JP7205940B2 - Solid phase reaction chip and measurement method using the same - Google Patents

Description

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

Type I allergy, which causes diseases such as bronchial asthma, allergic rhinitis, urticaria, or anaphylactic shock, is known to be caused by an excessive reaction between a specific antigen (allergen) and its IgE antibody. In clinical practice sites such as hospitals, tests are performed to detect IgE antibodies contained in test fluids in order to identify causative allergens that cause allergic diseases. Currently, methods for detecting allergen-specific IgE antibodies contained in sample liquids include CAP, which uses immunofluorescence as its measurement principle, and MAST, which uses immunochemiluminescence as its measurement principle. .

By the way, conventionally, in order to efficiently detect target substances such as nucleic acids, proteins, and glycolipids contained in a sample liquid, a binding substance having specific binding ability with the target substances is immobilized on a solid phase. , a solid-phase reaction chip is used that performs up to detection of a substance to be measured bound to a binding substance on the solid phase. Solid-phase reaction chips are also called biochips in the field of biochemistry, and known examples include mRNA chips, cDNA chips, micro PCR chips, and protein chips.

In recent years, immunological analysis microchips using antigen-antibody reactions have been proposed for the diagnosis of allergic diseases. Disclosed is a biochip analysis method that uses a mounted biochip to automate the process of detecting a reaction between a sample liquid and an antigen after collection of the sample liquid, and to obtain measurement results quickly.

CAP, which is a conventional technology, is a single-item method in which measurement is performed for each allergen in principle, and because a sample liquid is required for each measurement, the physical burden on the subject is large, and the throughput related to measurement is also low. not good. On the other hand, MAST is a multi-item method in which, for example, about 30 kinds of allergens are measured at once. Although the number of allergens that can be measured simultaneously is larger than that of the single item method, the operation is complicated and it takes about 5 hours to obtain measurement results.

Further, the technique described in Patent Document 1 requires a suction nozzle for sucking each liquid supplied from each nozzle such as a washing nozzle, an antibody nozzle, and a reagent nozzle. In addition to the time required, there was also the risk of insufficient suction power due to malfunction of the suction pump, and the possibility of contamination due to deposits adhering to the suction nozzle adhering to the biochip.

In view of such a situation, the applicant of the present application provided a liquid absorbing member on the outer periphery of the rotating body formed by laminating the first base portion and the second base portion, and accommodated the liquid absorbing member. A solid-phase reaction chip consisting of a cover member was developed and applied for a patent (Patent Document 2). The invention according to Patent Document 2 fixes a plurality of binding substances having specific binding ability to the substance to be measured contained in the specimen liquid on the surface of the second base facing the first base. This makes it possible to perform simultaneous multi-item measurement with a small amount of sample liquid. can be detected.

Japanese Patent Application Laid-Open No. 2011-13000 JP 2016-200431 A Patent No. 6107244 International publication WO2018/056454

The solid-phase reaction chip according to Patent Document 2 requires a cover member that accommodates a rotating body formed by laminating the first base portion and the second base portion. The cover member forms the outer shape of the solid-phase reaction chip and also serves to hold the rotating body via the liquid-absorbing member. However, as a result of repeated experiments, it became clear that the rotating body is displaced inside the cover member as it rotates at high speed, which affects the measurement accuracy. In addition, since the circular rotating body is in surface contact with the liquid absorbing member formed in an annular shape, there is also a problem that liquid pools occur on the contact surface, and the liquid absorbing ability is lowered.

The present invention has been made to solve such problems, and an object of the present invention is to overcome the drawbacks of the solid-phase reaction chip having a liquid-absorbing member inside, and to rapidly and simply operate the sample. An object of the present invention is to provide a solid-phase reaction chip capable of accurately detecting a substance to be measured contained in a liquid, and a measurement method using the same.

In order to solve the above problems, a fixed reaction chip according to the present invention is a solid-phase reaction chip formed as a rotating body by fitting and integrating an upper lid member and a lower lid member, wherein the upper lid member or an immobilizing section provided on the inner bottom surface side of any of the lower lid members for immobilizing a plurality of binding substances having specific binding ability to a substance to be measured contained in the sample liquid; and provided on the upper lid member. an opening for supplying the sample liquid to the surface of the immobilizing portion; and a liquid absorbing portion provided outside the immobilizing portion for absorbing the sample liquid supplied through the opening. , the immobilization part has a plurality of immobilization areas separated by raised parts that protrude toward the inner bottom surface facing each other, and each immobilization area has n×n rows of spots (where n is 2 to 6). an integer of ), each spot is recessed so that the binding substance can be immobilized, the outer edge of each immobilization region includes at least one vertex , and each immobilization region is It is characterized by being rotationally symmetrical .

Further, the measurement method according to the present invention includes a binding step of introducing the sample liquid through the opening into the solid phase reaction chip to bind the substance to be measured and the binding substance; an addition step of adding a reaction solution containing a physiologically active substance having an ability to specifically bind to the substance to be measured and having enzymatic activity with respect to the substance to be measured; The method is characterized by comprising a removing step of removing the washing liquid by rotating the reaction chip at a predetermined number of revolutions, and a measuring step of measuring the enzymatic activity in the physiologically active substance.

Further, in the measurement method according to the present invention, the sample liquid is introduced into the solid-phase reaction chip through the opening, and the substance to be measured and the binding substance immobilized on the immobilizing portion are separated from each other. and a labeled substance having specific binding ability to the substance to be measured and labeled with a signal-generating substance was bound to the binding substance in the first binding step It is characterized by comprising a second binding step of binding to the substance to be measured, and a measuring step of measuring a signal generated by the signal-generating substance.

