Analytical apparatus and analytical method

Abstract

To provide a material separation device, an analyzer, and a method for analysis which can accurately measure the amount of a measurement target object with a large molecular weight.SOLUTION: A material separation device 1 has a reaction container 2 of which inside storing a solution is separated into a first room 2a and a second room 2b by a dialysis membrane 4. A sample S is stored in the first room 2a and has a binding ability to specifically bind with a measurement target object T in the sample S with a larger molecular weight than the molecular weight cut off of the dialysis membrane 4. A coloring agent modified molecule M is stored in at least one of the first room 2a and the second room 2b. The coloring agent modified molecule M has a smaller molecular weight than the molecular weight cut off of the dialysis membrane 4.SELECTED DRAWING: Figure 1

Description

Technical Field

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[0001] The present disclosure , minutes relates to an analysis device and an analysis method.

Background Art

[0002] An immunoassay is an analysis method using an antibody-antigen reaction (special Patent document 1 reference (see). According to an immunoassay, an antigen can be detected with high sensitivity by using an antibody that specifically binds to the measurement target. Among them, a heterogeneous method that separates a labeled antigen (bound, B) bound to an antibody from a free labeled antigen (free, F) (B / F separation) is known as a highly sensitive measurement method. However, in order to perform B / F separation, a separate device is required, and the measurement system becomes complicated. Therefore, a method that does not require B / F separation is desired.

Prior Art Documents

Patent Documents

[0006] This invention was made under the circumstances described above, and it is possible to accurately quantitatively measure substances with large molecular weights. portion The objective is to provide analytical apparatus and analytical methods. [Means for solving the problem]

[0007] To achieve the above objectives, the first aspect of this disclosure relates analysis The device is A reaction vessel in which the interior containing the solution is divided into a first chamber and a second chamber by a dialysis membrane. and, A detection optical system is provided which irradiates at least one of the first and second chambers with excitation light to cause the dye to emit light, and detects the emission intensity and polarization degree of the portion irradiated with the excitation light, Equipped with, The specimen is in the first chamber. Manually or automatically They were taken into custody. A dye-modified molecule contained in the sample and having the ability to specifically bind to a target object whose molecular weight is larger than the molecular weight cutoff of the dialysis membrane is present in at least one of the first and second chambers. Manually or automatically They were taken into custody. The aforementioned dye-modified molecule has a smaller molecular weight than the fractional molecular weight of the dialysis membrane.

[0009] The analytical method relating to the third aspect of this disclosure is: Of the two chambers of the reaction vessel, which contains the solution and is separated by a dialysis membrane, the sample is placed in the first chamber. At least one of the first and second chambers contains a dye-modified molecule that has the ability to specifically bind to a metering object contained in the sample and having a molecular weight greater than the molecular weight cutoff of the dialysis membrane, and has a molecular weight smaller than the molecular weight cutoff of the dialysis membrane. death, At least one of the first chamber and the second chamber is irradiated with excitation light to cause the dye to emit light, and the emission intensity and polarization degree of the portion of the first chamber and the second chamber irradiated with the excitation light are detected. [Effects of the Invention]

[0010] According to the present disclosure, a measurement object with a large molecular weight can be accurately quantitatively measured.

Brief Description of the Drawings

[0011] [Figure 1] (A) is a perspective view showing the configuration of a reaction vessel of a substance separation apparatus according to Embodiment 1 of the present disclosure. (B) is a schematic diagram showing the substances accommodated in the reaction vessel of (A). [Figure 2] It is a flowchart showing the analysis process according to Embodiment 1 of the present disclosure. [Figure 3] It is a perspective view showing a modified example of the reaction vessel of the substance separation apparatus according to Embodiment 1 of the present disclosure. [Figure 4] It is a schematic diagram showing the configuration of an analyzer according to Embodiment 2 of the present disclosure. [Figure 5] It is a flowchart showing the analysis process according to Embodiment 2 of the present disclosure. [Figure 6] (A) and (B) are schematic diagrams showing examples of specimens and molecules introduced into the first reaction vessel and the second reaction vessel of the substance separation apparatus according to Embodiment 3 of the present disclosure. [Figure 7] It is a schematic diagram showing the configuration of an analyzer according to Embodiment 4 of the present disclosure. [Figure 8] It is a schematic diagram showing the configuration of an analyzer according to Embodiment 5 of the present disclosure. [Figure 9] It is a schematic diagram showing the configuration of an analyzer according to Embodiment 6 of the present disclosure.

Modes for Carrying Out the Invention

[0012] Embodiments according to the present disclosure will be described with reference to the drawings. Note that the present disclosure is not limited by the following embodiments and drawings. In the following embodiments, expressions such as "having", "including", or "containing" also include the meaning of "consisting of" or "composed of".

[0013] (Embodiment 1) As shown in Figure 1(A), the substance separation apparatus 1 according to this embodiment includes a reaction vessel 2. The inside of the reaction vessel 2, in which the solution L is contained, is divided into two chambers by a partition wall 3. One chamber is called the first chamber 2a, and the other chamber is called the second chamber 2b.

