WO2020195991A1 - Puce de réaction en phase solide et procédé de mesure utilisant cette dernière - Google Patents

Puce de réaction en phase solide et procédé de mesure utilisant cette dernière Download PDF

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WO2020195991A1
WO2020195991A1 PCT/JP2020/011313 JP2020011313W WO2020195991A1 WO 2020195991 A1 WO2020195991 A1 WO 2020195991A1 JP 2020011313 W JP2020011313 W JP 2020011313W WO 2020195991 A1 WO2020195991 A1 WO 2020195991A1
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substrate
solid
reaction
reaction chip
phase reaction
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PCT/JP2020/011313
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English (en)
Japanese (ja)
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義徳 鈴木
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インターメディック株式会社
常盤化学工業株式会社
有限会社Ｖｅｎｔｕｒｅ Ｌａｂ
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Priority to JP2021509067A priority Critical patent/JPWO2020195991A1/ja
Publication of WO2020195991A1 publication Critical patent/WO2020195991A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a solid phase reaction chip and a measurement method using the same.
  • Type I allergens that cause diseases such as bronchial asthma, allergic rhinitis, urticaria, or anaphylactic shock are caused by an overreaction of a specific antigen (allergen) with an IgE antibody against it.
  • Measurements that detect IgE antibodies contained in the sample solution are used to identify the causative allergen of allergic diseases.
  • CAP a method whose measurement principle is immunofluorescence
  • MAST a method called immunochemiluminescence.
  • CAP is a single-item method in which measurement is performed for each type of allergen, and a sample solution is required for each test item, which places a heavy physical burden on the subject and inferior in measurement throughput.
  • MAST is a multi-item method that can measure about 30 kinds of allergens at once, has a small burden on patients, and is excellent in throughput.
  • Patent Document 1 a biochip in which antigens of various allergens are mounted as independent spots is used to automate the reaction detection process between the sample solution and the antigen, and the measurement result can be obtained quickly.
  • Patent Document 1 An analysis method (Patent Document 1). This technique requires the use of measuring equipment that is complicated, expensive, and difficult to operate, and has a problem of low measurement accuracy. In particular, there is a problem that contamination occurs around the cleaning, antibody, and reagent supply nozzles, and the waste liquid suction nozzle.
  • Patent Document 2 A solid-state reaction chip (Patent Document 2) that has a rotating base and can quickly perform a specific reaction with a large number of binding substances having a specific binding potential and measure multiple items at the same time is disclosed.
  • This technology can perform multi-item inspections at the same time and is easy to operate, but it is added next because liquid such as sample liquid adheres to the outer periphery of the void in the reaction part and flows back into the void due to capillary action. There is a problem that the liquid to be discharged cannot be injected and measurement cannot be performed.
  • the present invention can measure multiple items at the same time with a small amount of sample solution, can detect the substance to be measured contained in the sample solution with a quick and simple operation, has a simple structure, is hard to break down, and has high measurement accuracy. It is an object of the present invention to provide a highly reliable solid-state reaction chip that does not make measurement impossible due to a failure such as backflow, and a measurement method using the same.
  • the present invention (1) A rotating body formed by laminating a first substrate and a second substrate, which is specific to an opening for introducing a sample solution into the first substrate and a substance to be measured contained in the sample solution. It has a reaction part having an immobilization part to which a binding substance having a binding ability is fixed, and a pillar part for forming a gap in which the sample liquid is developed, and a liquid such as a sample liquid can be discharged from the gap on the outer periphery of the reaction part.
  • a solid-state reaction chip to be discharged characterized in that it has at least one apex having an internal angle of 45 to 170 degrees on the outer circumference of each reaction portion.
  • the outer circumference of the main part consisting of the reaction part, the column part, and the opening of the first substrate is symmetry of any of 2 times rotational symmetry, 3 times rotational symmetry, 4 times rotational symmetry, and 5 times rotational symmetry.
  • the solid-state reaction chip according to Item 1 wherein the solid-state reaction chip is characterized by having at least one property.
  • the solid phase reaction chip is prepared.
  • a measurement method comprising: a removal step of removing a cleaning liquid by rotating at a predetermined rotation speed, and a measurement step of measuring an enzyme activity in a physiologically active substance. Is.