ADVANTAGE OF THE INVENTION According to the present invention, the drawbacks of the solid-phase reaction chip having a liquid-absorbing member inside can be overcome, and the solid-phase reaction chip can be rapidly and simply operated to accurately detect the substance to be measured contained in the sample liquid. A phase reaction chip and a measurement method using the same can be provided.

1 is a perspective view for explaining an overview of a solid phase reaction chip 100 according to this embodiment; FIG. 4 is a component diagram of each member constituting the solid-phase reaction chip 100. FIG. (a) is a plan view of the upper lid member 10 viewed from the inner bottom surface 11 side, and (b) is a side sectional view of the upper lid member 10. FIG. (a) is a plan view when the lower lid member 20 is viewed from the inner bottom surface 21 side, and (b) is a side sectional view of the lower lid member 20. FIG. FIG. 2 is a side sectional view of the solid-phase reaction chip 100 shown in FIG. 1; It is a flow chart explaining a measuring method concerning this embodiment. 2 is a block diagram illustrating a configuration example of an automatic measurement device 200; FIG. (a) is a plan view explaining an upper lid member 101 according to a modification, and (b) is a plan view explaining a lower lid member 301 according to a modification. (a) is a plan view explaining an upper lid member 102 according to a modification, and (b) is a plan view explaining a lower lid member 302 according to a modification. (a) is a perspective view explaining a solid phase reaction chip 50 according to a modification, and (b) is a perspective view explaining a solid phase reaction chip 60 according to a modification. FIG. 4 is a diagram showing the color development state of colorimetric spots measured by the measurement method according to the present embodiment. FIG. 4 is a diagram showing the color development state of colorimetric spots measured by the measurement method according to the present embodiment.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. It should be noted that the present invention is not limited to the following description, and can be modified as appropriate without departing from the gist of the present invention. Also, the drawings are schematic, and the ratio of each dimension may differ from the actual one. Specific dimensions should be determined with reference to the following description. In addition, it goes without saying that there are portions with different dimensional relationships and ratios between the drawings.

A solid-phase reaction chip according to the present invention is a solid-phase reaction chip formed as a rotating body by fitting and integrating an upper lid member and a lower lid member. An immobilization section provided on the bottom side for immobilizing a plurality of binding substances having specific binding ability to the substance to be measured contained in the sample liquid; It has an opening for supply and a liquid absorption part provided outside the immobilization part for absorbing the sample liquid supplied through the opening, and the immobilization part protrudes toward the opposite inner bottom surface. It has a plurality of immobilization areas separated by ridges, each immobilization area including at least one apex at its outer edge. A detailed description will be given below.

FIG. 1 is a perspective view for explaining an overview of the solid phase reaction chip 100 according to this embodiment, and FIG. 2 is a component diagram of each member constituting the solid phase reaction chip 100. A solid-phase reaction chip 100 according to this embodiment comprises an upper cover member 10 and a lower cover member 20 which is integrally fitted with the upper cover member 10 and forms a body of rotation together with the upper cover member 10. The upper cover member 10 and the lower cover member In the internal space 30 formed by the fitting of the 20, a liquid absorbing liquid for collecting the sample liquid, the reaction liquid, the washing liquid, etc. (hereinafter sometimes simply referred to as liquid) supplied through the opening 14 A member 40 is provided along an outer circumference 27 of the immobilizing portion 23, which will be described later. In the following description, a configuration in which the fixing portion 23 is provided on the inner bottom surface 21 side of the lower lid member 20 will be described. may be provided.

3(a) is a plan view of the upper lid member 10 viewed from the inner bottom surface 11 side, and FIG. 3(b) is a side sectional view of the upper lid member 10. FIG. The upper lid member 10 according to the present embodiment has a substantially perfect circular shape, and has an upper lid fitting wall along the peripheral edge (circumference) of the inner bottom surface 11 so that it can be fitted with the lower lid member 20 . 12 are erected. The inner bottom surface 11 of the upper lid member 10 is formed with a fixing portion facing surface 13 that has the same square shape as the fixing portion 23 of the lower lid member 20 in plan view and faces the fixing portion surface 24 . . The immobilizing portion facing surface 13 forms a gap with the immobilizing portion surface 24 by abutting a protuberant portion 26 provided on the immobilizing portion surface 24 to be described later, and supplies the liquid through the opening 14 . The applied liquid can be developed on the immobilizing part surface 24 . In addition, as shown in FIG. 3B, the fixing portion facing surface 13 can be formed with a predetermined protrusion width a from the inner bottom surface 11, or can be formed as a smooth surface on the surface of the inner bottom surface 11. Direct formation is also possible. In addition, the plane view shape of the immobilizing portion facing surface 13 may be the same shape as the plane view shape of the entire immobilizing portion 23, or may be a different shape. The opening 14 is provided substantially at the center of the surface of the upper lid member 10 and coaxially with the rotating shaft of the lower lid member 20 . The opening 14 has a taper-shaped opening and is electrically connected to the immobilizing portion facing surface 13 .

