JP2016156673A - Automatic analyzer and analytical method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an automatic analyzer and an analytical method capable of improving reaction speed between fine particles to which probe molecules in a sample solution are bonded and biological molecules which are analysis objects.SOLUTION: A congestion region 36a of magnetic particulates 38 is formed in a reaction zone 36 by a magnetic field generation device, and reaction liquid 39 is allowed to pass through the congestion region 36a of magnetic particulates 38 by a liquid sending device. Then, when the reaction liquid 39 passes through the reaction zone 36, a liquid sending direction is inverted, and the reaction liquid 39 is allowed to pass again through the congestion region.SELECTED DRAWING: Figure 2

Description

  The present invention relates to an automatic analyzer and an analysis method for analyzing a sample using a reaction between an antigen and an antibody.

  As a method for analyzing the type and abundance of a specific biomolecule contained in the sample solution to be analyzed, for example, a probe molecule (capture probe molecule) that captures the biomolecule to be analyzed in fine particles as a reaction support is used. After binding, the probe molecule reacts with the biomolecule to be analyzed and captured, and further reacted with the probe molecule (label molecule) to which the label is bound, and the target biomolecule is analyzed by quantifying the label. The method is known.

  On the other hand, when microparticles are used as a reaction support, the movement speed of microparticles in solution is orders of magnitude smaller than that of biomolecules to be analyzed. It is also known that the reaction rate is significantly reduced. For the purpose of accelerating the reaction on the fine particles, for example, Patent Document 1 (US Patent Application Publication No. 2009/0227044) encloses a reaction solution containing the fine particles in the flow path. A technique is disclosed in which a magnetic field in a reaction solution is moved by applying a varying magnetic field from the outside with an electromagnet, and the reaction solution is stirred.

US Patent Application Publication No. 2009/0227044

  However, when the reaction rate is improved by stirring the solution as in the prior art, the reacting molecules, that is, the microparticles to which the probe molecules are bonded and the biomolecule to be analyzed are substantially in the same direction along the flow of the solution. Since it moves, it is difficult to increase the relative movement speed related to the collision frequency between molecules that greatly contribute to the reaction speed, and the improvement of the reaction speed is limited and there is room for improvement.

  The present invention has been made in view of the above, and provides an automatic analyzer and an analysis method capable of improving the reaction rate between microparticles combined with probe molecules in a sample solution and biomolecules to be analyzed. Objective.

  In order to achieve the above object, the present invention provides a liquid feeding device that sends a reaction liquid mixed with a sample to be analyzed and a reagent containing magnetic fine particles used for analysis of the sample to the reaction flow path, and the reaction flow path A magnetic field generator for generating a magnetic field acting on the magnetic fine particles in a predetermined reaction region of the magnetic field, and the reaction liquid to pass through a dense region of the magnetic fine particles formed in the reaction region by the magnetic field generator. And a control device for controlling the liquid feeding device.

  It is possible to improve the reaction rate between microparticles bound to probe molecules in sample solution and biomolecules to be analyzed.

It is a figure which extracts and shows the reaction part of the analysis unit of 1st Embodiment. It is a figure which expands and shows the A section of FIG. 1 is a diagram schematically showing an overall configuration of an automatic analyzer according to a first embodiment. It is a figure which shows typically the structure of the analysis unit of the automatic analyzer shown in FIG. It is a figure which shows typically an example of the reaction which arises in a reaction liquid. It is a figure which shows typically the other example of the reaction which arises in a reaction liquid. It is a figure which shows the example of 1 structure of a magnetic field generation mechanism. It is a figure which shows the other structural example of a magnetic field generation mechanism. It is a figure which shows the further another structural example of a magnetic field generation mechanism. It is a figure explaining the effect in 1st Embodiment, and is a figure which compares the reaction rate when the reaction process of this Embodiment is applied to a reaction liquid when the reaction liquid is left still. It is a figure which shows the relationship between the ratio with respect to the reaction liquid of the magnetic fine particle in the dense area | region formed in the reaction area | region by 1st Embodiment, and reaction efficiency. It is a disassembled perspective view of the reaction flow path in 2nd Embodiment. It is a longitudinal cross-sectional view of the reaction flow path in 2nd Embodiment.

  Embodiments of the present invention will be described with reference to the drawings.

<First Embodiment>
A first embodiment of the present invention will be described in detail with reference to FIGS.

  FIG. 3 is a diagram schematically showing the overall configuration of the automatic analyzer according to the present embodiment.

