Material separation device, analyzer, and method for analysis

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]

[0001] The present disclosure relates to a material separation device, an analysis device, and an analysis method. [Background technology]

[0002] Immunoassay is an analytical method that utilizes an antibody-antigen reaction (see Non-Patent Documents 1 and 2). With immunoassay, an antibody that specifically binds to the object to be measured is used to detect the antigen with high sensitivity. Among them, the heterogeneous method, which separates the labeled antigen bound to the antibody (bound, B) from the free labeled antigen (free, F) (B / F separation), is known as a highly sensitive measurement method. However, a separate device is required to perform B / F separation, which makes the measurement system complicated, so a method that does not require B / F separation is desired. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 11-44688 Summary of the Invention [Problem to be solved by the invention]

[0004] In B / F separation, the solid-phase method is generally used, in which the antibody is immobilized and then washed. However, the solid-phase method has the following problems: (1) Reagents and surface treatments for fixation are required. (2) Antibody immobilization must be performed at a high density so that it can be distinguished from subsequent nonspecific binding. (3) The antibody fixation process takes time. (4) On the other hand, the state of antibody immobilization may vary depending on the target surface. (5) Measurement requires numerous steps, making it unsuitable for simple analysis.

[0005] On the other hand, dialysis has long been known as a B / F separation method that does not require washing. However, in the B / F separation method using dialysis, the molecular size of the antigen (measurement target) must be sufficiently small compared to the antibody. Therefore, this method is not suitable for accurately measuring proteins with large molecular weights, and the measurable measurement targets are limited.

[0006] The present invention has been made under the above circumstances, and an object of the present invention is to provide a substance separation apparatus, an analysis apparatus and an analysis method capable of accurately quantitatively measuring a measurement target having a large molecular weight. [Means for solving the problem]

[0007] In order to achieve the above object, a material separation device according to a first aspect of the present disclosure comprises: The reaction vessel includes an interior portion for accommodating a solution, the interior portion being divided into a first chamber and a second chamber by a dialysis membrane, A specimen is placed in the first chamber; A dye-modified molecule having a binding ability that specifically binds to a measurement target that is contained in the sample and has a molecular weight larger than the molecular weight cutoff of the dialysis membrane is contained in at least one of the first chamber and the second chamber; The dye-modified molecule has a molecular weight smaller than the molecular weight cutoff of the dialysis membrane.

[0008] An analysis apparatus according to a second aspect of the present disclosure, A material separation apparatus according to a first aspect; a detection optical system that irradiates at least one of a first chamber and a second chamber of a reaction vessel of the substance separation device with excitation light that causes a dye to emit light, and detects either the emission intensity of the portion irradiated with the excitation light or the emission intensity and the degree of polarization; Equipped with.

[0009] The analysis method according to the third aspect of the present disclosure includes: A reaction vessel contains a first chamber and a second chamber separated by a dialysis membrane, and a specimen is placed in the first chamber; At least one of the first chamber and the second chamber contains a dye-modified molecule that has a binding ability to specifically bind to a measurement target contained in the sample and has a molecular weight larger than the molecular weight cut-off of the dialysis membrane, and that has a molecular weight smaller than the molecular weight cut-off of the dialysis membrane. Effect of the Invention

[0010] According to the present disclosure, it is possible to accurately quantitatively measure a measurement target having a large molecular weight. [Brief description of the drawings]

[0011] [Figure 1] 1A is a perspective view showing a configuration of a reaction vessel of a substance separation device according to a first embodiment of the present disclosure, and FIG. 1B is a schematic diagram showing substances contained in the reaction vessel of FIG. [Diagram 2] 4 is a flowchart showing an analysis process according to the first embodiment of the present disclosure. [Diagram 3] FIG. 4 is a perspective view showing a modified example of the reaction vessel of the substance separation device according to the first embodiment of the present disclosure. [Figure 4] FIG. 11 is a schematic diagram showing a configuration of an analysis device according to a second embodiment of the present disclosure. [Diagram 5] 13 is a flowchart showing an analysis process according to a second embodiment of the present disclosure. [Figure 6] 13(A) and 13(B) are schematic diagrams showing examples of specimens and molecules that are put into a first reaction vessel and a second reaction vessel of a substance separation device according to a third embodiment of the present disclosure. [Figure 7] FIG. 11 is a schematic diagram showing a configuration of an analysis device according to a fourth embodiment of the present disclosure. [Figure 8] FIG. 11 is a schematic diagram showing a configuration of an analysis device according to a fifth embodiment of the present disclosure. [Figure 9] FIG. 13 is a schematic diagram showing a configuration of an analysis device according to a sixth embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The embodiments of the present disclosure will be described with reference to the drawings. Note that the present disclosure is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

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

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

[0015] As shown in FIG. 1(B), a sample S to be measured is contained in the first chamber 2a. The sample S contains an object to be measured T. Meanwhile, a 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 a binding ability to specifically bind with the object to be measured T.