4A is a plan view of the lower lid member 20 viewed from the inner bottom surface 21 side, and FIG. 4B is a side sectional view of the lower lid member 20. FIG. The lower lid member 20 according to the present embodiment has the same shape as the upper lid member 10, which is substantially a perfect circle, and can be fitted with the upper lid member 10 inside the peripheral edge (circumference) of the inner bottom surface 21. The lower lid fitting wall 22 is erected so as to be. An inner bottom surface 21 of the lower lid member 20 is formed with a fixed portion 23 having a square shape in plan view. An immobilization region 25 is formed on the immobilization surface 24 of the immobilization part 23 to immobilize a plurality of binding substances having specific binding ability to the substance to be measured. In this embodiment, the immobilizing portion surface 24 is divided into four immobilizing regions 25 by raised portions 26 that protrude toward the inner bottom surface 11 of the upper lid member 10 facing each other. As described above, the raised portion 26 provided on the immobilization portion surface 24 abuts against the immobilization portion-facing surface 13 of the upper lid member 10, thereby providing a gap between the immobilization portion surface 24 and the immobilization portion-facing surface 13. A gap is formed in , and the liquid supplied through the opening 14 is introduced into the gap (immobilization region 25). The outer edge 28 of each immobilization region 25 bounded by ridges 26 includes at least one vertex 230 . In this embodiment, one vertex 230 having an inner angle of 90° is provided on the outer edge portion 28 of each immobilization region 25, and the immobilization portion 23 has a rectangular shape as a plan view of the entire immobilization portion 23. It shows an example of Note that the vertices included in the outer edge portion 28 of the immobilization region 25 indicate an inner angle of 30° to 162°, and the number of vertices is not particularly limited as long as it is one or more. It is preferable that the four immobilizing regions 25 included in the immobilizing part 23 have the same planar shape and are rotationally symmetrical . By forming the four immobilization regions 25 included in the immobilization part 23 into such a shape, a stable rotation state can be obtained when the solid-phase reaction chip 100 is rotated. The supplied liquid can be spread uniformly in the gap between the immobilizing portion surface 24 and the immobilizing portion facing surface 13 . In the immobilization region 25, spots 250 having a diameter of about 100 μm to 1 mm are formed in 4×4 rows (16 spots) per region. The number of binding substances immobilized in the immobilization region 25 is not limited to the 64 types described above, and may be, for example, 2×2 rows (total 16 types) to 6×6 rows (total 144 types). It can be changed as appropriate according to the type of measurement. In addition, the method of immobilizing the binding substance is not based on the above-mentioned spotting, but rather, a U-bottomed (spherical), V-bottomed, flat-bottomed (round or square) shape, etc., is provided at a location corresponding to each immobilization position. A method of directly or indirectly immobilizing a binding substance in a depression (well) having a bottom may be employed.

By forming the outer edge portion 28 of the immobilizing region 25 of the immobilizing portion 23 provided on the inner bottom surface 21 of the lower lid member 20 into the above shape, it is possible to reduce the contact area with the liquid absorbing member 40. As a result, it is possible to reduce the occurrence of liquid puddles. By the way, as shown in FIG. 4(b), the width b of the protrusion from the inner bottom surface 21 of the immobilization portion 23 can be adjusted if the gap formed between the immobilization portion facing surface 13 and the immobilization portion facing surface 13 has a predetermined gap width. There is no limitation, and the height of the protuberance 26 may be taken into account, and it may be appropriately set according to the protrusion width a of the fixing portion facing surface 13 formed on the upper lid member 10 .

FIG. 5 is a side sectional view of the solid-phase reaction chip 100 shown in FIG. In this embodiment, the gap width c of the gap formed between the immobilizing part surface 24 and the immobilizing part facing surface 13 is preferably between 0.05 mm and 0.30 mm. , the gap width c is preferably between 0.08 mm and 0.15 mm. As described above, the gap width c can be changed as appropriate by adjusting the protrusion width b of the immobilizing portion 23, the protrusion width a of the immobilizing portion facing surface 13, and the height of the raised portion 26. Therefore, the amount of liquid supplied to the gap can be set to a desired amount.

The upper lid member 10 and the lower lid member 20 having the above-described structure are not particularly limited as long as they are impermeable to liquid and have non-adsorptive properties for proteins and the like. Examples include polyethylene, polycarbonate, and polyethylene terephthalate. , vinyl chloride, polystyrene, ABS resin, polyamide, tetrafluoroethylene, polypropylene, unsaturated polyester, epoxy and other plastic materials, for example, injection molding, insert molding, blow molding, extrusion molding, compression molding, or transfer molding It can be manufactured by a molding method such as molding. Although there are no particular restrictions on the color and transparency of the material, it is preferable that the upper cover member 10 has transparency and the lower cover member 20 is non-transparent in order to suppress reflection during measurement. Although there is no particular limitation on the integration of the upper lid member 10 and the lower lid member 20, the upper lid fitting wall 12 of the upper lid member 10 and the lower lid fitting wall 22 of the lower lid member 20 are ultrasonically coupled. Joining by welding, vibration welding, hot plate welding, high frequency welding, hot air welding, laser welding, welding such as welding, adhesion of adhesives, adhesive tapes, etc., mechanical fastening using tapping screws, bolts and nuts, etc. can achieve fitting and integration.

By the way, the liquid absorbing member 40 accommodated in the internal space 30 formed by fitting the upper lid member 10 and the lower lid member 20 is formed by joining thin plate-shaped members at both ends thereof. It can be formed as an annular member and is provided along the outside (periphery 27 ) of the immobilization portion 23 . The liquid absorbing member 40 is provided to absorb the liquid removed by the centrifugal force generated along with the rotation. Examples of materials used for manufacturing the absorbent member 40 include polyester fibers such as polyethylene terephthalate and polyethylene terebutylate; polyolefin fibers such as polyethylene and polypropylene; polyurethane; Materials having excellent water absorption and retention properties, such as fibers such as plant fibers such as pulp fibers, cotton fibers and hemp fibers, regenerated fibers such as silk fibers and rayon fibers, woven fabrics, non-woven fabrics and paper, can be used. In addition, it is possible to use cellulose-based materials such as pulp fibers and rayon fibers, and porous matrices composed of polysaccharides such as acetate (acetyl cellulose) produced by reacting wood pulp with acetic acid.

Next, a measurement method using the solid phase reaction chip 100 according to this embodiment will be described with reference to the flow chart of FIG. In this measurement method, a sample liquid is introduced into the solid-phase reaction chip 100 through the opening 14 to bind the substance to be measured and the binding substance; an addition step of adding a reaction solution containing a physiologically active substance having specific binding ability and enzymatic 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 of measuring the enzymatic activity in the physiologically active substance.