  In FIG. 3, an automatic analyzer 100 includes a sample container rack 2 in which a plurality of sample containers 1 that store biological samples such as blood and urine (hereinafter referred to as samples) are stored, and a rack that carries the sample container rack 2. For mixing the sample and the reagent with the transport line 3, the reagent container disk 5 in which a plurality of reagent containers 4 containing various reagents used for sample analysis are stored and kept warm and covered with the reagent disk cover 7 An incubator disk 9 in which a plurality of reaction containers 8 are housed, a sample dispensing mechanism 10 for dispensing a sample from the sample container 1 to the reaction container 8 of the incubator disk 9 by rotation driving and vertical driving, and a reagent disk cover 7 are provided. The reagent is transferred from the reagent container 4 to the reaction container 8 of the incubator disk 9 by rotational driving and vertical driving through the reagent disk cover opening 7a. A reagent dispensing mechanism 11 for dispensing, a reaction container stirring mechanism 14 for stirring the reaction liquid (mixed liquid of the sample and the reagent) housed in the reaction container 8, and a light emission induction reagent container 40 containing a light emission induction reagent; , A cleaning reagent container 41 containing a cleaning reagent, and a reaction liquid mixed in the reaction container 8 of the incubator disk 9 by rotation and vertical driving, a luminescence induction reagent container 40, or a reagent contained in the cleaning reagent container 41 The aspirating mechanism 17 for aspirating the liquid, the analysis unit 18 for performing analysis using the reaction liquid and the reagent aspirated by the aspirating mechanism 17, the dispensing operation of each part, and the analysis processing (reaction processing, measurement processing, etc.) by the analysis unit 18 The control part 19 which controls operation | movement of the automatic analyzer 100 whole containing this is roughly provided. The reagent disk 5 contains a light-emitting label reagent container for storing a reagent containing a light-emitting label, a magnetic particle reagent container for storing a reagent containing magnetic fine particles, and the like as the reagent container 4. The magnetic fine particles are not particularly limited, and examples thereof include those produced by dispersing ferrite in a polymer and those obtained by fixing dextran, polyethylene glycol or the like on the surface of the ferrite fine particles. As the luminescent label, a normal fluorescent dye such as Cy3, Cy5, FITC (fluorescein isothiocyanate) or a fluorescent protein such as phycoerythrin or allophycocyanin can be used.

  The automatic analyzer 100 also includes a reaction container / sample dispensing tip storage unit 13 in which a plurality of unused reaction vessels 8 and sample dispensing tips 10a are accommodated, and a standby reaction for replacement / replenishment thereof. Container / sample dispensing tip storage unit 12, disposal hole 15 for discarding used reaction vessel 8 and sample dispensing tip 10a, and transport mechanism for gripping and transporting reaction vessel 8 and sample dispensing tip 10a 16. The transport mechanism 16 is provided so as to be movable in the X-axis, Y-axis, and Z-axis directions (not shown), and the reaction container 8 stored in the reaction container / sample dispensing chip storage unit 13 is placed in the incubator disk 9. The used reaction container 8 is discarded into the disposal hole 15, or the unused sample dispensing tip 10a is transported to the tip mounting position 16a.

  FIG. 4 is a diagram schematically showing the configuration of the analysis unit of the automatic analyzer shown in FIG. Further, FIG. 1 is a diagram showing the reaction part of the analysis unit in an extracted manner, and FIG. 2 is a diagram showing the A part of FIG.

  In FIG. 4, the analysis unit 18 is a reaction process that promotes the reaction between the magnetic fine particles as the reaction support contained in the reaction solution sucked from the reaction vessel 8 of the incubator disk 9 by the suction mechanism 17 and the biomolecule to be analyzed. The reaction unit 30 that performs the measurement, the measurement unit 31 that performs the measurement process of the reaction liquid that has been subjected to the reaction process by the reaction unit 30, and the discharge unit 32b of the measurement unit 31 that sucks the reaction liquid from the suction unit 32a of the reaction unit 30 And a transfer mechanism 32 for transferring the reaction liquid by discharging the liquid.

  The reaction unit 30 includes a base substrate 30a, a reaction channel 35 formed on the substrate 30a, storage of the reaction solution sucked by the suction mechanism 17, a luminescence induction reagent, a cleaning reagent, and the like and the reaction channel 35. A reaction liquid supply unit 33 capable of supplying a liquid, a magnetic field generator 37 that is disposed in a predetermined reaction region 36 on the reaction flow path 35 and generates a magnetic field that acts on magnetic fine particles in the reaction liquid, and a reaction A switching valve 34 for switching the destination of the reaction liquid by the liquid feeding unit 33 and a suction part 32 a for sucking the reaction liquid by the transfer mechanism 32 are provided. In FIG. 4, each component of the reaction unit 30 is shown in a simplified manner for simplicity of illustration, and the details thereof will be described below.

