WO2019151542A1 - Procédé de mesure d'échantillon, couvercle de plaque à puits multiples, kit de mesure d'échantillon et dispositif de mesure d'échantillon - Google Patents

Procédé de mesure d'échantillon, couvercle de plaque à puits multiples, kit de mesure d'échantillon et dispositif de mesure d'échantillon Download PDF

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Publication number
WO2019151542A1
WO2019151542A1 PCT/JP2019/004318 JP2019004318W WO2019151542A1 WO 2019151542 A1 WO2019151542 A1 WO 2019151542A1 JP 2019004318 W JP2019004318 W JP 2019004318W WO 2019151542 A1 WO2019151542 A1 WO 2019151542A1
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WIPO (PCT)
Prior art keywords
sample
measuring
well
lid
state
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PCT/JP2019/004318
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English (en)
Japanese (ja)
Inventor
石川　陽一
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エイブル株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to a measurement method for measuring the state of a sample in a test tank, a multi-well plate lid suitable for measurement, a sample measurement kit, a sample measurement device, and more particularly, cell proliferation, bacteria, etc.
  • the present invention relates to a method for measuring a change in the state of a sample derived from the activity of a microorganism of the above by a fluorescence measurement method and an apparatus used for the method.
  • Patent Document 1 discloses a technique for measuring an oxygen concentration using a probe having a fluorescent material at the tip.
  • Patent Document 2 discloses a technique for measuring the state of a sample by using a fluorescent material provided on the bottom surface of a test tank.
  • One of the objects of the present invention is to provide a sample measurement method, a multi-well plate lid, which can use an instrument having a simple structure and can be applied to adhesion-dependent or floating cells, etc.
  • the object is to provide a sample measurement kit and a sample measurement apparatus.
  • a sample measurement method is a method for measuring the state of a liquid sample using a multi-well plate, comprising a step of preparing a multi-well plate in which a liquid sample is injected into each well; A step of covering the well plate and a step of measuring the state of the sample, wherein the lid has a plurality of protrusions corresponding to each well of the multi-well plate, and the protrusions are When the lid is attached to the multi-well plate, the tip of the multi-well plate is configured to come into contact with the sample while being separated from the bottom of each well. The step of measuring the state of the sample is performed by the multi-well plate.
  • the step of measuring the state of the sample may include measuring the amount of received fluorescence, time delay, or decay rate with respect to the irradiated excitation light.
  • the convex portion may have a flat tip portion and a rod-like support portion that supports the tip portion, and the phosphor may be provided at the tip portion.
  • the sample measurement method may further include a plurality of individual lids corresponding to the wells of the multiwell plate.
  • the step of measuring the state of the sample may include the individual lid.
  • the bottom of the well may be airtight.
  • the step of measuring the state of the sample may include measuring at least one of the dissolved oxygen concentration, the dissolved carbon dioxide concentration, pH, and turbidity of the sample.
  • the lid of the multi-well plate according to the present invention has a plurality of convex portions corresponding to each well of the multi-well plate, and a phosphor provided at the tip of the convex portion, and the convex portion is the lid Is attached to the multi-well plate, the tip of the multi-well plate is configured to come into contact with the sample injected into each well while being separated from the bottom of each well. The phosphor is irradiated with excitation light and the fluorescence is received.
  • the sample measurement kit according to the present invention includes the multiwell plate lid described above and a multiwell plate.
  • the sample measurement device according to the present invention includes the sample measurement kit described above.
  • the sample measurement method according to the present invention includes a step of preparing a test tank into which a liquid sample is injected, and a step of providing a phosphor in contact with the sample while being separated from the bottom of the test tank in the test tank. Measuring the state of the sample, and the step of measuring the state of the sample irradiates the phosphor with excitation light from the lower side of the test chamber and receives the fluorescence. including.
  • a sample measurement method capable of accurately measuring the state of a sample using a device having a simple structure. Further, it is possible to provide a multi-well plate lid that has a simple structure and can accurately measure the state of a sample. Furthermore, it is possible to provide a sample measurement method capable of accurately measuring the state of the sample using a device having a simple structure.