In the measurement method according to this embodiment, an example in which an IgE antibody that specifically binds to an allergen is used as a substance to be measured will be described. The allergen as a binding substance immobilized as a binding substance in the immobilization region 25 of the immobilization portion surface 24 of the immobilization portion 23 is not particularly limited, but for example, house dust 1 (2), Dermatophagoides farinae , Japanese cedar, cypress, alder (genus), birch (genus), Dactylis, ragweed, mugwort, Alternaria, Aspergillus, Malassezia (genus), cat (dandruff), dog (dandruff), cockroach, moth, latex, etc.・Other allergens such as milk, egg white, opomucoid, rice, wheat (fruit), buckwheat, soybeans, peanuts, apples, kiwi, sesame, beef, chicken, shrimp, crab, mackerel, salmon, tuna, etc. Any allergen can be selected as long as it can be examined as an allergen test item at a medical institution or the like.

Immobilization of the allergen to the immobilization region 25 can be performed by generally used physical adsorption or chemical bonding. For example, an allergen solution containing an allergen of a measurement item is placed at a desired position in the immobilization region 25, or a recess (well) having a bottom such as a U bottom (spherical shape), a V bottom, or a flat bottom (round shape, square shape). ), the allergen is immobilized on the immobilization region 25 by spotting or stamping. In this case, the allergen may be directly immobilized on the immobilization region 25, or the allergen may be immobilized via a spacer substance. Since a total of 64 spot positions are provided in the immobilization region 25 according to the present embodiment, 64 types of measurement items can be measured at once by immobilizing different allergens in each spot. can be done.

In addition, the physiologically active substance having the ability to specifically bind to the IgE antibody as the substance to be measured and having enzymatic activity is not particularly limited, but examples thereof include alkaline phosphatase, β-galactosidase, glucose oxidase, urease, Anti-IgE antibodies labeled with creatine kinase, uricase, glucose-6-phosphate dehydrogenase, peroxidase or the like are preferably used. In this embodiment, an example using an HRP-labeled antibody obtained by labeling an anti-IgE antibody 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 a chemiluminescent signal depending on the substrate. Substrates used to generate colorimetric signals include, for example, tetramethylbenzidine and its derivatives, o-phenylenediamine, triarylmethanes, imidazole leuco dyes, and the like. Examples of chemiluminescent signal generation include acridinium salts, dioxetanes, luciferin, lucigenin, oxalyl chloride, and the like.

After the liquid absorbing member 40 is housed in the internal space 30 formed by the upper lid member 10 and the lower lid member 20 in which the allergen is immobilized in the immobilization region 25, the lower lid member 20 and the upper lid member 10 are fitted and integrated. By doing so, the solid phase reaction chip 100 is constructed.

Then, the specimen liquid (30 μl to 60 μl) is supplied through the opening 14 of the upper cover member 10 (step S100). The sample liquid can be appropriately selected from biological samples such as whole blood, serum, plasma, tears, urine, saliva, spinal fluid, and the like. When the sample liquid is supplied to the opening 14, the sample liquid is spread over the entire immobilization region 25, and the IgE antibody having specific binding ability binds to the allergen, which is the binding substance immobilized on each spot. At this time, the solid-phase reaction chip 100 is preferably placed in a temperature environment of 35.degree. C. to 40.degree. For example, the IgE antibody can be efficiently bound to the allergen by performing a series of operations in a constant temperature bath capable of maintaining a temperature of around 37°C.

Next, for example, after supplying a washing liquid (30 μl to 60 μl) containing a surfactant such as tween 20 through the opening 14, the washing liquid is removed by rotating at a predetermined rotation speed (2000 to 2500 rpm/min). (step S101).

Next, in step S102, a reaction solution (30 μl to 60 μl) containing the HRP-labeled antibody is supplied through the opening 14 (step S102). After binding the HRP-labeled antibody to the IgE antibody bound to the allergen, for example, after supplying a washing solution (30 μl to 60 μl) containing a surfactant such as tween 20 through the opening 14, a predetermined number of rotations (2000 to The washing liquid is removed by rotating at 2500 rpm/min (step S103).

After the washing in step S103 is completed, a substrate such as tetramethylbenzidine (TMB) is added, and the spots emitting colorimetric signals are measured using imaging means such as CCD (step S104).

By the way, the measuring method described in the flow chart of FIG. 6 is performed manually, but the measurement can be automatically performed by using an automatic measuring device 200 having the configuration shown in FIG. 7, for example. The automatic measuring device 200 includes at least a dispensing unit 201 capable of aspirating and discharging a solution from a set reagent cartridge, and a centrifugal unit 201 that holds the solid-phase reaction chip 100 and rotates it at a predetermined number of revolutions per minute. A separating unit 202, an imaging unit 203 including an imaging unit such as a CCD, and an operation unit 204 including 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. , an image analysis unit 205 that analyzes an image (colorimetric signal emission spot, etc.) captured by the imaging unit 203, a measurement result generation unit 206 that generates measurement results from the analysis results of the image analysis unit 205, and these are integrated It is equipped with a controller 207 that performs physical control, and is configured so that actual operations by the inspector are not required except for setting the reagent cartridge or solid-phase reaction chip 100 to the apparatus.

According to the automatic measurement apparatus 200 configured as described above, the final measurement result can be obtained in about 20 minutes, so the throughput is high. Since it can be done, there is an advantage that a skilled technique is not required. In addition, since the device itself can be made smaller, it can be applied as a device for immediate examination at clinical sites.

In the above description of the measurement method, an example of an IgE antibody that specifically binds to an allergen is used as the substance to be measured, and an example of an allergen is described as a substance that is immobilized as a binding substance in the immobilization region 25. The invention is not limited to this, and the substance to be measured is, for example, a genotype derived from human papillovirus (HPV), and is immobilized on the immobilization region 32c of the second base 32 as a binding substance. Alternatively, a genomic DNA probe derived from HPV can be used. In this case, 13 high-risk types such as type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, etc. It is also possible to construct an assay system for detecting classified HPV.

