limited to such embodiments.

(1) Preparation of luminescent particles Solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) in a pH 7 MES (2-morpholinoethanesulfonic acid) buffer (manufactured by Kishida Chemical Co., Ltd.).
[Tris(2-thenoyltrifluoroacetone)(Bis(triphenylphosphineoxide)) europium(III)] which is a europium complex ]) (Central Techno Stock (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter abbreviated as "Eu(TTA) ₃ (TPPO) ₂ "), styrene monomer (manufactured by Kishida Chemical Co., Ltd.), 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Kasei Kogyo Co., Ltd., hereinafter abbreviated as "MPS"). ) were mixed to prepare reaction solution B. Reaction solution B was added into a four-necked flask containing solvent A, and 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 was set to 70° C., and nitrogen flow was further performed for 15 minutes. After heating and stirring the mixture, an aqueous solution containing potassium persulfate (hereinafter referred to as "KPS") (manufactured by Aldrich) was added to the reaction solution, and emulsion polymerization was carried out for 20 hours. After the polymerization reaction, the resulting suspension was ultrafiltered with about 4 L of ion-exchanged water using an ultrafiltration membrane with a molecular weight cut off of 100K, and the product was washed to obtain a dispersion of luminescent particles.

A dispersion of luminescent particles obtained by emulsion polymerization was separated and added to an aqueous solution in which 1% by mass of Tween 20 (manufactured by Kishida Chemical Co., Ltd.) was dissolved. After stirring for 10 minutes, a silane coupling agent, X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred overnight. After stirring, the dispersion was centrifuged, the supernatant was removed, and the precipitate was redispersed with pure water. Centrifugation and redispersion were performed three times or more to wash the product. The washed precipitate was redispersed in pure water. As described above, ligand-binding functional groups were introduced into particles 1 to 8. The mass ratio of the charged particles, pure water, and X12-1135 was 1:300:2.

(Preparation of anti-CRP antibody modified luminescent reagent)
0.25 mL of a 1.2 wt % particle dispersion corresponding to the synthesized luminescent particles was collected, and the solvent was replaced with 1.6 mL of a pH 6.0 MES buffer. 0.5 wt % of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimide were added to the particle MES buffer solution, and the mixture was reacted at 25° C. for 1 hour. After the reaction, the dispersion was washed with a pH 5.0 MES buffer, 100 μg/mL of anti-CRP antibody was added, and the anti-CRP antibody was allowed to bind to the particles at 25° C. for 2 hours. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with a phosphate buffer to obtain an anti-CRP antibody-modified luminescent reagent (also referred to as affinity particles) at a concentration of 0.3 wt%.

The binding of the antibody to the particles was confirmed by measuring the amount of decrease in the antibody concentration in the buffer solution to which the antibody was added using a BCA assay.

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

(Preparation of diluted solution)
A diluted solution was prepared by mixing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and PBS buffer at a volume ratio of 1:1.

Reaction between target substance and ligand (antigen-antibody reaction)
A solution containing CRP antigen mixed with HEPES buffer was prepared and heated to a temperature of 37°C. A luminescent reagent solution was added to the prepared mixture, and after stirring quickly, the polarization anisotropy was observed. The luminescence reagent was fixed at 0.01 mg/mL or 0.0025 mg/mL, and the CRP antigen concentration was examined at 0 to 1000 pM. The observation temperature was 37°C. Polarization anisotropy will be described later.

(Example 1)
After setting the specific value of polarization anisotropy R1 to 0.062, an antigen-antibody reaction was performed using apparatus 1, which will be described later, and the value of polarization anisotropy R was measured. The time when the polarization anisotropy R1 reached 0.062 was measured, a plot of time and CRP antigen concentration was created, and the CRP antigen concentration was evaluated. The concentration of the luminescent reagent was 0.01 mg/mL.

(Example 2)
The CRP antigen concentration was evaluated in the same manner as in Example 1, except that the value of the specific polarization anisotropy R1 was set to 0.043 and Apparatus 2, which will be described later, was used.

(Example 3)
After measuring the antigen-antibody reaction in the same manner as in Example 1, the results obtained by subtracting the value of polarization anisotropy R 5 minutes after the start of the reaction and the R value 1 minute after the start of the reaction, and 40 minutes after the start of the reaction. Evaluation was made by subtracting the value of the polarization anisotropy R after 1 minute from the start of the reaction.