  As shown in FIGS. 1 and 2, in the reaction unit 30, the reaction liquid feeding unit 33 includes a pump 33 a that feeds a reaction liquid 39 including magnetic fine particles 38 and the like in a reaction flow path 35, and a reaction liquid 39. The reaction liquid storage units 33b and 33c are used for storage. The switching valve 34 includes a switching valve 34a that switches the flow direction of the reaction liquid 39 with the pump 33a, the reaction liquid storage section 33b, and the reaction flow path 35, a reaction flow path 35, a reaction liquid storage section 33c, and a suction section 32a. And a switching valve 34b for switching the flow direction of the reaction liquid. Further, the magnetic field generation measure 37 is configured by magnets 37 a and 37 b arranged so as to face both sides of the reaction flow path 35 in the reaction region 36.

  As the pump 33a, for example, a peristaltic pump or a syringe pump suitable for transporting a small amount of droplets can be used. In order to pass the reaction liquid 39 through the reaction region 36 of the reaction flow path 35 a plurality of times, a pump capable of both intake and exhaust is preferable.

  FIG. 7 is a diagram illustrating a configuration example of a magnetic field generation mechanism.

  In FIG. 7, the magnets of the magnetic field generation device 37 are a pair of permanent magnets 137 a and 137 b (for example, neodymium magnets) disposed opposite to the positions sandwiching the reaction channel 35, and are permanent with respect to the reaction channel 35. The magnets 137a and 137b are disposed on an XY stage 130 that can move in a direction in which the magnets 137a and 137b are brought closer to or away from each other. In addition, the XY stage 130 is movable to a position where the reaction channel 35 is separated from the space between the permanent magnets 137a and 137b, that is, the space affected by the magnetic force of the permanent magnets 137a and 137b.

  When the reaction liquid 39 including the magnetic fine particles 38 is sent to the reaction flow path 35 via the switching valve 34a by the pressure control of the pump 33a, the reaction liquid 39 passes through the reaction region 36. At this time, when a magnetic field is formed in the reaction region 36 by the magnetic field generator 37, the magnetic fine particles 38 in the reaction solution 39 are trapped in the reaction region 36, and the dense region 36 a over the entire cross section of the reaction channel 35 is formed. Form. That is, when a fluctuating magnetic field is generated in the reaction region 36 by the operation of the XY stage 130, the magnetic fine particles 38 remain in the reaction region 36 in the reaction flow path 35 and take a distribution according to the magnitude and strength of the magnetic field. However, it is possible to stay for a long time while moving in the reaction region 36. Note that the spatial size of the reaction region 36 depends on the spatial size of the magnetic field formed by the magnetic field generator 37.

  By such a magnetic field generator 37, the magnetic fine particles 38 in the reaction liquid in the reaction flow path 35 are sufficiently held in the reaction region 36. For example, a glass capillary (manufactured by Hilgenberg) having an inner diameter of 0.72 mm as the reaction flow path 35 and two neodymium magnets having a width of 10 mm, a height of 20 mm, and a thickness of 5 mm as the magnetic field generator 37 are opposed to each other at an interval of 5 mm. Using the one placed on the stage (manufactured by Sigma Kogyo), 50 ul of the reaction liquid 29 in which 60 M of magnetic fine particles (manufactured by Ademtech) having a diameter of 300 nm were dispersed as the magnetic fine particles 28 was fed at a flow rate of 200 ul / minute to react. When the liquid 29 passes through the reaction region 36, the liquid feeding direction is reversed and the reaction solution 29 is passed again through the reaction region 36, through the dense region 36 a where the magnetic fine particles 28 are densely packed, and the magnetic field generator 37 is set to 0. When operated at 5 Hz and a moving distance of 5 mm, the retention rate of the magnetic fine particles 28 in the dense region 36a in the reaction region 36 in about 10 minutes is about 95%. There was.

  Further, when the magnetic fine particles 28 are held by the magnetic field generator 37, the magnetic field acting on the magnetic fine particles 28 is changed to a variable magnetic field by driving the XY stage, and the frequency of the variable magnetic field is set to 0.02 Hz or more. For example, even when the dense state of the magnetic fine particles 38 in the reaction region 36 becomes sufficiently long with respect to the reaction time, it is possible to suppress the reaction molecules from reacting with only some of the magnetic fine particles 38.

