Automated analyzer and automated analysis method

Abstract

This method provides a highly sensitive analytical method for measuring the concentration of a substance over a wide range, from low to high concentrations. [Solution] The automated analyzer comprises a light source unit 11, a reaction vessel 30, a detection unit 40, and a processing unit. The light source unit emits at least two lights of different wavelengths. The reaction vessel is capable of containing a reaction solution in which the substance to be measured and a reagent are mixed. The detection unit irradiates the reaction vessel with incident light emitted from the light source unit, and receives at least two different wavelengths of first and second emitted light emitted from the reaction vessel. The processing unit calculates the concentration of the substance to be measured based on the signal output from the detection unit. The first emitted light is fluorescence obtained by wavelength conversion of the incident light by the reagent. The detection unit has separation means for separating the first emitted light and the second emitted light and receiving them with a photodetector. The processing unit calculates the concentration of the substance to be measured from at least one of the two output signals output from the detection unit according to the emitted light.

Description

[Technical Field]

[0001] Embodiments disclosed herein and in the drawings relate to an automated analyzer and an automated analyzer method for analyzing components of a test sample using an antigen-antibody reaction. [Background technology]

[0002] In specimen testing methods utilizing antigen-antibody reactions, fluorescence polarization testing, which utilizes the polarization properties of fluorescence, is known. Fluorescence polarization testing involves irradiating a mixture (reaction solution) containing the test item (substance to be measured) with a fluorescent reagent with linearly polarized excitation light. The fluorescence intensity emitted from the reaction solution is then measured by polarization decomposition, and the degree of polarization (polarization anisotropy, or anisotropy) is evaluated. This anisotropy value is highly sensitive to the rotational motion of the object being measured, and this rotational motion depends on the size of the object. On the other hand, in antigen-antibody reactions, when the substance to be measured (antigen) is mixed with a reagent modified with an antibody, the antigen and antibody react specifically and bind, forming aggregates.

[0003] Therefore, by measuring anisotropy, it is possible to detect changes in the size of the substance being measured (aggregation reaction) with high sensitivity. The relationship between the change in the size of the substance being measured and the measured anisotropy depends on the concentration relationship between the substance and the reagent. If a calibration curve is obtained in advance using a known amount of reagent, showing the relationship between the concentration of the substance and the measured anisotropy, it is possible to calculate the concentration of the substance from the anisotropy measurement results. Patent Document 1 discloses an analytical apparatus using this fluorescence polarization method. On the other hand, Patent Document 2 discloses an apparatus that analyzes agglutination reactions by measuring the intensity of transmitted or scattered light emitted from the reaction solution. Furthermore, Patent Document 3 discloses an apparatus that analyzes by simultaneously measuring fluorescence polarization and the intensity of transmitted or scattered light. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 1692254 [Patent Document 2] Patent No. 6013796 [Patent Document 3] Japanese Patent Publication No. 2007-120976 [Overview of the project] [Problems that the invention aims to solve]

[0005] According to the fluorescence polarization method disclosed in Patent Document 1, very low concentrations of a substance can be detected with high sensitivity by optimally adjusting the amount of reagents mixed. However, a problem arises in that the measurable concentration range is limited to the low-concentration region when reagent conditions are adjusted for such high-sensitivity detection. If the concentration of the substance exceeds that concentration range, the anisotropy value saturates to a constant value regardless of the concentration of the substance, and the method loses sensitivity to the concentration of the substance. Conversely, if the amount of reagent is adjusted to measure in the high-concentration region, the measurement sensitivity in the low-concentration region decreases. Thus, the fluorescence polarization method has the problem of not being able to achieve both high sensitivity and a wide measurable concentration range.

[0006] On the other hand, Patent Document 2 discloses an automated analysis method that simultaneously measures transmitted light and scattered light, and selects the most suitable measurement method according to the concentration range of the substance being measured. In other words, the measurement range can be expanded by combining two measurement methods with different sensitivities. However, Patent Document 2 does not disclose the fluorescence polarization method. Furthermore, as pointed out in the text of Patent Document 2, unless it is known which measurement method can measure with high accuracy in which concentration range, combining different measurement methods is of no use.

[0007] Furthermore, Patent Document 3 discloses an immunoassay that simultaneously measures the intensity of fluorescence polarization and transmitted or scattered light to calculate the modification rate of modified proteins. However, in Patent Document 3, since some of the scattered light that is not wavelength-converted is received by the fluorescence polarization detector, the modified proteins are detected at an apparently higher level, making it impossible to calculate the modification rate with high accuracy.

[0008] The problem to be solved by the embodiments disclosed in this specification and the drawings is to provide an automatic analyzer and an automatic analysis method capable of highly sensitively analyzing the concentration of a measurement object over a wide range from a low concentration to a high concentration range. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problems. It is also possible to position the problems corresponding to the respective effects of the respective configurations shown in the embodiments described later as other problems.

Means for Solving the Problem

[0009] The automatic analyzer of the embodiment has a light source unit, a reaction vessel, a detection unit, and a processing unit. The light source unit emits at least two lights with different wavelengths. The reaction vessel can accommodate a reaction solution in which a measurement object and a reagent are mixed. The detection unit irradiates the reaction vessel with incident light radiated from the light source unit, and receives at least two different wavelengths of first emitted light and second emitted light respectively emitted from the reaction vessel. The processing unit calculates the concentration of the measurement object based on the signal output from the detection unit. The first emitted light is fluorescence whose wavelength is converted by the reagent from the incident light. The detection unit has separation means for separating the first emitted light and the second emitted light and receiving them with a photodetector. The processing unit calculates the concentration of the measurement object from at least one of the two output signals output from the detection unit according to the first emitted light and the second emitted light.

Effects of the Invention

[0010] According to the present invention, it is possible to provide an automatic analyzer capable of highly sensitively analyzing the concentration of a measurement object over a wide range from a low concentration to a high concentration range.

Brief Description of the Drawings

[0011] [Figure 1] A diagram showing an example of the measurement range of the fluorescence polarization method. [Figure 2] A schematic diagram under different concentration conditions of the state of the measurement object, phosphor, and its aggregate in the reaction solution. [Figure 3] A schematic diagram of the change in each signal obtained by two measurements of the fluorescence polarization method and the scattering method with respect to the concentration of the measurement object. [Figure 4] A schematic diagram of the photometric unit configuration of the automated analyzer in Example 1. [Figure 5] Detailed diagram of the fluorescence polarization measurement system in Example 1. [Figure 6] The spectrum of light emitted from the reaction vessel in Figure 5. [Figure 7] Film properties of the dichroic mirror in Figure 5. [Figure 8] The light spectrum after dichroic mirror separation in Figure 5. [Figure 9] Other film properties of the dichroic mirror in Figure 5. [Figure 10] The light spectra after separation of dichroic mirrors with different film characteristics, as shown in Figure 5. [Figure 11] A schematic diagram of the reagent, the sample to be measured, and its aggregates in Example 1. [Figure 12] A diagram showing the measurement and analysis flow in Example 1. [Figure 13] A schematic diagram showing the anisotropic signal obtained by fluorescence polarization and the depolarization signal obtained by scattering as a function of reaction time. [Figure 14] Detailed diagram of another embodiment 1 in Example 1. [Figure 15] The light spectrum after dichroic mirror separation in Figure 14. [Figure 16] The light spectra after separation of dichroic mirrors with different film properties, as shown in Figure 14. [Figure 17] Detailed diagrams of another embodiment 2 in Example 1. [Figure 18] Detailed diagram of another embodiment 3 in Example 1. [Figure 19] Detailed diagram of another embodiment 4 in Example 1. [Figure 20] Detailed diagram of another embodiment 5 in Example 1. [Figure 21] Detailed diagram of the fluorescence measurement system in Example 2. [Figure 22] Configuration diagram of the automated analyzer in Example 3.

Mode for Carrying Out the Invention

[0012] Hereinafter, an automatic analyzer and an automatic analysis method according to an embodiment will be described with reference to the drawings.

[0013] (Explanation of the principle and problems of the automatic analyzer) The automatic analyzer according to the present embodiment mixes a reagent with a test sample such as blood or urine collected from a specimen such as a human, and measures the concentration of a desired test item (analyte) contained in the test sample. The reagent contains a phosphor (fluorescent molecule), and the phosphor is modified with an antibody. This antibody specifically reacts with an antigen (analyte) contained in the test sample, and the reagent containing the phosphor aggregates via the antigen. By quantifying this degree of aggregation, the concentration of the analyte can be measured. Regarding this quantification, in the automatic analyzer according to the present embodiment, the polarization dependence of the fluorescence intensity is measured, and a parameter called the degree of polarization (anisotropy) is calculated to evaluate the degree of aggregation. For example, linearly polarized excitation light is irradiated onto the reaction solution, and regarding the fluorescence emitted from the reaction solution, the fluorescence intensity I orth ,

[0016] , , orth , , , para , para , ,

[0015] ,

[0017] , of the fluorescence polarized parallel to the polarization direction of the excitation light and the fluorescence intensity I orth of the fluorescence polarized orthogonally are measured. Using these two measurement results, anisotropy r is calculated according to the following formula (1).

