JP2013238420A - Sample analysis apparatus and sample analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample analysis apparatus and a sample analysis method which are capable of analysis excellent in sensitivity and repeatability by using a small amount of magnetic particles.SOLUTION: A flat substrate is used as a detection part. An image of a small amount of magnetic particles adsorbed in the detection part is taken every cycle of an analysis, and a light emission amount of the small amount of magnetic particles is acquired. A quantitative analysis of a substance to be analyzed is performed on the basis of an average light emission amount per one of the magnetic particles.

Description

  The present invention relates to a sample analyzer and a sample analysis method, and more particularly to a sample analyzer and a sample analysis method for analyzing an analysis sample derived from a biological fluid such as serum and urine.

  First, immunoassay will be described as an example of sample analysis. Immunoassay refers to the quantitative analysis of antibodies and antigens contained in samples derived from biological fluids such as serum and urine using specific binding between antigens and antibodies. Based on the results of immunoassays To diagnose the disease.

  As a typical immunoassay method, there is ELISA (Enzyme-Linked ImmunoSorbent Assay). In ELISA, an antibody (first antibody) against the antigen that is the analyte is immobilized on the bottom of a plastic container, and an analytical sample such as serum is placed in this container, and the antigen contained in the analytical sample is bound to the first antibody. Let Further, the antigen (second antibody) bound to the labeling substance is further bound to the antigen bound to the first antibody, and the antigen is quantitatively analyzed by detecting a signal emitted from the labeling substance. As a labeling substance, for example, a fluorescent substance is used. In this case, the fluorescence increases or decreases depending on the concentration of the second antibody bound to the antigen among the second antibodies bound to the labeling substance, and the antigen in the analysis sample is quantified by detecting this fluorescence with a photodetector. Can do.

  As a specific example of the immunoassay device, magnetic particles are used as the solid phase, and the first antibody is immobilized on the surface of the magnetic particles. A luminescent labeling substance is bound to the second antibody as a labeling substance. When the analyte (antigen) contained in the analysis sample is mixed with the magnetic particles on which the first antibody is immobilized, the antigen is bound to the magnetic particles via the first antibody. Further, when the second antibody is reacted, the luminescent labeling substance is bound to the magnetic particles via the second antibody, the antigen, and the first antibody. The concentration of the luminescent labeling substance that binds to the magnetic particles increases or decreases depending on the concentration of the antigen contained in the analysis sample.

  After the magnetic particles bound with the antigen are magnetically adsorbed at a predetermined position of the sample analyzer, the luminescent labeling substance bound to the magnetic particles is caused to emit light by applying a laser beam or the like. By detecting this luminescence and measuring the amount of luminescence, the antigen contained in the analysis sample can be quantitatively analyzed.

  In order to perform immunoassay with high sensitivity and reproducibility, magnetic particles bound with the antigen to be analyzed are magnetically adsorbed and captured at a predetermined position using a magnet or the like, and bound to the antigen during that time. Replace the solution containing the second antibody that is not. This operation is called B / F separation (Bound / Free separation).

In the above method, there is no restriction on the number of magnetic particles used for one analysis, but generally 10 ⁵ to 10 ⁶ particles are used per analysis. However, in order to reduce the analysis cost, a technique is required for quantitative analysis of a lower concentration analyte substance using a smaller number of magnetic particles. There is Patent Document 1 as a sample analysis method using a small number of magnetic particles.

Japanese Patent No. 4790026

  In the sample analysis method disclosed in Patent Document 1, a substrate having a large number of minute openings is used as the detection unit. Fine particles combined with the substance to be analyzed are filled in a minute opening on the substrate. Then, a light emission image of the fine particles filled one by one in the opening on the substrate is acquired. Here, when the magnetic particle is adsorbed to the minute opening of the detection unit using the detection unit disclosed in Patent Document 1, the solution remains in the opening when the solution is exchanged during the B / F separation. Thus, sufficient B / F separation could not be achieved. In addition, after one analysis cycle is completed, when the analyzed sample is discharged from the detection unit and the detection unit is cleaned, the magnetic particles and the solution remain in the opening, and the detection unit is sufficiently cleaned. Could not be achieved.

  An object of the present invention is to provide a sample analyzer and a sample analysis method capable of performing analysis with high sensitivity and reproducibility.

  In the present invention, a flat substrate is used as the particle capturing unit, and the analysis sample is continuously flowed to the particle capturing unit, whereby the same particle capturing unit is repeatedly used for different samples. More specifically, a sample solution containing magnetic particles is flowed into the flow channel, and the magnetic particles are captured by a magnet that is close to the particle capturing unit set in the flow channel. Therefore, sufficient B / F separation can be performed during the analysis cycle as compared with a substrate having a fine opening. Moreover, the particle | grain capture | acquisition part can fully be wash | cleaned after completion | finish of an analysis cycle.

  On the other hand, since the particle trapping part is a flat substrate, the magnetic adsorption position of each magnetic particle on the substrate is random. Therefore, an imaging unit that captures a bright field image of a small number of magnetic particles magnetically adsorbed on the particle trapping unit and a light detection unit that detects light emission of the small amount of magnetic particles are installed. From the bright field image of a small number of magnetic particles, the number of magnetic particles magnetically adsorbed on the detection unit is calculated, and the average light emission amount per magnetic particle is calculated using this value and the light emission amount of the minority particles. Quantitative analysis. Fluorescence or chemiluminescence can be used for light emission.