  For example, as an example of the magnetic fine particle 38, a material in which a single molecule of biotin is introduced onto a magnetic fine particle is prepared, and a combination of streptavidin and a fluorescent protein is used as a label material (labeled molecule), and the reaction is performed while changing the frequency of the variable magnetic field. By examining the number of magnetic fine particles introduced with label material in the region and examining in detail the relationship between the frequency of the varying magnetic field and the number of fine particles introduced with the label material, at what frequency the magnetic fine particles 38 are concentrated. It was examined whether it was necessary to move the region 36a in the reaction channel 35.

  As magnetic fine particles, magnetic beads with a mouse secondary antibody having a diameter of 300 nm (manufactured by Ademtech) are used, and a mouse biotinylated antibody (manufactured by Abcom) so that the molecular number ratio becomes 1/10 of the number of beads of the magnetic beads. ) Was reacted. As a reaction molecule, a 30 pM solution of phycoerythrin with streptavidin (Roche) was used. Then, the magnetic fine particle solution and the reaction molecule solution were mixed to prepare a 50 ul reaction liquid in which 60 M magnetic fine particles were dispersed.

  A glass capillary (made by Hilgenberg) having an inner diameter of 0.72 mm as the reaction channel 35, and two neodymium magnets having a width of 10 mm, a height of 20 mm, and a thickness of 5 mm as the magnetic field generator 37 are opposed to each other at an interval of 5 mm. 50 ul reaction liquid 29 in which 60 M magnetic fine particles (manufactured by Ademtech) having a diameter of 300 nm are dispersed as magnetic fine particles 28 is fed at a flow rate of 200 ul / min. When the liquid passes through the reaction region 36, the liquid feeding direction is reversed, and the reaction liquid 29 passes through the reaction region 36 again, and passes through the dense region 36a where the magnetic fine particles 28 are dense. At this time, the neodymium magnet of the magnetic field generator 37 is moved by the XY stage at a moving distance of 5 mm, and the frequency of the magnetic field acting on the magnetic fine particles is 2 Hz, 1 Hz, 0.5 Hz, 0.2 Hz, 0.1 Hz,. The number of fluorescent base points was measured at 05 Hz and 0.02 Hz. The number of fluorescent bright spots was measured using a 300 W xenon lamp (manufactured by Asahi Spectroscopy) as a light source, and a fluorescent image was obtained using a sCMOS camera (manufactured by Hamamatsu Photonics) and a fluorescent microscope (manufactured by Olympus). As a result, it can be seen that the number of fluorescent bright spots hardly fluctuates between 2 Hz and 0.02 Hz. Therefore, if the fluctuation magnetic field has a frequency of 0.02 Hz or more, the reaction is not biased toward some magnetic particles. I understood it.

  As the magnetic field generator 37, in addition to those using the permanent magnets 137a and 137b as described above, as shown in FIG. 8, electromagnets 237a and 237b are arranged facing each other, and the reaction channel 35 is arranged therebetween. A configuration or a configuration in which a conducting wire 337 is coiled as shown in FIG.

  Here, the reaction that occurs in the reaction solution in the reaction section or the like will be described. FIG. 5 is a diagram schematically showing an example of the reaction that occurs in the reaction solution, and FIG. 6 is a diagram schematically showing another example.

  For example, as shown in FIG. 5, a reaction liquid 39 sent to the reaction region 36 of the reaction flow path 35 is divided into a biomolecule 70 to be analyzed and a probe molecule (such as an antibody or a nucleic acid molecule that captures the biomolecule 70). The magnetic particle 28 to which the capture probe molecule 71) is bound and the capture probe molecule 73 to which the label 72 such as a fluorescent protein is bound (that is, the label molecule 74 composed of the label 72 and the capture probe 73) are included. At this time, the biomolecule 70 is captured by the capture probe 73 of the labeled molecule 74 to form a molecule to be reacted, and the biomolecule 70 of the molecule to be reacted becomes the capture probe molecule 71 of the magnetic particle 28. By being captured, a complex of the magnetic fine particle 28, the biomolecule 70 and the label 72 is formed.

  As another example, as shown in FIG. 6, a capture probe molecule 71 in which a reaction liquid stored in a reaction vessel 8 is combined with a biomolecule 70 to be analyzed and biotin (or avidin / streptavidin). And a capture probe molecule 73 to which a label 72 is bound (that is, a label molecule 74 composed of the label 72 and the capture probe 73). At this time, the biomolecule 70 is captured by the capture probe 73 and the capture probe 71 of the label molecule 74, thereby forming a reaction molecule 75 as a complex.

