JP6600861B2 - Solid phase reaction chip and measurement method using the same - Google Patents

Description

  The present invention relates to a solid-phase reaction chip and a measurement method using the same.

  It is known that type I allergy that causes diseases such as bronchial asthma, allergic rhinitis, urticaria, or anaphylactic shock is caused by an excessive reaction between a specific antigen (allergen) and an IgE antibody thereto. In each clinical site such as a hospital, in order to identify a causal allergen that is a cause of an allergic disease, a test for detecting IgE antibody contained in a sample liquid is performed. Currently, as a technique for detecting an allergen-specific IgE antibody contained in a sample liquid, a technique called CAP using an immunofluorescence method as a measurement principle or a technique called MAST using an immunochemiluminescence method as a measurement principle is used. .

  By the way, in order to efficiently detect substances to be measured such as nucleic acids, proteins, and glycolipids contained in a sample solution, a binding substance having specific binding ability with the substance to be measured is immobilized on a solid phase. A solid-phase reaction chip is used that performs up to detection of a measurement target substance bound to a binding substance on the solid phase. The solid phase reaction chip is also called a biochip in the field of biochemistry, and for example, an mRNA chip, a cDNA chip, a micro PCR chip, a protein chip, and the like are known.

  In recent years, an immunoassay microchip using an antigen-antibody reaction has also been proposed for the diagnosis of allergic diseases. For example, Patent Document 1 discloses various allergen antigens as independent spots, that is, spaced spots. A biochip analysis method has been disclosed in which a reaction detection process between a sample liquid and an antigen can be automated and a measurement result can be quickly obtained after the sample liquid is collected using the mounted biochip.

JP 2011-13000 A

  CAP, which is a conventional technique, is a single-item method in which measurement is performed for each type of allergen in principle. Since a sample solution is required for each measurement, the physical burden on the subject is large and the measurement throughput is also high. Not good. On the other hand, MAST is a multi-item method in which, for example, about 30 types of allergens are measured at a time. Although the number of allergens that can be measured simultaneously is large compared to the single item method, the operation is complicated and it takes about 5 hours to obtain the measurement result.

  Further, in the technique described in Patent Document 1, a suction nozzle for sucking each liquid supplied from each nozzle such as a cleaning nozzle, an antibody nozzle, and a reagent nozzle is necessary, and the suction of each liquid is completed. In addition to the time required, there was a possibility that the suction force was insufficient due to the malfunction of the suction pump, and that the adhered matter adhered to the suction nozzle adhered to the biochip and caused contamination.

  The present invention has been made in view of such a situation, and an object of the present invention is to enable simultaneous multi-item measurement with a small amount of sample liquid, and to be included in the sample liquid quickly and with a simple operation. It is to provide a solid phase reaction chip capable of detecting a measurement target substance and a measurement method using the same.

In order to solve the above problems, a solid-phase reaction chip according to the present invention includes a rotating body formed by laminating a first base portion and a second base portion with a predetermined stacking width; a cover member which is capable of accommodating formation of the rotary member, the Rukoto is sandwiched between the inner circumferential surface of the outer peripheral surface and the cover member of the rotating body, and inhibits liquid absorbing member a displacement of the rotary body And provided in a coaxial portion of the first base portion and the second base portion laminated with the cover member, and the first base portion and the second base portion. A measurement target substance contained in the sample liquid on a surface facing the first base part of the second base part, having an opening for introducing the sample liquid into a gap formed therebetween immobilization region fixed multiple binding substance in a square matrix having a specific ability to bind to is a plurality of locations forming, the air It is characterized in that the volume of the sample solution to be introduced can be changed by adjusting the stacking width of the second base portion and the first base portion.