[0014] r = (I para - I orth ) / (I para + 2I<​​​​​​​​​​​​​​​​​​​​A phosphor absorbs, excites, and emits light depending on the relative relationship between the polarization direction of the excitation light and the orientation (molecular axis) of the fluorescent molecule. Meanwhile, the phosphor undergoes rotational and translational motion due to Brownian motion in the reaction solution. If the phosphor's rotation is sufficiently slow to the fluorescence lifetime during the emission process, and the effect of rotation can be ignored, then light is absorbed and emitted along the molecular axis parallel to the excitation light. Therefore, the fluorescence intensity measured for polarization parallel to the excitation light is the highest. Conversely, if the phosphor's rotation is very fast compared to its fluorescence lifetime, the phosphor rotates randomly between light absorption and emission, resulting in unpolarized fluorescence. In an intermediate state, where the fluorescence lifetime and rotation are roughly equal, the phosphor maintains some degree of the excitation light's polarization direction while rotating, thus exhibiting polarization characteristics in the measured fluorescence. Measuring the anisotropy of fluorescence under these conditions allows for the capture of changes in the phosphor's rotational motion due to aggregation reactions, which is a key feature of fluorescence polarization spectroscopy. This rotational motion depends on the volume of the phosphor (e.g., the cube of its size), making it highly sensitive to changes in the size of the phosphor. This principle allows for highly sensitive measurement of the degree of aggregation in reaction solutions, even at very low concentrations.

[0018] However, since there is an upper limit to the value of this anisotropy r, measurements must be performed within a range that does not reach that upper limit. Figure 1 shows the results of measuring the concentration of a certain substance using fluorescence polarization. The horizontal axis represents the concentration of the substance in the reaction solution, and the vertical axis represents the anisotropy r. In Figure 1, in the region where the anisotropy r changes according to the concentration of the substance (the measurement range in the figure), there is a one-to-one correspondence between the measurement result of anisotropy r and the concentration of the substance, so the concentration of the substance can be calculated from the anisotropy r. However, when the anisotropy r reaches a certain upper limit r max Beyond a certain point, the value becomes constant with respect to the concentration of the measured substance (saturation region in the figure), making it impossible to calculate the concentration from the anisotropy value. In other words, the value of anisotropy r exceeds the upper limit r. max If it is less than r, the concentration can be quantified, maxIf the concentration exceeds this level, the anisotropy saturates, making it impossible to measure the concentration. In the example in Figure 1, it can be seen that the measurement range for concentration is approximately two orders of magnitude. The lower limit of the measurement range is determined by the signal-to-noise ratio (S / N) of the fluorescence intensity being measured.

[0019] Here, it is possible to shift the measurement range to the higher concentration side by adjusting the amount of phosphor reagent used in the reaction. However, in that case, the measurement sensitivity at low concentrations will be sacrificed, and the characteristics of fluorescence polarization method will not be utilized.

[0020] The upper limit of the anisotropy r mentioned above is r max The measured fluorescence intensity depends on which contributes more to the emission: the emission from the aggregated phosphor or the emission from the unaggregated phosphor (hereinafter referred to as "free phosphor"). Figure 2 schematically shows the state of the sample 1, phosphor 2, and their aggregate 3 in the reaction solution. Here, the rotation (rotation relaxation time) of the free phosphor 2 is assumed to be sufficiently shorter than the fluorescence lifetime of phosphor 2 (the rotation is fast). In other words, with phosphor 2 alone, the fluorescence becomes almost unpolarized due to the effect of rotation, and the measured anisotropy is very small.

[0021] Figure 2(a) shows a state where the number of phosphor 2 reagents is greater than the number of substance 1 being measured. In this case, the anisotropy is the sum of the contributions of two factors: the emission from the free phosphor 2 and the emission from the aggregated phosphors (aggregates 3). This situation corresponds to a region where the measured anisotropy changes depending on the concentration of substance 1, under the measurement range conditions shown in Figure 1.

[0022] On the other hand, Figure 2(b) shows that when the number of phosphors 2 is small relative to the sample 1, fluorescence is emitted almost entirely from aggregates 3 and measured. The anisotropy measured at this time takes almost the maximum value that can be taken under those conditions. This is the saturation region shown in Figure 1. In this state, even if the concentration of sample 1 becomes even higher, the measured fluorescence is still emitted from aggregates 3, so the anisotropy value does not change.

[0023] On the other hand, scattered light from aggregates is affected by anisotropy, and the polarization dependence of the scattered light changes. Therefore, the aggregation reaction can be evaluated by measuring the polarization dependence of the scattered light (for example, the depolarization component of the scattered light). As described in Patent Document 2, the measurement of scattered light (hereinafter referred to as the "scattering method") can measure at lower concentrations of the sample with higher sensitivity than the measurement of transmitted light (hereinafter referred to as the "transmission method"). In contrast, in this embodiment, the concentration ranges in which two measurement methods—depolarization of scattered light by the scattering method and fluorescence anisotropy (fluorescence polarization method)—can be measured with high sensitivity were experimentally clarified.

[0024] Figure 3 is a schematic plot based on experimental results, with the concentration of the measured substance on the horizontal axis and the signals of the fluorescence polarization method (change in anisotropy) and the scattering method (change in light intensity of the depolarized component) on the vertical axis, respectively. As shown in Figure 3, in the concentration range of region 1, the fluorescence polarization method is sensitive, and the signal is within the measurement range (anisotropy r) shown in Figure 1. <r max This is the region that falls within ). On the other hand, in this low-concentration region, the scattering method does not have sufficient sensitivity, so no significant signal is obtained. In contrast, the concentration range of region 2 (anisotropy r1≦r <r max In this region, fluorescence polarization is shown in Figure 1 as the saturation region (anisotropy r≧r max ) is close but still within the measurement range. Furthermore, the scattering method enters a region where signal changes due to aggregation reactions can be gradually observed. Moreover, in region 3, the fluorescence polarization method reaches the saturation region and the concentration change of the substance cannot be measured. On the other hand, the scattering method enters a region where signal changes can be sufficiently observed, and the concentration of the substance can be measured accurately. From these results, it can be concluded that by measuring low concentrations of substances using the fluorescence polarization method, switching to the scattering method when the fluorescence polarization method reaches the saturation region, and selecting the method with better sensitivity in between, or by using both results in combination, the concentration of the substance can be measured over a wide range, including relatively high concentrations.

[0025] Here, as a scattering method, measurements based on dynamic light scattering (DLS) may be performed. In the reaction solution, the measured substance (scattered particles) and their aggregates move in random directions (translational motion) due to Brownian motion. Smaller particles move relatively fast, and larger particles move relatively slowly. Therefore, if the time variation of the scattered light intensity emitted from the reaction solution is evaluated using the autocorrelation function, the former will be observed as a short correlation time, and the latter as a long correlation time. Generally, this autocorrelation function can be expressed as an exponential function such as exp(-Γτ) (τ: delay time), and using its decay coefficient Γ, the autocorrelation function for short correlation times will have a large Γ, and conversely, the autocorrelation function for long correlation times will have a small Γ. Therefore, by quantifying the aggregation reaction based on this decay coefficient Γ, it is also possible to measure the concentration of the measured substance. Experiments have confirmed that DLS can measure with higher sensitivity than the transmission method, and that the measurable concentration range can be confirmed. As a result, it has been confirmed that it can be combined with fluorescence polarization, similar to the depolarization of scattering. Therefore, as a scattering method, depolarized scattered light or DLS can be used. Alternatively, the intensity of scattered light emitted from the reaction solution at a specific angle may be measured.

[0026] Furthermore, the concentration range of measurement can be expanded by, for example, using two antibodies with different affinities to the antigen, labeling each with a different fluorescent agent, and measuring the luminescence characteristics of the fluorescent agent due to the binding of the antigen-antibody reaction according to the reaction time (e.g., changes in luminescence intensity, changes in anisotropy). In high-sensitivity measurements at low concentrations, the luminescence signal from the antibody with high affinity is measured and analyzed, while at high concentrations, the luminescence signal from the antibody with low affinity is measured and analyzed. In this case, by measuring and analyzing fluorescence signals at at least two different wavelengths, the concentration of the substance (antigen) from low to high concentrations can be measured.