[0014] A dialysis membrane 4 is provided in at least a portion of the partition wall 3. Solution L can pass between the first chamber 2a and the second chamber 2b through the dialysis membrane 4. As the dialysis membrane 4, a material resistant to solution L is used, and as described later, a material having a predetermined molecular weight cutoff (MWCO) in relation to the molecular weight of the object to be measured T and the dye-modified molecule M is used.

[0015] As shown in Figure 1(B), the sample S to be measured is contained in the first chamber 2a. The sample S contains the substance T to be measured. Meanwhile, the dye-modified molecule M is contained in at least one of the first chamber 2a and the second chamber 2b. The dye-modified molecule M is modified (labeled) with a fluorescent dye F as a dye. The dye-modified molecule M has the ability to specifically bind to the substance T to be measured.

[0016] The molecular weight of the substance T to be measured is greater than the molecular weight cutoff (MWCO) of the dialysis membrane 4. Therefore, the substance T to be measured remains contained in the first chamber 2a and does not move to the second chamber 2b. On the other hand, the pigment-modified molecule M has a molecular weight smaller than the MWCO of the dialysis membrane 4. Therefore, the pigment-modified molecule M can move between the first chamber 2a and the second chamber 2b via the dialysis membrane 4 unless it binds to the substance T to be measured. The pigment-modified molecule M that moves to the first chamber 2a binds to the substance T to be measured.

[0017] When the concentration of the substance T is high, many dye-modifying molecules M bind to the substance T in chamber 1 2a. The higher the concentration of the substance T, the greater the number of dye-modifying molecules M that bind to the substance T, and the lower the concentration of dye-modifying molecules M. On the other hand, when the concentration of the substance T is low, the number of dye-modifying molecules M (bound) that bind to the substance T in chamber 1 2a is small, and therefore the number of dye-modifying molecules M (free) that are not bound to the substance T increases.

[0018] Thus, the number of bound dye-modifying molecules M and the number of free dye-modifying molecules M change depending on the concentration of the substance T being measured. On the other hand, when excitation light that causes the fluorescent dye F to emit light is irradiated into the first chamber 2a or the second chamber 2b, the emission intensity P1 of the fluorescent dye F changes according to the concentration of the substance T being measured in the first chamber 2a. Therefore, by detecting the emission intensity P1, the concentration of the substance T being measured can be estimated.

[0019] Furthermore, the combined product (complex) of the substance T and the dye-modified molecule M cannot move to the second chamber 2b via the dialysis membrane 4 and remains in the first chamber 2a. On the other hand, the concentration of the free form becomes the same in both the first chamber 2a and the second chamber 2b at equilibrium after a certain period of time. Therefore, as shown in Figure 1(B), both the combined and free forms coexist in the first chamber 2a. This point must be taken into consideration when estimating the concentration of the substance T based on the luminescence intensity P1.

[0020] [Measurement targets, dye-modified molecules, and dialysis membranes] As the target substance T, a large molecule such as a protein can be selected. For example, ovalbumin and β-lactoglobulin are examples. If the target substance T is ovalbumin (molecular weight 43 kDa), then the dye-modifying molecule M can be selected, for example, a VHH (Variable domain of Heavy chain of Heavy chain antibody) antibody (molecular weight 15 kDa). In this case, the MWCO of the dialysis membrane 4 can be set to, for example, 20 kDa. If the target substance T is β-lactoglobulin (molecular weight 18.3 kDa), then the dye-modifying molecule M can be selected, for example, an aptamer (molecular weight 8 kDa). In this case, the MWCO of the dialysis membrane 4 can be set to, for example, 10 kDa. Alternatively, a Fab antibody may be used as the dye-modifying molecule M. In this way, the MWCO of the dialysis membrane 4 is determined based on the target substance T and the dye-modifying molecule M.

[0021] [Fluorescent dyes] A fluorescent dye F is a dye that emits fluorescence when excited by excitation light. Each fluorescent dye F has its own unique fluorescence lifetime. In this disclosure, depending on the molecular weight of the dye modification molecule M to be modified, a fluorescent dye F with a fluorescence lifetime of 1 to 10 nanoseconds, a fluorescent dye F with a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds, and a fluorescent dye F with a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds can be appropriately selected and used. For example, fluorescent dyes F with a fluorescence lifetime of 1 to 10 nanoseconds include fluorescein compounds such as indorenine, chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5 and 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein, as well as rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; registered trademarks or trade names include the Alexa Fluor series such as Alexa Fluor 488, BODIPY series, DY series, ATTO series, Dy Light series, Oyster series, HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, and Yakima. Examples include Yellow, VIC, HEX, R6G, Cy3, TAMRA, Rhodamine Red-X, Redmond Red, ROX, Cal Red, Texas Red, LC Red 640, Cy5, Cy5.5, and LC Red 705. Fluorescent dyes F with fluorescence lifetimes ranging from over 10 nanoseconds to 200 nanoseconds include naphthalene derivatives such as dialkylaminonaphthalenesulfonyl, and pyrene derivatives such as N-(1-pyrenyl)maleimide, aminopyrene, pyrenebutanoic acid, and alkynylpyrene. Furthermore, fluorescent dyes F with fluorescence lifetimes ranging from over 200 nanoseconds to 3,000 nanoseconds include metal complexes such as platinum, rhenium, ruthenium, osmium, and europium.