  According to the present invention, improvement in B / F separation efficiency, improvement in washing efficiency, improvement in measurement accuracy, improvement in reproducibility of measurement results, improvement in analysis sensitivity, and the like can be expected.

  Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

Schematic which shows an example of the sample analyzer by this invention. The figure which shows the particle | grain adsorption position in a flow path, and the positional relationship of a mirror. The figure which shows the particle | grain adsorption position in a flow path, and the positional relationship of a mirror. The figure which shows the particle | grain adsorption position in a flow path, and the positional relationship of a mirror. The figure which shows the particle | grain adsorption position in a flow path, and the positional relationship of a mirror. Schematic which shows the other example of the sample analyzer by this invention. Schematic which shows the other example of the sample analyzer by this invention. The figure which shows the relationship between an adsorbed particle density and the emitted light amount in a particle | grain adsorption position.

Embodiments of the present invention will be described below with reference to the drawings.
As an example of the sample analyzer, an immune analyzer using magnetic particles will be described. The present invention is not limited to immunological analyzers, but can be applied to any sample analyzer based on the principle of capturing magnetic particles by switching magnetic field strength using magnetic particles. For example, DNA sequencing devices, It can also be applied to chemical analyzers.

  FIG. 1 is a schematic diagram showing an example of a sample analyzer according to the present invention. In FIG. 1, the flow path 15 is connected to a sipper nozzle 24 and a pump 25 through a tube 21 and a tube 22. The sipper nozzle 24 can be moved by an arm 26, and a sample liquid container 32, a cleaning liquid container 33, and a buffer liquid container 40 are installed within the moving range.

  A valve 30 is provided in the tube 22 between the flow path 15 and the pump 25. The pump 25 is controlled by a controller 37 and can suck and discharge a predetermined amount of solution. The tube 22 further continues to the waste liquid container 34 via the tube 23.

  The flow path wall 14 of the flow cell serving as the detection unit is made of a transparent material in the visible light region, and a flow path 15 through which the solution flows is formed therein. Since the flow path wall transmits visible light, the flow state of the solution in the flow path can be observed from the outside. Instead of manufacturing the entire flow path wall 14 with a transparent material, a transparent material may be used only for a portion that transmits light.

  The flow cell wall is preferably made of a material that is substantially transparent to the wavelength of light emitted by the luminescent labeling substance bound to the surface of the magnetic particles adsorbed at the particle adsorption position in the flow cell. For example, it is preferably made of glass, quartz, plastic or the like.

  It is desirable that the flow path 15 is manufactured so that the width is 2 to 20 times the depth (that is, the thickness). As a result, the magnetic particles contained in the sample liquid introduced into the flow cell are easily spread in the width direction of the flow path, and the magnetic particles are easily adsorbed uniformly in the plane of the particle adsorption position 13.

  A laser light source 18 and a condenser lens 17 are installed around the lower portion of the flow path 15. The laser light emitted from the laser light source 18 is condensed by the condenser lens 17 and irradiated to the particle adsorption position 13 in the flow path 15.

  A magnet 11 is used as a magnetic field application means for magnetic adsorption of magnetic particles. When magnetically attracting magnetic particles to the particle adsorption position 13, the magnet 11 is moved directly below the flow path 15. For example, the magnet 11 is installed in a slide mechanism 16 that can move freely in the horizontal direction, and when the magnetic particles are magnetically adsorbed, the magnet 11 is moved directly below the flow path 15. When cleaning the inside of the flow path 15, the magnet 11 is moved to a position where the influence of the magnetic field on the magnetic particles can be sufficiently reduced. Instead of moving the magnet 11 in the horizontal direction with respect to the flow path using the slide mechanism 16, the magnet 11 may be moved in the vertical direction with respect to the flow path.

  The particle adsorption position 13 installed in the flow path of the flow cell is a flat surface that is difficult to get dirty, such as gold, platinum, carbon, etc., considering that the magnet 11 approaches the lower side and magnetic particles are magnetically adsorbed on the upper side. It is preferable to manufacture with a non-magnetic material capable of forming.

  The controller 37 includes an arm 26, a light detector 39, an imaging device 41, a mirror drive mechanism 42, a slide mechanism 16, a laser light source 18, an illumination 45, a pump 25, valves 30, 31, a data storage unit 48, and signal lines 38a to 38e. Are connected to each other and can be controlled.

  The magnetic particles 10 are magnetically attracted to the surface of the particle adsorption position 13 in the flow path 15 by the magnetic force generated by the magnet 11. When measuring the luminescence of the luminescent labeling substance bonded to the surface of the magnetic particle, the magnet 11 is moved away from the flow path 15 by the slide mechanism 16 so that no magnetic force acts on the magnetic particle 10 adsorbed at the particle adsorption position 13. At this time, the flow of the liquid in the flow path 15 is stopped, and the magnetic particles 10 are retained in the flow path 15 while being adsorbed at the particle adsorption positions 13. Here, by irradiating the magnetic particles 10 with laser light from a laser light source 18 installed in the lower part of the flow path 15, the luminescent labeling substance on the surface of the magnetic particles is caused to emit light. The amount of light emitted from this luminescent labeling substance is detected by the photodetector 39.

