JP2005172708A - Biochemical analyzing cartridge - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance reaction sensitivity by performing reaction treatment using a reduced amount of a reaction solution. <P>SOLUTION: A chamber for housing a biochemical analyzing unit and a housing chamber 37 for housing a tube 40 are provided to a cartridge 17. When the cartridge 17 is set to a reactor main body 19, the lid part 41 of the cartridge 17 is opened to draw out the tube 40 from the tube housing chamber 37 and the drawn-out tube 40 is attached to a tube pump 55. The reaction solution is circulated in the chamber by driving the tube pump 55. Since the tube 40 is used, a circulating flow channel can be shortened. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biochemical analysis cartridge that accommodates a biochemical analysis unit used for analysis of a DNA base sequence and the like.

  A biochemical analysis unit is used to perform biochemical analysis such as analysis of a base sequence of a biological substance, for example, DNA. The biochemical analysis unit forms a plurality of fine through holes in a substrate, and pushes an adsorbent substance such as a porous material into each of the through holes, thereby forming a plurality of adsorbent regions (hereinafter referred to as spot regions). This is called a microarray because a plurality of fine spot regions are arranged on the substrate. The biochemical analysis method using this biochemical analysis unit is a spotting method in which a specific binding substance (hereinafter referred to as a probe) serving as a reagent is spotted and immobilized on a plurality of spot regions of the biochemical analysis unit. From each of the steps, a reaction step in which a specific binding substance (hereinafter referred to as a target) as a specimen is infiltrated into the spot region to cause specific binding (binding of the probe and the target), and a unit for biochemical analysis, A data reading step of reading biochemical analysis data indicating the result of the specific binding reaction of the spot region, and a data analysis step of analyzing the read analysis data with a personal computer or the like (for example, Patent Document 1 and Patent Document 2).

  Since the probe serves as a reagent for examining the expression information of the target, a probe having a known molecular structure such as a base sequence or composition is used. Examples of probes include biological substances such as hormones, tumor markers, enzymes, antibodies, antigens, abzymes, receptors, other proteins, ligands, nucleic acids, cDNA, DNA, RNA, etc. It is a possible substance. The target has an unknown molecular structure, and hormones, tumor markers, enzymes, antibodies, antigens, abzymes, receptors, other proteins, ligands, nucleic acids, cDNA, DNA, mRNA, etc., are derived from living organisms. Extracted, isolated and collected, or chemically treated after this collection.

  When examining the base sequence, different types of probes are immobilized in each spot region of the biochemical analysis unit in the spotting step. Then, in the reaction step, a solution in which the target is dissolved in a solvent (hereinafter, simply referred to as “target solution”) is permeated into each spot region, so that the target and a probe having a complementary relationship with the target are uniquely identified. Tie them together.

  As a labeling method for labeling that specific binding has occurred, for example, a chemiluminescence method using light emission by a chemiluminescence reaction is used. In the chemiluminescence method, each reaction process of a target reaction process, an antigen-antibody reaction process, and a chemiluminescence reaction process is executed in order. The target reaction process is a process in which a target solution in which an antigen is bound to a target is permeated into each spot region to specifically bind the target and the probe. After this target reaction treatment, the biochemical analysis unit is washed to remove the target attached to the portion other than the spot region where the specific binding has occurred. Thereby, the target and the antigen remain only in the spot region where the specific binding has occurred.

  After the target solution is washed, an antigen-antibody reaction process is performed. In antigen-antibody reaction treatment, an enzyme antibody solution in which an enzyme (acts on a chemiluminescent substrate and decomposes it to emit light) and an antibody polymer (hereinafter referred to as enzyme antibody) in a solvent is permeated into the spot region. Then, the enzyme antibody and the antigen are bound by reacting the enzyme antibody solution with the antigen. After the antigen-antibody reaction process, the biochemical analysis unit is washed again, and the enzyme antibody is removed from the spot area where the antigen and the enzyme antibody are not bound.

  After washing the enzyme antibody, a chemiluminescent substrate solution containing a chemiluminescent substrate is permeated into each spot region to cause a chemiluminescent reaction. In the spot region where the antigen and the enzyme antibody are combined, the chemiluminescent substrate is decomposed by the action of the enzyme and emits light. The presence or absence of specific binding is detected by reading this light. As the detection means, for example, an imaging device such as a CCD image sensor that reads optical information is used.

  For example, a method using a shaking method is known as a method for allowing each of these reaction solutions to penetrate into each spot region. The shaking method is a method in which a reaction solution and a unit for biochemical analysis are placed in a reaction vessel, the vessel is shaken with a shaker, and the reaction solution is caused to flow in the reaction vessel to permeate the adsorptive region.

  By the way, in the shaking method, the reaction solution only flows in contact with the surface of the biochemical analysis unit, and therefore, the rate of shaking and substitution of the reaction solution into the adsorptive region is small, and the target in the reaction solution is Since the probability of encountering the probe in each spot region is low, there is a problem that there are few targets that specifically bind to the probe, and sufficient sensitivity cannot be obtained.

  Therefore, in order to eliminate such inconveniences, Patent Documents 1 and 2 propose a reactor (reaction apparatus) that automatically circulates and reacts a reaction solution in a reaction vessel. This reactor is provided with a mechanism for injecting the reaction solution, a circulation mechanism for circulating the reaction solution in the reaction vessel, a mechanism for discharging the reaction solution after the reaction, and the like.