(Example 4)
After setting a specific value of polarization anisotropy R1 to 0.076, an antigen-antibody reaction was performed using apparatus 1, which will be described later, and the value of polarization anisotropy R was measured. The time when the polarization anisotropy R1 reached 0.076 was measured, a plot of time and CRP antigen concentration was created, and the CRP antigen concentration was evaluated. Sodium alginate was added to the diluted solution to thicken it, and the concentration of the luminescent reagent was set to 0.0025 mg/mL.

(Comparative example 1)
Antigen-antibody reactions were measured in the same manner as in Example 3, and only the results obtained by subtracting the value of polarization anisotropy R 5 minutes after the start of the reaction and the R value 1 minute after the start of the reaction were evaluated.

(Comparative example 2)
The antigen-antibody reaction was measured in the same manner as in Example 3, and only the results obtained by subtracting the value of polarization anisotropy R 40 minutes after the start of the reaction and the R value 1 minute after the start of the reaction were evaluated.

(Product evaluation)
The products in Examples and Comparative Examples were evaluated as follows.
The shape of the product was evaluated using an electron microscope (S5500, manufactured by Hitachi High Technologies).

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

The concentration of the suspension in which the product was dispersed was evaluated using a gravimetric analyzer (Thermoplus TG8120 manufactured by Rigaku).

Apparatus 1 and apparatus 2 were used to measure R. The device 1 has the following configuration.
Prepare an LED light source with excitation light of 340 nm, insert a polarizing filter (manufactured by Sigma Koki Co., Ltd., NSPFU-30C) and a short pass filter (manufactured by Edmont Optics, Inc., 84-706) into the optical path, and irradiate a 1 cm square quartz cell. I set up an optical system that would allow me to do so. A polarizing filter (manufactured by Thorlabs, PIVISC050) and a bandpass filter (manufactured by Thorlabs, FB610-10) were set in a direction 90° to the incident light. In order to measure light emission in two directions, _IVV and _IVH , at the same time, two sets were prepared in which the polarizers were assembled in different ways in the 90° direction relative to the incident light. To detect polarized light, spectroscopic measurements were performed using QEPRo manufactured by Ocean Optics. A temperature control was set on the sample holder so that measurements could be taken at 37°C. The polarization anisotropy R was measured with the LED light source fixed at an output of 12 mW and an integration time of 3 seconds. The measurement interval was 15 seconds. From the emission spectrum of the obtained polarized light emission, the emission intensity in the wavelength range of 600 nm or more and 630 nm or less was applied to equation (4) to determine the polarization anisotropy R.

The apparatus 2 used to evaluate the polarization anisotropy R of the product and the CRP antigen-antibody reaction has the following configuration.

Prepare an LED light source with excitation light of 340 nm, insert a polarizing filter (manufactured by Sigma Koki Co., Ltd., NSPFU-30C) and a short pass filter (manufactured by Edmund Optics, Inc., 84-706) into the optical path, and place it in a quartz cell with an optical path length of 5 mm. I set up an optical system that can irradiate it. An excitation light cut filter (manufactured by Edmont Optics, 33-910) and a polarizing beam splitter (manufactured by Edmont Optics, 47-127) are used to pass the polarized light emitted from the sample into an optical path that is in a straight line with the excitation light across the sample. and a polarizer (manufactured by Sigma Koki Co., Ltd., SPF-30C-32) were set in this order to separate the polarized light into two directions. The separated polarized light emission (two directions) was detected using an avalanche photodiode (APD, manufactured by Hamamatsu Photonics Co., Ltd., C15522-3010SA). The temperature control of the sample holder was set so that measurements could be taken at 37°C. The polarization anisotropy R was measured with the LED light source fixed at an output of 60 mW and an integration time of 8 milliseconds. The measurement interval was 30 seconds. The obtained polarized light emission signal was measured with an oscilloscope and applied to equation (1) to determine the polarization anisotropy R.

Evaluation of non-specific aggregation inhibition of the product was performed as follows.
60 μl of a human serum solution diluted 15 times with a buffer solution was added to the luminescence reagent dispersion (3 mg/mL), and the mixture was incubated at 37° C. for 5 minutes. The absorbance at 527 nm was measured before and after keeping warm, and the amount of change in absorbance before and after keeping warm was measured three times. Table 2 shows the average value of three measurements. When the amount of change in the absorbance x 10,000 value was less than 1,000, it was evaluated that non-specific aggregation was suppressed, and when it was 1,000 or more, it was evaluated that non-specific aggregation was occurring.