  • FIG. 1 is a diagram for explaining a sample measurement kit according to the present embodiment.
  • FIG. 2 is a diagram for explaining the sample measurement kit according to the present embodiment.
  • FIG. 3 is a diagram for explaining the sample measuring apparatus according to the present embodiment.
  • FIG. 4 is a diagram for explaining the sample measurement method according to the present embodiment.
  • FIG. 5 is a diagram for explaining the sample measurement method according to the present embodiment.
  • FIG. 6 is a diagram for explaining a sample measurement kit according to a modification.
  • FIG. 7 is a diagram for explaining a sample measurement kit according to a modification.
  • FIG. 8 is a diagram for explaining a sample measurement kit according to a modification.
  • FIG. 9 is a diagram for explaining a sample measurement method according to a modification.
  • FIG. 10 is a diagram for explaining a sample measuring apparatus according to a modification.
  • the sample measurement kit 1 which concerns on this Embodiment is demonstrated.
  • the sample measurement kit 1 has a multi-well plate 10.
  • the multi-well plate 10 is a member in which a plurality of recesses (tanks) called wells are formed, and each well can be used as an independent test tank.
  • the multi-well plate 10 is a multi-well plate called a 96-well plate in which 96 wells 12 are provided.
  • the present invention is not limited to this, and it is also possible to apply a multiwell plate (microwell plate) having the number of wells of 6, 12, 24, 48, 384, 1536 or the like.
  • a multiwell plate microwell plate having the number of wells of 6, 12, 24, 48, 384, 1536 or the like.
  • the bottom of the well 12 of the multi-well plate 10 is flat.
  • the present invention is not limited to this, and it is also possible to use a multi-well plate in which the bottom of the well is U-shaped or V-shaped.
  • adjacent wells 12 of multi-well plate 10 are connected at the upper end.
  • the multi-well plate 10 has a structure in which a plurality of recesses are formed on the upper surface, and the lower end of the well 12 is independent.
  • the multi-well plate may be configured such that the lower ends of the wells are connected and the upper ends are independent (not shown).
  • at least the bottom of the multi-well plate 10 is made of a light transmissive material.
  • the sample measurement kit 1 can perform excitation light irradiation and fluorescence reception from the lower side of the multiwell plate 10 (details will be described later).
  • the sample measurement kit 1 has a lid 20.
  • the lid 20 is a member that is attached to the multiwell plate 10 and covers the upper surface side (opening side of the well 12) of the multiwell plate 10. In the present embodiment, the lid 20 is configured to cover the entire multi-well plate 10.
  • the lid 20 has a plurality of convex portions 22 corresponding to the wells 12 of the multi-well plate 10. As shown in FIG. 2, the convex portions 22 are provided on the top plate 24 of the lid 20, and when the lid 20 is attached to the multiwell plate 10, each convex portion 22 is inserted into the well 12. It is arranged so that. Note that the convex portion 22 is arranged such that when the lid 20 is attached to the multi-well plate 10, the tip 26 thereof is arranged away from the bottom of the well 12 (with a space), and the well 12. It is comprised so that it may contact with the sample S inject
  • the amount of the sample S injected into the well 12 is adjusted so that the sample S and the tip 26 are in contact (see FIG. 5).
  • the lid 20 has (substantially) the same outer shape as the multi-well plate 10 and is positioned by fitting the respective outer peripheral portions.
  • a phosphor 30 is provided at the tip 26 of the convex portion.
  • the specific composition of the phosphor 30 is not particularly limited, any substance that can be used as a sensor for measuring dissolved oxygen, dissolved carbon dioxide, pH, turbidity, etc. in a liquid can be used. .
  • the sample measuring apparatus 2 according to the present embodiment will be described with reference to FIGS. 3 (A) and 3 (B).
  • the sample measuring device 2 is a device for measuring the state of the sample using the sample measuring kit 1 described above.
  • the sample measuring device 2 has a measuring table 50.
  • the measurement table 50 is a light transmissive member on which the sample measurement kit 1 is placed.