[0022] To modify (label) molecule M with a fluorescent dye F, for example, the fluorescent dye F and molecule M can be directly covalently bonded, or bonded via a linker such as oligoethylene glycol and an alkyl chain. The fluorescent dye F has functional groups that can bond to carboxyl groups, amino groups, hydroxyl groups, thiol groups, and phenyl groups of the molecule. By reacting the functional groups of the fluorescent dye F and molecule M under known conditions, molecule M can be labeled with the fluorescent dye F. The number of molecules of fluorescent dye F used to modify one molecule can be arbitrarily selected. Preferably, there is one or more molecules per molecule, and it may be 2 to 5 molecules.

[0023] Next, an analysis method, i.e., an analysis process, using the substance separation apparatus 1 according to this embodiment will be described. It should be assumed that a dialysis membrane 4 having MWCO smaller than the molecular weight of the substance T to be measured and larger than the molecular weight of the dye-modified molecule M is installed on the partition wall 3, and that the solution L is contained in the reaction vessel 2.

[0024] In the analytical process, as shown in Figure 2, first, the sample S is placed in the first chamber 2a (step S1). Next, the dye-modified molecule M is placed in at least one of the first chamber 2a and the second chamber 2b (step S2). Then, a certain period of time is waited for (step S3; No). After the certain period of time has elapsed (step S3; Yes), the analytical method using the substance separation device 1 is terminated.

[0025] After a certain period of time, the dye-modified molecule M (bound form) bound to the substance T remains in the first chamber 2a within the reaction vessel 2 and does not move into the second chamber 2b. Therefore, for example, by transferring the solution L in the first chamber 2a or the second chamber 2b to a measurement cell, irradiating the measurement cell with excitation light that causes the fluorescent dye F to emit light, and detecting either the emission intensity P1 of the fluorescence from the measurement cell or the emission intensity P1 and polarization degree P2, it becomes possible to measure the concentration of the substance T, as will be described later.

[0026] As described in detail above, according to the substance separation apparatus 1 of this embodiment, a dialysis membrane 4 having a molecular weight cutoff (MWCO) smaller than the molecular weight of the substance T to be measured and larger than the molecular weight of the dye-modified molecule M that specifically binds to the substance T to be measured can be used to maintain the bound form in which the substance T to be measured and the dye-modified molecule M are bound in the first chamber 2a. Therefore, by detecting the emission intensity P1 and polarization degree P2 of the fluorescent dye F in at least one of the first chamber 2a and the second chamber 2b, the substance T with a large molecular weight can be accurately quantified.

[0027] By using the analytical device 5 according to this embodiment, B / F separation in an immunoassay becomes possible, for example, when the target substance T is the antigen and the dye-modified molecule M is the antibody. This eliminates the need for antibody immobilization.

[0028] The reaction vessel 2 itself can be used as the measurement cell. This eliminates the need to transfer solution L from the reaction vessel 2 to the measurement cell and prevents contamination of the sample S. In this case, the material of the reaction vessel 2 must be one that transmits excitation light and fluorescence.

[0029] The reaction vessel 2, which serves as the measurement cell, can have the configuration shown in Figure 3. The reaction vessel 2 shown in Figure 3 has a configuration in which a tubular second chamber 2b is inserted into a rectangular first chamber 2a. A tubular dialysis membrane 4 (dialysis tube) is formed on the wall of the second chamber 2b. The substance to be measured T is introduced into the first chamber 2a, and the dye-modified molecule M is introduced into the second chamber 2b. The dye-modified molecule M passes through the dialysis membrane 4 (dialysis tube) and enters the first chamber 2a, where it binds to the substance to be measured T. This reaction vessel 2 can also be made of a material that transmits excitation light and fluorescence.

[0030] (Embodiment 2) In this embodiment, an analytical apparatus 5 equipped with the reaction vessel 2 according to Embodiment 1 as a measuring cell will be described. As shown in Figure 4, the analytical apparatus 5 according to this embodiment comprises the reaction vessel 2 according to Embodiment 1 and a detection optical system 10. The reaction vessel 2 may be the one with the configuration shown in Figure 1(A) or the one with the configuration shown in Figure 3.

[0031] The detection optical system 10 irradiates the first chamber 2a of the reaction vessel 2 of the material separation device 1 with excitation light IL to cause the fluorescent dye F (see Figure 1(B)) to emit light, and detects either the emission intensity P1 of the fluorescent EL in the first chamber 2a, or the emission intensity P1 and the polarization degree P2.

[0032] The detection optical system 10 comprises a light source unit 11, an optical fiber 12, a lens 50, a reflector 13 positioned obliquely (hereinafter simply referred to as "obliquely") with respect to the optical axis direction of the exit end of the excitation light IL of the optical fiber 12, an optical fiber 14, and a detection unit 15.