  • the present invention it is possible to measure multiple items at the same time with a small amount of sample solution, the substance to be measured contained in the sample solution can be detected quickly and easily, the structure is simple, it is hard to break down, and the measurement accuracy is high.
  • FIG. 1 described in Patent Document 2 which is a prior art, when the shape of the portion corresponding to the main portion of the present invention is a circle or a shape close to a circle, the liquid to be excluded is contained in the outer peripheral portion of the void.
  • the solid-state reaction chip of Patent Document 2 Since the solid-state reaction chip of the present invention has an apex having a specific internal angle on the outer periphery of the reaction portion, the occurrence of backflow failure can be reduced to 0%, and a highly reliable and practical solid-state reaction chip can be provided.
  • FIG. 11 is a cross-sectional view taken along the line DD shown in FIG.
  • FIG. 11 is a cross-sectional view taken along the line EE shown in FIG.
  • FIG. 12 is a figure which shows the shape definition of the well shown in FIG. 12 and FIG.
  • It is an exploded view of the solid phase reaction chip shown in FIG. It is a perspective view of the solid phase reaction chip shown in FIG.
  • Solid-phase reaction chip 11 Opening 12 Peripheral part 13 Reaction part 13a, 13b Edge of outer circumference of reaction part 14 Immobilization part 15 Pillar part 16A, 16B, 16C, 16D Main part (opening 11, reaction part 13 and pillar part) Part containing 15) 17, 17a, 17b Top of the outer circumference of the reaction part 18 Waste liquid part 19 Absorbent 20 Second substrate 21 Smoothing part 22 Peripheral part 28 Waste liquid part 31 Void 100 First substrate 141 Binding substance 142 Well 200 Automatic measuring device 201 Dispensing unit 202 Centrifugation Separation unit 203 Imaging unit 204 Operation unit 205 Image analysis unit 206 Measurement result generation unit 207 Control unit
  • FIG. 2 is a plan view of the first substrate 100
  • FIG. 3 is a cross-sectional view of a portion AA of FIG.
  • the first substrate 100 shown in FIG. 2 has an opening 11, a reaction portion 13, an immobilization portion 14, a pillar portion 15, a waste liquid portion 18, and a peripheral portion 12.
  • FIG. 4 is a plan view of the second substrate 20
  • FIG. 5 is a cross-sectional view of a portion BB of FIG.
  • the second substrate 20 has a smoothing portion 21, a waste liquid portion 28, and a peripheral portion 22.
  • the portion (main portion 16A) including the opening 11, the reaction portion 13, and the pillar portion 15 of the first substrate 100 faces the smooth portion 21.
  • both substrates include a waste liquid portion, but a method of discharging the waste liquid to the outside of both substrates can also be used. In that case, the pillar portion 15 and the portion of the smoothing portion 21 facing the pillar portion 15 are formed to be in contact with each other, and both substrates can be laminated.
  • the first substrate 100 shown in FIG. 2 is an example having four-fold rotational symmetry, and since the four reaction units 13 are the same as each other, one of them will be described.
  • the line connecting the points 13a, 17a, 17b, and 13b is the outer circumference of the reaction unit 13.
  • Points 17a and 17b are vertices 17 on the outer circumference of the reaction unit 13.
  • the first substrate 100 has at least one apex 17 having an internal angle of 45 to 170 degrees on the outer periphery of the reaction unit 13.
  • the size of the internal angle is more preferably 90 to 160 degrees, further preferably 90 to 140 degrees.
  • the internal angle is too large, the difference in the magnitude of the centrifugal force applied to the liquid between the side and the apex becomes small, so that the waste liquid adhering to the outer periphery of the reaction unit 13 flows back due to the capillary phenomenon, and the next additive liquid is introduced. Not preferable because it makes it impossible. If the internal angle is too small, the size of the chip becomes too large, which is not preferable. Further, if the outer periphery of the reaction unit 13 has an apex 17 having an internal angle of 190 degrees or more, a liquid such as a sample solution adheres to the recess of the apex 17 and easily flows back, which is not preferable.
  • the shape of the outer periphery of the main portion 16A preferably has any one of two-fold rotational symmetry, three-fold rotational symmetry, four-fold rotational symmetry, and five-fold rotational symmetry, and three-fold rotational symmetry.