The present invention also provides, for example, preoperative infection test markers, liver function test markers, renal function test markers, autoimmune hepatitis markers, heart disease markers, sepsis markers, thyroid markers, or specific drugs contained in blood. etc. can also be used as substances to be measured.

Here, Table 1 shows (1) Syphilis TP, HIV Ag/Ab combo, Anti-HCV, and HBsAg as preoperative infection test markers, and (2) Anti-HBc and Anti-HBc as liver function test markers. HBs, Anti-HBe, HBeAg, Anti-HBc IgM, (3) BUN, CRE, UA as renal function test markers, (4) IgG, antinuclear antibody as autoimmune hepatitis markers, (5) (6) Procalcitonin and Presepsin as sepsis markers; , house dust, house dust mite, cat skin, egg white, Dermatophagoides mite, milk, wheat, soybean, cypress, cedar, (8) TSH, Free T4, TRAb as thyroid markers, (9) specific drugs contained in blood , the results of evaluating VCM, TEIC, and ABK by the above measurement method are shown. In Table 1, the substance to be measured that gave a superior colorimetric signal compared to the control (without the substance to be measured) was evaluated as “positive”, and even if it was not “positive”, it was Substances to be measured for which a superior difference was observed in the results were evaluated as “false positives.”

In addition, FIG. 11 shows (1) preoperative infection test markers (1. Syphilis TP, 2. HIV Ag/Ab combo, 3. Anti-HCV, 4. HBsAg), (2) liver function test markers (1 (3) renal function test markers (1.BUN, 2.CRE, 3.UA), (4) autoimmune hepatitis markers (1. IgG, 2. antinuclear antibody), and (5) cardiac disease markers (1. troponin, 2. myoglobin, 3. biochemical myocardial markers such as CK-MB, 4 . heart-type fatty acid-binding protein) are shown in the color development of the colorimetric spots obtained when measuring each of them. Figure 12 shows (6) sepsis markers (1. Procalcitonin, 2. Presepsin), (7) typical examples of allergies (allergens) (1. house dust, 2. house dust mite, 3. cat skin, 4. egg white, 5 (8) thyroid markers (1. TSH, 2. Free T4, 3. TRAb), and (9) blood The color development state of the colorimetric spots obtained when each of the specific drugs (1.VCM, 2.TEIC, 3.ABK) contained in is measured is shown.

As shown in Table 1 and Figures 11 and 12, (1) 2. in preoperative infection test markers HIV Ag/Ab combo and 4. HBsAg, and (7) typical examples of allergies (allergens), 4. 6. egg white; milk;7. 8. wheat; soybeans; and9. Although the result was false positive for some items such as cypress, superior colorimetric signals were observed compared to the control for all other items that were measured this time. From these results, it was shown that the measurement system using the solid phase reaction chip according to the present invention is suitable for so-called screening tests regardless of the type of measurement target.

According to the solid-phase reaction chip according to the present invention, which has such a wide range of application, other than the test markers such as AST (GOT), ALT (GPT), and γ-GTP, which are used as liver function test markers, as test markers for the same disease, various cancer markers such as liver cancer, stomach cancer, uterine cancer, esophageal cancer, kidney cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, hemorrhagic fever, Severe acute respiratory syndrome, Middle East respiratory syndrome, bird flu, bacillary dysentery, hepatitis E, hepatitis A, influenza, viral hepatitis, acute meningitis, etc. Application to infectious disease markers such as infectious diseases, designated infectious diseases, and new infectious diseases, or inflammatory markers such as leukocytes, type C-reactive protein, and serum amyloid A protein, is also expected to be applied to disease test markers other than those described above.

Furthermore, in addition to the above disease-related test markers, the present invention also includes, for example, insecticides, acaricides, nematicides, fungicides, herbicides, insecticide fungicides, rodenticides, plant growth regulators, repellents. pesticides, attractants, spreading agents, stimulants, narcotic analgesics, anabolic agents, diuretics, peptide and glycoprotein hormones and their congeners, blood doping, alcohol, marijuana, local anesthetics, cortico It is also possible to develop the test and measurement of doping drugs such as steroids (adrenal corticosteroids) and beta-blockers.

In the above description, as a preferred example of the measurement method using the solid-phase chip according to the present invention, an example using a physiologically active substance having an ability to specifically bind to the substance to be measured and having enzymatic activity is used. explained. A measurement system using an enzyme label is an excellent measurement system from the viewpoint of reaction specificity and signal amplification. However, since enzymes have large molecular weights, there are problems such as affecting the antigen-antibody reaction, and deactivation of the enzymes causing the system to cease functioning as a measurement system. The reproducibility of was sometimes lowered. Since the solid-phase reaction chip according to the present invention can also construct a measurement system using a labeling substance labeled with a signal-generating substance other than an enzyme, it will be described below.

In the measurement method using a labeling substance labeled with a signal-generating substance other than an enzyme, the sample liquid is introduced through the opening of the solid-phase reaction chip, and the substance to be measured and the immobilization portion are immobilized. a first binding step of binding the binding substance to the binding substance; and a measuring step of measuring the signal generated by the signal-generating substance. In this case, a washing step may be provided in which a washing liquid is added between each step or any of the steps and the washing liquid is removed by rotating the solid-phase reaction chip at a predetermined number of rotations. Here, the labeled substance having specific binding ability to the substance to be measured and labeled with the signal-generating substance includes any of radioisotopes, lanthanide elements, free radical derivatives, chemiluminescent substances and fluorescent luminescent substances. Proteins such as antibodies, protein A/G, lectins, avidin, and streptavidin labeled with , nucleic acids, or fragments thereof can be used. The binding of the signal-generating substance to the labeling substance is not particularly limited, and examples include covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption, chemical adsorption, and the like. Moreover, in order to prevent non-specific adsorption between the signal-generating substance and the labeling substance, a linker composed of an organic molecule such as a polyethylene glycol chain may be provided. As in the above description, when the substance to be measured is an IgE antibody, the labeled antibody is an antibody labeled with either a radioactive isotope, a lanthanide element, a free radical derivative, a chemiluminescent substance, or a fluorescent substance. Preferably, IgE antibodies are used.