[0033] The light source unit 11 comprises a light source 20, an excitation filter 21, and a coupling lens 22. The light source 20 emits excitation light IL. The excitation light IL enters the optical fiber 12 after passing through the excitation filter 21, which adjusts the wavelength of the excitation light IL, and the coupling lens 22.

[0034] The optical fiber 12 sends excitation light IL to the first chamber 2a of the reaction vessel 2 via the lens 50. The excitation light IL is focused in the liquid by the lens 50, and fluorescence EL is generated by the fluorescent dye F in the first chamber 2a near this focal point. The reflector 13 is positioned at an angle and diffusely reflects the excitation light IL that has passed through the reaction vessel 2. The fluorescence EL from the emission of the fluorescent dye F enters the optical fiber 14. The optical fiber 14 sends the fluorescence EL generated in the first chamber 2a to the detection unit 15.

[0035] The detection unit 15 comprises a coupling lens 25, a fluorescence filter 26, a liquid crystal element 27, and an imaging unit 28. The coupling lens 25 sends light emitted from the optical fiber 14 to the fluorescence filter 26. The fluorescence filter 26 allows light in the wavelength band of the fluorescent EL generated by the emission of the fluorescent dye F to pass through, while blocking light of other wavelengths. The liquid crystal element 27 adjusts the polarization direction of the transmitted fluorescent EL. The imaging unit 28 captures an image showing the fluorescent EL that has passed through the liquid crystal element 27. Based on this image, it is possible to detect either the emission intensity P1 of the fluorescent EL or the emission intensity P1 and the polarization degree P2.

[0036] The detection optical system 10 having the above configuration detects either the emission intensity P1 of the fluorescent EL in the first chamber 2a of the reaction vessel 2, or the emission intensity P1 and the polarization degree P2.

[0037] [Fluorescence emission intensity] When detecting the emission intensity P1 of a fluorescent EL, the liquid crystal element 27 is adjusted to transmit all polarization components of the incident fluorescent EL. This allows the imaging unit 28 to receive the entire beam of light from the fluorescent EL and detect the emission intensity P1. A calibration curve showing the relationship between the emission intensity P1 of the fluorescent EL and the concentration of the dye-modifying molecule M has been created in advance, and the analyzer 5 measures the concentration of the dye-modifying molecule M corresponding to the emission intensity P1 based on the calibration curve. The calibration curve is created by detecting the emission intensity P1 of the fluorescent EL in the first chamber 2a or the second chamber 2b while changing the concentrations of the known substance T and the dye-modifying molecule M.

[0038] [Degree of polarization of fluorescence] When detecting the polarization degree P2 of a fluorescent EL, the excitation light IL emitted from the light source unit 11 is linearly polarized. Of the received fluorescent EL, the polarization degree parallel to the excitation light IL is defined as I1, and the polarization degree perpendicular to the excitation light IL is defined as I2. The detection optical system 10 controls the liquid crystal element 27 of the detection unit 15 to detect these two fluorescence polarization intensities I1 and I2, and calculates the polarization degree P2 using the following formula. P2 = (I1 - I2) / (I1 + I2) The degree of polarization P2 indicates the degree of rotation of the dye-modified molecule M between excitation and emission of fluorescent EL. Molecules with small molecular weights rotate vigorously in solution due to Brownian motion, resulting in a low degree of polarization P2, while molecules with large molecular weights exhibit weaker Brownian motion, resulting in a higher degree of polarization P2. Therefore, a higher concentration of the substance T results in a higher degree of polarization P2, and a lower concentration results in a lower degree of polarization P2. A calibration curve showing the relationship between the degree of polarization P2 and the concentration of the substance T has been determined in advance. The analyzer 5 measures the concentration of the substance T corresponding to the degree of polarization P2 based on this calibration curve. This calibration curve is also created by detecting the degree of polarization P2 of the fluorescent EL in the first chamber 2a or the second chamber 2b while varying the concentrations of the substance T and dye-modified molecule M for combinations of known concentrations.

[0039] Incidentally, although the MWCO of the dialysis membrane 4 is smaller than the molecular weight of the substance T to be measured, MWCO is a statistical value, and there is variability in the pore size of the dialysis membrane 4. For this reason, a small amount of the substance T to be measured may pass through the dialysis membrane 4 and move to the second chamber 2b. In this case, the analyzer 5 may measure both the emission intensity P1 and the polarization degree P2 of the fluorescent EL, for example. For example, the emission intensity P1 of the fluorescent EL in the second chamber 2b reflects both the dye-modified molecules M (bound form) that are bound to the substance T to be measured and the dye-modified molecules M (free form) that are not bound to the substance T to be measured. Furthermore, the polarization degree P2 of the fluorescent EL in the second chamber 2b reflects the ratio of the concentration of the bound form to the concentration of the free form. Therefore, by estimating the total concentration of dye-modified molecules M based on the emission intensity P1 and estimating the ratio of bound to free forms based on the polarization degree P2, it becomes possible to measure the concentration of the substance T to be measured that is bound to the dye-modified molecules M with higher accuracy. To estimate the ratio of bound to free forms, it is necessary to know the amount of dye-modifying molecule M being introduced.