  Further, the reaction liquid 39 sent to the reaction region 36 of the reaction flow path 35 is configured to include the magnetic fine particles 28 bonded with avidin / streptavidin (or biotin) and the molecule 75 to be reacted. At this time, the avidin / streptavidin of the magnetic fine particle 28 and the biotin of the molecule 75 to be reacted (or the biotin of the magnetic fine particle 28 and the avidin / streptavidin of the molecule 75 to be reacted) bind to each other. A complex of 70 and label 72 is formed.

  The measurement unit 31 includes a base substrate 31a, a measurement channel 51 formed on the substrate 31a, a discharge unit 32b from which a reaction liquid conveyed from the reaction unit 30 by the transfer mechanism 32 is discharged, and a measurement channel. A liquid feeding unit (not shown) that feeds the reaction liquid in 51 and a magnetism in the reaction liquid reaction liquid that is disposed in a predetermined measurement region 52 on the measurement channel 51 and discharged to the discharge portion 32b. A magnetic field generator 57 that generates a magnetic field that acts on the fine particles, a measurement mechanism 101 that measures the magnetic fine particle group 52a formed in the measurement region 52 by the magnetic field generator 57, and a reaction liquid that has been measured by the measurement mechanism 101 And a discharge portion 61 for discharging the water.

  The measurement mechanism 101 includes a light source 60, a condensing lens 59 that condenses the light emitted from the light source 60, and a wavelength that is unnecessary for excitation of a label (fluorescent dye or fluorescent protein) from the light that has passed through the condensing lens 59. A filter 58 that blocks the light and transmits only the excitation light, a dichroic mirror 54 that reflects and transmits the excitation light from the filter 58, and an objective that irradiates the measurement region 52 with the excitation light reflected by the dichroic mirror 54 A filter 53 for removing reflected light of excitation light from the lens 53 and fluorescence emitted from the magnetic fine particle group 52a of the measurement region 52 by the excitation light, condensed by the objective lens 53, and transmitted through the dichroic mirror 53; A condensing lens 56 that condenses the fluorescent light that has passed through 55, and a photodetector 57 that detects the fluorescent light that has been condensed and imaged by the condensing lens 56. .

  As the light source 60, a light source suitable for excitation of a fluorescent dye used as a label may be selected. A xenon lamp, a mercury lamp, a halogen lamp, or the like may be used, and a laser or LED may be used. In the measurement region 52, a magnetic fine particle group 52 a in which the magnetic fine particles 38 are wide and thin and dense is formed by the magnetic field generator 57, and excitation light is irradiated through the objective lens 53. The fluorescence emitted from the magnetic fine particles 38 of the magnetic fine particle group 52 a by the excitation light and the excitation light reflected by the measurement region 52 are guided to the filter 55 via the objective lens 53 and the dichroic mirror 54. In the filter 55, only the reflected light of the excitation light is removed, and only the fluorescence is transmitted, and the light is condensed by the condenser lens 56, imaged on the sensor of the photodetector 57, and detected. As the photodetector 57, a CCD camera, a CMOS camera, a photomultiplier, an avalanche photodiode, or the like can be used. In addition, when detecting by a digital count method, it is preferable to obtain | require the number of fluorescent luminescent spots, and it is preferable to use the two-dimensional image sensor which has position resolution as a photodetector.

  The operation in the present embodiment configured as described above will be described.

  In the analysis process, the control unit 19 dispenses a sample from the sample container 1 accommodated in the sample container rack 2 to the reaction container 8 by the sample dispensing mechanism 10, and the reagent from the reagent container 4 accommodated in the reagent container disk 5. The reagent is dispensed into the reaction vessel 8 by the dispensing mechanism 11. Subsequently, the control unit 19 stirs the reaction liquid (mixed liquid of the sample and the reagent) mixed in the reaction container 8 of the incubator disk 9 by the reaction container stirring mechanism 14 and sends the solution to the analysis unit 18 by the suction mechanism 17. The reaction process and the measurement process are performed.

  In the reaction process, the control unit 19 causes the magnetic field generator 37 to form a dense region 36 a of the magnetic fine particles 38 in the reaction region 36, and causes the reaction solution 39 to pass through the dense region 36 a of the magnetic fine particles 38 using the liquid feeding device 33. When the reaction solution 29 passes through the reaction region 36, the liquid feeding direction is reversed and the reaction solution 29 is passed again through the dense region 36. At this time, the control unit 19 causes the magnetic field generator 37 to vary the magnetic field acting on the magnetic fine particles 38 at 0.5 Hz (more specifically, 0.02 Hz or more).

  In this way, the magnetic fine particles 38 used as the reaction support are captured by the magnetic field generation unit 37 to form a dense region 36 over the entire cross section of the reaction channel 35, and the biomolecule to be analyzed is placed in the dense region 36. The reaction between the magnetic fine particles and the molecule to be reacted can be promoted by passing the reaction liquid 29 containing the molecule to be reacted formed as a complex containing.