[0016] The molecular weight of the analyte T is larger than the molecular weight cutoff (MWCO) of the dialysis membrane 4. Therefore, the analyte T remains contained in the first chamber 2a and does not move to the second chamber 2b. On the other hand, the dye-modified molecule M has a molecular weight smaller than the MWCO of the dialysis membrane 4. Therefore, the dye-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 analyte T. The dye-modified molecule M that has moved to the first chamber 2a binds to the analyte T.

[0017] When the concentration of the analyte T is high, many dye-modified molecules M bind to the analyte T in the first chamber 2a. The higher the concentration of the analyte T, the greater the number of dye-modified molecules M that bind to the analyte T, and the lower the concentration of the dye-modified molecules M. On the other hand, when the concentration of the analyte T is low, the fewer the number of dye-modified molecules M (bound type) that bind to the analyte T in the first chamber 2a, and the greater the number of dye-modified molecules M (free type) that are not bound to the analyte T.

[0018] In this way, the number of bound dye-modified molecules M and the number of free dye-modified molecules M vary depending on the concentration of the object to be measured T. Meanwhile, the emission intensity P1, etc. of the fluorescent dye F when the first chamber 2a or the second chamber 2b is irradiated with excitation light that causes the fluorescent dye F to emit light varies depending on the concentration of the object to be measured T in the first chamber 2a. Therefore, by detecting the emission intensity P1, etc., the concentration of the object to be measured T can be estimated.

[0019] The bound substance (complex) between the analyte T and the dye-modified molecule M cannot move to the second chamber 2b through the dialysis membrane 4 and remains in the first chamber 2a, while the free concentration becomes the same in the first chamber 2a and the second chamber 2b in an equilibrium state after a certain time has elapsed, so that the bound and free forms coexist in the first chamber 2a, as shown in Fig. 1(B). The concentration of the analyte T based on the emission intensity P1 must be estimated with this in mind.

[0020] [Measurement target, dye-modified molecule, and dialysis membrane] For example, a large molecule such as a protein can be selected as the measurement object T. Examples include ovalbumin and β-lactoglobulin. When the measurement object T is ovalbumin (molecular weight 43 kDa), for example, a VHH (variable domain of heavy chain of heavy chain antibody) antibody (molecular weight 15 kDa) can be selected as the dye-modified molecule M. In this case, the MWCO of the dialysis membrane 4 can be, for example, 20 kDa. When the measurement object T is β-lactoglobulin (molecular weight 18.3 kDa), an aptamer (for example, molecular weight 8 kDa) can be selected as the dye-modified molecule M. In this case, the MWCO of the dialysis membrane 4 can be, for example, 10 kDa. Also, a Fab antibody may be used as the dye-modified molecule M. In this way, the MWCO of the dialysis membrane 4 is determined based on the measurement object T and the dye-modified molecule M.

[0021] [Fluorescent dye] The fluorescent dye F is a dye that emits fluorescence when excited by excitation light. Each fluorescent dye F has its own fluorescence lifetime. In the present disclosure, a fluorescent dye F having a fluorescence lifetime of 1 to 10 nanoseconds, a fluorescent dye F having a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds, or a fluorescent dye F having a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds can be appropriately selected and used depending on the molecular weight of the dye-modified molecule M to be modified. For example, fluorescent dyes F having a fluorescence lifetime of 1 to 10 nanoseconds include fluorescein compounds such as indolenine, chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5 and 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and 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. 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 a fluorescence lifetime of more than 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. Fluorescent dyes F with a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds include metal complexes of platinum, rhenium, ruthenium, osmium, and europium.