[0027] As described above, in this embodiment, two different wavelengths of light (fluorescence and scattered light, or two different fluorescence) are measured almost simultaneously, and the concentration of the substance is measured over a wide range from low to high concentrations by combining and analyzing the two signals that change according to the reaction time. [Examples]

[0028] Figure 4 is a schematic diagram of the photometric unit of the automated analyzer in Example 1. The detailed optical system will be described later. The photometric unit comprises, for example, a light source unit 10, a reaction vessel 30, a detection unit 40, and a processing unit 70. The light source unit 10 is equipped with a light source that emits light of two wavelengths. One wavelength is one that excites the phosphor contained in the reagent, and the other is one that is not absorbed by the phosphor. The wavelength of the light source can be appropriately selected to match the excitation wavelength of the phosphor in the reagent. For example, it may be light in the visible band to the near-infrared band with a wavelength of 400 to 1100 nm, or it may be light in the ultraviolet band with a wavelength of 400 nm or less, or light in the infrared band with a wavelength of 1100 nm or more. LEDs and lasers can be used as light sources, but it is desirable to use monochromatic light with a relatively narrow spectral width. The light source unit 10 is an example of a "light source unit". That is, the light source unit 10 emits at least two lights of different wavelengths.

[0029] Light emitted simultaneously from two light sources with different wavelengths passes through polarizers (linear polarizers) mounted on the light source unit 10 and enters the reaction solution 31 (Figure 5) contained in the reaction vessel 30 as linearly polarized incident light. The reaction solution 31 is a mixture of a test sample containing the substance to be measured and a reagent containing a phosphor modified with an antibody that specifically reacts with the substance to be measured. In the reaction solution 31, aggregates are formed by an antigen-antibody reaction depending on conditions such as the size and concentration of the substance to be measured and the phosphor, as well as the time elapsed since mixing the substance to be measured and the reagent (reaction time) and the temperature of the reaction solution. The reaction solution 31 is stirred by a stirring unit (not shown) after dispensing the test sample and reagent, so that the substance to be measured and the reagent are uniformly dispersed in the reaction solution. The reaction vessel 30 is an example of a "reaction vessel". That is, the reaction vessel 30 can contain a reaction solution containing a mixture of the substance to be measured and a reagent that specifically reacts with the substance to be measured. Light emitted from the light source unit 10 enters the reaction vessel 30 from the first surface and exits from the second surface opposite the first surface.

[0030] From the reaction vessel 30, fluorescence (first emission light) emitted when the reagent is excited by the incident light, and non-excited light (second emission light) scattered by the reagent are emitted. These lights are incident on the detection unit 40, and the excited light is cut off by an excitation light cut filter mounted on the detection unit 40. Next, both the fluorescence and the scattered non-excited light are separated by a polarizing beam splitter (hereinafter referred to as "PBS") mounted on the detection unit 40 into fluorescence and scattered light with a polarization component parallel to the polarization of the incident light (polarization preservation component) and fluorescence and scattered light with a polarization component orthogonal to the polarization of the incident light (polarization depolarization component), respectively, and are received by detectors mounted on the detection unit 40. The received light signals are sent to the processing unit 70, where the anisotropy r is calculated from the fluorescence intensity and the autocorrelation function is calculated from the scattered light intensity. By referring to a pre-prepared relationship between the calculation results and the concentration of the measured substance (calibration curve), the concentration of the measured substance can be output. The detection unit 40 is an example of a "detection unit". In other words, the detection unit 40 irradiates the reaction vessel 30 with incident light emitted from the light source unit 10, thereby receiving at least two different wavelengths of first and second emitted light emitted from the reaction vessel 30. The first emitted light is fluorescence obtained by wavelength conversion of the incident light by the reagent. The detection unit 40 has separation means for separating the first emitted light and the second emitted light and receiving them with a photodetector.

[0031] The processing unit 70 implements its functions, for example, by having a hardware processor (computer) execute a program stored in memory (not shown). A hardware processor refers to circuits such as a CPU (Central Processing Unit), GPU (Graphics Processing Unit), Application Specific Integrated Circuit (ASIC), programmable logic device (for example, a Simple Programmable Logic Device (SPLD) or Complex Programmable Logic Device (CPLD)), or Field Programmable Gate Array (FPGA).

[0032] The processing unit 70 is an example of a "processing unit". Specifically, the processing unit 70 calculates the concentration of the substance to be measured based on the signal output from the detection unit 40. The processing unit 70 calculates the concentration of the substance to be measured from at least one of the two output signals output from the detection unit 40 in accordance with the first and second emission light. The processing unit 70 evaluates the signal change of the output signal after an arbitrary time has elapsed since the start of the reaction in which the substance to be measured and the reagent are mixed, and calculates a first signal change based on the first emission light and a second signal change based on the second emission light. If the first signal change satisfies certain predetermined conditions, the concentration of the substance to be measured is calculated based on the first signal change; otherwise, the concentration of the substance to be measured is calculated based on the second signal change. The reagent contains scattering particles containing fluorescent molecules, the first signal change is a signal change based on fluorescence anisotropy, and the second signal change is a signal change based on the scattered light intensity or an autocorrelation function calculated from the time variation of the scattered light intensity. Another signal change involves a reagent containing at least two antibodies with different affinities, each labeled with a fluorescent molecule that emits light at a different wavelength. The first and second signal changes are based on the fluorescence intensity of the different wavelengths. Under predetermined conditions, the first and second signal changes are compared, and the concentration of the substance is calculated based on the larger signal change relative to the change in the substance's concentration or over time.

[0033] (Detailed explanation of the fluorescence polarization measurement system) Figure 5 will be used to provide a detailed explanation of the fluorescence polarization measurement system of Example 1. The light emitted from the light source 11 has a broad beam and is made nearly parallel by the collimator lens 12. Here, the light source 11 is an LED that emits excitation light with a wavelength of 340 nm.

[0034] Light passing through the collimator lens 12 passes through the short-pass filter 13, the polarizer 14, and the diagonally positioned dichroic mirror 15 which transmits excitation light and reflects red light, is reflected by the mirror 16, and is focused by the condenser lens 17 to irradiate the reaction solution 31 contained in the reaction vessel 30. At this time, the short-pass filter 13 has the characteristic of passing the excitation wavelength, and can cut out unwanted light other than the desired wavelength to be received by the detection unit 40. The polarizer 14 transmits only polarized light in the z direction as shown in Figure 5, is reflected by the mirror 16, and the reaction solution 31 is irradiated with vertically polarized light (y direction) as incident light. The z direction is the direction in which the condenser lens 17, reaction vessel 30, and collimator lens 41 are aligned in order. In other words, the z direction is the direction in which the light focused by the condenser lens 17 is directed toward the reaction vessel 30. The y direction is perpendicular to the z direction. The x direction is perpendicular to the zy-plane.

[0035] The reaction solution 31 contained in the reaction vessel 30 contains the aforementioned test sample, a reagent containing a phosphor, and aggregates thereof. When incident light is irradiated, wavelength-converted fluorescence and unconverted excitation light are emitted from the reaction solution 31. Here, the central wavelength of fluorescence is 611 nm.

[0036] The diffuse fluorescence and excitation light are roughly parallelized by the collimator lens 41 and reach the excitation light cut filter 42. At this time, the excitation light cut filter 42 has the characteristic of passing through the desired wavelength received by the detection unit 40, and can cut out the excitation light of wavelength 340 nm that was not wavelength converted. Next, the fluorescence is separated into horizontally (x-direction) polarized light and vertically (y-direction) polarized light by the PBS 43. Light incident on the separation surface of the PBS 43 with s-polarization is reflected at the separation surface, passes through the polarizer 44 and bandpass filter 46 which are set up so that the reflected linearly polarized light is transmitted to the maximum extent, is focused by the condenser lens 47, and the detector 48 measures the fluorescence intensity I of the polarization component parallel to the polarization of the incident light. para It receives light. Therefore, the detector 48 only receives linearly polarized light in the y direction.

[0037] Furthermore, light incident on the separation surface of PBS43 with p-polarization passes through the separation surface and then passes through the polarizer 54, which is positioned to transmit the maximum amount of linearly polarized light. Fluorescence at a wavelength of 611 nm passes through the diagonally positioned dichroic mirror 55, is focused by the condenser lens 57, and the detector 58 measures the fluorescence intensity of the polarization component perpendicular to the polarization of the incident light. orth It receives light. Therefore, detector 58 only receives linearly polarized light in the x direction.