(Performance evaluation)
The particle size of the synthesized luminescent reagent was approximately 100 nm, and it exhibited strong red luminescence with excitation light of 340 nm.

As a result of the non-specific aggregation suppression evaluation, the change in absorbance was below the specified value (the amount of change in the value of absorbance x 10000 was 1000 or less), confirming that the particles were capable of suppressing non-specific adsorption.

FIG. 5 shows the measurement results of Example 1, in which the R1 time is plotted on the horizontal axis and the measured CRP concentration is plotted on the vertical axis. From the figure, it can be seen that when R1 is set to 0.062, the time for the R value to reach 0.062 becomes longer depending on the CRP concentration. From FIG. 5, it can be confirmed that it is possible to measure the CRP antigen measurement range from 1000 pM to 5 pM in a measurement time of 400 seconds.

The results of Examples 1 to 4 are shown in Table 1. The initial polarization anisotropy R0 measured using only the luminescent reagent in Examples 1 and 3 examined using apparatus 1 was 0.0585, and the polarization anisotropy R0 examined using apparatus 2 was 0. It was .0388. This difference reflects the difference in light receiving sensitivity of each polarization component of the device, and does not affect the essence of the measurement. In Example 4, as a result of a study using the apparatus 1, the initial polarization anisotropy R0 measured using only the luminescent reagent was 0.0740. The reason why R0 of Example 4 is larger than that of Example 1 is because sodium alginate, which has a thickening effect, was added to the reagent. On the other hand, the saturated polarization anisotropy value R2 was determined from a sample left overnight using a large excess of CRP antigen (100,000 pM). As a result, in Example 1, Example 3, Example 4, Comparative Example 1, and Comparative Example 2 examined using Apparatus 1, the initial polarization anisotropy R2 measured only with the luminescent reagent was 0.1150. , the polarization anisotropy R2 examined using Apparatus 2 was 0.0900. In all Examples, it was confirmed that the set value of polarization anisotropy R1 was between R0 and R2.

In Example 1, as a result of measurement with R1 set to 0.062, it was confirmed that the range of CRP antigen detection concentration was 5 pM to 1000 pM.

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

The difference in this example is considered to be due to the difference in the sensitivity of the measuring instrument and the setting conditions for the R1 value.

In Example 3, the antigen-antibody reaction was further divided and evaluated at two measurement times. From the results of Example 3, when the measurement time is as short as 5 minutes, measurement sensitivity in the low concentration region (approximately 5 pM) cannot be obtained (from 50 pM to 1000 pM), and on the contrary, when the measurement time is as long as 40 minutes, The antigen-antibody reaction in the high concentration region (approximately 1000 pM) was saturated and did not exceed the R2 value (5 pM to 200 pM). In other words, it has been found that the measurement range becomes narrow when the quantitative evaluation results are measured at one time as in the comparative example, but by measuring the R value at multiple measurement times and performing quantitative evaluation, a wide measurement range (5 pM to 1000 pM) can be achieved. It turns out that it is possible to measure

In Example 4, the concentration of the luminescent reagent used was lowered and the evaluation was performed with the addition of sodium alginate as a thickener. The R2 value was set at 0.0760. As a result, it was confirmed that the CRP antigen detection concentration range was 0.05 pM to 100 pM in measurements up to 600 seconds. By lowering the concentration of the luminescent reagent, even a small antigen-antibody reaction can cause a large change in polarization anisotropy, and by adding a thickening agent, the effect of promoting the agglutination reaction of particles in the luminescent reagent allows for a wide range of It has been found that in addition to range measurements, it is possible to perform measurements with high sensitivity and in a short time.

From the above, it has become clear that the analysis method according to the present embodiment is a method that can measure the CRP antigen, which is a target substance, with high sensitivity and in a wide measurement range with one reagent composition.

Therefore, when the analysis method according to this embodiment is used, measurement can be performed in a wide measurement range without adjusting many test reagents depending on the concentration of the target substance. Generally, preparing a large number of reagents for the same type of target substance in an analysis device is not only complicated, but also often poses a problem because it affects the installation space. Furthermore, if quantification fails because the concentration exceeds the measurement concentration range, it is necessary to dilute and adjust the sample again, which increases the cost for one measurement. Therefore, the analysis method according to this embodiment, which has the possibility of covering a wide measurement range with a small number of types of reagents, is useful as a sample testing method.