[0040] Furthermore, in the analyzer 5, excitation light IL may be irradiated into the second chamber 2b, and either the emission intensity P1 of the fluorescent EL in the second chamber 2b or the emission intensity P1 and polarization degree P2 may be detected to measure the concentration of the substance T to be measured. It is also possible to estimate the concentration of the substance T to be measured based on both the detection result from the first chamber 2a and the detection result from the second chamber 2b. In addition, although the first chamber 2a and the second chamber 2b are separated by a dialysis membrane 4, a light-shielding layer may also be placed between the first chamber 2a and the second chamber 2b. This makes it possible to make the optical signal obtained from the first chamber 2a and the optical signal from the second chamber 2b independent, thereby enabling more accurate monitoring of the fluorescence signals in each chamber.

[0041] Next, we will explain the analysis method using the analytical device 5, that is, the analytical process.

[0042] As shown in Figure 5, the analytical process according to this embodiment includes the same steps S1 to S3 as the analytical process described above (see Figure 2). After a certain period of time has elapsed (step S3; Yes), excitation light IL that causes the fluorescent dye F to emit light is irradiated into at least one of the first chamber 2a and the second chamber 2b of the reaction vessel 2, and either the emission intensity P1 of the fluorescent EL in the portion of the first chamber 2a and the second chamber 2b irradiated with excitation light IL, or the emission intensity P1 and polarization degree P2 is detected (step S4). Subsequently, the concentration of the substance to be measured T is determined from this detection result based on various calibration curves. After step S4 is completed, the analyzer 5 terminates the analytical process.

[0043] According to this embodiment, by using the reaction vessel 2 as a measurement cell, it becomes unnecessary to transfer the solution, and contamination of the sample can be prevented.

[0044] (Embodiment 3) As shown in Figures 6(A) and 6(B), the substance separation apparatus 1 according to this embodiment comprises a first reaction vessel 2A and a second reaction vessel 2B. In this case, for example as shown in Figure 6(A), sample S1 can be placed in the first chamber 2a of the first reaction vessel 2A, and a dye-modified molecule M1 that binds to the substance to be measured T1 can be placed in at least one of the first chamber 2a and the second chamber 2b. Furthermore, sample S2 can be placed in the first chamber 2a of the second reaction vessel 2B, and a dye-modified molecule M1 can be placed in at least one of the first chamber 2a and the second chamber 2b. After a certain period of time has elapsed since the samples were added, excitation light IL that causes the fluorescent dye F to emit light is irradiated into at least one of the first chamber 2a and the second chamber 2b of the first reaction vessel 2A. At the same time, the emission intensity P1 of the fluorescent EL in the portion of the first chamber 2a and the second chamber 2b irradiated with excitation light IL, or the emission intensity P1 and polarization degree P2 are detected. At the same time, excitation light IL that causes the fluorescent dye F to emit light is irradiated into at least one of the first chamber 2a and the second chamber 2b of the second reaction vessel 2B. At the same time, the emission intensity P1 of the fluorescent EL in the portion of the first chamber 2a and the second chamber 2b irradiated with excitation light IL, or the emission intensity P1 and polarization degree P2 are detected. By doing this, the concentrations of the target substance T of different samples S1 and S2 can be measured at once. This makes it possible to compare the concentrations of the target substance T between sample S1 and sample S2.

[0045] Furthermore, for example, as shown in Figure 6(B), sample S1 can be placed in the first chamber 2a of the first reaction vessel 2A, and a dye-modified molecule M1 that binds to the target substance T1 can be placed in at least one of the first chamber 2a and the second chamber 2b. Sample S1 can also be placed in the first chamber 2a of the second reaction vessel 2B, and a dye-modified molecule M2 that binds to the target substance T2 can be placed in at least one of the first chamber 2a and the second chamber 2b. After a certain period of time has elapsed since placement, excitation light IL can be irradiated in the same manner as in Figure 6(A). By detecting either the emission intensity P1 of the fluorescent EL in the first reaction vessel 2A, or the emission intensity P1 and polarization degree P2, and by detecting either the emission intensity P1 of the fluorescent EL in the second reaction vessel 2B, or the emission intensity P1 and polarization degree P2, the concentrations of different types of target substances T1 and T2 in sample S1 can be measured at once. This makes it possible to compare the concentrations of target substances T1 and T2 in sample S1 of the same type.

[0046] In this way, by preparing multiple reaction vessels 2A and 2B, it becomes possible to simultaneously measure the concentration of the target substance T with different combinations of the target substance T and the dye-modified molecule M.

[0047] (Embodiment 4) As shown in Figure 7, the analyzer 5 according to this embodiment includes a detection optical system 10 capable of simultaneously detecting either the emission intensity P1 of the fluorescent EL in the first reaction vessel 2A, or the emission intensity P1 and polarization degree P2, and either the emission intensity P1 of the fluorescent EL in the second reaction vessel 2B, or the emission intensity P1 and polarization degree P2. The detection optical system 10 includes a first light source unit 11A, a second light source unit 11B, an optical fiber 12A, an optical fiber 12B, and two lenses 50.