  • the measurement table 50 may have a structure that supports the entire surface of the multiwell plate 10, but may have a structure that supports a part of the measurement table 50 (for example, a frame shape that supports only the outer peripheral portion).
  • the measurement table 50 is provided with a positioning mechanism (not shown), and the sample measurement kit 1 is arranged at a predetermined position on the measurement table 50.
  • the sample measuring device 2 has a plurality of light emitting / receiving modules 52.
  • the light emitting / receiving module 52 includes a mechanism for irradiating the fluorescent material 30 provided at the tip of the convex portion 22 with excitation light and receiving the fluorescence.
  • the light receiving / emitting module 52 includes a light emitting element 54 and a light receiving element 56.
  • the light emitting element 54 irradiates the phosphor 30 with excitation light.
  • the phosphor 30 is excited by receiving the excitation light from the light emitting element 54 and generates fluorescence (including phosphorescence) when returning to the ground state.
  • the light receiving element 56 receives the fluorescence generated by the phosphor 30 and converts it into an electrical signal.
  • the sample measuring device 2 includes a plurality of light emitting / receiving modules 52, and each light receiving / emitting module 52 is provided so as to correspond to each well 12 of the multiwell plate 10. Thereby, the state of the sample S in each well 12 can be measured.
  • the sample measuring device 2 has a control unit 58.
  • the control unit 58 blinks the light emitting element 54 at a predetermined cycle, converts the fluorescence received by the light receiving element 56 into an electrical signal, and transmits the electrical signal to the processing unit 60.
  • the processing unit 60 performs various calculations for deriving the state of the sample S based on the light emission information of the excitation light from the light emitting / receiving module 52 (light emitting element 54) and the electrical signal transmitted from the control unit 58. Do.
  • the phosphor detects the dissolved oxygen concentration
  • the decay of the fluorescence is accelerated by the dissolved oxygen, the intensity is weakened, and the time delay between the excitation light and the fluorescence is reduced. If the time delay between the excitation light and the fluorescence becomes small, the phase delay of the light reception period with respect to the light emission period becomes small. From this, it becomes possible to obtain the dissolved oxygen concentration of the sample S based on the phase delay of the light receiving period with respect to the light emitting period.
  • the method of measuring the phase lag value as a method for obtaining the temporal difference between excitation light and fluorescence
  • a method of measuring the decay rate at which fluorescence decays after emission of excitation light is known, Either method may be used.
  • the sample measuring device 2 can also be configured to detect the intensity of the fluorescence together and use it for measuring the state of the sample S.
  • the sample measuring device 2 can be configured to measure the state of the sample S using image recognition processing.
  • the sample measurement method according to the present embodiment will be described taking a toxicity test of the reagent X against the cell C (adhesion-dependent cell) as an example.
  • the sample measurement method according to the present embodiment first includes a step of culturing adhesion-dependent cells C in each well 12 of the multi-well plate 10 (step S10). Specifically, as shown in FIG.
  • the medium M adjusted for cell culture is injected into each well 12 of the multi-well plate 10 and the cells are seeded, and left in a CO 2 incubator. .
  • the cells C can be adhered and cultured in the wells 12 (bottom surface and inner surface) of the multi-well plate 10.
  • the reagent X is added to each well 12 (step S20).
  • the reagent may be added to the medium M as shown in FIG. 5C, or may be added after removing the medium or after replacing the medium. Thereby, a multi-well plate in which the liquid sample S is injected into each well can be prepared.
  • the sample measurement method according to the present embodiment includes a step of covering the multiwell plate 10 with a lid 20 (Step S30). In this step, the tip 26 of the convex portion 22 provided on the lid 20 is brought into contact with the sample S as shown in FIG. Thereby, the phosphor 30 provided at the tip 26 can be brought into contact with the sample S.
  • the sample measurement method according to the present embodiment includes a step (step S40) of measuring the state of the sample S. In this step, as shown in FIG.
  • the phosphor 30 is irradiated with excitation light from the lower side of the multi-well plate 10, and the fluorescence of the phosphor 30 is received from the lower side of the multi-well plate 10.
  • the state of the sample S is measured by performing a predetermined calculation.