（式（１）中、
Ｉ_ＶＶ・・・第一の偏光で励起したときの、第一の偏光と振動方向が平行な発光成分の発光強度
Ｉ_ＶＨ・・・第一の偏光で励起したときの、第一の偏光と振動方向が直交する発光成分の発光強度
Ｉ_ＨＶ・・・第一の偏光と振動方向が直交する第二の偏光で励起したときの、第二の偏光と振動方向が直交する発光成分の発光強度
Ｉ_ＨＨ・・・第一の偏光と振動方向が直交する第二の偏光で励起したときの、第二の偏光と振動方向が平行な発光成分の発光強度
Ｇ・・・補正値
である。）
（方法８）
前記測定工程は、繰り返し行われることを特徴とする方法１から方法７のいずれかに記載の解析方法。
（方法９）
前記繰り返し行われる前記測定工程において、第ｎ回目の測定工程ではじめて前記Ｒが前記Ｒ１を超えた場合、第ｎ－１回目の測定工程の終了時間から第ｎ＋１の測定工程の開始時間の間のいずれかの数値を前記Ｔ１とすることを特徴とする方法８に記載の解析方法。
（方法１０）
前記繰り返し行われる測定工程は、（ａ＋ｂ）秒周期で繰り返されることを特徴とする方法８または方法９に記載の解析方法、ただし、ａは測定に要する時間、ｂは測定間隔である。
（方法１１）
前記繰り返し行われる前記測定工程において、はじめて前記Ｒが前記Ｒ１に達したときに、該Ｒ１があらかじめ定められた値であるＲ２を超えた場合、前記ａとｂの少なくともいずれか一方を短く設定することを特徴とする方法８から方法１０のいずれかに記載の解析方法。
（構成１）
標的物質と結合する発光試薬を用いて、偏光異方に関する値（Ｒ）を測定することで、前記標的物質の濃度を算出する解析装置であって、
前記標的物質を含む試料と、前記発光試薬を混合して、混合液とする混合部、
前記混合のときから時間Ｔ経過後の混合液の前記Ｒを測定する測定部、および
制御部
を有し、
前記制御部は
前記測定部において、前記ＲがＲ１に達したときをＴ１とし、該Ｔ１に基づいて前記標的物質の濃度を算出することを特徴とする解析装置、
ただし、標的物質と混合していない前記発光試薬について測定される前記ＲをＲ０としたとき、
Ｒ１＞Ｒ０を満たす。 The disclosure of this embodiment includes the following methods and configurations.
(Method 1)
An analysis method for calculating the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance, the method comprising:
a mixing step of mixing the sample containing the target substance and the luminescent reagent to form a mixed solution; and a measuring step of measuring the R of the mixed solution after a time T has elapsed from the time of the mixing.
has
In the measurement step, the time when the R reaches R1 is defined as T1, and the concentration of the target substance is calculated based on T1, and further, the luminescence reagent that is not mixed with the target substance is measured. When R is R0, R1>R0 is satisfied.
analysis method.
(Method 2)
The analysis method according to method 1, characterized in that R1-R0≧0.0001 is satisfied.
(Method 3)
The analysis method according to either method 1 or method 2, characterized in that R0≧0.001.
(Method 4)
The analysis method according to any one of Methods 1 to 3, wherein the luminescent reagent contains luminescent particles.
(Method 5)
The analysis method according to any one of methods 1 to 4, wherein the luminescent particles contain a europium complex.
(Method 6)
The analysis method according to any one of Methods 1 to 5, wherein the luminescent reagent contains a ligand that binds to the target substance.
(Method 7)
The analysis method according to any one of methods 1 to 6, wherein the R is defined by r in the following formula (1).