[0048] The internal configurations of the first light source unit 11A and the second light source unit 11B are the same as those of the light source unit 11 in Figure 4. The first light source unit 11A emits a first excitation light IL1 that causes the first fluorescent dye F1 to emit light. The second light source unit 11B emits a second excitation light IL2, which has a different wavelength from the first excitation light IL1 and causes the second fluorescent dye F2 to emit light. The optical fiber 12A sends the first excitation light IL1 emitted from the first light source unit 11A to the first chamber 2a of the first reaction vessel 2A via the lens 50. The optical fiber 12B sends the second excitation light IL2 emitted from the second light source unit 11B to the first chamber 2a of the second reaction vessel 2B via the lens 50.

[0049] The dye-modified molecule M contained in the first chamber 2a of the first reaction vessel 2A is modified with a first fluorescent dye F1 that emits light when irradiated with the first excitation light IL1. Therefore, the dye-modified molecule M that binds to the object T near the focusing position of the first excitation light IL1 by the lens 50 in the first chamber 2a of the first reaction vessel 2A emits fluorescence EL1. Similarly, the dye-modified molecule M contained in the first chamber 2a of the second reaction vessel 2B is modified with a second fluorescent dye F2 that emits light when irradiated with the second excitation light IL2. Therefore, the dye-modified molecule M that binds to the object T near the focusing position of the second excitation light IL2 by the lens 50 in the first chamber 2a of the second reaction vessel 2B emits fluorescence EL2.

[0050] The detection optical system 10 includes a diagonally positioned reflecting mirror 13A, a similarly diagonally positioned reflecting mirror 13B, an optical fiber 14A, an optical fiber 14B, a detection unit 15, and a control unit 16. The reflecting mirror 13A diffusely reflects the first excitation light IL1 that passes through the first reaction vessel 2A. Similarly, the reflecting mirror 13B diffusely reflects the second excitation light IL2 that passes through the second reaction vessel 2B. Meanwhile, the fluorescent EL1 from the dye-modified molecule M in the first chamber 2a of the first reaction vessel 2A is focused onto the optical fiber 14A by the lens 50, and the optical fiber 14A sends the light containing the fluorescent EL1 generated in the first chamber 2a of the first reaction vessel 2A to the detection unit 15. Similarly, the fluorescent EL2 from the dye-modified molecule M in the first chamber 2a of the second reaction vessel 2B is focused onto the optical fiber 14B by the lens 50, and the optical fiber 14B sends the light containing the fluorescent EL2 generated in the first chamber 2a of the second reaction vessel 2B to the detection unit 15.

[0051] The detection unit 15 individually detects either the emission intensity P1 of the fluorescent EL1 in the first chamber 2a of the first reaction vessel 2A, or the emission intensity P1 and polarization degree P2, and either the emission intensity P1 of the fluorescent EL2 in the first chamber 2a of the second reaction vessel 2B, or the emission intensity P1 and polarization degree P2. The control unit 16 controls the first light source unit 11A to emit the first excitation light IL1 and the second excitation light IL2 from the second light source unit 11B to stop emission during the first sampling time. The control unit 16 also controls the second light source unit 11B to emit the second excitation light IL2 and the first excitation light IL1 from the first light source unit 11A to stop emission during the second sampling time. The control unit 16 controls the first light source unit 11A, the second light source unit 11B, and the detection unit 15 so that the first sampling time and the second sampling time are repeated. As a result, the detection unit 15 acquires either the emission intensity P1 of the fluorescent EL1 in the first chamber 2a of the first reaction vessel 2A, or the emission intensity P1 and polarization degree P2, during the first sampling time, and acquires either the emission intensity P1 of the fluorescent EL2 in the first chamber 2a of the second reaction vessel 2B, or the emission intensity P1 and polarization degree P2.

[0052] The detection unit 15 may also include a dichroic mirror for separating fluorescent EL1 and fluorescent EL2, an image sensor for detecting the intensity of fluorescent EL1, and an image sensor for detecting the intensity of fluorescent EL2. In this way, the dichroic mirror separates fluorescent EL1 and fluorescent EL2, and the respective image sensors can individually detect either the luminescence intensity P1 of the first reaction vessel 2A or the luminescence intensity P1 and polarization degree P2, and either the luminescence intensity P1 of the second reaction vessel 2B or the luminescence intensity P1 and polarization degree P2.

[0053] Similar to Embodiment 3 described above, the luminescence intensity P1 and polarization degree P2 may be detected in the second chamber 2b of the first reaction vessel 2A and the second reaction vessel 2B, or in both the first chamber 2a and the second chamber 2b. In addition, although the first chamber 2a and the second chamber 2b are separated by the dialysis membrane 4, a light-shielding layer may also be placed between the first chamber 2a and the second chamber 2b. This makes it possible to separate the optical signal from the first chamber 2a and the optical signal from the second chamber 2b, thereby enabling more accurate monitoring of the fluorescence signals in each chamber.