[0022] To modify (label) molecule M with fluorescent dye F, for example, fluorescent dye F and molecule M may be directly covalently bonded to each other or may be bonded via a linker such as oligoethylene glycol or an alkyl chain. Fluorescent dye F has a functional group capable of binding to a carboxyl group, an amino group, a hydroxyl group, a thiol group, a phenyl group, or the like of a molecule. Molecule M can be labeled with fluorescent dye F by reacting the respective functional groups of fluorescent dye F and molecule M under known conditions. The number of molecules of fluorescent dye F modifying one molecule can be selected arbitrarily. It is preferably one or more molecules per molecule, and may be 2 to 5 molecules.

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

[0024] In the analysis process, as shown in Fig. 2, first, a specimen S is placed in the first chamber 2a (step S1). Then, dye-modified molecules M are placed in at least one of the first chamber 2a and the second chamber 2b (step S2). After that, a certain period of time is awaited (step S3; No). After the certain period of time has passed (step S3; Yes), the analysis method using the substance separation device 1 is terminated.

[0025] After a certain time has passed, the dye-modified molecule M (bound type) bound to the object to be measured T remains in the first chamber 2a and does not move into the second chamber 2b in the reaction vessel 2. 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 in the measurement cell or the emission intensity P1 and the polarization degree P2, it becomes possible to measure the concentration of the object to be measured T, as described below.

[0026] As described in detail above, the substance separation device 1 of this embodiment uses a dialysis membrane 4 having a molecular weight cut-off (MWCO) smaller than the molecular weight of the object to be measured T and larger than the molecular weight of the dye-modified molecule M that specifically binds to the object to be measured T, and can retain the binding type in which the object to be measured T and the dye-modified molecule M are bound in the first chamber 2a.Therefore, by detecting the emission intensity P1 and the polarization degree P2 of the fluorescent dye F in at least one of the first chamber 2a and the second chamber 2b, the object to be measured T, which has a large molecular weight, can be accurately quantitatively measured.

[0027] By using the analysis device 5 according to the present embodiment, for example, B / F separation in an immunoassay in which the measurement object T is an antigen and the dye-modified molecule M is an antibody can be performed. 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 the solution L from the reaction vessel 2 to the measurement cell, and prevents contamination of the specimen S. In this case, the material of the reaction vessel 2 must be one that transmits the excitation light and the fluorescence.

[0029] The reaction vessel 2 serving as the measurement cell can be configured as shown in FIG. 3. The reaction vessel 2 shown in FIG. 3 has a configuration in which a tubular second chamber 2b is inserted into a rectangular parallelepiped first chamber 2a. A tubular dialysis membrane 4 (dialysis tube) is formed on the wall of the second chamber 2b. The object to be measured T is placed in the first chamber 2a, and the dye-modified molecule M is placed in the second chamber 2b. The dye-modified molecule M passes through the dialysis membrane 4 (dialysis tube) and enters the first chamber 2a, and binds to the object 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 analysis device 5 including the reaction container 2 according to the above-mentioned embodiment 1 as a measurement cell will be described. As shown in Fig. 4, the analysis device 5 according to this embodiment includes the reaction container 2 according to the above-mentioned embodiment 1 and a detection optical system 10. As the reaction container 2, the one having the configuration shown in Fig. 1(A) may be used, or the one having the configuration shown in Fig. 3 may be used.

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

[0032] The detection optical system 10 comprises a light source unit 11, an optical fiber 12, a lens 50, a reflector 13 arranged obliquely (hereinafter simply referred to as "obliquely") with respect to the optical axis direction of the emission 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 includes 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 passes through the excitation filter 21 that adjusts the wavelength of the excitation light IL and the coupling lens 22, and enters the optical fiber 12.

[0034] The optical fiber 12 sends the 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 the focusing point. The reflecting mirror 13 is positioned at an angle and diffusely reflects the excitation light IL that has passed through the reaction vessel 2. The fluorescence EL generated by 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 includes a coupling lens 25, a fluorescent filter 26, a liquid crystal element 27, and an imaging unit 28. The coupling lens 25 sends the light emitted from the optical fiber 14 to the fluorescent filter 26. The fluorescent filter 26 passes light in the wavelength band of the fluorescent EL generated by the emission of the fluorescent dye F and blocks 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 degree of polarization P2.

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

[0037] [Fluorescence emission intensity] When detecting the emission intensity P1 of the fluorescent EL, the liquid crystal element 27 is adjusted so that all polarized components of the incident fluorescent EL are transmitted. This allows the imaging unit 28 to receive all the light beams of 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-modified molecule M is prepared in advance, and the analysis device 5 measures the concentration of the dye-modified molecule M corresponding to the emission intensity P1 based on the calibration curve. The calibration curve is prepared by detecting the emission intensity P1 of the fluorescent EL in the first chamber 2a or the second chamber 2b for a combination of the measurement object T, the concentration of which is known, and the dye-modified molecule M while changing their concentrations.