[0038] The anisotropy r can be calculated from the light intensity obtained from the two detectors 48 and 58 using equation (1) or (2) above. Here, avalanche photodiodes (hereinafter referred to as "APDs") are used for detectors 48 and 58, but high-sensitivity light sensors such as photomultiplier tubes (hereinafter referred to as "PMTs") or MPPCs (Multi-Pixel Photon Counters) may also be used.

[0039] (Detailed explanation of the scattered light measurement system) The light emitted from light source 21, although smaller than that from light source 11, has a slight spread and is made nearly parallel by the collimator lens 22. Here, light source 21 is a semiconductor laser (hereinafter referred to as "LD") that emits red light with a wavelength of 635 nm.

[0040] Light passing through the collimator lens 22 is transmitted through the polarizer 23, reflected by the dichroic mirror 15, reflected by the mirror 16 (similar to the fluorescence polarization measurement system described above), and focused by the condenser lens 17 to irradiate the reaction solution 31 contained in the reaction vessel 30. At this time, the polarizer 23 transmits only light polarized in the z direction, and the reaction solution 31 is irradiated with light polarized in the y direction as incident light.

[0041] The incident light is scattered by the reagents and aggregates in the reaction solution 31 and emitted from the reaction vessel 30 as scattered light.

[0042] The diffused light is roughly parallelized by the collimator lens 41 and transmitted through the excitation light cut filter 42. Next, the scattered light that has passed through the excitation light cut filter 42 is separated into horizontally polarized light and vertically polarized light by the PBS 43, similar to fluorescence. Scattered light incident on the separation surface of the PBS 43 with s polarization is reflected by the separation surface, but this light is unwanted in scattered light measurements and is cut off by the bandpass filter 46. This is because the detector 48 does not receive light other than the desired fluorescence mentioned above.

[0043] On the other hand, scattered light incident on the separation surface of PBS43 with p-polarization passes through the separation surface and then through the polarizer 54. Next, the scattered light that has passed through the polarizer 54 is reflected by the dichroic mirror 55 and focused by the condenser lens 67, and the detector 68 measures the scattered light intensity I of the polarization component perpendicular to the polarization of the incident light. orth The detector 68 receives light. Therefore, only linearly polarized light in the x-direction is received by the detector 68. Here, an APD is used for the detector 68, but other high-sensitivity light sensors may also be used.

[0044] The dichroic mirror 55 is an example of a "separation means for the detection unit." That is, the dichroic mirror 55 reflects the longer wavelength light and transmits the shorter wavelength light of two lights of different wavelengths. The PBS 43 is an example of a "polarization separation element." That is, the PBS 43 separates the first emitted light, which is emitted from the reaction solution by linear polarization, into a direction parallel to and perpendicular to the polarization direction of the linear polarization, and receives each of them with a photodetector.

[0045] (Effective placement of detectors for scattered light measurement) The arrangement of the detector 68 in Figure 5 will be explained in detail, along with the spectrum from the optical system described above.

[0046] Figure 6 shows the spectrum of light emitted from the reaction vessel 30. The solid line in Figure 6 represents the fluorescence spectrum with a central wavelength of 611 nm, which has been wavelength-converted by the excitation light from the light source 11, and the dotted line represents the scattered light with a central wavelength of 635 nm, which is emitted from the light source 21 and scattered by the reaction solution 31. The light emitted from the reaction solution 31 is the combined spectrum of these two types of light.

[0047] Figure 7 shows the reflective film characteristics of the dichroic mirror 55. The solid line in Figure 7 is the reflectance of s-polarized light, the dotted line is the reflectance of p-polarized light, and the dashed line is the average reflectance. The wavelength at which the average reflectance is 50% is called the cut-off wavelength of this dichroic mirror.

[0048] Figure 8 shows the spectrum of the light after separating the dichroic mirror 55 in Figure 5. The solid line is the light transmitted through the dichroic mirror 55, and the dotted line is the light reflected by the dichroic mirror 55. They are respectively condensed by the condenser lenses 57 and 67 and received by the detectors 58 and 68. The desired light detected by the detector 58 is fluorescence with a central wavelength of 611 nm, and the scattered light with a central wavelength of 635 nm that can become noise light is not received, so a high-precision signal can be obtained. On the contrary, the desired light detected by the detector 68 is scattered light with a central wavelength of 635 nm, and the fluorescence with a central wavelength of 611 nm that can become noise light is not received, so a high-precision signal can also be obtained here. If weak noise light becomes a problem, although not shown in Figure 5, band-pass filters may be installed in front of the condenser lenses respectively.

[0049] Thus, the light emitted from the reaction vessel 30 becomes two lights with different wavelengths. When the central wavelength on the long wavelength side (635 nm) is A, the central wavelength on the short wavelength side (611 nm) is B, and the above-mentioned cut-off wavelength is C, an optical arrangement in which the dichroic mirror (Figure 7) having the characteristic of A - C < C - B is installed to be incident with s-polarized light, and a detector for obtaining the desired light on the long wavelength side (635 nm) is installed at the reflection position is the optimal arrangement (Figure 5).

[0050] On the other hand, when a detector for scattered light is installed at the transmission position of the dichroic mirror 55 and a detector for fluorescence polarization is installed at the reflection position, as shown in Figure 8, mainly only fluorescence is received by the detector for scattered light, and on the contrary, mainly only scattered light is received by the detector for fluorescence polarization. Therefore, a detector for receiving long wavelength light needs to be installed at the reflection position of the dichroic mirror 55.

[0051] Next, consider the case where a dichroic mirror with the film characteristics shown in Figure 9 is used in the optical system of Figure 5. The condition is AC > CB. In this case, the spectrum of the light after dichroic mirror separation is shown in Figure 10. Since fluorescence is mixed in the light received by detector 68, it is necessary to install a bandpass filter (not shown) in front of detector 68 to cut out the fluorescence.

[0052] (Explanation of the principle of fluorescence polarization measurement) Here, the reagents used in this embodiment will be described. Normally, fluorescent molecules are small in size, and it is difficult to accurately measure scattered light with reagents composed solely of fluorescent molecules. In this regard, as shown below, the reagents used in this embodiment have a configuration that allows for the measurement of both fluorescence and scattered light. Figure 11 is a schematic diagram of the reagents used in this embodiment. Figure 11(a) shows a reagent in which fluorescent molecules 2 are accumulated inside the scattering particles 5. By modifying the scattering particles 5 with an antibody 4 that specifically reacts with the substance to be measured 1, both the fluorescent molecules and the scattering particles react specifically with the substance to be measured 1, generating aggregates 6.

[0053] When the aforementioned excitation light is irradiated here, the excitation light is absorbed by the fluorescent molecule 2, and fluorescence is generated. The anisotropy of the fluorescence changes because the rotational relaxation time of the fluorescent molecule changes before and after the formation of the aggregate 6. On the other hand, when non-excitation light is irradiated, the non-excitation light does not interact with the fluorescent molecule 2 and is scattered by the scattering particles 5. The polarization characteristics of the incident light of the scattered light change depending on the scattering concentration of the reagent and the size and shape of the aggregate. That is, the light intensity of the depolarization component of the scattered light changes. Thus, in this embodiment, the reagent is equipped with the function to measure two signals, fluorescence polarization and scattering, and two measurements are made possible by irradiating with light of different wavelengths, excitation light and non-excitation light, respectively. For example, by simultaneously illuminating with light of different wavelengths mounted on the light source unit 10 and mixing the material to be measured with the reagent, two signals, fluorescence anisotropy and the depolarization component of the scattered light, can be continuously measured.

[0054] Alternatively, the reagent configuration may be as shown in Figure 11(b). Both the fluorescent molecule 2 and the scattering particle 5 are modified with an antibody 4 that specifically reacts with the substance to be measured 1. The antigen-antibody reaction generates aggregates 6 that sandwich the substance to be measured 1 between the scattering particle 5 and the fluorescent molecule 2. The formation of aggregates slows down the rotational relaxation time of the fluorescent molecule, and as a result, the anisotropy of the measured fluorescence changes. Similarly, the polarization characteristics of the scattering also change due to the formation of aggregates. Therefore, as in the case of Figure 11(a), the anisotropy of fluorescence and the depolarization component of the scattered light can be measured simultaneously using this reagent, and the aggregation reaction can be evaluated.

[0055] Here, the reagent phosphor 2 can be appropriately selected and designed considering its characteristics such as absorption wavelength, emission wavelength, fluorescence efficiency, and fluorescence lifetime, as well as its combination with the antibody 4 that specifically reacts with the analyte 1. In particular, it is desirable to use a phosphor 2 that has a fluorescence lifetime of approximately the same order as the rotational relaxation time estimated from the size of the analyte 1 and the size of the aggregate 6. The particles containing the phosphor are luminescent particles that accumulate europium complexes, and for example, latex particles with a diameter of 10 to 500 nm may be used. The size and concentration of scattering particles also affect the anisotropy of fluorescence. Therefore, it is desirable to design the reagent considering the signal-to-noise ratio balance of both the fluorescence polarization method and the scattering method.