  The shape of the magnet 11 is basically a rectangular parallelepiped, but may be another shape such as a cylinder or a prism. As a method for arranging the magnets, either a method in which the N pole and the S pole are arranged in a horizontal direction and substantially parallel to the flow cell, or a method in which the N pole and the S pole are arranged in a vertical direction and arranged substantially perpendicular to the flow cell is desirable. The magnet can be a permanent magnet or an electromagnet.

  The adsorption distribution of the magnetic particles 10 in the flow path 15 includes two types of forces acting on the magnetic particles, that is, the magnetic force received from the magnetic field of the magnet 11 disposed on the lower side of the flow path 15, and a sample containing the magnetic particles. It is determined by the drag received from the flow field when the liquid is introduced into the flow cell. When the drag force received by the magnetic particles from the flow field is greater than the magnetic force received from the magnetic field, the magnetic particles are not magnetically attracted to the particle adsorption position 13 and are discharged out of the flow path together with the sample liquid. Accordingly, it is necessary to optimize the flow rate of the solution. For example, the magnitude of the magnetic field in the flow path 15 is preferably 0.1 to 0.5T. Moreover, the flow rate of the liquid at that time is preferably 0.05 to 0.10 m / s.

  The light detector 39 may be any device that can detect light emitted from the luminescent labeling substance on the surface of the magnetic particles. For example, a CCD camera, a photomultiplier tube, a photomultiplexer, or the like is preferably used. The imaging device 41 may be any device that can acquire a bright field image of the magnetic particles 10 through the objective lens 44, and for example, a CCD camera is preferably used.

  An objective lens 44, a mirror 43, and a mirror driving mechanism 42 are installed around the upper side of the flow path 15. The mirror driving mechanism 42 has a function of adjusting the holding angle of the mirror 43. When the holding angle of the mirror 43 is held at 45 degrees with respect to the flow path 15 by the mirror driving mechanism 42, light emitted from the magnetic particles 10 at the particle adsorption position 13 in the flow cell passes through the objective lens 44 and the mirror 43. An optical path to the photodetector 39 is set. On the other hand, when the angle of the mirror 43 is maintained at 90 degrees with respect to the flow path 15 by the mirror driving mechanism 42, the light emitted from the magnetic particles 10 at the particle adsorption position 13 is transmitted to the imaging device 41 via the objective lens 44. The optical path to reach is set.

  The mirror driving mechanism 42 may have a function of translating the mirror 43 with respect to the flow path 15 in addition to the function of adjusting the holding angle of the mirror 43 as described above. When the mirror 43 is moved parallel to the flow path 15 by the mirror drive mechanism 42 while the angle of the mirror 43 is held at 45 degrees with respect to the flow path 15, and the mirror 43 is installed above the objective lens 44, An optical path through which light emitted from the magnetic particle 10 at the particle adsorption position 13 reaches the photodetector 39 via the objective lens 44 and the mirror 43 is set. Further, the mirror 43 is moved parallel to the flow path 15 by the mirror drive mechanism 42 while the angle of the mirror 43 is maintained at 45 degrees with respect to the flow path 15, and the mirror 43 is installed at a position away from the objective lens 44. In this case, an optical path from which the light from the magnetic particle 10 at the particle adsorption position 13 reaches the imaging device 41 via the objective lens 44 is set.

  Note that the installation position of the photodetector 39 and the installation position of the imaging device 41 may be exchanged. That is, the photodetector 39 may be disposed at the position of the imaging device 41 illustrated in FIG. 1, and the imaging device 41 may be disposed at the position of the photodetector 39.

  A cleaning liquid for cleaning the inside of the tube 21, the flow path 15, and the tube 22 is accommodated in the cleaning liquid container 33. The sample solution container 32 contains a sample solution obtained by mixing an analysis sample with a magnetic particle solution and a predetermined reagent and reacting the sample at a constant temperature (for example, 37 ° C.) for a predetermined time. A buffer solution for filling the inside of the tube 21, the flow path 15, and the tube 22 is accommodated in the buffer solution container 40.

  Next, the analysis sample, magnetic particles, magnetic particle solution, luminescent labeling substance, and sample solution will be described.

  An analysis sample is a sample derived from a biological fluid such as serum or urine. When the analysis sample is serum, the substance to be analyzed is a tumor marker, a thyroid-related substance, a hormone, a myocardial marker, an infectious disease-related substance, or the like.

  The magnetic particles are, for example, particles obtained by coating iron oxide particles exhibiting ferromagnetism with a polymer. The particle size of the magnetic particles is preferably in the range of 0.01 μm (micrometer) to 200 μm (micrometer), and more preferably in the range of 1 μm to 10 μm. The specific gravity is preferably in the range of 1.3 to 1.5. A substance having the property of specifically binding the substance to be analyzed, for example, an antibody having a property of specifically binding to an antigen, is bound to the surface of the magnetic particle.

  The magnetic particle solution is a solution in which the above magnetic particles are uniformly dispersed in a buffer solution. As the buffer solution, phosphate buffer solution, HEPES buffer solution, PIPES buffer solution, glycine buffer solution, Tris buffer solution and the like can be used.

The luminescent labeling substance is preferably the following substance.
(1) A labeling substance used in fluorescence immunoassay. For example, an antibody labeled with fluorescein isothiocyanate.
(2) A labeling substance used in chemiluminescence immunoassay. For example, an antibody labeled with an acridinium ester.
(3) A labeling substance used in chemiluminescent enzyme immunoassay. For example, an antibody labeled with a chemiluminescent enzyme using luminol or an adamantyl derivative as a luminescent substrate.