[0038] [Fluorescence polarization] When detecting the degree of polarization P2 of the fluorescence EL, the excitation light IL emitted from the light source unit 11 is linearly polarized. Of the received fluorescence EL, the degree of polarization parallel to the excitation light IL is designated as I1, and the degree of polarization perpendicular to the excitation light IL is designated as I2. The detection optical system 10 controls the liquid crystal element 27 of the detection unit 15 to detect the two fluorescence polarization intensities I1 and I2, and calculates the degree of polarization P2 using the following equation. P2 = (I1 - I2) / (I1 + I2) The polarization degree P2 indicates the degree of rotation of the dye-modified molecule M from excitation to emission of the fluorescence EL. Molecules with small molecular weight rotate vigorously in the solution due to Brownian motion, so the polarization degree P2 is low, and molecules with large molecular weight have weak Brownian motion, so the polarization degree P2 increases. Therefore, if the concentration of the object to be measured T is high, the polarization degree P2 increases, and if the concentration is low, the polarization degree P2 decreases. A calibration curve showing the relationship between the polarization degree P2 and the concentration of the object to be measured T is obtained in advance. The analysis device 5 measures the concentration of the object to be measured T corresponding to the polarization degree P2 based on this calibration curve. This calibration curve is also created by detecting the polarization degree P2 of the fluorescence EL in the first chamber 2a or the second chamber 2b for a combination of the object to be measured T and the dye-modified molecule M, the concentrations of which are known, while changing their concentrations.

[0039] Incidentally, the MWCO of the dialysis membrane 4 is smaller than the molecular weight of the analyte T, but the MWCO is a statistical value, and the size of the pores in the dialysis membrane 4 varies. For this reason, a small amount of the analyte T may pass through the dialysis membrane 4 and move to the second chamber 2b. In this case, the analysis device 5 may measure, for example, both the emission intensity P1 of the fluorescence EL and the polarization degree P2. For example, the emission intensity P1 of the fluorescence EL in the second chamber 2b reflects both the dye-modified molecule M (bound type) that is bound to the analyte T and the dye-modified molecule M (free type) that is not bound to the analyte T. Furthermore, the polarization degree P2 of the fluorescence EL in the second chamber 2b reflects the ratio of the concentration of the bound type to the concentration of the free type. Therefore, by estimating the total concentration of the dye-modified molecule M based on the emission intensity P1 and estimating the ratio of the bound type to the free type based on the polarization degree P2, it becomes possible to measure the concentration of the analyte T that is bound to the dye-modified molecule M with higher accuracy. In order to estimate the ratio between the bound and free forms, it is necessary to know the amount of dye-modified molecule M that is added.

[0040] The analyzer 5 may irradiate the second chamber 2b with the excitation light IL, detect either the emission intensity P1 of the fluorescence EL in the second chamber 2b, or the emission intensity P1 and the polarization degree P2, and measure the concentration of the object T. It is also possible to estimate the concentration of the object T based on both the detection result of the first chamber 2a and the detection result of the second chamber 2b. The first chamber 2a and the second chamber 2b are separated by the dialysis membrane 4, and in addition to this, a light-shielding layer may be disposed 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, making it possible to monitor the fluorescent signal of each chamber with higher accuracy.

[0041] Next, an analysis method using the analysis device 5, that is, an analysis process, will be described.