When a radioactive isotope is used as a signal-generating substance, for example, ¹²⁵ I, ¹³¹ I, ³ H, etc. can be mentioned, and ¹²⁵ I is preferably used from the viewpoint of sensitivity and half-life. When oxidized, iodine readily binds to tyrosine residues, histidine residues, etc. in antibodies (proteins) as a new electron reagent, and is thus suitable for labeling.

When using a lanthanide element as a signal-generating substance, elements with atomic numbers from 57 to 71 (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) can be mentioned. Among them, Eu (Europium) chelate with atomic number 63 has a large wavelength difference between the excitation light (340 nm) and the synchrotron radiation (615 nm), and the synchrotron radiation (fluorescence) lifetime is long, so time-resolved fluorescence measurement is performed. be able to.

When a free radical derivative is used as a signal-generating substance, examples thereof include piperidine-N-oxide derivatives, pyrrolidine-N-oxide derivatives, oxazolidine-N-oxide derivatives, etc. Labeling labeled with these free radical derivatives The ESR spectrum change of the antibody is measured as an index.

When a chemiluminescent substance is used as a signal-generating substance, examples thereof include luminol derivatives, acridinium salts, dioxetanes, luciferin, lucigenin, oxalyl chloride, indoxyl derivatives, etc. Labeled antibodies labeled with these chemiluminescent substances is subjected to a chemiluminescence reaction in the presence of enzymes such as microperoxidase and peroxidase in the presence of H ₂ O ₂ or under alkaline conditions in the presence of H ₂ O ₂ and measured.

When a fluorescent light-emitting substance is used as the signal-generating substance, any of fluorescent dyes, quantum dots, or fluorescent particles (beads) composed of fluorescent dyes or quantum dots and particles can be used. In addition, the fluorescence-emitting substance referred to here is a visible light to near-infrared light having a wavelength within the range of 400 nm to 1100 nm when excited by ultraviolet light to near-infrared light having a wavelength within the range of 200 nm to 700 nm. It shows synchrotron radiation (luminescence).

Examples of fluorescent dyes include fluorescein-based dye molecules, rhodamine-based dye molecules, ALexa Fluor (manufactured by Invitrogen)-based dye molecules, BODIPY (manufactured by Invitrogen)-based dye molecules, cascade-based dye molecules, coumarin-based dye molecules, and eosin-based dye molecules. Examples include dye molecules, NBD-based dye molecules, pyrene-based dye molecules, cyanine-based dye molecules, aromatic hydrocarbon-based molecules, and the like.

Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein, 6 -carboxy-2',4,7,7'-tetrachlorofluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy - rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, sulforhodamine B, sulforhodamine 101 and ALex Fluor 350, ALex Fluor 405, ALex Fluor 430, ALex Fluor 488, ALex Fluor ５００、ＡＬｅｘ Ｆｌｕｏｒ ５１４、ＡＬｅｘ Ｆｌｕｏｒ ５３２、ＡＬｅｘ Ｆｌｕｏｒ ５４６、ＡＬｅｘ Ｆｌｕｏｒ ５５５、ＡＬｅｘ Ｆｌｕｏｒ ５６８、ＡＬｅｘ Ｆｌｕｏｒ ５９４、ＡＬｅｘ Ｆｌｕｏｒ ６１０、ＡＬｅｘ Ｆｌｕｏｒ ６３３、ＡＬｅｘ Ｆｌｕｏｒ ６３５、ＡＬｅｘ Ｆｌｕｏｒ ６４７、ＡＬｅｘ Ｆｌｕｏｒ ６６０、ＡＬｅｘ Ｆｌｕｏｒ ６８０、 ＡＬｅｘ Ｆｌｕｏｒ ７００、ＡＬｅｘ Ｆｌｕｏｒ ７５０、ＢＯＤＩＰＹ ＦＬ、ＢＯＤＩＰＹ ＴＭＲ、ＢＯＤＩＰＹ ４９３／５０３、ＢＯＤＩＰＹ ５３０／５５０、ＢＯＤＩＰＹ ５５８／５６８、ＢＯＤＩＰＹ ５６４／５７０、ＢＯＤＩＰＹ ５７６／５８９、ＢＯＤＩＰＹ ５８１／５９１、ＢＯＤＩＰＹ ６３０／６５０、ＢＯＤＩＰＹ 650/665 (manufactured by Invitrogen), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7, HiLyte Fluor (registered trademark, manufactured by Anaspec), Dylight 594 (registered trademark, Thermo Scientific company) dye molecules, ATTO 594 (registered trademark, manufactured by ATTO-TEC), MFP 594 (registered trademark, manufactured by Mobitec), 5,10,15,20-tetraphenylporphinetetrasulfonic acid, zinc 5,10 , 15,20-tetraphenylporphinetetrasulfonic acid, phthalocyaninetetrasulfo acid, zinc phthalocyaninetetrasulfonic acid, N,N-Bis(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butyl-phenyl)-perylene-3,4,9,10- 4 , 4′,4″,4′″-[(1,3,8,10-tetrahydro-1,3,8,10-tetraoxoperylo[3,4-cd:9,10-c′d′] In addition to dipyran-5,6,12,13-tetrayl) tetralis (oxy)] tetrakis and the like, merocyanine, organic nitrogen compounds acridine, pyranine, pyromethene, luciferin, DCM (4-(dicyanomethylene)-2-methyl- 6-(4-dimethylaminostyryl)-4H-pyran), stilbenes, etc. may also be mentioned. In addition, these fluorescent dyes may be used singly or in combination.