[0054] The combination of the substance to be measured T and the dye-modified molecule M contained in the first reaction vessel 2A and the second reaction vessel 2B can be the combination of substance T1 and dye-modified molecule M1, as shown in Figure 6(A). Alternatively, these combinations can also be the combination of substances T1, T2 and dye-modified molecules M1, M2, as shown in Figure 6(B).

[0055] In the analytical apparatus 5 according to this embodiment, the detection unit 15 is common to both the first reaction vessel 2A and the second reaction vessel 2B. This makes it possible to miniaturize the analytical apparatus 5 and reduce manufacturing costs.

[0056] (Embodiment 5) As shown in Figure 8, the analytical apparatus 5 according to this embodiment includes a first reaction vessel 2A and a second reaction vessel 2B as the reaction vessel 2. The analytical apparatus 5 includes a detection optical system 10 that individually detects either the emission intensity P1 of the fluorescent EL in the first reaction vessel 2A, or the emission intensity P1 and polarization degree P2, and either the emission intensity P1 of the fluorescent EL in the second reaction vessel 2B, or the emission intensity P1 and polarization degree P2.

[0057] The detection optical system 10 includes a light source unit 11, optical fibers 12A and 12B, reflectors 13A and 13B, optical fibers 14A and 14B, a first detection unit 15A, a second detection unit 15B, and a lens 50.

[0058] The light source unit 11 irradiates the first chamber 2a of the first reaction vessel 2A with excitation light IL via the optical fiber 12A through the lens 50, while simultaneously irradiating the first chamber 2a of the second reaction vessel 2B with excitation light IL via the optical fiber 12B through the lens 50. The excitation light IL causes the dye-modified molecules M in the first chambers 2a of the first reaction vessel 2A and the second reaction vessel 2B to emit light near its focal point. The excitation light IL that has passed through the first chamber 2a of the first reaction vessel 2A is diffusely reflected obliquely by the reflector 13A, and the excitation light IL that has passed through the first chamber 2a of the second reaction vessel 2B is diffusely reflected obliquely by the reflector 13B.

[0059] The excitation light IL reflected by the reflector 13A and the fluorescent EL generated in the first reaction vessel 2A are sent to the first detection unit 15A via the lens 50 and optical fiber 14A. The excitation light IL reflected by the reflector 13B and the fluorescent EL generated in the second reaction vessel 2B are sent to the second detection unit 15B via the lens 50 and optical fiber 14B. The configuration of the first detection unit 15A and the second detection unit 15B is the same as the configuration of the detection unit 15 shown in Figure 4. The first detection unit 15A detects either the emission intensity P1 of the fluorescent EL in the first reaction vessel 2A, or the emission intensity P1 and polarization degree P2. The second detection unit 15B detects either the emission intensity P1 of the fluorescent EL in the second reaction vessel 2B, or the emission intensity P1 and polarization degree P2.

[0060] The combination of the substance to be measured T and the dye-modified molecule M contained in the first reaction vessel 2A and the second reaction vessel 2B can be the combination of substance T1 and dye-modified molecule M1, as shown in Figure 6(A). Alternatively, these combinations can also be the combination of substances T1, T2 and dye-modified molecules M1, M2, as shown in Figure 6(B).

[0061] In the analytical apparatus 5 according to this embodiment, the light source unit 11 is shared between the first reaction vessel 2A and the second reaction vessel 2B. The excitation light IL may be irradiated to the second chamber 2b, or to both the first chamber 2a and the second chamber 2b.

[0062] (Embodiment 6) The analytical apparatus 5 according to this embodiment differs from embodiments 4 and 5 in the configuration of the detection optical system 10. As shown in Figure 9, the detection optical system 10 comprises a light source unit 11, an optical fiber bundle 17, a solution probe 18, and a detection unit 15.

[0063] The light source unit 11 emits excitation light IL. The optical fiber bundle 17 is composed of multiple optical fibers 17a to 17g bundled together. One end of the central optical fiber 17a is connected to the light source unit 11, and one end of the outer optical fibers 17b to 17g are connected to the detection unit 15. The other ends of the optical fibers 17a to 17g are connected to the solution probe 18. Note that one end of the optical fiber 17a is bifurcated and may be used, connected to both the light source unit 11 and the detection unit 15.

[0064] The solution probe 18 comprises a collimator lens 30, a sample opening 31, and a diagonally positioned reflector 32. The collimator lens 30 focuses the excitation light IL emitted from the optical fiber 17a and directs it into the sample opening 31. The sample opening 31 is configured to allow the object to be measured T and dye-modified molecules M to enter from the outside. The object to be measured T and dye-modified molecules M that enter the sample opening 31 emit light due to the focused excitation light IL, generating fluorescence EL. The reflector 32 diffusely reflects the excitation light IL. The solution probe 18 may also be covered with a light-shielding tube. The light-shielding tube has a black interior to prevent stray light from entering the solution probe 18 from the outside. By covering the solution probe 18 with this light-shielding tube, it becomes possible to monitor the fluorescence signal with high accuracy without being affected by the conditions of other rooms.