  • the movement during rotation is stable, and since the sample solution can be uniformly spread in four directions, a large number of immobilization portions 14 are provided, and a large number of inspection items can be measured at the same time. It is preferable because it can be done.
  • the number of vertices 17 of each reaction unit 13 is preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 to 2.
  • the ratio of the lengths of both sides (long side / short side) forming 17 of the vertices is preferably 1 to 8, more preferably 1 to 4, and even more preferably 1 to 2. If the ratio is too large, the rotation of the substrate is likely to be disturbed and the development of the sample solution or the like becomes uneven, which is not preferable.
  • the number of sides is preferably 3 to 24, more preferably 6 to 12.
  • the difference in centrifugal force between the apex and the sides becomes small, and the liquid easily flows back, which is not preferable.
  • 50% or more of the outer peripheral length of the reaction unit 13 is a straight line. Even if it is a curve, if the vertical distance of each point on the curve from the straight line connecting both ends is 0.09 or less as a ratio (mutation) to the length of the straight line connecting both ends, it can be regarded as a straight line.
  • a straight line is preferable because the centrifugal force becomes non-uniform and it is easy to prevent backflow.
  • a plurality of binding substances 141 having a specific binding ability to the measurement target substance contained in the sample solution are fixed to the immobilization portion 14 of the first substrate 100 of the present embodiment.
  • a method for fixing the binding substance 141 there are a method of adhering the binding substance on a flat surface and a method of holding the binding substance 141 in the wells 142 as shown in FIGS. 12 and 13 described later, but the fixing is reliable and the measurement accuracy is accurate.
  • the well method is more preferable in that the amount can be increased.
  • the shape of the well 142 may be any shape as long as it is recessed from the surroundings, but the shape of a part of the spherical surface is excellent in that it has excellent immobilization reliability and position accuracy, and can speed up and improve the accuracy of measurement such as color development.
  • the radius Wr of the spherical surface forming the well 142 shown in FIG. 14 is preferably 0.4 to 1.0 mm, more preferably 0.5 to 0.8 mm.
  • the diameter Wd of the well 142 on the substrate is preferably 0.2 to 1.0 mm, more preferably 0.3 to 0.6 mm, and its depth Wf is preferably 0.02 to 0.08 mm, 0.04 to 0. .06 mm is more preferred.
  • the number of the fixing portions 14 of the first substrate 100 is preferably 24 to 128, more preferably 36 to 96.
  • a spotting method injection method
  • a microstamping method soft lithography
  • FIG. 1 A perspective view of the solid-state reaction chip 10 according to the embodiment of the present invention in which the first substrate 100 and the second substrate 20 are combined is shown in FIG. 1, and a cross-sectional view is shown in FIG.
  • This cross-sectional view is a cross-sectional view corresponding to the positions AA in FIG. 2 and BB in FIG.
  • the cross-sectional view of FIG. 6 is shown in a state in which the solid phase reaction chip 10 shown in FIG. 1 is turned upside down (the first substrate 100 is on the lower side and the second substrate is on the upper side).
  • the smoothing portion 21 of the second substrate 20 and the reaction portion 13 and the pillar portion 15 of the first substrate 100 face each other, and the pillar portion 15 is higher than the reaction portion 13, so that the reaction portion 13 and the second A gap 31 is formed between the substrate 20 and the smooth portion 21.
  • the height hz of the gap 31 is preferably 0.05 to 0.30 mm, more preferably 0.10 to 0.20 mm. If this height hz is too small, it becomes difficult for the sample liquid or the like to flow and the measurement accuracy becomes low, and if it is too large, a large amount of the sample liquid is required, which is not preferable.
  • the smoothing portion 21 of the second substrate 20 preferably has the same shape as the main portion 16A.
  • plastic resin is preferable because it is easy to manufacture and handle.
  • plastic resin polyethylene, polypropire, polycarbonate, polyethylene terephthalate, polyvinyl chloride, polystyrene, ABS resin, polyamide, polytetrafluoroethylene, unsaturated polyester, epoxy resin and the like can be used, and polyethylene, polypropire, polycarbonate, polyethylene and the like can be used. Telephthalate, polyvinyl chloride and polystyrene are preferred.