  The luminescent labeling substance is specifically bound to the substance to be analyzed by an appropriate means, and light is emitted by an appropriate means.

  The sample solution is obtained by mixing an analysis sample containing a substance to be analyzed with a magnetic particle solution and a luminescent labeling substance and reacting them at a constant temperature for a fixed time.

Next, an analysis cycle of the sample analyzer disclosed in the present invention will be described.
Prior to the start of the analysis cycle, an analysis sample containing the substance to be analyzed, a magnetic particle solution, and a luminescent labeling substance are mixed in advance in the reaction unit 36 and reacted at a constant temperature for a predetermined time to prepare a sample liquid. . One cycle of analysis starts when the sample solution container 32 containing the sample solution reacted in the reaction unit 36 is set at a predetermined position. One cycle of analysis includes a sample liquid suction period, a magnetic particle adsorption period, an image capturing period, a light emission detection period, a cleaning period, a reset period, and a preliminary suction period.

  In the sample liquid suction period, the slide mechanism 16 is operated by a signal from the controller 37, and the magnet 11 is moved to the lower part of the particle adsorption position 13 in the flow path 15. The valve 30 is set in an open state, and the valve 31 is set in a closed state. The arm 26 is operated by a signal from the controller 37 and the sipper nozzle 24 is inserted into the sample liquid container 32. In response to a signal from the controller 37, the pump 25 performs a certain amount of suction operation. The sample solution in the sample solution container 32 is sucked after the buffer solution in the tube 21 and flows into the tube 21. In this state, the pump 25 is stopped, the arm 26 is operated, the sipper nozzle 24 is driven, and the cleaning mechanism 35 is passed. At this time, the tip of the sipper nozzle 24 is washed.

  In the magnetic particle adsorption period, the pump 25 is sucked at a constant speed by a signal from the controller 37. In the meantime, the sample solution in the tube 21 passes through the flow path 15. Since the magnet 11 is installed below the particle adsorption position 13 in the flow path 15, the magnetic particles 10 contained in the sample liquid are attracted in the direction of the magnet 11 and the surface of the particle adsorption position 13 in the flow path 15. Is magnetically adsorbed.

  In the image capturing period, the mirror driving mechanism 42 is driven by a signal from the controller 37 to adjust the position of the mirror 43, and an optical path from the particle adsorption position 13 to the imaging device 41 via the objective lens 44 is selected. The illumination 45 is turned on, and the illumination light is irradiated to the particle adsorption position 13 in the flow path 15. In this state, an image of the magnetic particle 10 magnetically attracted to the particle adsorption position 13 is captured by the imaging device 41 through the objective lens. At this time, by selecting the magnification of the objective lens 44, the area of the particle adsorption position 13 captured by one image and the number of the magnetic particles 10 are adjusted. Further, the spatial resolution of the image to be captured is adjusted by adjusting the spatial resolution of the objective lens 44. Here, when the value indicating the spatial resolution of the objective lens 44 is smaller than the particle size of the magnetic particles, the magnetic particles can be identified one by one in the captured image.

  In the light emission detection period, the slide mechanism 16 is driven to move the magnet 11 away from the flow path 15. The mirror driving mechanism 42 is driven to adjust the position of the mirror 43, and an optical path from the particle adsorption position 13 to the photodetector 39 via the objective lens 44 is selected. Subsequently, the laser light is irradiated from the laser light source 18 by a signal from the controller 37, and the laser light is irradiated to the magnetic particles 10 adsorbed at the particle adsorption position 13 through the condenser lens 17. At this time, the luminescent labeling substance bonded to the surface of the magnetic particle 10 at the particle adsorption position 13 emits light. The wavelength of the emitted light is selected by an optical filter and detected by a photodetector 39. At this time, the light receiving field of the photodetector 39 is the same as the imaging field of view by the imaging device 41. Therefore, the light detector 39 detects the emission intensity generated from the same magnetic particle group as the magnetic particle group captured by the imaging device 41 during the previous image capturing period. After a predetermined time elapses, the laser beam irradiation is stopped. During the light emission detection period, the sipper nozzle 24 is moved by the arm 26 to pass through the cleaning mechanism 35.

  Note that the order of the image capturing period and the light emission detection period may be switched so that the image capturing period comes after the light emission detection period.

  In the cleaning period, the cleaning liquid is sucked from the cleaning liquid container 33 by the pump 25, and the cleaning liquid is passed through the flow path 15 through the tube 21. At this time, since the magnet 11 is moved away from the flow path, the magnetic particles 10 are not held on the particle adsorption position 13 but are discharged out of the flow path together with the cleaning liquid.

  In the reset period, the valve 30 is closed, the valve 31 is opened, and the pump 25 is discharged. The liquid in the pump 25 is discharged to the waste liquid container 34.

  In the preliminary suction period, the arm 26 is driven to insert the sipper nozzle 24 into the buffer solution container 40. Next, after closing the valve 31 and opening the valve 30, the pump 25 is driven to suck the buffer solution in the buffer solution container 40, and the buffer solution is put into the tube 21 and the flow path 15 through the sipper nozzle 24. Meet. After the pre-aspiration period, the next analysis cycle can be performed.