[0065] The fluorescent EL incident on the optical fibers 17b to 17g via the collimator lens 30 is sent to the detection unit 15. The detection unit 15 detects either the emission intensity P1 of the fluorescent EL in the first reaction vessel 2A, or the emission intensity P1 and the polarization degree P2.

[0066] Thus, by using a detection optical system 10 that includes an optical fiber bundle 17 and a solution probe 18 in which a collimator lens 30 and a reflector 32 are integrated, detection can be easily performed, thereby improving the efficiency of the detection work and shortening the detection time.

[0067] In this example, the optical fiber bundle 17 consisted of seven optical fibers 17a to 17g, but the number of optical fibers bundled together can be any number. Furthermore, the excitation light IL may be transmitted using multiple optical fibers.

[0068] In the above embodiment, the reaction vessel 2 is provided with a first reaction vessel 2A and a second reaction vessel 2B. However, there may be three or more reaction vessels 2. In this case, it becomes possible to detect the same type of target substance T contained in three or more samples, or to detect three or more types of target substances T contained in a sample S.

[0069] In the above embodiment, molecule M was modified with a fluorescent dye F, but this is not the only option. For example, molecule M may be modified with a phosphorescent dye. Other dyes may also be used, as long as they can be detected by the detection optical system.

[0070] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention. [Explanation of Symbols]

[0071] 1. Substance separation apparatus, 2. Reaction vessel, 2A. First reaction vessel, 2B. Second reaction vessel, 2a. First chamber, 2b. Second chamber, 3. Partition wall, 4. Dialysis membrane, 5. Analytical apparatus, 10. Detection optical system, 11. Light source unit, 11A. First light source unit, 11B. Second light source unit, 12, 12A, 12B. Optical fiber, 13, 13A, 13B. Reflecting mirror, 14, 14A, 14B. Optical fiber, 15. Detection unit, 15A. First detection unit, 15B. Second detection unit, 16. Control unit, 17. Optical fiber bundle, 17a~17g. Optical fiber, 18. Solution probe, 20. Light source, 21. Excitation filter, 22. Coupling lens, 25. Coupling lens, 26. Fluorescence filter, 27. Liquid crystal element, 28. Imaging unit, 30. Collimator lens, 31. Sample opening unit, 32. Reflecting mirror, 50. Lens, EL Fluorescence, F, F1, F2: Fluorescent dye, IL: Excitation light, IL1: First excitation light, IL2: Second excitation light, L: Solution, M, M1, M2: Dye-modified molecule, S, S1, S2: Sample, T, T1, T2: Target of measurement

Claims

1. A reaction vessel in which the interior containing the solution is divided into a first chamber and a second chamber by a dialysis membrane, The device comprises a detection optical system that irradiates at least one of the first and second chambers with excitation light to cause the dye to emit light, and detects the emission intensity and polarization degree of the portion irradiated with the excitation light, The specimen is placed in the first chamber manually or automatically. A dye-modified molecule having the ability to specifically bind to a target object contained in the sample and having a molecular weight greater than the fractional molecular weight of the dialysis membrane is manually or automatically placed in at least one of the first and second chambers. The aforementioned dye-modified molecule has a molecular weight smaller than the molecular weight of the dialysis membrane. Analyzer.

2. The reaction vessels include a first reaction vessel and a second reaction vessel, each having its interior divided into a first chamber and a second chamber by the dialysis membrane. The first reaction vessel and the second reaction vessel contain samples of different types and contain dye-modified molecules of the same type, The first reaction vessel and the second reaction vessel contain samples of the same type, and also contain dye-modified molecules of different types. The analytical apparatus according to claim 1.

3. The detection optical system is A first light source unit that irradiates the first reaction vessel with first excitation light to cause the first dye to emit light, A second light source unit that irradiates the second reaction vessel with a second excitation light that causes the second dye to emit light, A detection unit that individually detects the luminescence intensity and polarization degree in the first reaction vessel and the luminescence intensity and polarization degree in the second reaction vessel, Equipped with, The dye-modified molecule contained in the first reaction vessel is modified with the first dye, The dye-modified molecule contained in the second reaction vessel is modified with the second dye. The analytical apparatus according to claim 2.

4. The detection optical system is A light source unit that irradiates the first and second reaction vessels with excitation light to cause the dye to emit light, A first detection unit for detecting the luminescence intensity and polarization degree in the first reaction vessel, A second detection unit for detecting the luminescence intensity and polarization degree in the second reaction vessel, Equipped with, The analytical apparatus according to claim 2.

5. Of the first and second chambers of the reaction vessel, which contains the solution and is separated by a dialysis membrane, the sample is placed in the first chamber. At least one of the first and second chambers contains a dye-modified molecule that has the ability to specifically bind to a metering object contained in the sample and having a molecular weight greater than the molecular weight cutoff of the dialysis membrane, and has a molecular weight smaller than the molecular weight cutoff of the dialysis membrane. The first chamber and the second chamber are irradiated with excitation light to cause the dye to emit light, and the emission intensity and polarization degree of the portion of the first chamber and the second chamber irradiated with the excitation light are detected. Analysis method.

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