  • the second substrate 20 is preferably opaque in order to improve the measurement accuracy of light emission and the like.
  • the allergens that induce type I allergens used as the binding substance 141 include house dust 1 (2), Dermatophagoides farinae, cedar, hinoki, hannoki (genus), dandruff (genus), camogaya, butakusa, yomogi, alternaria, and aspergillus.
  • Sample solutions include whole blood, serum, plasma, tears and the like.
  • the object to be measured is preferably an allergen-specific antibody, and particularly preferably an IgE antibody.
  • the physiologically active substance having a specific binding ability to the IgE antibody as the measurement target substance and having enzymatic activity is not particularly limited, but for example, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, urease, and creatine kinase. , Urease, glucose-6-phospate dehydrogenase, peroxidase and the like labeled anti-IgE antibody can be used.
  • the object to be measured was a genotype derived from human papillomavirus (HPV) and a genomic DNA probe derived from the binding substance HPV, and 16 types, 18 types, 31 types, 33 types, 39 types, 45 types, and 51 types, 13 types of high-risk types such as 52 type, 56 type, 58 type, 59 type, 68 type and 82 type and 7 types such as 6 type, 11 type, 42 type, 43 type, 53 type, 54 type and 70 type It is also possible to construct it as a measurement system for detecting HPV classified as a low-risk type of virus.
  • HPV human papillomavirus
  • a colorimetric signal and a chemical luminescence signal can be used for detection.
  • Tetramethylbenzidine and its derivatives, o-phenylenediamine, triarylmethanes, imidazole leuco pigments, etc. are used as substrates used to generate a colorimetric signal, and acridinium salts and dioxetane are used to generate a chemical luminescence signal.
  • luciferin, lucigenin, oxalyl chloride and the like can be used.
  • An aqueous solution containing a surfactant can be used as the cleaning liquid, and a nonionic surfactant such as polyoxyethylene sorbitan is preferable as the surfactant. It is preferable to place a water-retaining substance such as paper, natural fiber, synthetic fiber, acetate fiber, or sponge on the waste liquid portions 18 and 28 because the waste liquid can be more reliably retained.
  • the amount of the sample solution, the washing solution, and the HRP-labeled antibody solution introduced by the measurement method using the solid phase reaction chip 10 of the present embodiment is preferably 20 to 80 ⁇ l, and all of them are added through the opening 11.
  • the measurement method consists of the following 7 steps. (S1) A binding step of injecting a sample solution to bind the substance to be measured and the binding substance 141, and (S2) a step of rotating the solid phase reaction chip 10 at a predetermined rotation speed of 200 to 2500 rpm to remove the reaction solution.
  • S3 A step of adding an HRP-labeled antibody solution, which is a reaction solution containing a physiologically active substance having a specific binding ability and enzymatic activity to the substance to be measured bound to the binding substance 141, (S4) solid phase reaction.
  • the measurement can be automatically performed by using the automatic measuring device 200 having the configuration shown in FIG.
  • the automatic measuring device 200 includes a dispensing unit 201 capable of sucking and discharging a solution from a reagent cartridge, a centrifuge 202 that rotates at a predetermined rotation speed while holding the solid phase reaction chip 10, and a CCD.
  • An image analysis unit 205 that analyzes (emission spots), a measurement result generation unit 206 that generates measurement results from the analysis results, and a control unit 207 that controls in an integrated manner are provided to the reagent cartridge or the apparatus of the solid phase reaction chip 10. It is possible to eliminate the actual operation by the inspector except for the set of.
  • the final measurement result can be obtained in about 20 minutes, so that the throughput is high, and the operation can be performed according to the screen displayed via the operation unit 204. It has the advantage of not requiring technology.
  • the device itself can be miniaturized and can be used as a device for immediate clinical examination.
  • the solid phase reaction chip of the embodiment according to the present invention can be used as a platform capable of detecting and measuring autoimmune diseases, cancer markers, infectious diseases, myocardial markers, etc., in addition to the above measurement examples.