[0056] Next, Figure 12 shows the measurement and analysis flow in this embodiment. First, in step S1, the substance to be measured and reagents are dispensed into the reaction vessel 30 to produce a reaction solution 31 and start the agglutination reaction. Next, in step S2, the reaction solution 31 is irradiated with excitation light and non-excitation light, and the anisotropy r of the fluorescence and the light intensity of the depolarization component of the scattered light are measured simultaneously. This measurement in S2 is performed continuously at a certain time interval with respect to the reaction time from the start of the agglutination reaction. In step S3, it is determined whether a predetermined reaction time has elapsed. The predetermined reaction time may be set to the time when the agglutination reaction has sufficiently converged. Alternatively, it may be set to any time (for example, 5 minutes) within the range in which the change in the reaction can be measured. Even if the reaction has not yet sufficiently converged, the concentration of the substance to be measured can be determined from the reaction speed by analyzing the change in signal within that time. If it is determined in step S3 that the predetermined reaction time has not elapsed, the measurement in S2 is repeated. The number of times measurements are performed within the reaction time can be arbitrarily set according to the post-measurement analysis process.

[0057] If it is determined in step S3 that a predetermined reaction time has elapsed, the measurement is terminated and the analysis flow proceeds to step S4 and beyond. First, in step S4, the anisotropy r of the reaction solution 31 is evaluated based on the measurement results of the fluorescence polarization method. For example, the anisotropy r may be evaluated from the result of the last measurement within the reaction time. This anisotropy r is compared with a first predetermined value r1. Here, the predetermined value r1 corresponds to, for example, r1 shown in Figure 3. If the measured anisotropy r is less than r1 (region 1 in Figure 3, step S4; NO), in step S5, the concentration of the substance is calculated from the fluorescence polarization calibration curve based on the anisotropy r result. For example, the solid line in Figure 3 is the fluorescence polarization calibration curve (first calibration curve). Using a standard sample containing a substance whose concentration is known in advance, the correspondence between the concentration of the substance and the anisotropy value can be measured under the same measurement conditions as in step S1, and this can be used as the first calibration curve.

[0058] On the other hand, if the anisotropy r is r1 or greater (step S4; YES), then proceed to step S6, and the second predetermined value r max Compare this predetermined value r. max For example, as shown in Figure 3, rmax This corresponds to the measured anisotropy r being r max If the value is less than (region 2 in Figure 3, step S6; NO), the concentration of the substance is calculated in step S7. In step S7, the concentration may be calculated from either of the calibration curves, using the measurement results of both the fluorescence polarization method and the scattering method. Alternatively, depending on the measured signal, a measurement method that is more sensitive may be selected based on the results of a previously measured calibration curve, and the concentration of the substance may be calculated from the measurement results of the selected measurement method. Here, for example, the dashed line in Figure 3 is the calibration curve for the scattering method (second calibration curve). The second calibration curve can also be obtained in advance using a standard sample. In region 2 of Figure 3, the first calibration curve and the second calibration curve can be compared, and the one with a sufficiently large signal-to-noise ratio and a large change in signal in response to changes in the concentration of the substance can be selected.

[0059] Here, Figure 13 shows a schematic representation of the anisotropy signal obtained by fluorescence polarization and the signal of the depolarization component light intensity obtained by scattering, as a function of reaction time. The condition for proceeding to step S7 is that the anisotropy r measured by fluorescence polarization is r1 ≤ r <r max This is the case. Since the measured anisotropy value is close to the saturation region shown in Figure 1, even if the concentrations of the substances being measured are different (concentration ρ2 > concentration ρ1), the fluorescence polarization method yields almost the same results for reaction time, as shown in Figures 13(a) and 13(b). On the other hand, the scattering method results corresponding to Figures 13(a) and 13(b) of the fluorescence polarization method are shown in Figures 13(c) and 13(d), respectively. When the concentration of the substance being measured is low, the scattering method does not yield a sufficient change in signal, as shown in Figure 13(c). In this case, the concentration can be calculated from the measurement results of the fluorescence polarization method shown in Figure 13(a). On the other hand, when the concentration of the substance being measured is higher than in the case of Figure 13(c), a sufficient change in signal can be obtained with the scattering method, for example, as shown in Figure 13(d). In this case, the concentration of the substance being measured can be calculated based on the measurement results of the scattering method. In other words, from the two measurement results of the fluorescence polarization method and the scattering method, it is possible to select a measurement method that yields a large change in signal, that is, a measurement method that is more sensitive to the concentration of the substance being measured.

[0060] Also, the predetermined values ​​r1 and r maxThis may be determined based on the first calibration curve (Figure 1 or Figure 3). Furthermore, r max This can be determined based on the anisotropy saturation value. Alternatively, the anisotropy value corresponding to the concentration where the sensitivity of the scattering method is higher than that of the fluorescence polarization method can be determined. max This is also acceptable. In this case, r max Let =r1, and steps S6 and S7 may be omitted. In step S4, r≧r1(=r max If the measurement result is r≧r in step S6, proceed to step S8. max If so (Step S6; YES), in Step S8, the concentration of the substance is calculated using the measurement results of the scattering method.

[0061] Basically, fluorescence polarization is more sensitive than scattering in low-concentration regions. Therefore, to make the most of the low-concentration sensitivity of fluorescence polarization, the amount of reagent dispensed may be adjusted to match the detection limit at which the lowest concentration of the substance can be measured under the conditions of the aforementioned apparatus configuration. Furthermore, it is desirable to adjust the amount of reagent dispensed so that fluorescence polarization covers the low-concentration region and scattering covers the high-concentration region, and that their respective measurement ranges overlap. Then, as a switching point for analysis using the two measurement methods, if the anisotropy is within the measurement range (region 1 in Figure 3) (step S5), the concentration of the substance is preferentially calculated using fluorescence polarization. Conversely, if the anisotropy is in the saturation region (region 3 in Figure 3) (step S8), the scattering method is used. If it is region 2 in Figure 3 (step S7), the more sensitive of either fluorescence polarization or scattering is used within the range of region 2. In this example, two measurement results, one from fluorescence polarization and one from scattering, are obtained and can be appropriately selected during analysis from step S4 onward.

[0062] Furthermore, in the automated analyzer of this embodiment, it is also possible to add a separate light-receiving system to measure light with a polarization component (polarization preservation component) parallel to the polarization of the incident light (=unexcited light). This is transmitted light that travels in a straight line and is transmitted without interacting with the reaction solution 31, and it is also possible to measure the agglutination reaction as a change in transmitted light intensity. For example, depending on the concentration of the substance to be measured, all three measurement results of fluorescence polarization (fluorescence), scattering (scattered light), and transmission (transmitted light) may be used. As pointed out in Patent Document 2, the transmission method has lower sensitivity at lower concentrations than the scattering method, but can measure up to high concentrations. Therefore, it is also possible to obtain measurement results of the three measurement methods up to step S3 and analyze them according to the switching point based on the calibration curve measured in advance, such as using fluorescence polarization for low concentrations, scattering for medium concentrations, and transmission for high concentrations. By combining the three measurement methods, it is possible to further expand the measurement range with high precision. As an example, it is possible to measure the concentration of the substance with a dynamic range of 6 orders of magnitude or more.

[0063] Here, the calibration curve signal may not be the fluorescence anisotropy r or the light intensity value of the depolarization component of scattered light itself, but rather the change from the start of the reaction. For example, fluorescence can be evaluated by setting the anisotropy immediately after the start of the reaction to r0, and evaluating the change in anisotropy from the start of the reaction Δr (=r-r0), or r / r0. On the other hand, scattered light can be evaluated using ΔI (=I-I0), or I / I0. Here, I0 and I are the light intensity of the depolarization component of scattered light immediately after the start and thereafter, respectively.

[0064] The following describes a modified version of the detection unit 40 in Example 1.

[0065] (Another form of Example 1) Figure 14 shows the configuration of the detection unit 40 located after the reaction vessel 30 in another form 1 of Example 1. The configuration up to the polarizer 54 is the same as in Figure 5. Linearly polarized light in the horizontal direction (x direction) that has passed through the polarizer 54 is incident on the dichroic mirror 55 as p-polarized light and is color-separated. The characteristics of the dichroic mirror 55 at this time are shown in Figure 7. The transmitted fluorescence passes through the bandpass filter 56, is focused by the condenser lens 57, and is received by the detector 58. In contrast, the scattered light reflected by the dichroic mirror 55 is focused by the condenser lens 67 and is received by the detector 68.