  2 and 3 are views showing the positional relationship between the particle adsorption position in the flow path and the mirror, and are diagrams for explaining an example of switching the optical path by moving the mirror.

  In the case of the arrangement of FIG. 2, an optical path from the particle adsorption position 13 to the imaging device 41 via the objective lens 44 is set. The mirror drive mechanism 42 is driven, and the mirror 43 is moved in parallel with the flow path 15 and installed at a position off the optical path. The illumination 45 is turned on to illuminate the magnetic particles 10 adsorbed at the particle adsorption position 13 with illumination light. In this state, a bright field image of the magnetic particle 10 adsorbed at the particle adsorption position 13 can be captured by the imaging device 41.

  In the arrangement of FIG. 3, the mirror drive mechanism 42 is driven, and the mirror 43 is placed above the particle adsorption position 13 and the objective lens 44. The installation angle of the mirror 43 is preferably 45 degrees with respect to the surface of the particle adsorption position 13. At this time, an optical path from the particle adsorption position 13 to the photodetector 39 through the objective lens 44 and the mirror 43 is set. The illumination 45 is turned off, and the luminescent labeling substance on the surface of the magnetic particle 10 is caused to emit light by a predetermined method. In this state, the light emitted from the luminescent labeling substance on the surface of the magnetic particles is introduced into the photodetector 39 through the objective lens 44 and the mirror 43, and is coupled to the magnetic particles in the bright field image captured by the imaging device 41. The total amount of luminescence of the luminescent labeling substance can be acquired.

  4 and 5 are diagrams showing the positional relationship between the particle adsorption position in the flow path and the mirror, and are diagrams for explaining an example of switching the optical path by changing the angle of the mirror.

  In the arrangement of FIG. 4, an optical path from the particle adsorption position 13 to the imaging device 41 via the objective lens 44 is set. The mirror drive mechanism 42 is driven, the installation angle of the mirror 43 is set to 90 degrees with respect to the flow path 15, and the mirror 43 is installed at a position off the optical path. Then, the illumination 45 is turned on to irradiate the magnetic particles 10 adsorbed at the particle adsorption position 13 with illumination light. In this state, a bright field image of the magnetic particle 10 adsorbed at the particle adsorption position 13 can be captured by the imaging device 41.

  In the case of the arrangement of FIG. 5, the mirror driving mechanism 42 is driven, and the installation angle of the mirror 43 is set to 45 degrees with respect to the surface of the particle adsorption position 13. At this time, an optical path from the particle adsorption position 13 to the photodetector 39 through the objective lens 44 and the mirror 43 is set. The illumination 45 is turned off, and the luminescent labeling substance on the surface of the magnetic particles is caused to emit light by a predetermined method. In this state, light emitted from the luminescent labeling substance on the surface of the magnetic particles is introduced into the photodetector 39 through the objective lens 44 and the mirror 43, and the magnetic particles existing in the bright field image captured by the imaging device 41 are reflected on the magnetic particles. The total amount of luminescence of the bound luminescent labeling substance can be acquired.

  FIG. 6 is a schematic view showing another embodiment of the sample analyzer according to the present invention. In the sample analyzer of this example, the laser light source was arranged on the photodetector side with respect to the flow path.

  The laser light from the laser light source 18 is reflected by the fixed mirror 50 and irradiated onto the magnetic particles 10 adsorbed by the objective lens 44 at the particle adsorption position 13 of the flow cell. Light emitted from the magnetic particles 10 is detected by the photodetector 39 via the objective lens 44 and the mirror 43. In this arrangement, both the fixed mirrors 46 and 50 arranged in the illumination unit 47 need to be half mirrors. The mirror 43 driven by the mirror driving mechanism 42 is, for example, a dichroic mirror that transmits laser light as excitation light and reflects light emission such as fluorescence generated from the magnetic particles 10.

  In the case of the sample analyzer shown in FIG. 1, the magnet 11 is interposed between the laser light source 18 and the flow cell. Therefore, when irradiating the magnetic particles 10 at the particle adsorption position 13 with the excitation laser light from the laser light source 18, it is necessary to move the magnet 11 outside the optical path of the laser light. Since the laser light from the laser light source 18 is irradiated to the flow cell from the illumination unit 47 as shown in FIG. 6, the magnetic particles 10 can be fixed to the particle adsorption position 13 by the magnet 11 even when the light emission amount is measured. The stability of the measurement can be increased. Further, it is not necessary to configure the particle adsorption position 13 with a transparent material.

  In the apparatus configuration shown in FIG. 1 or FIG. 6, the mirror 43 may be a fixed half mirror and the mirror driving mechanism 42 may be omitted.

  In the above embodiment, the two detectors, that is, the photodetector and the imaging device are used. However, by using the imaging device having a function of acquiring the light emission amount, the photodetector and the imaging device can be combined with one detector. It is also possible to use both. In that case, it is possible to acquire the bright field image and the light emission amount of the magnetic particles without switching the optical path using the mirror drive mechanism. Here, the imaging device having a function of acquiring the light emission amount is, for example, an imaging device having a function of calculating luminance values of all pixels (pixels) in a captured image and calculating a sum of the luminance values. Point to.

  FIG. 7 is a schematic view showing another embodiment of the sample analyzer according to the present invention. The sample analyzer of the present embodiment captures a bright-field image and acquires a light emission amount with a single imaging device.