[0042] As shown in Fig. 5, the analysis process according to this embodiment includes steps S1 to S3, which are the same as the analysis process described above (see Fig. 2). After a certain time has elapsed (step S3; Yes), at least one of the first chamber 2a and the second chamber 2b of the reaction vessel 2 is irradiated with excitation light IL that causes the fluorescent dye F to emit light, and either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL from the portion of the first chamber 2a and the second chamber 2b irradiated with the excitation light IL is detected (step S4). After that, the concentration of the measurement target T is obtained from this detection result based on various calibration curves. After step S4 is completed, the analysis device 5 ends the analysis 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 Fig. 6(A) and Fig. 6(B), the substance separation device 1 according to this embodiment includes a reaction vessel 2 including a first reaction vessel 2A and a second reaction vessel 2B. In this case, for example, as shown in Fig. 6(A), a specimen S1 can be placed in the first chamber 2a of the first reaction vessel 2A, and a dye-modified molecule M1 that binds to a measurement target T1 can be placed in at least one of the first chamber 2a and the second chamber 2b. Furthermore, a specimen 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 time has elapsed since the introduction, at least one of the first chamber 2a and the second chamber 2b of the first reaction vessel 2A is irradiated with excitation light IL that causes the fluorescent dye F to emit light, and either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the portion of the first chamber 2a and the second chamber 2b irradiated with the excitation light IL is detected, and at least one of the first chamber 2a and the second chamber 2b of the second reaction vessel 2B is irradiated with excitation light IL that causes the fluorescent dye F to emit light, and either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the portion of the first chamber 2a and the second chamber 2b irradiated with the excitation light IL is detected, whereby the concentrations of the measurement targets T of the different types of specimens S1 and S2 can be measured at once. This makes it possible to compare the concentrations of the measurement targets T between the specimens S1 and S2.

[0045] Also, for example, in the case shown in FIG. 6(B), the specimen S1 can be placed in the first chamber 2a of the first reaction vessel 2A, the dye-modified molecule M1 that binds to the measurement target T1 can be placed in at least one of the first chamber 2a and the second chamber 2b, and the specimen S1 can be placed in the first chamber 2a of the second reaction vessel 2B, and the dye-modified molecule M2 that binds to the measurement target T2 can be placed in at least one of the first chamber 2a and the second chamber 2b. After a certain time has elapsed since the placement, the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the first reaction vessel 2A can be detected by irradiating the excitation light IL in the same manner as in FIG. 6(A), and the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the second reaction vessel 2B can be detected, so that the concentrations of the measurement targets T1 and T2 of different types in the specimen S1 can be measured at once. This allows the concentrations of the measurement targets T1 and T2 in the same type of specimen S1 to be compared.

[0046] In this way, by preparing a plurality of reaction vessels 2A, 2B, it becomes possible to simultaneously measure the concentrations of the analyte T in different combinations of the analyte T and the dye-modified molecules M.

[0047] (Embodiment 4) 7, the analysis device 5 according to this embodiment includes a detection optical system 10 that can simultaneously detect either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the first reaction container 2A, and either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the second reaction container 2B. 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 the configuration of the light source unit 11 in FIG. 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 that 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 a first excitation light IL1. Therefore, the dye-modified molecule M that binds to the measurement 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. 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 a second excitation light IL2. Therefore, the dye-modified molecule M that binds to the measurement 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 reflecting mirror 13A arranged at an angle, a reflecting mirror 13B arranged at an angle in the same manner, 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 transmitted through the first reaction vessel 2A. Similarly, the reflecting mirror 13B diffusely reflects the second excitation light IL2 transmitted through the second reaction vessel 2B. Meanwhile, the fluorescence EL1 from the dye-modified molecule M in the first chamber 2a of the first reaction vessel 2A is collected by the lens 50 into the optical fiber 14A, and the optical fiber 14A sends the light including the fluorescence EL1 generated in the first chamber 2a of the first reaction vessel 2A to the detection unit 15. Similarly, the fluorescence EL2 from the dye-modified molecule M in the first chamber 2a of the second reaction vessel 2B is collected by the lens 50 into the optical fiber 14B, and the optical fiber 14B sends the light including the fluorescence EL2 generated in the first chamber 2a of the second reaction vessel 2B to the detection unit 15.

[0051] The detection unit 15 detects either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL1 in the first chamber 2a of the first reaction vessel 2A, and either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL2 in the first chamber 2a of the second reaction vessel 2B, individually. The control unit 16 controls the first light source unit 11A to emit the first excitation light IL1 and the second light source unit 11B to stop emitting the second excitation light IL2 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 light source unit 11A to stop emitting the first excitation light IL1 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, in the detection unit 15, at the first sampling time, either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL1 in the first chamber 2a of the first reaction container 2A is acquired, and at the second sampling time, either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL2 in the first chamber 2a of the second reaction container 2B is acquired.

[0052] The detection unit 15 may include a dichroic mirror that separates the fluorescence EL1 from the fluorescence EL2, an image sensor that detects the intensity of the fluorescence EL1, and an image sensor that detects the intensity of the fluorescence EL2. In this way, the fluorescence EL1 and the fluorescence EL2 are separated by the dichroic mirror, and the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the first reaction container 2A and the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the second reaction container 2B can be detected individually by the respective image sensors.