  In the reactor described in Patent Document 1, a solution inlet and an outlet for detachably attaching a pipe are provided in the reaction vessel, and when the reaction solution is injected, the reaction solution stored in the tank is supplied into the reaction vessel. Then, the reaction solution is injected into the reaction vessel, and then the pipe is removed and a circulation pipe connected to a pump is attached to both ports, and the reaction solution is circulated in the reaction vessel by operating the pump. I am letting. The circulation is a flow-through method in which the spot region arranged on the substrate passes from one side to the other side.

  In the invention described in Patent Document 2, the biochemical analysis unit is accommodated in a cartridge and handled in consideration of the ease of handling of the biochemical analysis unit. This cartridge is provided with a chamber for storing a biochemical analysis unit. This chamber also serves as a reaction chamber in which the reaction solution is circulated and reacted. When this cartridge is used, it is not necessary to handle the unit as it is when the biochemical analysis unit is loaded into or removed from the reactor. Such a cartridge is also provided with a solution inlet and a solution outlet for injecting the reaction solution and circulating the reaction solution. When the cartridge is loaded into the reactor main body, the solution inlet and the solution outlet are connected to a pair of tubes for injecting or circulating the reaction solution.

JP 2003-227825 A International Publication No. 01/045843 Pamphlet

  However, in the reactor described in the above publication, the circulation of pipes and tubes for circulation becomes longer, the circulation capacity increases, a large amount of reaction solution is required, and a pump with a large discharge pressure is required, which increases costs. Become. Further, when the circulation volume is increased, there arises a disadvantage that the concentration of the reaction solution is lowered and the reaction sensitivity is deteriorated.

  The present invention can reduce the consumption of the reaction solution, improve the reaction sensitivity with a small amount of the reaction solution, and can reduce the cost of the reactor on the side set using an inexpensive pump. It is an object to provide a cartridge for use.

  In order to achieve the above object, a biochemical analysis cartridge is provided with a tube attached to a tube pump incorporated in a reactor main body. The tube has elasticity, and both ends thereof are connected to a circulation inlet and a circulation outlet that pass through a wall that partitions the tube storage chamber and the chamber. After the cartridge is set in the reactor main body to circulate the reaction solution, between both ends of the tube is set in a tube pump. This set is completed by hanging between the tube ends around the tube pump rotor. The tube pump rotates the rotor, sequentially crushes the tube by the rotation of the rotor, pushes out the reaction solution in the rotation direction of the rotor, and sucks the reaction solution at a vacuum pressure generated when the crushed tube is restored. The circulation inlet and the circulation outlet may be provided on the same wall among the walls constituting the chamber, or may be provided on different walls.

  When not in use, the tube provided in the cartridge becomes an obstacle. Therefore, it is desirable to provide a tube storage chamber inside the cartridge, separated from the chamber, and store the tube therein. At the time of use, it is necessary to be able to easily take out the tube from the tube storage chamber. Therefore, it is preferable to provide a structure in which a lid can be opened and closed outside the cartridge and the tube storage chamber is exposed by opening the lid.

  A switching valve may be built in the cartridge. In this case, a switching valve having at least four ports is used. The four ports are ports connected to the internal supply port, the internal discharge port, the external supply port, and the external discharge port, respectively. The internal supply port and the internal discharge port are ports for supplying the reaction solution into the chamber from the outside of the chamber or discharging the liquid in the chamber to the outside of the chamber, and two of the four ports of the switching valve. It is connected to each mouth. The external supply port and the external discharge port are connected to the remaining two ports of the switching valve. The switching valve is connected to the internal supply port and the internal discharge port to form a circulation position for circulating the reaction solution in the chamber, and the internal supply port and the internal discharge port are connected to the external supply port and the external discharge port. The flow path of the reaction solution is switched to a supply / discharge position that simultaneously forms a flow path connected to each other. The internal supply port and the internal discharge port may be provided on a wall different from the wall provided with the circulation inlet and the circulation outlet, or may be provided on the same wall. Instead of providing the storage chamber, a switching valve may be provided, or both the storage chamber and the switching valve may be provided.

  Moreover, when using the chemiluminescence method which is an indirect labeling method, each solution of a target solution, an enzyme antibody solution, and a chemiluminescent substrate solution is selectively used sequentially as a reaction solution. In the direct labeling method in which a labeling substance is included in the target solution, a target solution containing a labeling substance such as a fluorescent substance or a radioactive substance is used.

  Since the biochemical analysis cartridge of the present invention is provided with a tube for circulating the reaction solution, as described in the prior art, compared to using a pipe or tube externally connected to the reaction vessel or the reactor body. The circulation path length can be shortened, the consumption of the reaction solution can be suppressed, and the reaction solution can be reduced, so that the reaction sensitivity can be improved. Further, since a tube pump can be used, the cost of the reactor can be reduced. Furthermore, in the invention provided with the storage portion for storing the tube, it is not disturbed when not in use, and the handling becomes simple.

  As shown in FIG. 1, the biochemical analysis method using the biochemical analysis unit 10 includes a spotting process, a reaction process, a data reading process, and a data analysis process. The reaction process includes four processes including a target reaction process, a target washing process, an antigen-antibody reaction process, an antibody washing process, and a chemiluminescence reaction process. The biochemical analysis unit 10 has a plurality of fine through holes 12 formed in a matrix on a substrate 11 and a plurality of spot regions 14 formed by press-fitting a membrane 13 as an adsorptive substance into each through hole 12. It is a flow-through type.