Examples of quantum dots include quantum dots containing group II-VI compounds, group III-V compounds, group IV elements, and the like, and these compounds may be used singly or in combination. May be combined.

Specific examples include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaAs, Si, Ge, and the like. In addition, we prepared a core/shell type quantum dot in which these quantum dots were used as the core and shelled with other nanomaterials, and furthermore, the surface was coated with organic substances with high biocompatibility such as polyethylene glycol and amphiphilic polymers. A coated one can also be used in the present invention.

Examples of fluorescent particles composed of fluorescent dyes or quantum dots and particles include inorganic particles such as silica and alumina, and resin particles (resin polymers) composed of organic polymers such as polystyrene and poly(meth)acrylic acid ester. Examples include those in which either fluorescent dyes or quantum dots are bonded, partially bonded, or encapsulated inside particles. The method of introducing the fluorescent dye into the resin particles is not particularly limited, and may be a method of binding the fluorescent dye to the monomer that is the raw material of the resin and then polymerizing the monomer, or a method of forming a polymer and then adding the fluorescent dye to the polymer. A method of binding, a method of mixing a monomer and a fluorescent dye to perform polymerization and binding of the fluorescent dye at the same time, a method of imparting resin polymerizability to the fluorescent dye itself, and the like can be employed. The average particle size of the fluorescent particles is not particularly limited as long as it does not affect the immune reaction and provides sufficient brightness, but is preferably in the range of 1 nm to 100 μm. An example of the fluorescent particles is a combination of particles and a fluorescent material that emits visible to near-infrared radiant light (luminescence) when excited by ultraviolet to near-infrared light. There is no particular limitation, and for example, a combination of the above lanthanoid element and resin particles may be used.

Fluorescent particles that can be used in the present invention can be prepared by a known method, and commercially available fluorescent particles may be used. For example, Patent Document 3 describes a method for synthesizing resin particles for fluorescent dye labeling, in which a fluorescent dye is bound to a resin particle made of a resin other than by a covalent bond. Fluorescent particles can be made.

Further, in recent years, attention has been focused on substances having aggregation-induced luminescence activity as a solution to self-quenching (concentration quenching) of fluorescent dyes encapsulated in resin particles. Patent Document 4 discloses 1,1-dimethyl-2,3,4,5-tetraphenylsilole-encapsulating polystyrene fine particles, 1,1,2,3,4,5-hexaphenylsilole-encapsulating polystyrene fine particles, 1,1- Particles made of a network polymer such as dimethyl-2,5-dianisyl-3,4-diphenylsilol-encapsulating polystyrene fine particles and 1,1,2,3,4,5-hexaphenylsilole-encapsulating polystyrene fine particles, which exhibit aggregation-induced luminescence activity Fluorescent resin microparticles containing compounds (siloles) are described. In addition to siloles, for example, tetraphenylethylene derivatives and the like are also known as aggregation-induced luminescence active compounds. , it is expected that the measurement can be performed with higher efficiency than when using conventional π-conjugated fluorescent materials.

In this way, by using a labeling substance labeled with a signal-generating substance such as a radioactive isotope, a lanthanide element, a free radical derivative, a chemiluminescent substance, or a fluorescent luminescent substance, it is possible to achieve the same extent as in a system for measuring enzyme activity. Or a more efficient measurement system can be constructed. In addition, when a radioactive isotope, a lanthanoid element, a free radical derivative, or a fluorescence-emitting substance is used as a signal-generating substance, unlike a system for measuring enzyme activity, there is no need to add a substrate (solution) in the measurement step. This allows for faster and more reproducible measurements.

[Modification]
In the description of the above embodiment, the outer edge portions 28 of the four immobilization regions 25 included in the immobilization portion 23 have one vertex with an inner angle of 90°, and the planar view shape of the immobilization portion 23 as a whole is A rectangular example has been described. However, the present invention is not limited to this shape, and for example, other polygonal shapes, such as a triangular shape and an octagonal shape, are possible as the planar view shape of the entire fixing portion. FIG. 8(a) shows an example of the upper lid member 101 in which the opening 141 and the fixing portion facing surface 131 are formed, and FIG. 251 is provided with one vertex 232 having an inner angle of 60° on each outer edge portion, and is a configuration example of the lower lid member 301 provided with the fixing portion 231 having a triangular shape as an overall planar view. 9A shows an example of the upper lid member 102 in which the opening 142 and the fixing portion facing surface 132 are formed, and FIG. 10 is a structural example of a lower lid member 302 including a fixing portion 233 having two vertices 234 with an inner angle of 135° on each outer edge of a fixing region 252 and having an octagonal shape as a whole in plan view. . The examples shown in FIGS. 4, 8, and 9 are only examples, and in the present invention, there are The line type of the side can be selected as appropriate, and the number of immobilization regions formed on the surface of the immobilization portion is not limited based on these selections.

Furthermore, in the present invention, the shape in which the upper lid member and the lower lid member are fitted together can be changed as appropriate. For example, as shown in FIG. 10(a), a solid-phase reaction chip 50 in which an upper cover member 103 and a lower cover member 303 having a square shape in plan view are fitted and integrated, or as shown in FIG. A combination of the solid-phase reaction chip 60 in which the upper lid member 104 and the lower lid member 304 having a hexagonal shape in plan view are fitted and integrated is also possible. The combination of the immobilized portion and the lid member can be appropriately selected in consideration of various conditions such as high measurement accuracy, reproducibility, ease of manufacture, and manufacturing cost.