  The apparatus configuration shown in FIG. 7 corresponds to a configuration obtained by removing the photodetector 39, the mirror 43, and the mirror driving mechanism 42 from the sample analyzer shown in FIG. First, the illumination 45 is turned on, and the illumination light is irradiated to the magnetic particles 10 adsorbed at the particle adsorption position 13 via the fixed mirror (half mirror) 46 and the objective lens 44. In this state, a bright field image of the magnetic particle 10 adsorbed on the particle adsorption position 13 is captured by the imaging device 41. At this time, since the particles are irradiated with a sufficient amount of illumination light, the light receiving sensitivity of the imaging device 41 is preferably set low. Next, the illumination 45 is turned off, and the luminescent labeling substance bonded to the surface of the magnetic particle 10 is caused to emit light by a predetermined method. This luminescence is introduced into the imaging device 41 via the objective lens 44, and the amount of luminescence of the luminescent labeling substance is acquired. At this time, since light emitted from the light-emitting labeling substance bonded to the surface of the particles is extremely weak, it is preferable to set the light receiving sensitivity of the imaging device 41 to be high.

  Next, an embodiment of the data processing method will be described with reference to FIG. The area of the particle adsorption position captured by the bright field image is set in advance. For example, the area is set to 100 μm □ (that is, 100 μm × 100 μm). The image acquired by the imaging device 41 is stored in the data storage unit 48. Next, the image is taken out to the arithmetic processing unit 49 and subjected to image processing to calculate the number of magnetic particles included in the image. For example, when the number of captured particles is 500, the density of adsorbed particles is 500 particles / 100 μm □. The calculated adsorption particle density is stored in the data storage unit. The number of magnetic particles calculated at this time is the number of all magnetic particles picked up and imaged by being adsorbed at the particle adsorption position regardless of whether or not the luminescent labeling substance is bound. Therefore, the calculated adsorbed particle density is the density of all the imaged magnetic particles including the magnetic particles to which the luminescent labeling substance is not bound.

  Next, after the optical path is switched by the mirror 43, the light emission amounts of all the magnetic particles included in the area of the same particle adsorption position as when the bright field image is captured are acquired. The amount of light emission is expressed by a value such as the amount of current detected by a photodetector (unit is count), for example, 2,500 counts / 100 μm □. The detected light emission amount is stored in the data storage unit 48.

  The adsorbed particle density and the light emission amount are extracted from the data storage unit 48 to the arithmetic processing unit 49, and the arithmetic processing unit 49 calculates the average light emission amount per magnetic particle from these two values. In the above example, 2,500 counts / 500 pieces = 5 counts / piece. The calculated average light emission amount per magnetic particle is stored in the data storage unit 48.

The substance to be analyzed in the immunoassay can take a very wide range of concentrations in biological fluids such as serum. Therefore, the immunoassay device is required to have a wide detection dynamic range (for example, on the order of 10 ⁶ ). In the present invention, the dynamic range of detection can be widened, for example, by the following method.

By defining the particle size of the magnetic particles, the area of the imaging target region of the bright field image to be captured, the number of magnetic particles captured in one bright field image, that is, the density of adsorbed particles is adjusted. For example, the density of adsorbed particles is adjusted to a range of 0 to 10,000 particles / 100 μm □ (10 ⁴ order). On the other hand, the concentration of the luminescent labeling substance that binds to the surface of the magnetic particles is increased or decreased according to the increase or decrease of the concentration of the analysis target substance contained in the analysis sample. As a result, the light emission amount of each magnetic particle is also increased or decreased. The light emission amount of one magnetic particle is adjusted by defining the detection sensitivity of the photodetector and the exposure time. For example, the light emission amount of one magnetic particle is adjusted to a range of 0 to 100 count / piece (10 ² order). With the above adjustment, in the present invention, it is possible to secure a dynamic range of detection over the order of 10 ^{4 on the} adsorbed particle density, 10 ^{2 on} the light emission amount of one magnetic particle, and 10 ⁶ in total.

  Here, fluorescent immunoassay (when laser light is irradiated from below the particle adsorption position), fluorescent immunoassay (when laser light is irradiated through the objective lens), chemiluminescent immunoassay, chemiluminescent enzyme immunoassay The difference in the analysis cycle in each analysis method will be described.

  First, in the fluorescence immunoassay method, the analysis cycle in the case of irradiating laser light from below the particle adsorption position (first fluorescence immunoassay method) will be briefly described using the apparatus configuration shown in FIG. 1 as an example. To do.

  In the sample liquid suction period, the magnet 11 is moved to the lower part of the particle adsorption position 13 in the flow path 15. Then, the sipper nozzle 24 is inserted into the sample solution container 32, and the sample solution is sucked to flow into the tube 21. In the subsequent magnetic particle adsorption period, the sample liquid is passed through the flow path 15. Thereby, the magnetic particles 10 are magnetically attracted to the particle adsorption position 13 in the flow path 15. In this case, a labeling substance used in the fluorescence immunoassay is bound to the surface of the magnetic particle 10. Next, in the image capturing period, the mirror driving mechanism 42 adjusts the position of the mirror 43 to select an optical path from the particle adsorption position 13 to the imaging device 41. Thereafter, the illumination 45 is turned on, and the particle adsorption position 13 is irradiated with illumination light via the mirror 46. In this state, the imaging device 41 captures a bright field image of the magnetic particle 10.