(In formula (1),
I _VV ... Emission intensity of the luminescent component whose vibration direction is parallel to the first polarized light when excited with the first polarized light I _VH ... The first polarized light when excited with the first polarized light Emission intensity of a luminescence component whose vibration direction is orthogonal I _HV ...Emission intensity of a luminescence component whose vibration direction is orthogonal to the second polarized light when excited with a second polarized light whose vibration direction is orthogonal to the first polarized light _IHH ... Emission intensity of a light emitting component whose vibration direction is parallel to the second polarized light when excited with the second polarized light whose vibration direction is orthogonal to the first polarized light G... Correction value. )
(Method 8)
The analysis method according to any one of methods 1 to 7, wherein the measurement step is performed repeatedly.
(Method 9)
In the repeatedly performed measurement process, if the R exceeds R1 for the first time in the n-th measurement process, the period between the end time of the (n-1)th measurement process and the start time of the n+1-th measurement process. The analysis method according to method 8, characterized in that any numerical value is set as the T1.
(Method 10)
The analysis method described in Method 8 or Method 9, characterized in that the repeatedly performed measurement step is repeated at a period of (a+b) seconds, where a is the time required for measurement and b is the measurement interval.
(Method 11)
In the repeatedly performed measurement step, when the R reaches the R1 for the first time and the R1 exceeds a predetermined value R2, at least one of the a and b is set short. The analysis method according to any one of methods 8 to 10, characterized in that:
(Configuration 1)
An analysis device that calculates the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance,
a mixing unit that mixes the sample containing the target substance and the luminescent reagent to form a mixed solution;
a measuring unit that measures the R of the mixed liquid after a time T has elapsed from the time of the mixing, and a control unit;
An analysis device characterized in that the control section calculates the concentration of the target substance based on T1 when the R reaches R1 in the measurement section;
However, when the R measured for the luminescent reagent not mixed with the target substance is R0,
R1>R0 is satisfied.

1 particle substrate 2 hydrophilic layer 3 europium complex

Claims (12)

By measuring the value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance,
An analysis method for calculating the concentration of the target substance, comprising:
a mixing step of mixing the sample containing the target substance and the luminescent reagent to form a mixed solution; and a measuring step of measuring the R of the mixed solution after a time T has elapsed from the time of mixing.
has
In the measurement step, the time when the R reaches R1 is defined as T1, and the concentration of the target substance is calculated based on T1, and further, the luminescence reagent that is not mixed with the target substance is measured. When the above R is R0,
Characterized by satisfying R1>R0,
analysis method. The analysis method according to claim 1, characterized in that R1-R0≧0.0001 is satisfied. The analysis method according to claim 1, characterized in that R0≧0.001. The analysis method according to claim 1, wherein the luminescent reagent contains luminescent particles. 5. The analysis method according to claim 4, wherein the luminescent particles include a europium complex. The analysis method according to claim 1, wherein the luminescent reagent contains a ligand that binds to the target substance. The analysis method according to claim 1, wherein the R is determined by r in the following formula (1).

(In formula (1),
I _VV ... Emission intensity of the luminescent component whose vibration direction is parallel to the first polarized light when excited with the first polarized light I _VH ... The first polarized light when excited with the first polarized light Emission intensity of a luminescence component whose vibration direction is orthogonal I _HV ...Emission intensity of a luminescence component whose vibration direction is orthogonal to the second polarized light when excited with a second polarized light whose vibration direction is orthogonal to the first polarized light _IHH ... Emission intensity of a light emitting component whose vibration direction is parallel to the second polarized light when excited with the second polarized light whose vibration direction is orthogonal to the first polarized light G... Correction value. ) The analysis method according to claim 1, wherein the measuring step is performed repeatedly. In the repeatedly performed measurement process, if the R exceeds R1 for the first time in the n-th measurement process, the period between the end time of the (n-1)th measurement process and the start time of the n+1-th measurement process. The analysis method according to claim 8, wherein any numerical value is set as the T1. The analysis method according to claim 8, wherein the repeatedly performed measurement step is repeated at a period of (a+b) seconds, where a is the time required for the measurement and b is the measurement interval. In the repeatedly performed measurement step, when the R reaches the R1 for the first time and the R1 exceeds a predetermined value R2, at least one of the a and b is set short. The analysis method according to claim 8, characterized in that: By measuring the value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance,
An analysis device that calculates the concentration of the target substance,
a mixing unit that mixes the sample containing the target substance and the luminescent reagent to form a mixed solution;
a measuring unit that measures the R of the mixed liquid after a time T has elapsed from the time of the mixing, and a control unit;
An analysis device characterized in that the control section calculates the concentration of the target substance based on T1 when the R reaches R1 in the measurement section;
However, when the R measured for the luminescent reagent not mixed with the target substance is R0,
R1>R0 is satisfied.