  FIG. 10 and FIG. 11 are diagrams for explaining the effects in the present embodiment. FIG. 10 shows the reaction rates when the reaction solution is left standing and when the reaction process of the present embodiment is applied to the reaction solution. FIG. 11 and FIG. 11 are diagrams showing the relationship between the reaction efficiency and the ratio of magnetic fine particles to the reaction solution in the dense region formed in the reaction region according to the present embodiment.

  In order to obtain the results of FIGS. 10 and 11, the following experiment was conducted.

  First, a glass capillary (made by Hilgenberg) having an inner diameter of 0.72 mm as the reaction channel 35 and two neodymium magnets having a width of 10 mm, a height of 20 mm, and a thickness of 5 mm as the magnetic field generator 37 are opposed to each other at an interval of 5 mm. Using the one placed on the stage (manufactured by Sigma Kogyo), 50 ul of the reaction liquid 29 in which 60 M of magnetic fine particles (manufactured by Ademtech) having a diameter of 300 nm were dispersed as the magnetic fine particles 28 was fed at a flow rate of 200 ul / minute to react. When the liquid 29 passes through the reaction region 36, the liquid feeding direction is reversed, and the reaction solution 29 is passed again through the reaction region 36, passing through the dense region 36 a where the magnetic fine particles 28 are densely packed, and the magnetic field variation by the magnetic field generator 37. Were operated at a frequency of 0.5 Hz and a moving distance of the neodymium magnet of 5 mm.

  For the magnetic fine particles, magnetic fine particles with mouse secondary antibody (Ademtech G0503, anti-mouse IgG coated) were used, and mouse biotinylated antibody (R & D mAb ER-PR8 biotin) was added to the number of magnetic fine particles. After adjusting the concentration so that the concentration ratio of the mouse biotinylated antibody was 10: 1, both were mixed and allowed to stand for 1 hour to immobilize one molecule of the antibody with biotin on one magnetic fine particle. R-phycoerythrin with streptavidin (Roche LumiGrade Ultrasensitive) is used as the fluorescent label material, and 2 pM / 50 ul of biotin-introduced magnetic particles are placed in a reaction tube. After removal, a fluorescent label material solution 30 pM · 50 ul was added to prepare a reaction solution. Two reaction liquids (50 ul) were prepared. Put one reaction solution in a syringe (manufactured by Terumo), attach it to a syringe pump (Fusion Touch 200 manufactured by Isis), connect the syringe outlet and the capillary with a silicone rubber tube, and flow from the syringe to the capillary at a flow rate of 200 ul / min. 50 ul of the reaction solution was fed. A silicon rubber tube was connected to the other end of the capillary about 20 cm, and the end of the tube was opened.

  When the reaction solution approached the neodymium magnet, it was observed that the magnetic fine particles in the reaction solution were trapped and moved in the capillary tube in accordance with the movement of the neodymium magnet. When the reaction solution passes through the magnet, the syringe pump is switched to the suction mode, and the reaction solution is moved in the opposite direction, so that it passes again through the vicinity of the magnet. Passed multiple times. The total reaction time was 10 minutes. Ten minutes after the start of liquid feeding, the reaction liquid was collected in the reaction container from the open end of the tube. On the other hand, another reaction solution was allowed to react by allowing to stand for 10 minutes. After the reaction, after collecting the magnetic fine particles in the reaction solution, 5 ul of reaction buffer is added and dispersed, and a PDMS (Polydimethylpolysiloxane) sheet with a 5 um thickness of 8 um x 8 mm with a 50 um thickness is made into a slide glass. The sample was sealed in an observation cell formed with an 18 mm × 18 mm cover glass, placed on a columnar neodymium magnet having a diameter of 50 mm, placed on a fluorescence microscope, and the number of fluorescent bright spots was measured. The objective lens was 20 times, and the size of one field of view was 0.667 × 0.667 mm. After observing the visual field, the number of bright spots per visual field was averaged and calculated.

  As shown in FIG. 10, compared to the case where the reaction solution is allowed to react by standing, a dense region of magnetic fine particles as a reaction support is formed as in this embodiment, and the reaction solution passes through the dense region. When the reaction is carried out, the number of fluorescent bright spots is about 10 times larger, and therefore the reaction rate is about 10 times faster. As described above, it has been clarified that the reaction rate can be remarkably increased by using the biomolecule analysis method of the present invention.