Further, the measurement method according to the present invention is formed so that the first base portion and the second base portion are stacked with a predetermined stacking width, and the rotating body can be accommodated. a cover member which is, by Rukoto is sandwiched between the inner peripheral surface of the cover member and the outer peripheral surface of the rotating body, and a suppressing liquid absorbing member a displacement of the rotary member, and the cover member laminated The specimen is provided in a gap formed between the first base portion and the second base portion, which is provided in a coaxial portion between the first base portion and the second base portion. An opening for introducing a liquid, and a surface of the second base part facing the first base part has a specific binding ability to a measurement target substance contained in the sample liquid. immobilization region fixed multiple binding substance in a square matrix is a plurality of locations forming, of the sample liquid to be introduced into the gap with The amount for the solid-phase reaction chip can be changed by adjusting the stacking width of the second base portion and the first base portion, introducing the sample liquid through the opening And a binding step for binding the substance to be measured and the binding substance, and a reaction solution containing a physiologically active substance having a specific binding ability for the substance to be measured bound to the binding substance and having an enzyme activity An addition step of adding a cleaning solution, a removal step of removing the cleaning solution by rotating a solid-phase reaction chip at a predetermined rotation speed after introducing the cleaning solution through the opening, and the enzyme activity in the physiologically active substance. And a measuring step for measuring.

  According to the present invention, a solid-phase reaction chip capable of simultaneous multi-item measurement with a small amount of sample liquid and capable of detecting a measurement target substance contained in the sample liquid quickly and with a simple operation, and the same A measurement method using can be provided.

It is the perspective view (a) and top view (b) for demonstrating the external appearance of the solid-phase reaction chip concerning this embodiment. It is a component diagram of each member which a solid-phase reaction chip comprises. It is the top view (a) explaining the 1st base part which comprises a rotary body, and the top view (b) explaining the 2nd base part. It is sectional drawing explaining the cross-sectional shape of a rotary body. It is a figure explaining the capacity | capacitance adjustment of the space | gap which consists of a 1st base part and a 2nd base part. It is a flowchart explaining the measuring method which concerns on this embodiment. It is a block diagram explaining the structural example of an automatic measuring apparatus.

  Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. In addition, this invention is not limited to the following description, In the range which does not deviate from the summary of this invention, it can change suitably. Further, the drawings are schematic, and the ratio of each dimension may be different from the actual one. Specific dimensions should be determined in consideration of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

  The solid phase reaction chip according to the present invention includes a rotating body formed by laminating the first base portion and the second base portion, a cover member formed to accommodate the rotating body, A liquid absorbing member is provided between the outer peripheral surface of the rotating body and the inner peripheral surface of the cover member, and the first base portion is interposed between the first base portion and the second base portion via the cover member. An opening for introducing the sample liquid into a gap formed between the first base portion and the surface of the second base portion facing the first base portion with respect to the measurement object contained in the sample liquid A plurality of binding substances having specific binding ability are immobilized. This will be described in detail below.

  FIG. 1 is a perspective view (a) and a plan view (b) for explaining an overview of a solid phase reaction chip 100 according to the present embodiment. FIG. 2 is a component diagram of each member constituting the solid-phase reaction chip 100. FIG. 3 is a plan view (a) for explaining the first base portion 31 constituting the rotating body 30 according to the present embodiment and a plan view (b) for explaining the second base portion 32. FIG. 4 is a cross-sectional view illustrating the cross-sectional shape of the rotating body 30. The solid phase reaction chip 100 includes a cover member 10 including an upper cover 11 and a lower cover 12, and a rotating body 30 formed by laminating a first base portion and a second base portion described later. When the rotating body 30 is accommodated in the cover member 10, the liquid absorbing member 20 is provided between the outer peripheral surface of the rotating body 30 and the inner peripheral surface of the cover member 10 (lower cover 12).

  The upper cover 11 has a substantially circular shape and is formed so as to be able to be fitted to the lower cover 12. The peripheral edge portion 11a of the upper cover 11 is formed in an annular shape so as to have a predetermined width toward the inside in the radial direction, and is connected to the disc portion 11c via the tapered portion 11b. At the center of the disc portion 11c, an upper cover opening portion 11d communicating with an opening portion provided in a first base portion of the rotating body 20 described later is formed, and the rotating body is interposed via the upper cover opening portion 11d. A sample solution, a reaction solution, a washing solution, and the like can be supplied to the sample 20. The sample liquid, the reaction liquid, and the cleaning liquid may be collectively referred to as a liquid.