  In the light emission detection period, the magnet 11 is moved away from the flow path 15 so as not to block the optical path of the laser light, and the position of the mirror 43 is adjusted by the mirror driving mechanism 42, so that the particle adsorption position 13 reaches the photodetector 39. Select the optical path. Thereafter, the laser beam is irradiated from the laser light source 18 to the magnetic particles 10, and the fluorescence emitted from the labeling substance bonded to the surface of the magnetic particles is detected by the photodetector 39. After the detection is completed, the laser beam irradiation is stopped. The flow path 15 and the particle adsorption position 13 are preferably made of a material that is substantially transparent to the wavelength of the laser beam, such as glass, quartz, or plastic.

  In the subsequent cleaning period, the cleaning liquid is sucked through the tube 21 and passed through the flow path 15. In the reset period, the liquid in the pump 25 is discharged to the waste liquid container 34. In the preliminary suction period, the buffer solution is sucked, and the tube 21 and the flow path 15 are filled with the buffer solution.

  Next, in the fluorescence immunoassay method, an analysis cycle in the case of irradiating laser light through the objective lens (second fluorescence immunoassay method) will be described by taking the apparatus configuration shown in FIG. 6 as an example. In this case, only the operation in the luminescence detection period is different from the first fluorescence immunoassay, and the operation in the other period is the same as the first fluorescence immunoassay.

  During the light emission detection period of the second fluorescence immunoassay, the position of the magnet 11 may be kept away from the flow path 15 or may be kept close to the lower part of the particle adsorption position 13 in the flow path 15. The position of the mirror 43 is adjusted by the mirror drive mechanism 42 to select an optical path from the particle adsorption position to the photodetector 39. Then, laser light is irradiated from the laser light source 18 to the magnetic particles 10 through the fixed mirror 50 and the objective lens 44, and the fluorescence emitted from the labeling substance bonded to the surface of the magnetic particles is detected by the photodetector 39. After the detection is completed, the laser beam irradiation is stopped. It is assumed that the laser light emitted from the laser light source 18 passes through the mirrors 43 and 46.

  The analysis cycle of the chemiluminescence immunoassay is different from the first fluorescence immunoassay in the luminescence detection period, but the operation in the other periods is substantially the same as the first fluorescence immunoassay. Note that a labeling substance used in the chemiluminescence immunoassay is bound to the surface of the magnetic particle 10 magnetically adsorbed at the particle adsorption position 13 in the flow path 15 during the magnetic particle adsorption period.

  During the luminescence detection period of the chemiluminescence immunoassay, the position of the mirror 43 is adjusted by the mirror drive mechanism 42 while the magnet 11 is kept close to the lower part of the particle adsorption position 13 in the flow path 15, and the particle adsorption position 13 To the light detector 39 is selected. Thereafter, the luminescent reagent is sucked from the luminescent reagent container 51 and introduced into the flow path 15 via the tube 21. The luminescent reagent is a reagent that induces chemiluminescence of the labeling substance. Then, the chemiluminescence emitted from the labeling substance bonded to the surface of the magnetic particles is detected by the photodetector 39.

  The analysis cycle of the chemiluminescent enzyme immunoassay is different from the first fluorescence immunoassay in the luminescence detection period, but the operation in other periods is substantially the same as the first fluorescence immunoassay. . Note that a labeling substance used in the chemiluminescent enzyme immunoassay is bound to the surface of the magnetic particle 10 magnetically adsorbed at the particle adsorption position 13 in the flow path 15 during the magnetic particle adsorption period.

  During the luminescence detection period of the chemiluminescent enzyme immunoassay, the position of the mirror 43 is adjusted by the mirror drive mechanism 42 while the magnet 11 is kept close to the lower part of the particle adsorption position 13 in the flow path 15, and the particle adsorption position. The optical path from 13 to the photodetector 39 is selected. Thereafter, the luminescent reagent is sucked from the luminescent reagent container 51 and introduced into the flow path 15 via the tube 21. A luminescent reagent is a luminescent substrate that emits light by reacting with an enzyme in a labeling substance. The chemiluminescence emitted from the luminescent reagent that has reacted with the enzyme in the labeling substance bound to the surface of the magnetic particles is detected by the photodetector 39.

  Next, specific application examples and effects of the sample analyzer and sample analysis method of the present invention will be described. Here, the sample analyzer shown in FIG. 1 and magnetic particles having an average particle diameter of 2.8 μm were used. The width of the particle adsorption position 13 in the flow cell in the solution flow direction was 5 mm. The width of the magnet in the solution flow direction was 3 mm. The magnet 11 was installed from the lower side of the flow cell in the range of 3 mm width from the coordinate 2 mm to 5 mm with the origin at the upstream end of the particle adsorption position 13. Two types of sample solutions, sample solution a and sample solution b, were prepared. Sample liquid a and sample liquid b contain magnetic particles and an analysis object. The sample solution a and the sample solution b contain the same concentration of magnetic particles, but the concentration of the substance to be analyzed in the sample solution a is higher than that of the sample solution b.