  • Example 1 ⁇ Preparation of binding substance solution, blocking solution, cleaning solution> Four kinds of extracted and purified allergens (sugi, mite, egg white, milk) were prepared in a solution of 10 ⁇ g / ml each to obtain binding substance solutions -1 to 4. Dissolve 20.923 g of 3-morpholinopropanesulfonic acid, 2.922 g of sodium chloride, 1.0 g of sodium azide, 0.3% of Microside I, 1.0 g of TritonX100, 10 g of BSA, and 2.36 g of sodium hydroxide in 1 L of water. Block solution-1 was obtained.
  • Example 2 Preparation of sample H-1 of solid phase reaction chip>
  • the first substrate 100 shown in FIGS. 2 and 3 and the second substrate 20 shown in FIGS. 4 and 5 were made of plastic.
  • the outer shape of the main portion 16A of the first substrate 100 and the smooth portion 21 of the second substrate 20 is octagonal and has four-fold rotational symmetry.
  • the linear distance between 13b and 13g in FIG. 2 is 20 mm.
  • Each reaction portion 13 of the first substrate has two vertices 17 (17a and 17b) having an internal angle of 140 degrees. Since the four reaction units 13 are the same, one reaction unit 13 will be described.
  • the internal angles of the vertices 17a and 17b are both 140 degrees.
  • the sides 13a-17a and 13b-17b of the apex are straight lines having a length of 15 mm, and 17a-17b are arcs having a length of 3.7 mm. Since the variation of the arc from the straight line is 0.074, which can be regarded as a straight line, the ratio of the length of the straight line to the total length of the outer circumference of the reaction unit 13 is 100%.
  • the peripheral portions of both substrates are 40 mm in length and 35 mm in width.
  • the height hz of the void 31 of the reaction unit 13 is 0.15 mm.
  • the immobilization portions 15 are arranged at equal intervals of 1.5 mm in the vertical and horizontal directions, and a total of 64 are fixed.
  • the main portion 16A of the first substrate 100 and the smooth portion 21 of the second substrate 20 have the same shape, and the outer peripheral shapes of the peripheral portions 12 and 22 of both substrates are the same and have a shape that can be combined with each other.
  • the block liquid-1 described in Example 5 was sprayed. And dried.
  • the solid-state reaction chip of Example 2 in which polyurethane was placed as an absorbent material 19 on the waste liquid portion 18, the second substrate 20 was covered on the first substrate 100, and the bonding substances 141 were immobilized on the peripheral portions 12 and 22. 10 samples H-1 were obtained.
  • the structure of sample H-1 of the solid phase reaction chip 10 of Example 2 is shown in FIGS. 15 and 16.
  • Example 3 ⁇ Preparation of solid phase reaction chip samples H-2 to 6> As shown in FIG. 7, the outer circumference of the reaction unit 13 is two straight lines, an isosceles triangle having the same length on both sides of 13a-17a and 13b-17a, the internal angle of the apex 17a is 160 degrees, and the peripheral edge.
  • AX-AX in FIG. 7 is a position corresponding to AA in FIG. 2.
  • a sample H-3 of the solid phase reaction chip 10 of Example 3 was prepared.
  • the size of the internal angle is 170 degrees, 90 degrees (main part 16C shown in FIG.
  • FIG. 8 and AY-AY in FIG. 8 are positions corresponding to AA in FIG. 2), 60 degrees (main part 16D shown in FIG. 9), respectively.
  • AZ-AZ in FIG. 9 is a position corresponding to AA in FIG. 2), except that the temperature is set to 45 degrees, the solid-phase reaction of Example 3 is carried out in the same manner as the sample H-3 of the solid-phase reaction chip 10.
  • Samples H-2, 4, 5, and 6 of chip 10 were prepared.
  • the size, shape, and distance from the axis of rotational symmetry of the column portion 15 are all the same for the samples H-1 to 6 of the solid-phase reaction chip 10.
  • Comparative Example 1 ⁇ Preparation of solid phase reaction chips C-1 to 3 of Comparative Example> Comparative Example Solid Reaction Chip Similar to Sample H-1 of Solid Reaction Chip 10 Described in Example 2 except that the outer shape of the main part of the first substrate and the smooth part of the second substrate is circular with a diameter of 25 mm.
  • Sample C-1 was prepared. The outer peripheral length of each reaction part is 13.1 mm.