  The lower cover 12 has a hollow shallow cylindrical shape that does not have a top surface, and the rotating body 30 and the liquid absorbing member 20 are accommodated in an internal space formed after fitting with the upper cover 11. The material used for the manufacture of the upper cover 11 and the lower cover 12 is not particularly limited as long as it does not transmit liquid and has non-adsorbability with respect to proteins and the like. For example, polyethylene, polycarbonate, Plastics such as polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, tetrafluoroethylene, polypropylene, unsaturated polyester, and epoxy can be used. As the upper cover 11, it is preferable to select a material that has a transparent form after manufacture so that the liquid passing through the upper cover opening 11d can be easily confirmed from the outside.

  The liquid absorbing member 20 is formed as an annular member in which members formed in a thin plate shape are joined at both ends, and the annular portion peripheral wall 20a is formed on the outer peripheral surface 30a of the rotating body 30 and the inner periphery of the lower cover 12. It arrange | positions so that it may be clamped between the surfaces 12a. The liquid absorbing member 20 is provided for suppressing the displacement of the rotating body 30 in the internal space of the cover member 10 and for absorbing liquid that is removed by the centrifugal force generated with the rotation of the rotating body 30 as will be described later. It is done. Examples of the material used for manufacturing the liquid absorbing member 20 include polyester fibers such as polyethylene terephthalate and polyethylene terbutyrate, polyolefin fibers such as polyethylene and polypropylene, composite fibers obtained by combining these fibers, and pulp fibers. In addition, fibers such as vegetable fibers such as cotton fibers and hemp fibers, fibers such as recycled fibers such as silk fibers and rayon fibers, woven fabrics, non-woven fabrics, and papers can be used. Moreover, the porous matrix which consists of polysaccharides, such as what has cellulose as a main component, such as a pulp fiber and rayon fiber, and acetate (acetylcellulose) produced by making acetic acid act on wood pulp can be used. In the description of the present embodiment, an example in which the absorbing member 20 is configured as an annular member has been described. It is also possible to have a shape.

  As illustrated in FIGS. 2 and 3, the rotating body 30 is configured by stacking a first base portion 31 and a second base portion 32. The first base portion 31 and the second base portion 32 are formed in a concentric disk shape, and as shown in FIG. 4, in the stacked state, the first base portion 31 and the second base portion A gap 30b is formed between the part 32 and the part 32.

  The first base portion 31 includes a taber-shaped opening 31c in an annular portion 31b provided at a substantially central position of a disc portion 31a having a diameter of about 22 mm, and is supplied via the upper cover opening 11d. The liquid can be introduced into the gap 30b between the first base portion 31 and the second base portion 32. Since the upper surface of the annular portion 31b is disposed in close contact with the back surface of the disk portion 11c of the upper cover 11, the liquid supplied through the upper cover opening portion 11d leaks to the surface of the first base portion 31. Never leave.

  The second base portion 32 is formed on a surface facing the first base portion, and a liquid receiving portion 32b provided at a substantially central position of a disc portion 32a having the same diameter as the first base portion 31. And an immobilization region 32c for immobilizing a plurality of binding substances having specific binding ability to the measurement object. The liquid receiving part 32b is formed in communication with the opening 31c of the first base part 31, and expands the liquid introduced through the opening 31c in the immobilization region 32c. In the fixed region 32c, spots having a diameter of about 100 μm to 1 mm are formed in 4 × 4 rows (16 spots) per region, and a total of 64 spots are spaced apart from the entire second base portion 32. It is configured to be possible. That is, in the present embodiment, 64 kinds of measurement items can be measured at a time by immobilizing the binding substance in all 64 spots. Any material can be used as a material used for manufacturing the immobilization region 32c as long as the binding substance can be immobilized directly or indirectly. However, when the second base portion 32 is integrally molded, In consideration of mechanical strength, it is preferable to use plastics such as polyethylene, polycarbonate, polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, tetrafluoroethylene, polypropylene, unsaturated polyester, and epoxy. In this case, if the first base part 31 is also made of the same material as that of the second base part 32, the manufacturing cost can be reduced and the design quality can be maintained.