  First, the mirror 43 is held at the position shown in FIG. 2, an optical path from the particle adsorption position 13 through the objective lens 44 to the imaging device 41 is selected, and a bright field image of the magnetic particle 10 adsorbed on the particle adsorption position 13 is captured. did. From this image, the density of adsorbed particles at the particle adsorbing position 13 (unit: pieces / 100 μm □) was calculated. Next, the mirror 43 is held at the position shown in FIG. 3, the optical path from the particle adsorption position 13 through the objective lens 44 and the mirror 43 to the photodetector 39 is selected, and the particle adsorption position in the same field of view as the bright field image. The amount of light emitted from the light-emitting labeling substance on the surface of the magnetic particle 10 adsorbed on 13 was measured (unit: count / 100 μm □). This operation was performed a plurality of times for the sample solution a and the sample solution b in which the concentration of the magnetic particles and the concentration of the analysis target substance are not sufficiently large.

  FIG. 8 is a diagram showing the relationship between the adsorbed particle density and the amount of luminescence. A plot (indicated by 61 in FIG. 8) showing the relationship between the density of adsorbed particles and the amount of luminescence in the sample liquid a was a straight line. The slope of the straight line 61 indicates the average light emission amount per magnetic particle in the sample liquid a, and in this example, it was 5 counts / particle. On the other hand, as a result of performing the same measurement with the sample liquid b, the plot (indicated by 62 in FIG. 8) showing the relationship between the adsorbed particle density and the light emission amount is also a straight line, and the slope of the straight line 62 is 3 counts / particle. became.

  From the above results, it can be seen that the average amount of luminescence per magnetic particle is the same for the same sample solution, regardless of the position at which the particles are adsorbed. In addition, it can be seen that in the sample solutions having different analysis target substance concentrations, the average light emission amount per magnetic particle increases or decreases in accordance with the increase or decrease of the analysis target substance concentration. Therefore, according to the method disclosed in the present example, based on the average light emission amount per magnetic particle calculated using the bright field image and the light emission amount of a small number of magnetic particles adsorbed at the particle adsorption position, Quantitative analysis of the analyte in the liquid can be performed.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

DESCRIPTION OF SYMBOLS 10 Magnetic particle 11 Magnet 13 Particle adsorption position 14 Flow path wall 15 Flow path 16 Slide mechanism 17 Condensing lens 18 Laser light source 21 Tube 22 Tube 23 Tube 24 Shipper nozzle 25 Pump 26 Arm 30 Valve 31 Valve 32 Sample liquid container 33 Cleaning liquid container 34 Waste liquid container 35 Cleaning mechanism 36 Reaction unit 37 Controller 38 Signal line 39 Photo detector 40 Buffer liquid container 41 Imaging device 42 Mirror drive mechanism 43 Mirror 44 Objective lens 45 Illumination 46 Fixed mirror 47 Illumination unit 48 Data storage unit 49 Arithmetic processing unit 50 Fixed mirror 51 Luminescent reagent container

Claims (12)

A capture unit for capturing particles to be measured;
An illumination unit that irradiates the capture unit with illumination light; and
An imaging unit that captures particles captured by the capturing unit and irradiated by the illumination light; and
A light emitting means for emitting particles captured by the capturing unit;
A light detection unit for detecting a light emission amount of the particles emitted by the light emitting means within the same field of view as the imaging unit;
The number, area, or density of particles in the image captured by the imaging unit is calculated, and analysis is performed based on the calculated number, area, or density of the particles and the amount of luminescence detected by the light detection means. An arithmetic processing unit;
A sample analyzer comprising: The sample analyzer according to claim 1,
The sample analyzer is characterized in that the imaging unit also serves as the light detection unit. The sample analyzer according to claim 1,
Sample analysis comprising a mirror for switching an optical path disposed so as to be movable or adjustable in angle in an optical path connecting the capturing unit and the imaging unit or in an optical path connecting the capturing unit and the light detection unit apparatus. The sample analyzer according to claim 1,
A sample analyzer comprising a half mirror for dividing and guiding an image and light emission of particles captured by the capturing unit into the imaging unit and the light detection unit. The sample analyzer according to claim 1,
The light emitting means is an excitation light source that irradiates the particles with excitation light,
The sample analyzer according to claim 1, wherein the particles emit fluorescence when irradiated with the excitation light. The sample analyzer according to claim 1,
The light emitting means is a chemiluminescent substrate added to the particles,
The sample analyzer according to claim 1, wherein the particles are chemiluminescent by the chemiluminescent substrate. The sample analyzer according to claim 1,
The sample analysis apparatus, wherein the imaging unit captures a bright field image of the particles. The sample analyzer according to claim 1,
The particles are magnetic particles;
A sample analyzer comprising: a magnet; and a drive unit that drives the magnet between a position near and far from the capturing unit. Capturing the particles to be measured in the capturing unit;
Irradiating the particles captured by the capturing unit with illumination light; and
Capturing an image of the particles irradiated with the illumination light;
Emitting the particles captured by the capturing unit; and
Detecting the amount of light emitted by particles captured in the capturing unit and existing in the same field of view as the image;
Calculating the number of particles in the image;
A step of calculating an average light emission amount per particle from the calculated number of particles and the detected light emission amount;
A sample analysis method characterized by comprising: The sample analysis method according to claim 9, wherein
In the step of causing the particles to emit light, the sample analysis method is characterized by emitting fluorescence by irradiating the particles with excitation light. The sample analysis method according to claim 9, wherein
In the step of causing the particles to emit light, a sample analysis method, wherein a chemiluminescent substrate is added to cause the particles to chemiluminescence. The sample analysis method according to claim 9, wherein
A sample analysis method, wherein the particles are magnetic particles, and the particles are captured by the capturing unit by a magnetic force.

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