  It is explanatory drawing of the unit 10 for biochemical analysis. In the data reading process, the biochemical analysis unit 10 is photoelectrically read by a detection device including a CCD image sensor described later. Therefore, the material of the substrate 11 is preferably a material that attenuates light in order to prevent light scattering, and metal, ceramic, plastic, or the like is used. The purpose of preventing light scattering is that light emitted from a certain spot region 14 passes through the substrate wall and reaches an adjacent spot region, so that light is emitted from the spot region that does not emit light. It is to prevent misrecognition that it looks like it is. By using a material having high efficiency for attenuating light, erroneous recognition is prevented, and highly reliable analysis data can be obtained. The light attenuation rate (decrease rate of the light intensity emitted from the spot area) is preferably 1/10 or less, and 1/100 or less, in the adjacent spot area. More preferably.

  The thickness of the substrate 11 is preferably in the range of 50 to 1000 μm, more preferably in the range of 100 to 500 μm. As the metal, copper, silver, gold, zinc, lead, aluminum, titanium, tin, chromium, iron, nickel, cobalt, tantalum, or the like can be used. Alternatively, an alloy such as stainless steel or brass can be used, but is not necessarily limited thereto. Examples of the ceramic include alumina and zirconia, but are not necessarily limited thereto.

  The use of plastic as the substrate material is preferable because it facilitates the formation of the through-holes, but there are cases where light attenuation is less likely to occur. Therefore, in order to further attenuate the light, it is preferable to fill the plastic with particles such as carbon, metal oxide particles and glass fiber and to disperse these inside the plastic. Examples of the metal oxide particles include, but are not necessarily limited to, silicon dioxide, alumina, titanium dioxide, iron oxide, and copper oxide.

  Examples of the method for forming the through hole 12 include a punching method, a discharge pulse method, an etching technique method (photolithography method), an electrolytic etching method, a method of irradiating a substrate with a laser such as an excimer laser, a YAG laser, and the like. Is not to be done. Each of these forming methods is appropriately selected according to the material of the substrate.

In order to increase the density of the through holes 12, the area of the opening of the through holes is preferably less than 5 mm ² , more preferably less than 1 mm ² , more preferably less than 0.3 mm ² , and 0.01 mm ^2. Less than is more preferable. Most preferably, it is 0.0001 mm ² or more. Further, when the through hole has a substantially circular shape, the diameter is preferably 30 μm to 300 μm.

The arrangement pitch P of the through holes 12 (distance from the center of two adjacent holes to the center) P is preferably in the range of 50 μm to 3000 μm, and the distance L from the ends of the two adjacent through holes 12 is: It is preferable to be in the range of 10 μm to 1500 μm. The density of the through holes 12 is preferably 10 / cm ² or more, more preferably 100 / cm ² or more, further preferably 500 / cm ² or more, and most preferably 1000 / cm ² or more and 50000. / Cm ² or less.

  The substrate 11 in which the through holes 12 are formed is subjected to a surface treatment in order to enhance the cleaning effect. When a metal or an alloy (for example, stainless steel) is used as the material of the substrate 11, the surface treatment is performed by at least one method such as corona discharge, plasma discharge, or anodizing. By this surface treatment, a highly hydrophilic surface treatment layer is formed on the substrate 11.

  After the surface treatment is performed, an adhesive is applied to the surface of the substrate 11 where the membrane 13 is press-fitted. The method of applying the adhesive is not particularly limited, but can be performed by roll coating, wire bar coating, dip coating, blade coating, air knife coating, or the like. Styrene butadiene rubber and acrylonitrile butadiene rubber are preferably used as the adhesive, but are not limited thereto. In order to prevent impurities from being generated in a later step, it is preferable to remove the excess adhesive by a method of scraping it off with a blade or by thermally decomposing it with laser light irradiation. In the present invention, the substrate surface treatment and the adhesive coating step may be omitted.

  After the adhesive is applied, the membrane 13 is pressed into the through hole 12. For the membrane 13, a porous material or a fiber material is preferably used. Further, a porous material and a fiber material can be used in combination. The membrane 13 used in the present invention may be a porous material (organic, inorganic, organic / inorganic) or a fiber material (organic, inorganic), or a mixture thereof. The thickness of the membrane 13 is not particularly limited, but it is preferable to use a membrane in the range of 50 μm to 200 μm (0.05 mm to 0.20 mm). Moreover, it is preferable to use a thing with the volume porosity C of the range of 10%-90%, and it is preferable to use what has the average hole diameter of the micropore which comprises a space | gap in the range of 0.1 micrometer-50 micrometers. The volume porosity C is expressed as a percentage of the volume sum of countless pores with respect to the apparent volume of the adsorbent material.

  The organic porous material is not particularly limited, but a polymer is preferably used. Examples of polymers include cellulose derivatives (eg, nitrocellulose, regenerated cellulose, cellulose acetate, cellulose acetate, cellulose acetate butyrate), aliphatic polyamides (eg, nylon 6, nylon 6,6, nylon 4,10, etc.), polyolefins (Eg, polyethylene, polypropylene, etc.), chlorine-containing polymers (eg, polyvinyl chloride, polyvinylidene chloride, etc.), fluororesins (eg, polyvinylidene fluoride, polytetrafluoride, etc.), polycarbonate, polysulfone, alginic acid And derivatives thereof (for example, alginic acid, calcium alginate, alginic acid / polylysine polyion complex, etc.), collagen, etc., and copolymers and composites (mixtures) of these polymers can also be used. That. In the present invention, it is preferable to use porous nylon from the viewpoint of water absorption.

  The inorganic porous material is not particularly limited, but is preferably a metal (eg, platinum, gold, iron, silver, nickel, aluminum, etc.), an oxide such as a metal (eg, alumina, silica, titania, Zeolite), metal salts (eg, hydroxyapatite, calcium sulfate, etc.), and composites thereof. A carbon porous material such as activated carbon is also preferably used.