  As described above, the gap 30 b is formed between the first base portion 31 and the second base portion 32. The volume of the liquid that can be accommodated in the gap 30b can be changed using the stacking width 30c between the disc portion 31a of the first base portion 31 and the disc portion 32a of the second base portion 32 as a guide. it can. Specifically, the lamination width 30c is preferably between 0.05 mm and 0.30 mm, and among these, the lamination width 30c is more preferably between 0.08 mm and 0.15 mm. As shown in FIG. 5A, when the lamination width 30c is 0.08 mm, the capacity of the gap 30b is about 30 μl. As shown in FIG. 5B, when the lamination width 30c is 0.1 mm, The capacity of the gap 30b is about 40 μl. As shown in FIG. 5C, when the stacking width 30c is 0.15 mm, the capacity of the gap 30b can be changed to about 60 μl. Thus, the rotating body 30 according to the present embodiment can change the sample volume to be provided as appropriate, and the sample volume required for one measurement is very small, about 30 μl to about 60 μl. The physical burden on the subject involved in the collection can be reduced.

  Next, a measurement method using the solid phase reaction chip 100 according to this embodiment will be described with reference to the flowcharts of FIGS. In this measurement method, a sample liquid is introduced into the solid-phase reaction chip 100 through the opening, and a binding step for binding the measurement target and the binding substance, and the measurement target substance bound to the binding substance are performed. An addition step of adding a reaction solution containing a physiologically active substance having specific binding ability and enzyme activity; a removal step of removing the reaction solution by rotating the solid-phase reaction chip at a predetermined rotation speed; And a measuring step for measuring enzyme activity in the active substance.

  In the measurement method according to the present embodiment, an example in which the measurement target is an IgE antibody that specifically binds to an allergen will be described. The allergen immobilized as a binding substance on the immobilization region 32c of the second base part 32 is not particularly limited. For example, house dust 1 (2), genus gourd, cedar, cypress, alder ( Genus), white birch (genus), camouflage, ragweed, mugwort, alternaria, aspergillus, malassezia (genus), cat (dandruff), dog (dandruff), cockroach, moth, latex, etc., allergens, milk, Allergen test items in medical institutions such as egg white, opomcoid, rice, wheat (fruit), buckwheat, soybean, peanut, apple, kiwi, sesame, beef, chicken, shrimp, crab, mackerel, salmon, tuna Any allergen can be selected as long as it can be consulted.

  Immobilization of the allergen to the immobilization region 32c can be performed by physical adsorption or chemical bonding generally used. For example, the allergen solution containing the measurement item allergen is spotted at a desired position in the immobilization region 32c using a microspotter or the like, thereby immobilizing the allergen in the immobilization region 32c. In this case, the allergen may be directly immobilized on the immobilization region 32c, or the allergen may be immobilized via a spacer substance. Since a total of 64 spot positions are provided in the immobilization region 32c according to this embodiment, it is possible to measure 64 types of measurement items at a time by spotting different allergens at each spot. it can. And the rotating body 30 is comprised by laminating | stacking the 1st base part 31 on the 2nd base part 32 by which the allergen was fix | immobilized by the fixed area | region 32c. At this time, the stacking width 30c between the first base portion 31 and the second base portion 32 is set to a predetermined width (for example, 0.05 mm to 0.30 mm, more preferably 0.08 mm to 0.15 mm). By doing so, the liquid volume to be provided can be adjusted.

  In addition, the physiologically active substance having specific binding ability to the IgE antibody as a measurement target substance and having an enzyme activity is not particularly limited. For example, alkaline phosphatase, β-galactosidase, glucose oxidase, urease, It is preferable to use an anti-IgE antibody labeled with creatine kinase, uricase, glucose-6-phosphate dehydrogenase, peroxidase or the like. In this embodiment, an example using an HRP-labeled antibody in which an anti-IgE antibody is labeled with horseradish peroxidase (HRP) will be described. When an HRP-labeled antibody is used, the generated signal can be obtained as a colorimetric signal or chemiluminescence signal depending on the substrate. As a substrate used for generation of a colorimetric signal, for example, tetramethylbenzidine and its derivatives, o-phenylenediamine, triarylmethanes, imidazole leuco dyes, and the like can be used. Examples of the generation of the chemiluminescence signal include acridinium salts, dioxetanes, luciferin, lucigenin, oxalyl chloride and the like.