[0053] As in the third embodiment, the emission intensity P1 and the 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. The first chamber 2a and the second chamber 2b are separated by the dialysis membrane 4, and in addition to this, a light-shielding layer may 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, making it possible to monitor the fluorescent signal of each chamber with higher accuracy.

[0054] The combination of the measurement target T and the dye-modified molecule M contained in the first reaction container 2A and the second reaction container 2B can be a combination of the measurement target T1 and the dye-modified molecule M1 as shown in Fig. 6(A). In addition, these combinations can also be a combination of the measurement targets T1, T2 and the dye-modified molecules M1, M2 as shown in Fig. 6(B).

[0055] In the analysis device 5 according to the present embodiment, the first reaction container 2A and the second reaction container 2B share the detection unit 15. This makes it possible to reduce the size of the analysis device 5 and the manufacturing costs.

[0056] (Embodiment 5) 8, the analysis device 5 according to this embodiment includes a first reaction container 2A and a second reaction container 2B as the reaction containers 2. The analysis device 5 includes a detection optical system 10 that individually detects either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the first reaction container 2A, and either the emission intensity P1 or the emission intensity P1 and the polarization degree P2 of the fluorescence EL in the second reaction container 2B.

[0057] The detection optical system 10 includes a light source unit 11, optical fibers 12A and 12B, reflecting mirrors 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 and the lens 50, while irradiating the first chamber 2a of the second reaction vessel 2B with excitation light IL via the optical fiber 12B and the lens 50. The excitation light IL causes the dye-modified molecules M in the first chamber 2a of the first reaction vessel 2A and the second reaction vessel 2B to emit light near the focusing position. The excitation light IL that passes through the first chamber 2a of the first reaction vessel 2A is diffusely reflected in an oblique direction by the reflecting mirror 13A, and the excitation light IL that passes through the first chamber 2a of the second reaction vessel 2B is diffusely reflected in an oblique direction by the reflecting mirror 13B.

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

[0060] The combination of the measurement target T and the dye-modified molecule M contained in the first reaction container 2A and the second reaction container 2B can be a combination of the measurement target T1 and the dye-modified molecule M1 as shown in Fig. 6(A). In addition, these combinations can also be a combination of the measurement targets T1, T2 and the dye-modified molecules M1, M2 as shown in Fig. 6(B).

[0061] In the analysis device 5 according to this embodiment, the first reaction container 2A and the second reaction container 2B share the light source unit 11. 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 analyzer 5 according to this embodiment differs from the above-described embodiments 4 and 5 in the configuration of the detection optical system 10. As shown in FIG. 9, the detection optical system 10 includes 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 configured by bundling a plurality of optical fibers 17a to 17g. Of these, one end of the central optical fiber 17a is connected to the light source unit 11, and one end of the peripheral optical fibers 17b to 17g are connected to the detection unit 15. Meanwhile, the other ends of the optical fibers 17a to 17g are connected to the solution probe 18. Note that the optical fiber 17a may have one end that is bifurcated and connected to both the light source unit 11 and the detection unit 15.

[0064] The solution probe 18 includes a collimator lens 30, a sample opening 31, and a reflecting mirror 32 arranged at an angle. The collimator lens 30 focuses the excitation light IL emitted from the optical fiber 17a and causes it to enter the sample opening 31. The sample opening 31 is configured to allow the measurement object T and the dye-modified molecule M to enter from the outside. The measurement object T and the dye-modified molecule M that enter the sample opening 31 emit light due to the focused excitation light IL, generating fluorescence EL. The reflecting mirror 32 diffusely reflects the excitation light IL. The solution probe 18 may be covered with a light-shielding tube. The inside of the light-shielding tube is painted black to prevent stray light from the outside from entering the solution probe 18. By covering the solution probe 18 with this light-shielding tube, it becomes possible to monitor the fluorescent signal with high accuracy without being affected by the state of other rooms.

[0065] The fluorescence 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 fluorescence EL in the first reaction container 2A, or the emission intensity P1 and the degree of polarization P2.

[0066] In this way, detection can be easily performed by using the detection optical system 10 equipped with the optical fiber bundle 17 and the solution probe 18 integrated with the collimator lens 30, the reflector 32, etc., thereby making it possible to improve the efficiency of the detection work and shorten the detection time.