  Also, organic fiber materials and inorganic fiber materials are not particularly limited. For example, organic fiber materials such as the cellulose derivatives and aliphatic polyamides, and inorganic fiber materials such as glass fibers and metal fibers can be used. Further, in order to increase the strength of the membrane 13, a fiber material that is insoluble in a solvent that dissolves the porous material may be mixed.

  The press-fitting of the membrane 13 is performed by intermittently pressing from above and below with a press plate in a state where the substrate 11 and the membrane 13 are overlapped. When an organic porous material and / or an organic fiber material is used for the membrane 13, the temperature of the substrate 11 is raised by heating the press plate and allowing it to penetrate into the substrate 11. Thereby, the membrane 13 is softened and becomes easy to push. A roller may be used instead of the press plate. Thus, the spot region 14 is formed by pushing the membrane 13 into the through hole 12.

  Specifically, as shown in FIG. 2, the flow-through area 28 in which a plurality of spot regions 14 are arranged has a substantially rectangular shape, and each spot region 14 is divided into blocks each having a substantially rectangular outline every predetermined number. It is regularly partitioned. The size of the substrate 11 is, for example, 70 mm in length and 90 mm in width. The size of each block 43 is about 4 mm square, for example. These blocks 43 are arranged in a matrix, and the number thereof is, for example, 10 vertically and 12 horizontally. The specifications of the flow-through area 28 such as the size and number of the blocks 43, the size of each spot area 14, and the arrangement pitch are determined so as to correspond to the specifications of the detection device 23 described later. Reference numeral 32 denotes a positioning hole for attaching the biochemical analysis unit 10 to a biochemical analysis cartridge 17 described later. In this example, each spot region 14 is partitioned in units of blocks, but such partitioning is not necessary. For example, the spot regions 14 are arranged at the same pitch over the entire flow-through area 28. May be.

  In the spotting step, a solution containing different types of probes (hereinafter referred to as probe solutions) is spotted on each spot region 14 of the biochemical analysis unit 10 using a spotter. The spotter is provided with a spot pin 15 having a split groove formed at the tip and spotting a probe solution. A plurality of types of probe solutions dispensed on the well plate are sucked up by the spot pins 15, and the sucked-up probe solutions are spotted on the spot regions 14. Thereafter, the probe is fixed to the spot region 14 by irradiating each spot region 14 with ultraviolet rays or the like. In this way, the biochemical analysis unit 10 to which the probe is fixed is accommodated in a biochemical analysis cartridge (hereinafter simply referred to as a cartridge) 17 having a chamber 16 and is sent to the reaction process in that form.

  In the reaction process, five processes are performed in the order of target reaction process, target cleaning process, antigen-antibody reaction process, antibody cleaning process, and chemiluminescence reaction process. In the target reaction process, the probe is reacted with the target serving as the specimen using the reactor 18. The reactor 18 includes a reactor main body 19, a circulation mechanism 20, and a supply / discharge mechanism 21. The reactor main body 19 includes a cartridge storage chamber 22 in which the cartridge 17 is detachably set. The reactor main body 19 also serves as a heat retaining case for keeping the temperature in the chamber 16 at a predetermined temperature.

  The target solution used in the experiment is made by a reaction solution preparation device. The reaction solution preparation apparatus prepares a target solution by dissolving a target bound to an antigen in a solvent.

  The circulation mechanism 20 causes the target solution to circulate in a chamber 16 provided inside the cartridge 17 and to cause a reaction. The supply / discharge mechanism 21 supplies the target solution to the chamber 16 and discharges the target solution after the reaction.

  The target solution in circulation penetrates into each spot region 14 from the lower surface side in the drawing and flows to the upper surface side in the drawing. As a result, the target solution penetrates into each spot region 14 having a probe complementary to the target. During this permeation, a specific binding reaction occurs between the target and the probe. The target solution that has passed through each spot region 14 is discharged from the upper surface side of each spot region 14 to the outside of the chamber 16 by the circulation mechanism 20, and the target solution discharged from the chamber 16 is again returned to the lower surface side of each spot region 14. Injected.

  After performing this target reaction process, the cleaning liquid is supplied to the chamber 16 to perform the target cleaning process. In the target washing treatment, the target bound to the antigen is removed from the portion other than the spot region 14 where the specific binding reaction has occurred. After washing the target, an antigen-antibody reaction process is performed. In the antigen-antibody reaction process, an enzyme antibody solution is supplied to the chamber 16 so that the enzyme antibody solution penetrates into each spot area. In the spot region 14 where the target remains specifically bound, an antigen-antibody reaction occurs, and the enzyme antibody binds to the antigen bound to the target. After the antigen-antibody reaction process, a cleaning solution is supplied to the chamber 16 to perform the antibody cleaning process. The enzyme antibody is removed from portions other than the spot region 14 where the antigen-antibody reaction has occurred by this antibody washing treatment.

  After performing this antibody washing process, a chemiluminescence reaction process is performed. In this process, a chemiluminescent substrate solution containing a chemiluminescent substrate is supplied to the chamber 16 so that each spot region 14 penetrates the chemiluminescent substrate solution. Thereafter, data reading is performed with the cartridge 17 loaded in the reactor body 19. The cartridge 17 is provided with a detection window described later, and the biochemical analysis data is photoelectrically read from the biochemical analysis unit 10 in the cartridge 17 through the detection window. This reading is performed by the detection device 23. The detection device 23 includes a plurality of optical fibers 24 set one by one in each spot area 14 and an imaging unit 25 that images each spot area 14 through the optical fiber 24. Then, the read data is analyzed in the data analysis process to identify the target.