  • a sample C-2 of the solid phase reaction chip of Comparative Example was prepared in the same manner as the sample H-3 of the solid phase reaction chip 10 of Example 3 except that the size of the internal angle was set to 30 degrees each.
  • a sample C-3 of the solid phase reaction chip of Comparative Example was prepared in the same manner as the sample H-3 of the solid phase reaction chip 10 of Example 3 except that 13a and 13b of FIG. 7 were connected by a straight line. This is a sample in which the reaction part does not have an apex but corresponds to an internal angle of 180 degrees.
  • Example 4 ⁇ List of solid phase reaction chips> Table 1 shows the number of vertices 17 of each reaction unit 13 in the samples H-1 to 6 of the solid phase reaction chip 10, the size of the internal angle on the outer circumference of each reaction unit 13, and the sample C of the solid phase reaction chip of Comparative Example. The points corresponding to them in -1 to C-3 are shown. The size, shape, and distance from the axis of rotational symmetry of the column portion are as shown in Samples H-1 to 6 of the solid-state reaction chip 10 of Examples 2 and 3, and Samples C-2 and 3 of the fixed reaction chip of Comparative Example. All are the same.
  • Example 6 ⁇ Measurement of backflow> 40 ⁇ l of serum, which is a sample, is injected into the openings 11 of the first substrates of the solid reaction chips 10 of Examples 2 and 3 and the samples H-1 to 6 of the solid reaction chips 10 and the samples C-1 and 3 of the solid reaction chips of Comparative Example. Then, after incubating at 37 ° C. for 10 minutes, the mixture was rotated at a rotation speed of 1500 rpm for 3 seconds, centrifuged, and the magnitude of backflow was visually observed. An experiment was also conducted in which the rotation was performed for 10 seconds instead of 3 seconds. Each experiment was repeated 3 times. The experimental results are shown in Table 3.
  • Samples H-1 to H-1 of the solid-phase reaction chip 10 of Examples 2 and 3 were preferred because they generated less backflow than samples C-1 and 3 of the solid-state reaction chip of Comparative Example. Even in the sample of this example, the sample H-6 of the solid phase reaction chip 10 generated one backflow in the experiment for 3 seconds, and was slightly inferior to the samples H-1 to 5. However, there is no practical problem because the actual product rotates for 10 seconds.
  • Example 7 ⁇ Sample H-7 of solid phase reaction chip> Sample H except that the immobilization portion 14 is a well 142, the depth Wf of the well 142 is 0.05 mm, the diameter Wd is 0.5 mm, and the radius Wr is a part of a spherical surface of 0.65 mm. Sample H-7 was prepared in the same manner as in -1. The backflow test described in Example 6 was performed, and there was no backflow for 3 seconds and 10 seconds.
  • FIG. 11 is a partially enlarged view of the reaction portion 13 of the first substrate 100 in the CC line range shown in FIG.
  • FIGS. 12, 13 and 14 show a structural example of the sample H-7 used in Example 7 in the partially enlarged view of the reaction unit 13 shown in FIG.
  • FIG. 12 is a sectional view taken along line DD shown in FIG. 11
  • FIG. 13 is a sectional view taken along line EE shown in FIG.
  • FIG. 14 is a diagram illustrating the shape of the well 142 shown in FIGS. 12 and 13.
  • 15 and 16 show structural examples of the solid phase reaction chip 10 used in the sample H-7
  • FIG. 15 is an exploded view of the solid phase reaction chip 10 corresponding to FIG. 6, and
  • FIG. 16 is an exploded view. It is a perspective view of the solid phase reaction chip 10 corresponding to FIG.
  • the well 142 is formed with a partial recess in the shape of a spherical surface on the substrate, for example, with the radius Wr of the spherical surface, the diameter Wd on the substrate, and the depth Wf in the plate thickness direction of the substrate. ..
  • the binding substance 141 is inserted into each well 142 and immobilized.
  • a method for immobilizing the binding substance 141 in the well 142 for example, a spotting method (injection method) or a microstamping method (soft lithography) can be used.