[0067] Although the optical fiber bundle 17 is formed by bundling seven optical fibers 17a to 17g, the number of optical fibers to be bundled may be any number. Also, the excitation light IL may be transmitted through a plurality of optical fibers.

[0068] In the above embodiment, a first reaction container 2A and a second reaction container 2B are provided as the reaction containers 2. However, there may be three or more reaction containers 2. In this way, it becomes possible to detect the same type of measurement target T contained in three or more specimens, or to detect three or more types of measurement target T contained in a specimen S.

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

[0070] The above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not by the embodiment. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Explanation of symbols]

[0071] 1 substance separation device, 2 reaction vessel, 2A first reaction vessel, 2B second reaction vessel, 2a first chamber, 2b second chamber, 3 partition wall, 4 dialysis membrane, 5 analysis device, 10 detection optical system, 11 light source section, 11A first light source section, 11B second light source section, 12, 12A, 12B optical fiber, 13, 13A, 13B reflector, 14, 14A, 14B optical fiber, 15 detection section, 15A first detection section, 15B second detection section, 16 control section, 17 optical fiber bundle, 17a to 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 section, 30 collimator lens, 31 sample opening section, 32 reflector, 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 molecules, S, S1, S2 specimen, T, T1, T2 measurement target

Claims

1. A reaction vessel is provided with an interior portion for accommodating a solution, the interior portion being divided into a first chamber and a second chamber by a dialysis membrane, A specimen is contained in the first chamber; a dye-modified molecule having a binding ability to specifically bind to a measurement target substance contained in the sample and having a molecular weight larger than the molecular weight cutoff of the dialysis membrane is contained in at least one of the first chamber and the second chamber; The dye-modified molecule has a molecular weight smaller than the molecular weight cutoff of the dialysis membrane. Material separation equipment.

2. the reaction vessels include a first reaction vessel and a second reaction vessel, the interior of each of which is partitioned into the first chamber and the second chamber by the dialysis membrane; The first reaction vessel and the second reaction vessel contain different types of specimens and the same type of dye-modified molecule, The first reaction vessel and the second reaction vessel contain the same type of specimen and different types of dye-modified molecules.

2. The material separation device of claim 1.

3. A material separation device according to claim 1; a detection optical system that irradiates at least one of a first chamber and a second chamber of a reaction vessel of the substance separation device with excitation light that causes a dye to emit light, and detects either the emission intensity of the portion irradiated with the excitation light or the emission intensity and the degree of polarization of the portion irradiated with the excitation light; An analytical device comprising:

4. the detection optical system detects the luminescence intensity and the degree of polarization of the portion irradiated with the excitation light. The analytical device according to claim 3 .

5. A material separation device according to claim 2; a first light source unit that irradiates a first reaction container of the substance separation device with first excitation light that causes a first dye to emit light; a second light source unit that irradiates a second reaction container of the substance separation device with second excitation light that causes a second dye to emit light; a detection unit that detects either the luminescence intensity in the first reaction vessel or the luminescence intensity and the degree of polarization in the first reaction vessel, and either the luminescence intensity in the second reaction vessel or the luminescence intensity and the degree of polarization in the second reaction vessel, individually; 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. Analyzer.

6. A material separation device according to claim 2; a light source unit that irradiates a first reaction container and a second reaction container of the substance separation device with excitation light that causes a dye to emit light; a first detection unit that detects either the luminescence intensity in the first reaction vessel or the luminescence intensity and the degree of polarization in the first reaction vessel; a second detection unit that detects either the luminescence intensity in the second reaction vessel or the luminescence intensity and the degree of polarization in the second reaction vessel; An analytical device comprising:

7. A reaction vessel contains a first chamber and a second chamber separated by a dialysis membrane, and a specimen is contained in the first chamber; At least one of the first chamber and the second chamber contains a dye-modified molecule having a binding ability to specifically bind to a measurement target contained in the sample and having a molecular weight larger than the molecular weight cutoff of the dialysis membrane, and having a molecular weight smaller than the molecular weight cutoff of the dialysis membrane. Analysis method.

8. The method further includes irradiating at least one of the first chamber and the second chamber with excitation light that causes a dye to emit light, and detecting either the emission intensity of a portion of the first chamber and the second chamber that is irradiated with the excitation light, or the emission intensity and the degree of polarization of the portion of the first chamber and the second chamber that is irradiated with the excitation light. The analytical method according to claim 7.

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