  Further, the inventor of the present application has found that the reaction efficiency between the molecule to be reacted in the reaction solution and the surface of the magnetic fine particle (such as the capture probe molecule) is greatly influenced by the volume ratio of the magnetic fine particle 38 in the dense region 36a. The relationship between the volume fraction (%) of magnetic fine particles in the dense region 36a and the reaction efficiency was investigated. As a result, as shown in FIG. 11, it was found that the reaction efficiency rapidly increases when the volume ratio of the magnetic fine particles in the dense region 36a exceeds 50%. This is presumably because the probability that the reacted molecules collide with the magnetic fine particles 38 within the time of passing through the dense region 36a is increased due to the short distance between the magnetic fine particles 38 in the dense region 36a. Therefore, in the present embodiment, the volume ratio of the magnetic fine particles 38 in the dense region 36a is set to 50% or more.

  In the measurement process, the control unit 19 causes the magnetic field generator 57 to form the magnetic fine particle group 52a in the measurement region 52, measures the number of fluorescent bright spots (or the amount of fluorescent light) with the measurement mechanism 101, and stores the measurement result. Store in the area.

  The effect in this Embodiment comprised as mentioned above is demonstrated.

  When the reaction rate is improved by agitating the reaction mixture in which the sample and the reagent are mixed as in the prior art, the reacting molecules, that is, the fine particles combined with the probe molecules and the biomolecule to be analyzed are in the solution. Since it moves in the same direction along the flow, it is difficult to increase the relative movement speed related to the collision frequency of molecules that greatly contribute to the reaction speed, and the improvement of the reaction speed is limited and there is room for improvement. .

  In contrast, in the present embodiment, a magnetic field generator that generates a magnetic field that acts on magnetic fine particles is arranged in a predetermined reaction region of a reaction channel through which a reaction liquid in which a sample and a reagent are mixed is sent. In addition, a magnetic particle generator forms a dense region of magnetic fine particles in the reaction region, and a reaction process is performed in which the reaction solution is passed through the dense region. Therefore, analysis is performed with fine particles bound to probe molecules in the sample solution. The reaction rate with the target biomolecule can be dramatically improved, and the analysis throughput can be greatly improved.

  In this embodiment, a case is described in which a fluorescent dye or a fluorescent protein is used as a label, and the fluorescence of the label formed in a complex with the target biomolecule is measured to quantify the target biomolecule. However, the present invention is not limited to this, and it is conceivable to use fine particles having a specific charge as a label. That is, by using a molecule containing fine particles having a specific charge as a labeling molecule, fixing the magnetic fine particles on the electrode, and then quantifying the amount of fine particles having a specific charge on the electrode from the amount of charge detected by the electrode The concentration information of the biomolecule to be analyzed can be obtained. For example, an antibody that recognizes a biomolecule to be analyzed immobilized on a polystyrene bead surface can be used as a labeled molecule. The measurement is performed by inserting an electrode having magnetic fine particles fixed on the surface and a counter electrode into a buffer such as phosphate buffered saline (PBS), and a bias of about minus 5 volts to plus 5 V between both electrodes. The amount of the labeled molecule on the electrode (that is, the amount of the biomolecule to be analyzed) can be determined by measuring the current flowing between both electrodes with an ammeter such as a picoammeter. .

<Second Embodiment>
A second embodiment of the present invention will be described in detail with reference to FIGS.

  In the first embodiment, one reaction channel is used. In the present embodiment, a plurality of reaction channels are used in parallel.

  12 is an exploded perspective view of the reaction channel, and FIG. 13 is a longitudinal sectional view of the reaction channel. In the figure, the same members as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

  As shown in FIGS. 12 and 13, the plurality of reaction flow paths 235 of the present embodiment are composed of an upper plate glass 235 a, a lower plate glass 235 b, and a PDMS sheet sandwiched and joined between the upper plate glass 235 a and the lower plate glass 235 b. 235c.

  The upper plate glass 235a, the lower plate glass 235b, and the PDMS sheet 235c are configured to have a length direction dimension of about 128 mm and a width direction dimension of about 60 mm. The holes 238 are formed so as to be accurately aligned by matching. The upper glass plate 235a is formed with a thickness t≈0.175 mm, the lower glass plate 235b is formed with a thickness t≈1.0 mm, and the PDMS sheet 235c is formed with a thickness t≈0.05 mm.

  In the PDMS sheet 235c, a plurality (six in this embodiment) of hollow portions 239 extending in the length direction are arranged in parallel, and the PDMS sheet 235c includes the upper plate glass 235a, the lower plate glass, 235b, As a result, the hollow portions 239 become a plurality of reaction channels 235 through which the reaction solution is fed.