  As shown in FIG. 3, the cartridge 17 includes an upper half part 26 and a lower half part 27. In the upper and lower half portions 26 and 27, as shown in detail in FIG. 4, an upper concave portion 29 and an upper concave portion 30 that are recessed by one step are formed at portions facing the flow-through area 28 of the biochemical analysis unit 10. Thus, a predetermined space is secured between the inner walls of the upper and lower recesses 29 and 30 and the flow-through area 28. The chamber 16 is configured as a rectangular space in plan view by combining the upper and lower recesses 29 and 30, and the upper and lower recesses 29 and 30 are partitioned vertically by the biochemical analysis unit 10.

  The lower half 27 is provided with positioning pins 31. The biochemical analysis unit 10 is configured so that the flow-through area 28 is accommodated in the chamber 16 by inserting these pins 31 into the positioning holes 32. Positioning is performed. Grooves 34 for attaching packings 33 are formed around the upper and lower recesses 29 and 30, respectively. The chamber 16 is watertight by sandwiching the upper and lower half portions 26 and 27 with a pair of stoppers 35.

  In the upper and lower half portions 26 and 27, a tube storage chamber 37 is provided next to the chamber 16 with the partition wall 36 interposed therebetween. The tube storage chamber 37 is also configured by combining the upper and lower half portions 26 and 27. The tube storage chamber 37 is provided with a circulation inlet 38 and a circulation outlet 39 exposed. The circulation inlet 38 communicates with the lower recess 30, and one end 40 a of the tube 40 is connected to this. On the other hand, the circulation outlet 39 communicates with the upper recess 29, and the other end 40 b of the tube 40 is connected to this. The circulation inlet 38 and the circulation outlet 39 are provided on the wall 16 a corresponding to one side of the rectangular outline of the chamber 16 in plan view. The tube 40 constitutes a circulation path for circulating the reaction solution between the upper recess 29 and the lower recess 30.

  The tube 40 is formed of a material having elasticity, and is stored in the tube storage chamber 37 by folding an intermediate portion between the one end 40a and the other end 40b. The tube storage chamber 37 is provided at a position adjacent to the wall 16a provided with the circulation inlet 38 and the circulation outlet 39 and is isolated from the outside. For this reason, the lid part 41 which can be opened and closed is provided in the part which comprises an upper half part so that the tube 40 can be pulled out simply. The middle part of the drawn tube 40 is set in a tube pump for circulation described later in detail. The lid portion 41 is formed with a notch portion 42 for allowing the tube 40 to escape, so that the tube 40 can be closed with the tube pump set in the tube pump. Instead of the cutout portion 42, the whole may be opened.

  The configuration of the lid 41 may be a configuration in which a hinge is provided and attached by a separate member from the upper half 26, or a part of the upper half 26 is thinned and formed so as to be foldable. An integrated pull-top lid may be used. In the case of an integrated configuration, the upper half 26 may be formed of a resin material. Further, not only the upper half portion 26 but also the lower half portion 27 may be formed of a resin material. As the resin material, it is preferable to use, for example, ABS (acrylonitrile butadiene styrene) resin, PP (polypropylene) resin, or the like. In addition, as the tube 40, it is preferable in terms of durability to use a silicon tube, a composite tube of a fluorine resin and a fluorine-based elastomer, or the like.

  Further, the cartridge 17 is provided with a solution injection port 44 and an outflow port 45 exposed to the outside on a wall 16b adjacent to the wall 16a. The solution injection port 44 is connected to the upper recess 29, and the solution outlet 45 is connected to the lower recess 30. The reaction solution and the cleaning solution are supplied or discharged using these ports 44 and 45. The position of the cartridge 17 where the solution injection port 44 and the outflow port 45 are provided is not limited to the wall 16b adjacent to the wall 16a, but may be provided on the same wall 16a. You may provide in the wall of the opposite side. Moreover, you may provide in the upper surface and lower surface instead of the outer periphery of the cartridge 17.

  A detection window 47 is provided at a position facing the flow-through area 28 of the top plate 46 of the upper half 26. The detection window 47 includes a plurality of openings formed at positions corresponding to the spot regions 14. The diameter of each opening corresponds to the diameter of the spot region 14.

  As shown in FIG. 5, the cartridge 17 is loaded into the reactor main body 19. The reactor main body 19 includes a main body base 51 and an upper lid 50. A cartridge housing chamber 22 is provided inside the main body base 51 and the upper lid 50. The cartridge storage chamber 22 is formed in accordance with the outer shape of the cartridge 17, and a notch 53 that exposes the solution inlet 44 and the outlet 45 to the outside of the reactor body 19 is provided at each side end portion thereof. Is provided. The upper lid 50 is placed over the main body base 51 so as to cover the loaded cartridge 17. In this state, the upper lid 50 and the main body base 51 are crimped by a crimping mechanism (not shown) to hold the cartridge 17 in the cartridge storage chamber 22 and close the detection window 47 of the cartridge 17.

  A tube pump 55 is fixed to the main body base 51 next to the cartridge storage chamber 22. The tube pump 55 includes a tube case 56 and a motor 57. The tube case 56 includes a base plate 58 and a detachable lid 59. A disk-shaped rotor 61 is rotatably attached to the bottom of the base plate 58 at the bottom of the recessed portion 60 that is recessed by one step. The rotor 61 has a configuration in which a plurality of rollers 62 are attached to the outer periphery, and rotates in one direction by driving a motor 57. The inner wall which comprises the recessed part 60 becomes a substantially U shape, and the rotor 61 rotates inside the circular arc wall among the U-shaped inner walls. A gap is formed between the arc wall and the rotor 61. The intermediate portion of the tube 40 is fitted along the gap, and then the lid 59 is attached to complete the setting of the tube 40.