  • Example 8 ⁇ Measurement method> 40 ⁇ l of serum as a sample was injected into the opening 11 of the first substrate 100, incubated at 37 ° C. for 10 minutes, and then rotated at a rotation speed of 1500 rpm for 10 seconds for centrifugation. 40 ⁇ l of HRP-labeled anti-human IgE antibody, which is a labeled antibody, was injected through the opening 11 of the first substrate 100 and then incubated at 37 ° C. for 5 minutes. 40 ⁇ l of the cleaning liquid-1 was injected through the opening 11 of the first substrate 100, rotated at 1500 rpm for 10 seconds, and centrifuged. This washing was carried out a total of 3 times.
  • Example 9 Serum was measured by the measuring method described in Example 8 using the samples H-1 to 7 of the solid phase reaction chips 10 of Examples 2 and 3 and Example 7 according to the present invention. Normal measurement results were obtained for all of the samples H-1 to 7 of the solid phase reaction chip 10. Further, the sample H-7 of the solid phase reaction chip 10 of Example 7 in which the well 142 is provided in the immobilization portion 14 has higher measurement reproducibility than the samples H-1 to 6 having a planar structure of Examples 2 and 3. I liked it more.

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Abstract

L'invention concerne une puce de réaction en phase solide (10) constituée d'un corps rotatif formé par stratification d'un premier substrat (100) et d'un second substrat (20), la puce comprenant : une ouverture (11) permettant de diriger un fluide échantillon vers le premier substrat (100) ; une partie de réaction (13) comprenant une partie immobilisation (14) au niveau de laquelle un matériau de liaison présentant la capacité de se lier spécifiquement à une substance cible de mesure dans le fluide échantillon est immobilisé ; et un montant (15) permettant de former un espace dans lequel le fluide échantillon est expansé, la puce comprenant un fluide de sorte que le fluide échantillon soit éjecté depuis l'espace dans la périphérie de la partie réaction (13), la périphérie de chaque partie réaction (13) comprenant au moins un sommet présentant un angle intérieur de 45 à 170 °.
PCT/JP2020/011313 2019-03-25 2020-03-14 Puce de réaction en phase solide et procédé de mesure utilisant cette dernière WO2020195991A1 (fr)

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JP2002071684A (ja) * 2000-08-25 2002-03-12 Wako Pure Chem Ind Ltd 多項目生体成分測定用試験具及びその製造方法
JP2010520991A (ja) * 2007-03-09 2010-06-17 中国人民解放軍軍事医学科学院微生物流行病研究所 複合的な検出のためのイムノクロマトグラフィのストリップディスクおよびそれを用いる検出方法
WO2012001972A1 (fr) * 2010-06-30 2012-01-05 株式会社メタボスクリーン Puce microchimique, procédé pour la produire et procédé pour l'utiliser
WO2015174429A1 (fr) * 2014-05-15 2015-11-19 タカノ株式会社 Puce d'analyse et appareil d'analyse d'échantillon
JP2016200431A (ja) * 2015-04-08 2016-12-01 株式会社パートナーファーム 固相反応チップ及びこれを用いた測定方法
WO2019044379A1 (fr) * 2017-08-28 2019-03-07 国立大学法人大阪大学 Récipient, procédé de fabrication correspondant et procédé de détection de substance de test

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002071684A (ja) * 2000-08-25 2002-03-12 Wako Pure Chem Ind Ltd 多項目生体成分測定用試験具及びその製造方法
JP2010520991A (ja) * 2007-03-09 2010-06-17 中国人民解放軍軍事医学科学院微生物流行病研究所 複合的な検出のためのイムノクロマトグラフィのストリップディスクおよびそれを用いる検出方法
WO2012001972A1 (fr) * 2010-06-30 2012-01-05 株式会社メタボスクリーン Puce microchimique, procédé pour la produire et procédé pour l'utiliser
WO2015174429A1 (fr) * 2014-05-15 2015-11-19 タカノ株式会社 Puce d'analyse et appareil d'analyse d'échantillon
JP2016200431A (ja) * 2015-04-08 2016-12-01 株式会社パートナーファーム 固相反応チップ及びこれを用いた測定方法
WO2019044379A1 (fr) * 2017-08-28 2019-03-07 国立大学法人大阪大学 Récipient, procédé de fabrication correspondant et procédé de détection de substance de test

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