[0066] Figure 15 shows the light spectrum after separation by the dichroic mirror 55 in Figure 14. At detector 58, the desired light is fluorescence with a central wavelength of 611 nm, and scattered light with a central wavelength of 635 nm, which can be noise, is cut off by the bandpass filter 56. On the other hand, at detector 68, the desired light is scattered light with a central wavelength of 635 nm, and fluorescence with a central wavelength of 611 nm, which can be noise, is sufficiently reduced and not received, so a high-precision signal can be obtained without installing a bandpass filter.

[0067] Furthermore, Figure 16 shows the light spectrum after separation by the dichroic mirror 55 when the characteristics of the dichroic mirror 55 are changed as shown in Figure 9. In this case, fluorescence and scattered light are separated and incident on each detector, and the amount of light that can become noise is sufficiently small, so the bandpass filter 56 shown in Figure 14 does not need to be installed.

[0068] (Another form of Example 1, Part 2) Figure 17 shows the configuration of the detection unit 40 located after the reaction vessel 30 in another embodiment 2 of Example 1. The polarizer 14 in Figure 5 is installed rotated 90° with respect to the y-axis, and transmits only polarized light in the x-direction, which is reflected by the mirror 16, so that the reaction solution 31 is irradiated with light polarized in the x-direction.

[0069] Light incident on the detection unit 40 is separated into horizontally polarized and vertically polarized light by the PBS 43. Light incident on the separation surface of the PBS 43 in s-polarized form is reflected by the separation surface and passes through the polarizer 44, which is positioned so that the transmitted linearly polarized light is transmitted to the maximum extent. Fluorescence with a wavelength of 611 nm is incident on the diagonally positioned dichroic mirror 55 in s-polarized form, is transmitted, focused by the condenser lens 47, and received by the detector 48. At this time, the characteristics of the dichroic mirror 55 are as shown in Figure 7. In contrast, the scattered light reflected by the dichroic mirror 55 is focused by the condenser lens 67 and received by the detector 68.

[0070] The spectrum after separation using the dichroic mirror 55 is generally similar to that shown in Figure 8, and the noise at each detector is weak. If the characteristics of the dichroic mirror 55 are as shown in Figure 9, the spectrum will be generally similar to that shown in Figure 10, and it will be necessary to install a bandpass filter or longpass filter that cuts out fluorescence at a wavelength of 611 nm at the reflection position of the dichroic mirror 55.

[0071] (Another form of Example 1 3) Figure 18 shows the configuration of the detection unit 40 located after the reaction vessel 30 in another embodiment 3 of Example 1. The optical path reflecting the PBS 43 is the same as in Figure 17 up to the polarizer 44.

[0072] Fluorescence with a wavelength of 611 nm is incident on the diagonally positioned dichroic mirror 55 in p-polarized light, is transmitted through it, passes through the bandpass filter 46, is focused by the condenser lens 47, and is received by the detector 48. At this time, the characteristics of the dichroic mirror 55 are as shown in Figure 7. In contrast, the scattered light reflected by the dichroic mirror 55 is focused by the condenser lens 67 and received by the detector 68.

[0073] Since the spectrum after the dichroic mirror 55 separates is generally the same as that shown in FIG. 15, it is necessary to cut the scattered light of 635 nm with the band-pass filter 46. When the dichroic mirror 55 has the characteristic of A - C > C - B at the aforementioned wavelength as shown in FIG. 9, the p-polarized light incident on the dichroic mirror 55 is separated, and the spectrum after separation is generally the same as that shown in FIG. 8, and the noise at each detector is weak.

[0074] (Another form 4 of Example 1) FIG. 19 shows the configuration of the detection unit 40 located after the reaction vessel 30 of another form 4 of Example 1. Up to the excitation light cut filter 42, it is the same as FIG. 5.

[0075] The scattered light with a wavelength of 635 nm is reflected by the obliquely arranged dichroic mirror 55, transmitted through the polarizer 64, condensed by the condenser lens 67, and received by the detector 68. Here, since the light of the desired polarization at the detector 68 is the light polarized in the x direction, it is installed in such an arrangement that the light polarized in the z direction does not pass through the polarizer 64. At this time, when using the dichroic mirror with the characteristics shown in FIG. 7, the spectrum after the dichroic mirror 55 separates is generally the same as that shown in FIG. 8. Next, the fluorescence transmitted through the dichroic mirror 55 is divided into horizontally polarized light and vertically polarized light by the PBS 43, and is received by the detectors for performing fluorescence polarization measurement respectively.

[0076] As described above, it is necessary to install a detector for receiving light on the long wavelength side at the reflection position of the dichroic mirror 55. Also, the light emitted from the reaction vessel 30 becomes two lights with different wavelengths. Let the central wavelength on the long wavelength side be A, the central wavelength on the short wavelength side be B, and the aforementioned cut wavelength be C. When incident on the dichroic mirror with s-polarization, it is desirable to use a dichroic mirror with the characteristic of A - C < C - B, and when incident with p-polarization, it is desirable to use a dichroic mirror with the characteristic of A - C > C - B.

[0077] (Another form 5 of Example Ⅰ) Figure 20 shows the configuration of the detection unit 40 located after the reaction vessel 30 in another embodiment 5 of Example 1. The optical path passing through the PBS 43 is the same as in Figure 5 up to the polarizer 54.

[0078] The linearly polarized light in the horizontal direction (x direction) that passes through the polarizer 54 is focused by the condenser lens 57 and received by the detector 58. In contrast, the light reflected by the PBS 43 is linearly polarized light in the vertical direction (y direction) that passes through the polarizer 44, and is focused by the condenser lens 47 and received by the detector 48.

[0079] Here, fluorescence and scattered light are separated by PBS43 solely by the difference in polarization and received by the detector, so the light intensity I obtained by simultaneously illuminating light sources of different wavelengths para , I orth This is the fluorescence light intensity I Fpara , I Forth and the light intensity of scattered light I Spara , I Sorth The two are added together, and it is not possible to measure them separately. Therefore, the light sources 11 and 21 are controlled to be turned on alternately or sequentially, and the fluorescence and scattered light are separated and measured by their respective detectors accordingly.

[0080] For example, when the light source 11 is first turned on, fluorescence is emitted from the reaction vessel 30, and the light intensity I obtained by detectors 48 and 58 is measured. Fpara , I Forth Anisotropy can be determined from this. Next, when the light source 21 is turned on, scattered light is emitted from the reaction vessel 30, and the scattered component is the light intensity I obtained by the detector 58. Sorth As a result, since the detector 58 receives fluorescence and scattered light alternately or sequentially, it is possible to acquire two signals, one by fluorescence polarization and the other by scattering, almost simultaneously. The PBS used here corresponds to fluorescence and scattered light of different wavelengths and can efficiently separate s-polarization and p-polarization.

[0081] At this time, depending on the conditions of the reagents contained in the reaction solution 31 and the concentration of the substance being measured, the difference in intensity between the fluorescence and scattered light received by the detector may become large, causing the light intensity of one of the detectors to saturate. Therefore, saturation can be prevented by reducing the output of the light source causing saturation compared to the other light sources, or by shortening the operating time of the light source causing saturation. Generally, as the concentration increases, the output of scattered light tends to increase, so the output of light source 21 is often reduced.

[0082] The above describes sequentially turning on light sources 11 and 21, and receiving the fluorescence and scattered light alternately or sequentially with the detector. At this time, the detector alternately or sequentially outputs signals generated from the fluorescence light intensity and the scattered light intensity.

[0083] Alternatively, array sensors such as CCDs or CMOS sensors can be used as detectors. Array sensors are equipped with RGB color filters, wavelength cut filters, or attenuation filters for each light-receiving area, making it possible to simultaneously acquire light of different wavelengths emitted from the reaction solution. In this case, it is also possible to place attenuation filters for each wavelength, eliminating the need for the control described above.

[0084] In this embodiment, LEDs and LDs are used as light sources, but a high-power SLD (Super Luminescent Diode) or a solid-state laser emitting YAG harmonics with a spectral width suitable for DLS, which have an excitation wavelength suitable for the emission wavelength of the phosphor, or a gas laser such as He-Ne may also be used.

[0085] When the desired light received by the photodetector is such that the cut-off wavelength C is the wavelength at which the reflectance of the dichroic mirror is 50%, the wavelength of the longer-wavelength light among the two different-wavelength lights is A, and the wavelength of the shorter-wavelength light is B, if A - C < C - B, the light may be incident on the dichroic mirror with s-polarization and color-separated, and if A - C > C - B, the light may be incident on the dichroic mirror with p-polarization and color-separated. Of the light intensity of the photodetector receiving the first emitted light and the light intensity of the photodetector receiving the second emitted light, the output of the first light source forming the emitted light with the smaller light intensity may be greater than the output of the second light source, or the gain or exposure time of the photodetector receiving the emitted light with the smaller light intensity may be greater than that of the other photodetector.