  In the upper glass plate 235a, an inlet 236 for injecting a reaction solution is provided at a position corresponding to one end of each reaction channel 235, and the other end of each reaction channel 235 is provided at the other end. At the corresponding position, a discharge port 237 for discharging the reaction liquid is provided.

  As shown in FIG. 13, plate-like permanent magnets 337a and 337b constituting a magnetic field generating device are arranged along the upper side surface of the upper plate glass and the lower side surface of the lower plate glass, and the same as in the first embodiment. A drive mechanism (not shown) that drives the permanent magnets 337a and 337b to approach or separate from each reaction channel 235 is provided.

  The PDMS sheet 235c can be easily created by drawing a plurality of hollow portions 239 on a polydimethylsiloxane (PDMS) sheet along the hollow portions 239 with a laser. In addition, a polymer sheet having a plurality of hollow portions 239 can be easily prepared by preparing a mold with metal or the like and pouring a polymer solution into the mold.

  Other configurations are the same as those of the first embodiment.

  In the present embodiment configured as described above, the same effects as those of the first embodiment can be obtained.

  Moreover, in this Embodiment, reaction of a some sample can be performed simultaneously by multi-izing a reaction flow path.

  In addition, it is easy to simultaneously perform dispersion and position control of magnetic fine particles with a single magnetic field generator for a plurality of reaction channels.

  In addition, this invention is not limited to each above-mentioned embodiment, Various modifications are included. For example, the above-described embodiment has been described in detail for easy understanding of the present invention, and is not necessarily limited to one having all the configurations described.

DESCRIPTION OF SYMBOLS 1 ... Sample container, 2 ... Sample container rack, 3 ... Rack conveyance line, 4 ... Reagent container, 5 ... Reagent container disk, 8 ... Reaction container, 9 ... Incubator Disc 10: Sample dispensing mechanism 11 ... Reagent dispensing mechanism 14 ... Reaction vessel stirring mechanism 17 ... Suction mechanism 18 ... Analysis unit 19 ... Control unit 30 ... Reaction unit, 30a, 31a ... Substrate, 31 ... Measurement unit, 32 ... Transfer mechanism, 32a ... Suction unit, 32b ... Discharge unit, 33 ... Reaction liquid feed Liquid unit, 33a ... pump, 33b ... reaction liquid storage unit, 33c ... reaction liquid storage unit, 34 ... switching valve, 35 ... reaction flow path, 36 ... reaction region, 36a ... Dense area, 37 ... Magnetic field generator, 38 ... Magnetic particles, 39 · Reaction, 100 ... automatic analyzer, 101 ... measuring mechanism

Claims (7)

A liquid feeding device for sending a reaction liquid mixed with a sample to be analyzed and a reagent containing magnetic fine particles used for analysis of the sample to the reaction flow path;
A magnetic field generator for generating a magnetic field acting on the magnetic fine particles in a predetermined reaction region of the reaction channel;
An automatic analyzer comprising: a control device that controls the liquid feeding device so that the reaction liquid passes through a dense region of the magnetic fine particles formed in the reaction region by the magnetic field generator. . The automatic analyzer according to claim 1, wherein
The said control apparatus controls the said liquid feeding apparatus so that the said reaction liquid may be passed through the dense area | region of the said magnetic microparticles in multiple times, The automatic analyzer characterized by the above-mentioned. The automatic analyzer according to claim 1, wherein
The automatic analyzer according to claim 1, wherein a volume occupied by the magnetic fine particles in the reaction region is larger than a volume occupied by the reaction solution. The automatic analyzer according to claim 1, wherein
The reaction solution is
The magnetic microparticles to which capture probe molecules for capturing biomolecules to be analyzed contained in the sample are bound;
An automatic analyzer comprising: a labeled molecule formed of a label and a capture probe molecule; and a reacted molecule formed as a complex composed of the biomolecule. The automatic analyzer according to claim 1, wherein
The reaction solution is
The magnetic fine particles;
A target molecule formed as a complex comprising a capture probe molecule for capturing a biomolecule to be analyzed contained in the sample, a label molecule formed by the biomolecule and the capture probe molecule, and The automatic analyzer characterized by including. A procedure for sending a reaction solution in which a sample to be analyzed and a reagent containing magnetic fine particles used for analysis of the sample are mixed to the reaction channel;
Generating a magnetic field acting on the magnetic fine particles in a predetermined reaction region of the reaction flow path, and forming a dense region of the magnetic fine particles in the reaction region;
And a procedure for passing the reaction solution through the reaction region. The analysis method according to claim 6,
The analysis method, wherein the reaction solution passes through the reaction region a plurality of times.

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