As described above, according to the present invention, the cover member that was conventionally required is abolished, and the immobilization region for immobilizing the binding substance is configured to have at least one vertex at the outer edge thereof, so that the binding substance can be absorbed inside. A solid-phase reaction chip capable of overcoming the drawbacks of solid-phase reaction chips equipped with a liquid component and capable of detecting a substance to be measured contained in a sample liquid with high accuracy by rapid and simple operation, and measurement using the same can provide a method.

Reference Signs List 10, 101, 102, 103, 104 upper lid member 11, 21 inner bottom surface 12 upper lid fitting wall 13, 131, 132 fixing portion facing surface 14, 141, 142 opening 20, 301, 302, 303 , 304 lower lid member, 22 lower lid fitting wall, 23, 231, 233 immobilization portion, 24 immobilization portion surface, 25, 251, 252 immobilization region, 26 raised portion, 27 outer periphery, 28 outer edge portion, 30 inside Space, 40 liquid absorption member, 50, 60, 100 solid phase reaction chip, 200 automatic measurement device, 201 dispensing unit, 202 centrifugal separation section, 203 imaging section, 204 operation section, 205 image analysis section, 206 measurement result generation section , 207 control unit, 230, 232, 234 vertex, 250 spot

Claims (20)

A solid-phase reaction chip formed as a rotating body by fitting and integrating an upper lid member and a lower lid member,
an immobilization unit provided on the inner bottom surface side of either the upper lid member or the lower lid member for immobilizing a plurality of binding substances having specific binding ability to a substance to be measured contained in a sample liquid;
an opening provided in the upper lid member for supplying the sample liquid to the surface of the immobilizing portion;
a liquid absorbing part provided outside the immobilizing part and absorbing the sample liquid supplied through the opening,
The immobilization portion has a plurality of immobilization regions separated by raised portions that protrude toward the inner bottom surface facing each other, and each immobilization region has n×n rows of spots (where n is 2 to 6). an integer), and each spot has a recess so that the binding substance can be immobilized,
A solid-phase reaction chip, wherein the outer edge of each immobilization region includes at least one vertex , and each immobilization region is formed rotationally symmetrically . 2. The solid-phase reaction chip according to claim 1, wherein all of the immobilizing regions have the same planar shape. 2. The solid-phase reaction chip according to claim 1, wherein said spot is formed with a size having a diameter ranging from 100 microns to 1 mm . 4. The solid phase reaction chip according to any one of claims 1 to 3, wherein the binding substance is an allergen that induces type I allergic reaction. 5. The solid phase reaction chip according to claim 4, wherein the substance to be measured is an allergen-specific antibody. 6. The solid phase reaction chip according to claim 5, wherein said allergen-specific antibody is an IgE antibody. 4. The solid phase reaction chip according to any one of claims 1 to 3, wherein the binding substance is a genomic DNA probe derived from human papillomavirus. 8. The solid phase reaction chip according to claim 7, wherein the substance to be measured is a genotype derived from human papillomavirus. The substance to be measured is a preoperative infection test marker selected from Syphilis TP, HIV Ag/Ab combo, Anti-HCV, and HBsAg, according to any one of claims 1 to 3. Solid phase reaction chip as described. 4. Any one of claims 1 to 3, wherein the substance to be measured is a liver function test marker selected from Anti-HBc, Anti-HBs, Anti-HBe, HBeAg, and Anti-HBc IgM. The solid-phase reaction chip described in the paragraph. 4. The solid phase reaction chip according to any one of claims 1 to 3, wherein the substance to be measured is a renal function test marker selected from BUN, CRE, and UA. 4. The solid phase reaction chip according to any one of claims 1 to 3, wherein the substance to be measured is an autoimmune hepatitis marker selected from IgG and antinuclear antibodies. 4. The substance to be measured according to any one of claims 1 to 3, wherein the substance to be measured is a heart disease marker selected from troponin, myoglobin, a biochemical myocardial marker, and cardiac fatty acid binding protein. Solid state reaction chip. The solid phase reaction chip according to any one of claims 1 to 3, wherein the substance to be measured is a sepsis marker selected from procalcitonin and presepsin. 4. The solid phase reaction chip according to any one of claims 1 to 3, wherein the substance to be measured is a thyroid marker selected from TSH, Free T4, and TRAb. 4. The solid phase reaction chip according to any one of claims 1 to 3, wherein the substance to be measured is a drug in blood selected from VCM, TEIC, and ABK. For the solid phase reaction chip according to any one of claims 1 to 16,
a binding step of introducing the specimen liquid through the opening and binding the substance to be measured and the binding substance;
an adding step of adding a reaction solution containing a physiologically active substance having a specific binding ability to the substance to be measured bound to the binding substance and having enzymatic activity;
a removing step of removing the washing liquid by rotating the solid-phase reaction chip at a predetermined number of rotations after introducing the washing liquid through the opening;
and a measuring step of measuring the enzyme activity in the physiologically active substance. For the solid phase reaction chip according to any one of claims 1 to 16,
a first binding step of introducing the sample liquid through the opening and binding the substance to be measured and the binding substance immobilized on the immobilizing portion;
A second binding step of binding a labeling substance having specific binding ability to the substance to be measured and labeled with a signal-generating substance to the substance to be measured bound to the binding substance in the first binding step; a binding step;
and a measuring step of measuring a signal generated by the signal-generating substance. 19. The method of measurement according to claim 18, wherein the signal-generating substance is any of a radioactive isotope, a lanthanide element, a free radical derivative, a chemiluminescent substance, or a fluorescent luminescent substance. 20. The measurement method according to claim 19, wherein the fluorescent light-emitting substance is any one of a fluorescent dye, a quantum dot, or a fluorescent particle composed of a fluorescent dye or a quantum dot and a particle.

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