Example

[0086] (fluorescence x fluorescence) FIG. 21 shows the configuration of the automatic analyzer in Example 2. The configuration of the optical system is generally the same as that in Example 1, but two lights with different wavelengths emitted from the light source unit 110 are irradiated onto the reaction solution 131.

[0087] The reaction solution 131 is composed of two antibodies with different affinities for the antigen and different fluorescent labels corresponding to each of them, and each phosphor emits fluorescence with different wavelengths in response to the above two excitation lights. In this example, the difference in the binding of the antigen-antibody reaction according to the reaction time is utilized to measure the change in the emission intensity of the two phosphors. For example, a method of measuring the concentration of the analyte by measuring the change in the intensity of the fluorescence (light enhancement or quenching) generated by the occurrence of energy transfer due to the proximity of the distance between the reagents by the aggregation reaction may be applied. Here, it is assumed that the reagents are appropriately mixed under appropriate conditions according to the measurement method.

[0088] Hereinafter, a detailed description of the measurement system for measuring the concentration of the analyte from the change in the emission intensity of two different phosphors will be given.

[0089] (Detailed description of the measurement of the first fluorescence (short wavelength side)) Light emitted from the light source 111 is made into nearly parallel light by the collimator lens 112. Here, as an example, the light source 111 is an LED with a wider spectral width than a laser. The light that has passed through the collimator lens 112 and the short-pass filter 113 passes through the dichroic mirror 115, is reflected by the mirror 116, and is focused by the condenser lens 117 to irradiate the reaction liquid 131 contained in the reaction vessel 30. At this time, the short-pass filter 113 has the characteristic of passing through the excitation wavelength, and can cut out unwanted light other than the desired wavelength light that is received by the detection unit 140 emitted by the LED.

[0090] A first fluorescence is emitted from the reaction vessel 30, corresponding to the excitation wavelength. This is the shorter wavelength fluorescence of the two fluorescence spectroscopy

[0091] (Detailed explanation of the second fluorescence (long wavelength) measurement system) Light emitted from the light source 121 is made into nearly parallel light by the collimator lens 122. For example, the light source 121 is an LD with a narrow spectral width. The light that has passed through the collimator lens 122 is reflected by the dichroic mirror 115, and then reflected by the mirror 116, as described above, and focused by the condenser lens 117 to irradiate the reaction liquid 131 contained in the reaction vessel 30.

[0092] A second fluorescence is emitted from the reaction vessel 30, corresponding to the excitation wavelength. This is the longer wavelength fluorescence of the two fluorescence spectroscopy

[0093] (Effective arrangement for fluorescence measurement) Regarding the arrangement after the dichroic mirror 155 in FIG. 21, it will be described in detail. The light emitted from the reaction vessel 130 becomes two fluorescences of different wavelengths. Let the central wavelength of the second fluorescence be A, the central wavelength of the first fluorescence be B, and when the average reflectance of s-polarized light and p-polarized light is 50% due to the reflection film characteristics of the dichroic mirror, the wavelength is the cut wavelength C. Consider an optical arrangement where the light is incident on a dichroic mirror having the characteristic of A - C < C - B, and a detector for obtaining the second fluorescence is installed at the reflection position (FIG. 21).

[0094] The light of the desired wavelength detected by the detector 158 is the first fluorescence, and since the second fluorescence that can become noise light is mixed, it is necessary to cut the unnecessary second fluorescence with the band-pass filter 156. On the other hand, the light of the desired wavelength detected by the detector 168 is the second fluorescence, and since the first fluorescence that can become noise light is weak, it is not necessary to install a band-pass filter here and a high-precision signal can be obtained. Even if weak noise light becomes a problem, although not shown in FIG. 21, a band-pass filter for cutting the weak first fluorescence may be installed in front of the condenser lens 167.

[0095] On the other hand, when a detector for obtaining the second fluorescence is installed at the transmission position of the dichroic mirror 155 and a detector for obtaining the first fluorescence is installed at the reflection position, mainly only the second fluorescence is received by the detector installed at the reflection position, and the desired first fluorescence is not received. Therefore, it is necessary to install a detector for receiving the second fluorescence at the reflection position of the dichroic mirror 155.

[0096] Next, consider the case when a dichroic mirror having a film characteristic under the condition of A - C > C - B is used in the optical system of FIG. 21. In this case, since the first fluorescence is mixed in the light received by the detector 168, it is necessary to install a band-pass filter (not shown) for cutting the first fluorescence in front of the detector 168.

[0097] <00004As described above, a detector that receives long-wavelength light is installed at the reflection position of the dichroic mirror 155, and when short-wavelength light, which is noise light, is mixed in with the received light, a bandpass filter that cuts out the short-wavelength light is installed in front of the detector.

[0098] (Another form of Example 2) The above describes a method for measuring the concentration of a substance from the change in emission intensity of two different phosphors. However, a method of measuring the concentration of a substance from the polarization anisotropy of two different phosphors is also acceptable. Alternatively, a method of measuring the concentration of a substance from the polarization anisotropy of one phosphor and from the change in emission intensity of the other phosphor is also acceptable.

[0099] The former method of measuring the concentration of a substance from the polarization anisotropy of two different phosphors uses roughly the same optical system configuration as shown in Figure 5. However, in order to measure the polarization anisotropy of two different phosphors, it is necessary to install dichroic mirrors at the PBS reflection position as well as the PBS transmission position to separate fluorescence of different wavelengths. In this case, it is necessary to add detectors to receive the reflected light of the separated light. However, as explained in other form 5 of Example 1, the number of detectors can be reduced and the optical system can be simplified by adding control to alternately or sequentially light sources of different wavelengths.

[0100] The latter method, which measures the concentration of a substance from the change in polarization anisotropy and the change in the emission intensity of a phosphor, uses an optical system configuration that is roughly the same as that shown in Figure 19. The polarization anisotropy is measured by the intensity I received by detectors 48 and 58, respectively. para , I orth The change in the emission intensity of the other phosphor is calculated from the above, and measured by the detector 68. At this time, the polarizer 64 placed in front of the detector 68 is no longer needed, and the polarizer (not shown) after the light source that excites the fluorescence received by the detector 68 is also no longer needed.

[0101] In this embodiment, LEDs and LDs are used as light sources, but solid-state lasers that emit high-power SLDs or YAG harmonics, or gas lasers such as He-Ne, which have excitation wavelengths suitable for the emission wavelength of phosphors, may also be used. [Examples]

[0102] (Automated analysis device integrated into a rotating disk) Figure 22 is a diagram showing the configuration of the automatic analyzer 1000 in this embodiment. As disclosed in Japanese Patent Publication No. 5908954, the automatic analyzer 1000 includes an analysis unit 200 and a control unit 280 that controls the analysis unit 200. The control unit 280 controls the measurement flow in the photometric unit, receives signals output from the photometric unit, and controls the processing unit 70 and memory 290 to perform data transfer, processing, and storage. The automatic analyzer 1000 also includes a display unit 300 that displays the results processed by the processing unit 70.

[0103] The control unit 280 implements its functions, for example, by having a hardware processor (computer) execute a program stored in the memory 290. The hardware processor includes, for example, a CPU. The control unit 280 is an example of a "control unit". Specifically, the control unit 280 lights up at least two light sources that emit light of different wavelengths simultaneously, alternately, or sequentially. The detection unit receives the first emitted light and the second emitted light simultaneously, alternately, or sequentially, according to the lighting time of the light sources.

[0104] Memory 290 can be implemented using, for example, semiconductor memory elements such as RAM (Random Access Memory) or flash memory, a hard disk, or an optical disk. Memory 290 stores, for example, various data related to the measurement (such as calibration curves). This data may be stored not in memory 290 (or in addition to memory 290) but in an external memory that the automated analyzer 1000 can communicate with. The external memory is controlled by a cloud server that manages the external memory, for example, by accepting read and write requests from the cloud server.

[0105] The display unit 300 may be, for example, a liquid crystal display, a CRT (Cathode Ray Tube), or an organic EL (Electroluminescence) display. The display unit 300 may also be a display device (for example, a tablet terminal) that can communicate wirelessly with the automated analyzer 1000.