  First, as shown in FIG. 2, the rotating body 30 including the second base portion 32 and the first base portion 31 in which the allergen is immobilized in the immobilization region 32c is set on the liquid absorbing member 20, The solid phase reaction chip 100 is configured by housing the rotating body 30 and the liquid absorbing member 20 in the cover member 10 including the upper cover 11 and the lower cover 12.

  Then, the sample liquid (30 μl to 60 μl) is supplied through the upper cover opening 11d of the upper cover 11 (step S100). The sample fluid can be appropriately selected from whole blood, serum, plasma, tear fluid and the like. When the sample liquid is supplied to the upper cover opening 11 d, the sample liquid is introduced into the liquid receiving part 32 b of the second base part 32 through the opening part 31 c of the first base part 31. The sample liquid that has reached the liquid receiving portion 32b is developed over the entire immobilization region 32c, and an IgE antibody having specific binding ability binds to the allergen that is a binding substance immobilized on each spot.

  Next, for example, after supplying a cleaning liquid (30 μl to 60 μl) containing a surfactant such as tween 20 through the upper cover opening 11d, the cleaning liquid is rotated at a predetermined rotational speed (2000 to 2500 rpm / min). Is removed (step S101).

  Next, in step S102, a reaction solution (30 μl to 60 μl) containing an HRP-labeled antibody is supplied through the upper cover opening 11d (step S102). After the HRP-labeled antibody is bound to the IgE antibody bound to the allergen, for example, a cleaning solution (30 μl to 60 μl) containing a surfactant such as tween 20 is supplied through the upper cover opening 11d, and then a predetermined rotational speed ( The cleaning liquid is removed by rotating at 2000 to 2500 rpm / min) (step S103).

  After completion of the cleaning in step S103, a substrate such as tetramethylbenzidine (TMB) is added, and the spot emitting a colorimetric signal is measured using an imaging means such as a CCD (step S104).

  Incidentally, although the measurement method described in the flowchart of FIG. 6 is manual, for example, the measurement can be automatically performed by using the automatic measurement apparatus 200 having the configuration shown in FIG. The automatic measuring apparatus 200 includes a dispensing unit 201 capable of sucking and discharging a solution from at least a set reagent cartridge, and a centrifugal rotating at a predetermined number of revolutions per minute while holding the solid-phase reaction chip 10. An operation unit 204 including a separation unit 202, an imaging unit 203 including an imaging unit such as a CCD, an input unit such as a touch panel, and a display unit such as an LCD (Liquid Crystal Display) for displaying device information or measurement results. And an image analysis unit 205 that analyzes an image (colorimetric signal emission spot) captured by the imaging unit 203, a measurement result generation unit 206 that generates a measurement result from the analysis result by the image analysis unit 205, and these And a control unit 207 for controlling the actual operation by the inspector other than setting the reagent cartridge or the solid-phase reaction chip 100 in the apparatus. Is configured to be unnecessary.

  According to the automatic measuring apparatus 200 having the above configuration, the final measurement result can be obtained in about 20 minutes, so that the throughput is high, and the operation can be intuitively performed according to the screen displayed via the operation unit 204. This is advantageous because it does not require skilled skills. In addition, since the device itself can be reduced in size, it can be applied as a device for immediate clinical examination.

  In the description of the measurement method, the measurement target is an IgE antibody that specifically binds to the allergen, and the allergen is assumed to be immobilized as a binding substance in the immobilization region 32c of the second base 32. However, the present invention is not limited to this, and the measurement object is, for example, a genotype derived from human papillow virus (HPV), and the second base 32 is immobilized. It is also possible to use a genomic DNA probe derived from HPV as a substance immobilized on the region 32c. In this case, there are 13 types of high-risk types such as 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, etc. It is also possible to construct as a measurement system for detecting HPV to be classified.

  Thus, the solid-phase reaction chip according to the present invention can be used as a platform capable of detecting and measuring, for example, autoimmune diseases, cancer markers, infectious diseases, myocardial markers, etc. in addition to those described in the above measurement examples. Yes, there is no limit to the type of inspection / measurement application.