  The motor 57 is attached to the main plate 58, and the contour projects from the main body base 51. When the motor 57 is driven by an external operation, the rotor 61 rotates, and the plurality of rollers 62 sequentially crush the tube 40 as the rotor 61 rotates. Accordingly, the tube pump 55 pushes out the reaction solution in the rotation direction of the rotor 61 and sucks the reaction solution with a vacuum pressure generated when the crushed tube 40 is restored. The wall of the tube case 56 facing the cartridge 17 is formed with an open portion or a cutout portion so as to allow both ends of the tube 40 to escape.

  A supply / discharge mechanism 21 is connected to the solution inlet 44 and the outlet 45 exposed from the reactor body 19. As shown in FIG. 6, the supply / discharge mechanism 21 includes a switching valve 65, a supply mechanism 66, and a discharge mechanism 67. The supply mechanism 66 has a pair of supply valves B1 to B5 and supply tanks T1 to T5 arranged in each of the pipes 70a to 70e radially arranged around the main 70f. The main valve 70f and a switching valve to be described in detail later. The third port 65c of 65 is connected by a single pipe 71, and the solution stored in any one of the supply tanks T1 to T5 is opened by opening the corresponding supply valves B1 to B5. A supply flow path for supplying to the chamber 16 is formed. The discharge mechanism 67 includes a discharge valve 72, a discharge tank 74, and a pipe 75 that connects these, and a discharge channel that discharges the solution remaining in the chamber 16 to the discharge tank 74 after each treatment. Is forming.

  The target solution is stored in the supply tank T1, and the cleaning liquid is stored in the supply tank T2 and the supply tank T4, respectively. The cleaning liquid stored in the supply tank T2 is for removing the target that did not specifically bind, and the cleaning liquid stored in the supply tank T3 is for removing the enzyme antibody that did not bind in the antigen-antibody reaction. It is. The supply tank T3 stores an enzyme antibody solution, and the supply tank T5 stores a chemiluminescent substrate solution.

  The supply valves B1 to B5 are switched to an open position where any of the valves opens the supply flow path during supply, and to a closed position where the supply flow path on the supply tank side is closed during discharge. The discharge valve 72 is switched to an open position for opening the discharge flow path during discharge, and to a closed position for closing the discharge flow path on the discharge tank side during supply.

  The switching valve 65 is provided with four ports 65a to 65d. The solution inlet 44 is connected to the first port 65a, and the solution outlet 45 is connected to the second port 65b by pipes. The third port 65c is connected to the downstream end of the supply flow path of the supply mechanism 66, and the fourth port 65d is connected to the upstream end of the discharge flow path of the discharge mechanism 67 by a pipe. The switching valve 65 switches the flow path between the circulation position and the supply / discharge position by an external operation. At the supply / discharge position, the first port 65a and the third port 65c are connected, the supply flow path of the supply mechanism 66 is connected to the solution injection port 44, and the second port 65b and the fourth port 65d are connected to each other. The solution outlet 45 is connected to the discharge flow path of the discharge mechanism 67. At the circulation position, as shown in FIG. 7, the first port 65a and the second port 65b are connected, and the solution inlet 44 and the solution outlet 45 are directly connected to form a circulation channel.

  The work procedure in the reaction process will be described. The cartridge 17 is handled in a form in which the tube 40 is stored in the tube storage chamber 37. Next, the upper lid 50 of the reactor main body 19 is opened to set the cartridge 17 in the cartridge storage chamber 22, and then the lid portion 41 of the cartridge 17 is opened to pull out the tube 40. The intermediate portion of the tube 40 is set on the tube pump 55. The setting is completed by opening the lid 59 of the tube case 56, fitting the intermediate portion of the tube 40 along the gap on the outer periphery of the rotor 61, and then attaching the lid 59. After the lid 59 of the tube case 56 is closed, the lid 41 of the cartridge 17 is closed, and finally the upper lid 50 of the reactor body 19 is attached.

  Thereafter, in order to perform the target reaction process, the switching valve 65 is switched to the supply / discharge position. When switched to the supply / discharge position, the solution inlet 44 is connected to the supply flow path of the supply mechanism 66, and the discharge flow path of the discharge mechanism 67 is connected to the solution outlet 45. Thereafter, the valve B1 of the supply mechanism 66 is switched to the open position, and the valve 72 of the discharge mechanism 67 is switched to the open position. Finally, by driving the tube pump 55, the target solution is supplied from the supply tank T1 to the chamber 16 inside the cartridge 17. The other valves B2 to B5 are in the closed position.

  After the target solution is filled in the chamber 16, the switching valve 65 is switched to the circulation position. As a result, the solution inlet 44 and the solution outlet 45 are connected to form a circulation channel. At this time, the valves B1, 72 of the supply / discharge mechanisms 66, 67 are also switched to the closed position. Thereafter, the tube pump 55 is driven. Thereby, the tube pump 55 pushes out the target solution in the rotation direction of the rotor 61 and sucks the target solution with the vacuum pressure generated when the crushed tube 40 is restored. The pushed target solution flows into the lower recess 30 of the chamber 16 through the circulation inlet 38, and the target solution is sucked from the upper recess 29 toward the tube pump 55 through the circulation outlet 39. By circulating the target solution for a certain period of time, the circulating target solution permeates the spot regions 14 from the lower recesses 30 and flows in one direction toward the upper recesses 29. It penetrates into each spot area 14. During this permeation, a specific binding reaction occurs between the target and the probe. The direction in which the target solution is circulated is not limited to the one direction described above, and the tube pump 55 may be reversed and circulated in the reverse direction, or the tube pump 55 may be circulated alternately in the forward and reverse directions. Also good.