[0106] The analysis unit 200 includes, for example, a rotatable disk 210 and a plurality of reaction vessels 30 arranged on the circumference of the disk 210. The photometric unit may consist of a photometric unit 220a that performs fluorescence polarization and a photometric unit 220b that performs scattering. The analysis unit 200 also includes a first dispensing unit 230 for dispensing a sample such as a standard sample or a substance to be measured into the reaction vessel 30, a first reagent dispensing unit 240 for dispensing a first reagent that reacts with components contained in the sample, and a second reagent dispensing unit 250 for dispensing a second reagent that is paired with the first reagent. The analysis unit 200 also includes a stirring unit 260 for stirring a mixture of the sample and reagent, and a washing and drying unit 270 for aspirating the mixture from the reaction vessel 30 after measurement and washing and drying the inside of the reaction vessel 30. Therefore, the automated analyzer 1000 can continuously perform a series of steps, from dispensing, stirring, measuring, aspirating, washing, and drying of the sample and reagents, while rotating the disk 210. The reaction vessel 30 is housed in a constant temperature bath, and the temperature of the reaction solution is kept constant.

[0107] In the apparatus configuration described above, measurement and analysis are performed according to the flow shown in Figure 12. In this embodiment, measurements are taken when the reaction vessel 30 passes through the respective photometric units 220a and 220b. Measurements are performed multiple times during the rotation of the disk 210 as a function of reaction time to evaluate anisotropy and the depolarization component of scattering. As in this embodiment, the fluorescence polarization method and the scattering method may be measured almost simultaneously and independently. Furthermore, the switching points between the fluorescence polarization method and the scattering method in post-measurement processing, and the calibration curves corresponding to each measurement method are stored in the memory 290, and the control unit 280 reads the conditions as needed and executes them according to the flow shown in Figure 12.

[0108] Here, the photometric unit may also include a photometric unit 22c (not shown) that performs the transmission method. Furthermore, as described in Example 1, multiple combinations are possible, such as using one photometric unit 220a for both fluorescence polarization and scattering methods, and performing the transmission method with a photometric unit 220b.

[0109] Thus, it is desirable that the present invention utilizes a reagent capable of measuring both fluorescence polarization and scattering, and that the amount of reagent be adjusted so that the respective measurement ranges overlap. As an effect of the present invention, while maintaining high sensitivity by fluorescence polarization, a wide concentration range can be measured in a single flow as shown in Figure 12 by appropriately selecting and analyzing multiple measurement results, including scattering and even transmission methods. Here, the scattering method is a means of measuring scattered light, and as explained above, several methods can be considered, such as the depolarization component of unexcited light, the light intensity emitted at a certain scattering angle, or the autocorrelation function calculated from the time variation of the scattered light intensity. Transmission light intensity by the transmission method may also be included.

[0110] The present invention aims to expand the range of measurable concentrations by combining a measurement method that measures aggregation due to antigen-antibody reactions as a change in rotational motion using fluorescence polarization and a measurement method that measures it as a change in scattering cross-section or translational motion using scattering. The former is volume-dependent and therefore, in principle, more sensitive to minute changes than the latter. Utilizing this principle, aggregation changes due to antigen-antibody reactions are measured independently using two different methods based on different physical phenomena. By appropriately selecting the two measurement results according to the concentration of the substance being measured, the measurement range can be expanded.

[0111] Although embodiments of the present invention have been described above with reference to exemplary embodiments, it should be understood that the present invention is not limited to the embodiments described above. The scope of the appended claims should be considered to be interpreted in the broadest sense to include all such variations and equivalent structures and functions. [Explanation of Symbols]

[0112] 1. Measured object 2. Phosphors 3, 6 aggregates 4 Antibodies 5 scattering particles 10, 110 Light source section 11, 21, 111, 121 light source 12, 22, 41, 112, 122, 141 collimator lenses 13, 113 Short-pass filters 14, 23, 44, 54, 64 polarizers 15, 55, 155, 115 Dichroic Mirrors 16, 116 Mirror 17, 47, 57, 67, 117, 157, 167 condenser lenses 30 reaction vessel 31, 131 Reaction solution 40, 140 detection unit 42, 142 Excitation light cut filter 43 Polarizing Beam Splitter (PBS) 46, 56, 156 bandpass filters 48, 58, 68, 158, 168 detectors 70 Processing Unit 200 Analysis Department 210 discs 220a, 220b Photometering section 230 Dispensing Section 240 First reagent dispensing section 250 Second reagent dispensing section 260 Stirring section 270 Washing and drying section 280 Control Unit 290 memory 300 Display 1000 automatic analyzer

Claims

1. A light source that emits at least two light sources of different wavelengths, A reaction vessel capable of containing a reaction solution in which a substance to be measured and a reagent that specifically reacts with the substance to be measured are mixed, A detection unit that receives at least two different wavelengths of first emitted light and second emitted light emitted from the reaction vessel by irradiating the reaction vessel with incident light emitted from the light source unit, A processing unit that calculates the concentration of the measured substance based on the signal output from the detection unit. It has, The first emitted light is fluorescence obtained by wavelength conversion of the incident light by the reagent, The detection unit has separation means for separating the first emitted light and the second emitted light and receiving them with a photodetector. The processing unit calculates the concentration of the substance to be measured from at least one of the two output signals output from the detection unit in response to the first emitted light and the second emitted light. Automatic analyzer.

2. The aforementioned processing unit, The signal change of the output signal after a certain time has elapsed since the start of the reaction in which the measured substance and the reagent were mixed is evaluated. The first signal change based on the first emitted light and the second signal change based on the second emitted light are calculated, If the first signal change satisfies a predetermined condition, the concentration of the measured substance is calculated based on the first signal change; if the predetermined condition is not met, the concentration of the measured substance is calculated based on the second signal change. The automated analyzer according to claim 1.

3. The aforementioned reagent contains scattering particles that encapsulate fluorescent molecules. The first signal change described above is a signal change based on fluorescence anisotropy, The second signal change described above is a signal change based on the scattered light intensity, or an autocorrelation function calculated from the time variation of the scattered light intensity. The automated analyzer according to claim 2.

4. The reagent comprises at least two antibodies with different affinity, and each antibody is labeled with a fluorescent molecule that emits light at a different wavelength. The first signal change and the second signal change are signal changes based on the fluorescence intensity of fluorescence of different wavelengths. The automated analyzer according to claim 2.

5. The separation means of the detection unit is a dichroic mirror in which, of two lights of different wavelengths, the longer wavelength light is reflected and the shorter wavelength light is transmitted. An automated analyzer according to any one of claims 1 to 4.

6. The light source unit has a linear polarizer, and at least one of the lights emitted from the light source unit is linearly polarized to irradiate the reaction solution. The detection unit has a polarization separation element and separates the first emitted light ejected from the reaction liquid by the linear polarization into a direction parallel to the polarization direction of the linear polarization and a direction perpendicular to it, and receives each of them with the photodetector. An automated analyzer according to any one of claims 1 to 3.

7. The desired light received by the photodetector is defined as follows: when the cut-off wavelength C is the wavelength at which the reflectance of the dichroic mirror is 50%, and of two different wavelengths of light, the wavelength of the longer wavelength is A and the wavelength of the shorter wavelength is B, If A-C < C-B, the dichroic mirror is incident with s-polarized light to separate the colors; if A-C > C-B, the dichroic mirror is incident with p-polarized light to separate the colors. The automated analyzer according to claim 5.

8. Of the light intensity of the photodetector receiving the first emitted light and the light intensity of the photodetector receiving the second emitted light, The output of the first light source that forms emitted light with low light intensity is greater than the output of the second light source, or the gain or exposure time of the photodetector that receives the emitted light with low light intensity is greater than that of the other photodetector. An automated analyzer according to any one of claims 1 to 4.

9. The system further includes a control unit that alternately or sequentially lights up at least two light sources emitting light of different wavelengths, The detection unit receives the first emitted light and the second emitted light alternately or sequentially, according to the lighting time of the light source. An automated analyzer according to any one of claims 1 to 4.

10. Light emitted from the light source unit enters the reaction vessel from the first surface and exits from the second surface opposite the first surface. An automated analyzer according to any one of claims 1 to 4.

11. The predetermined conditions involve comparing the first signal change and the second signal change, and calculating the concentration of the measured substance based on the signal change that is larger in relation to the concentration change of the measured substance or the passage of time. The automated analyzer according to claim 2 or 3.

12. An irradiation step in which a reaction solution, which is a mixture of the substance to be measured and a reagent that specifically reacts with the substance to be measured, is irradiated with at least two lights of different wavelengths, A light receiving step of separating and receiving at least two first and second emitted light beams of different wavelengths emitted from the reaction solution, A processing step which calculates the concentration of the measured substance based on the signal output by the light receiving step, It has, The processing step involves calculating the concentration of the substance to be measured from at least one of the changes in the signal based on the first emitted light and the changes in the signal based on the second emitted light. Automatic analysis method.

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