  As described above, according to the present invention, simultaneous multi-item measurement with a small amount of sample liquid is possible, and a measurement target substance contained in the sample liquid can be detected quickly and easily. A phase reaction chip and a measurement method using the same can be provided.

DESCRIPTION OF SYMBOLS 10 Cover member 11 Upper cover 11a Peripheral part 11b Tapered part 11c Disk part 11d Upper cover opening part 12 Lower cover 12a Lower cover inner peripheral surface 20 Liquid absorption member 20a Annular part peripheral wall 30 Rotor 30a Outer peripheral surface 30b Gap 30c Lamination width 31 1st base part 31a Disk part 31b Ring part 31c Opening part 32 2nd base part 32a Disk part 32b Liquid receiving part 32c Immobilization area 100 Solid phase reaction chip 200 Automatic measuring device 201 Dispensing unit 201
202 Centrifugal Separator 203 Imaging Unit 204 Operation Unit 205 Image Analysis Unit 206 Measurement Result Generation Unit 207 Control Unit

Claims (10)

A rotating body formed by laminating a first base portion and a second base portion with a predetermined stacking width;
A cover member formed so as to accommodate the rotating body;
The Rukoto is sandwiched between the inner peripheral surface of the cover member and the outer peripheral surface of the rotating body, and a liquid absorbing member to suppress the displacement of the rotating body,
Provided in the coaxial part of the first base part and the second base part laminated with the cover member, and formed between the first base part and the second base part Having an opening for introducing the sample liquid into the gap,
A plurality of binding substances having specific binding ability to the measurement target substance contained in the sample liquid are fixed in a square matrix on the surface of the second base part facing the first base part. Multiple fixed areas are formed,
The volume of the sample liquid introduced into the gap can be changed by adjusting the stacking width of the first base part and the second base part. . The lamination width is a width between 0.05 mm and 0.30 mm.
The solid phase reaction chip according to claim 1. The stacking width is between 0.08 mm and 0.15 mm.
The solid phase reaction chip according to claim 2, wherein: The first base part and the second base part are any of polyethylene, polycarbonate, polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, tetrafluoroethylene, polypropylene, unsaturated polyester, or epoxy. Manufactured from
The solid-phase reaction chip according to any one of claims 1 to 3, wherein The binding substance is an allergen that induces a type I allergic reaction
The solid-phase reaction chip according to any one of claims 1 to 4, wherein The substance to be measured is an allergen-specific antibody
The solid phase reaction chip according to claim 5. The allergen-specific antibody is an IgE antibody
The solid phase reaction chip according to claim 6. The binding substance is a genomic DNA probe derived from human papillow virus
The solid phase reaction chip according to any one of claims 1 to 7, wherein The substance to be measured is a genotype derived from human papillow virus
The solid-phase reaction chip according to claim 8. A rotating body formed by laminating a first base portion and a second base portion with a predetermined stacking width;
A cover member formed to accommodate the rotating body;
A liquid absorbing member that suppresses displacement of the rotating body by being sandwiched between the outer peripheral surface of the rotating body and the inner peripheral surface of the cover member;
Provided in the coaxial part of the first base part and the second base part laminated with the cover member, and formed between the first base part and the second base part Having an opening for introducing the sample liquid into the gap formed,
A plurality of binding substances having specific binding ability to the measurement target substance contained in the sample liquid are fixed in a square matrix on the surface of the second base part facing the first base part. Multiple fixed areas are formed,
The volume of the sample liquid introduced into the gap can be changed by adjusting the stack width of the first base part and the second base part,
A binding step of introducing the sample liquid through the opening and binding the substance to be measured and the binding substance;
An addition step of adding a reaction solution containing a physiologically active substance having a specific binding ability to the measurement target substance bound to the binding substance and having enzyme activity;
A removal step of removing the cleaning liquid by introducing the cleaning liquid through the opening and then rotating the solid-phase reaction chip at a predetermined number of rotations;
And a measuring step for measuring the enzyme activity in the physiologically active substance.
Measuring method characterized by