  After the reaction is completed, the cleaning solution is supplied and the target solution is discharged. In the supply / discharge operation, the switching valve 65 is switched to the supply / discharge position. Next, the valve B2 of the supply mechanism 66 is switched to the open position, and the valve 72 of the discharge mechanism 67 is switched to the open position. Then, by driving the tube pump 55, the cleaning liquid is supplied from the supply tank T2, and the target solution is discharged from the chamber 16 to the discharge tank 74 so as to be pushed out by the cleaning liquid.

  After performing the target reaction process, a target cleaning process is performed. By this processing, the biochemical analysis unit 10 is washed, and the target and the antigen are removed from portions other than the spot region 14 where the specific binding reaction has occurred. In order to perform cleaning, when the switching valve 65 is switched to the circulation position, the valves B2 and 72 of the supply / discharge mechanisms 66 and 67 are switched to the closed position, and the tube pump 55 is driven, the cleaning liquid is supplied from the supply tank T2 to the cartridge 17. Then, cleaning is performed by circulating in the circulation path.

  After the target washing treatment, an antigen-antibody reaction treatment is performed. In this process, the switching valve 65 is switched to the supply / discharge position, the valve B3 is opened, the valve 72 of the discharge mechanism 67 is switched to the open position, and the tube pump 55 is driven to transfer the enzyme antibody solution from the supply tank T3. While being supplied, it is pushed out by this solution and the cleaning liquid is discharged to the discharge tank 74.

  After the antigen-antibody reaction treatment, the antibody washing treatment is performed. In this process, the switching valve 65 is switched to the supply / discharge position, the valves B3, 72 of the supply / discharge mechanisms 66, 67 are switched to the closed position, the valve B4 is then opened, and the valve 72 of the discharge mechanism 67 is set to the open position. By switching and driving the tube pump 55, the second cleaning liquid is supplied from the supply tank T4 and pushed out by the cleaning liquid, and the enzyme antibody solution is discharged to the discharge tank 74. After that, when the switching valve 65 is switched to the circulation position, the valves B4 and 72 of the supply / discharge mechanisms 66 and 67 are switched to the closed position, and the tube pump 55 is driven, the cleaning liquid circulates in the circulation path, thereby cleaning the second time. Is done.

  In order to further improve the effect of the second washing, it is preferable that a so-called blocking material is infiltrated into the biochemical analysis unit 10 before the enzyme antibody solution is supplied. By doing so, in the washing step, the enzyme antibody that has not been bound by the antigen-antibody reaction is more easily peeled off, and the washing effect is further improved.

  After the antibody washing process, a chemiluminescence reaction process is performed. In this process, in order to infiltrate the chemiluminescent substrate solution into each spot region, the switching valve 65 is switched to the supply / discharge position, the valve B5 is opened, and the valve 72 of the discharge mechanism 67 is switched to the open position. By driving, the chemiluminescent substrate solution is supplied from the supply tank T5 and pushed out by this solution, and the cleaning liquid in the circulation path is discharged to the discharge tank 74.

  After the injection of the chemiluminescent substrate solution, the enzyme decomposes the chemiluminescent substrate and the chemiluminescent substrate emits light. After the injection of the chemiluminescent substrate solution, data reading is performed with the cartridge 17 loaded in the reactor body 19.

  As shown in FIG. 8, the detection device 23 includes an imaging unit 25 including a CCD image sensor 80 that receives light emitted from a chemiluminescent substrate and photoelectrically converts the light, and a light guide 81 attached thereto. . The imaging unit 25 includes a CCD package 82 in which the CCD image sensor 80 is packaged, and a cooling unit 83 that cools the CCD package 82. Since the weak light must be detected when reading the data of the biochemical analysis unit 10, the CCD image sensor 80 is cooled to 0 ° C. or less by the cooling unit 83.

  The cooling unit 83 is obtained by attaching a heat sink 86 to a case body 85 in which a Peltier device 84 is incorporated. As is well known, the Peltier element 84 is an element utilizing the “Peltier effect” in which heat is transferred from one semiconductor to the other when two kinds of semiconductors are joined to a thin metal and current is passed through the semiconductor. This is used for cooling a CPU of a computer because no noise is generated. The CCD package 82 is accommodated in the case main body 85 and attached to a heat transfer plate 87 joined to the heat absorption surface of the Peltier element 84. The case body 85 is filled with dry nitrogen to prevent condensation. The Peltier element 84 is attached such that its heat radiating surface is in contact with the case main body 85, and the absorbed heat is radiated by the case main body 85 and the heat sink 86.

  The light guide 81 is composed of a plurality of optical fibers 24, and is arranged such that one end of each optical fiber 24 faces the light receiving surface and the other end faces the spot region. A position fixing member 88 for fixing the arrangement position of each optical fiber 24 so as to correspond to the arrangement of each spot region 14 is attached to the end facing the biochemical analysis unit 10.

  In the reaction process, the upper surface of the cartridge 17 including the detection window 47 is covered with the upper lid 50. When the reaction and the cleaning process are completed, the upper lid 50 is removed, and the detection window 47 is exposed to the outside. In this state, the end face of the light guide 81 is placed on the detection window 47 so that each optical fiber 24 faces each opening of the detection window 47 formed corresponding to each spot region 14. Light emitted from each spot region 14 is input to each optical fiber 24 through each opening.

  In the spot region 14 where the specific binding reaction has occurred, the labeling substance remains and therefore emits light. On the other hand, no light is emitted in the spot region 14 where no specific binding reaction occurs. The CCD image sensor 80 acquires image data indicating the result of the specific binding reaction of each spot region 14. In the data analysis step, this image data is analyzed as biochemical analysis data.

  In addition, since the tube 40 of the cartridge 17, 90, 100 for biochemical analysis of the above embodiment is subjected to biochemical analysis using the indirect labeling method and the chemiluminescence method, the target solution, the enzyme antibody solution, and Although used to circulate a reaction solution such as a chemiluminescent substrate solution, the present invention is not limited to this, and may be a tube for circulating any one of these reaction solutions, Alternatively, a direct labeling method in which a labeling substance is included in the target solution may be used, and a tube that circulates only the target solution containing the labeling substance may be used.

  In the above embodiment, the switching valve 65 for circulation is provided in the reactor 18, but the switching valve 65 for circulation may be provided in the cartridge 90 as shown in FIG. In this case, an external supply port 91 and an external discharge port 92 for connection to the supply / discharge mechanism are provided to be exposed to the outside of the cartridge 90. Further, the reactor main body 19 is provided with a motor and an actuator that serve as a driving source for switching the switching position of the switching valve 65. Then, it is only necessary to provide engagement / disengagement means by which the drive unit of the drive source can be engaged / disengaged with the drive input unit of the switching valve 65 by attaching / detaching the cartridge 90 to / from the reactor body 19. As the engagement / disengagement means, for example, in the case where the structure of the switching valve uses a rotational force to switch the flow path, a key portion 95 is provided at the tip of the output shaft 94 of the motor 93 as shown in FIG. The part 95 may be configured to engage with the drive input part 96 of the switching valve 65. Of course, if the set direction and the engagement direction are aligned so that the output shaft of the drive source is automatically engaged with the drive input portion of the switching valve by setting the cartridge 90 on the main body base 51, the cartridge 90 will be described. This is preferable because the set operation becomes simple.

  Moreover, in the said embodiment, although it has the structure which accommodates all the tubes 40 in the tube storage chamber 37, and opens and pulls out the cover part 41, in this invention, as shown in FIG. In addition, a pair of openings 102 and 103 are provided in the outer cover 101 of the cartridge 100, a part 104 of the intermediate part of the tube 40 is exposed to the outside from these openings 102 and 103, and the remaining intermediate part is stored in the tube storage chamber It is good also as composition to do. In this case, a pulling mechanism is provided inside the cartridge 100, and the tube 40 is pulled toward the inside of the cartridge 17 by the pulling mechanism. When using, it is only necessary to hold the part 104 of the tube 40 and pull it out against the pulling force of the pulling mechanism and attach it to the tube pump.

  Furthermore, the tube 40 may be configured by combining a plurality of tubes of different materials. For example, the portion that is not pulled out of the cartridge 17 is formed of a resin material or a metal material, and the portion that is pulled out or the portion that is attached to the tube pump is connected to a tube formed of an elastic material. Also good.

  Further, in each of the above embodiments, the tube storage chamber 37 for storing all or part of the tube 40 is provided in the cartridge 17, but this may be omitted and the tube 40 may be attached to the outside of the cartridge 17.

It is a flowchart which shows the whole process of the biochemical analysis method using the unit for biochemical analysis. It is explanatory drawing which shows the unit for biochemical analysis, and has expanded and shown a part. It is a disassembled perspective view which shows a cartridge. It is sectional drawing which shows the principal part of a cartridge and a reactor main body. It is a disassembled perspective view which shows a reactor main body. It is explanatory drawing of the reactor which shows a flow path when supplying or discharging | emitting a reaction solution. It is explanatory drawing of the reactor which shows a flow path when circulating a reaction solution. It is explanatory drawing which shows the outline of a detection apparatus. It is explanatory drawing which shows other embodiment which incorporated the switching valve for circulation in the cartridge. FIG. 10 is a main part perspective view showing a means for connecting a circulation switching valve to a reactor-side drive source in the example described in FIG. 9. It is a perspective view showing an example in which a part of the tube is exposed to the outside and the tube is pulled out by holding the part.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Biochemical analysis unit 14 Spot area 16 Chamber 17, 90, 100 Cartridge 18 Reactor 19 Reactor main body 28 Flow through area 37 Tube storage chamber 40 Tube 55 Tube pump 65 Switching valve

Claims (3)

A cartridge for biochemical analysis comprising a chamber for containing a flow-through type biochemical analysis unit formed with a plurality of spot regions through which a reaction solution passes, and detachably loaded in the reactor,
A biochemical analysis cartridge characterized in that a tube connected to the chamber and detachably attached to a tube pump incorporated in the reactor is provided to circulate the reaction solution by driving the tube pump. .   The cartridge for biochemical analysis according to claim 1, further comprising a storage portion for storing the tube when the tube is detached from the reactor.   Circulating the reaction solution in the chamber, and a supply / discharge position for forming a flow channel for supplying the reaction solution into the chamber from outside and a flow channel for discharging the liquid in the chamber to the outside The cartridge for biochemical analysis according to claim 1 or 2, further comprising a switching valve for switching the flow path of the reaction solution with a circulation position for forming a flow path.

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