  • the reagent X is toxic to the cell C
  • the activity of the cell C is reduced and the consumption of dissolved oxygen in the sample S is reduced.
  • the reagent X is not toxic to the cell C
  • the activity of the cell C is maintained for a certain period, and the dissolved oxygen of the sample S is consumed. From this, by measuring the amount of dissolved oxygen in the sample S, it is possible to detect whether or not the reagent X is toxic to the cells C and to what extent.
  • the phosphor 30 for measuring the state of the sample S is provided at the tip of the convex portion 22 of the lid 20. This eliminates the need to provide a phosphor in the well 12 of the multi-well plate 10, so that the composition of the inner surface of the well 12 can be made uniform, and it becomes easy to culture adhesion-dependent cells and the like. Observation from the bottom surface of the multiwell plate 10 (microscopic observation, etc.) becomes easy.
  • the phosphor 30 is irradiated with excitation light and received with fluorescence from the lower side of the multi-well plate 10 (via the bottom surface of the multi-well plate 10).
  • the state of the sample S is accurately measured using the lid 20 having a simple structure. It becomes possible. Further, in the present embodiment, the lid 20 is provided with the phosphors 30 (convex portions 22) corresponding to the respective wells 12, and the samples S in all the wells 12 are simply attached to the lid 20. It becomes possible to measure the state. Therefore, the process of measuring the state of the sample S can be performed efficiently. In the present embodiment, fluorescence and excitation light are transmitted through the cell C. For this reason, there is a concern that the measurement may be affected in cells in which the cells are congested in the well.
  • the state of the sample S it is possible to accurately measure the state of the sample S by ending the culturing process and starting the measuring process within a range where the cells do not become congested.
  • a method for measuring the state of a sample based on excitation light and fluorescence there are a method using the intensity of fluorescence and a method measuring based on the phase difference and disappearance time of fluorescence.
  • a method for measuring the state of the sample based on the phase difference and disappearance time of the fluorescence it can be made less susceptible to changes in the intensity of the fluorescence, and cell C culture has progressed. Even in the state, the state of the sample S can be accurately measured.
  • FIG. 6 is a diagram for explaining a first modification of the embodiment to which the present invention is applied.
  • the lid 60 has a convex portion 62.
  • the convex portion 62 includes a flat tip portion 64 and a rod-like support 66 that supports the tip portion 64.
  • the cross-sectional area of the tip part 64 is larger than the cross-sectional area of the support body 66 in the cross section orthogonal to the length direction.
  • FIG. 7 is a diagram for explaining a second modification of the embodiment to which the present invention is applied.
  • the sample measurement kit has an individual lid 70.
  • the individual lid 70 is a plate-like member having a through hole formed in the center.
  • the individual lid 70 is disposed in each well 12, and the convex portion 22 of the lid 20 passes through the through hole.
  • the individual lid 70 is configured so that the specific gravity is lighter than that of the sample S. Thereby, the individual lid 70 is disposed in the vicinity of the liquid surface of the sample S.
  • the individual lid 70 Since the individual lid 70 is disposed in the vicinity of the liquid surface of the sample S, the gas-liquid contact area between the sample S and the gas phase is reduced, so that the gas can be dissolved into the sample S or the sample S can be changed into the gas phase. Gas emission can be reduced. Thereby, since the influence of the gas phase can be reduced and a change in the state of the sample S can be measured, for example, information in which the activity of the cell C is reflected more accurately can be measured.
  • the individual lid 70 can also be configured such that the bottom of the well 12 is airtight. For example, by making the individual lid 70 the same shape as the inner shape of the well 12, the region below the individual lid 70 of the well 12 can be kept airtight. Thereby, the influence of the gas phase can be further reduced.
  • the sample measurement kit may be configured to have an individual lid 74 as shown in FIG.
  • the individual lid 74 is fixed to the convex portion 22 of the lid 20.
  • the individual lid 74 can be disposed above the liquid surface of the sample S, or can be disposed so as to be in contact with the sample S.
  • the well 13 has a tapered shape whose diameter decreases toward the bottom. By adjusting the attachment position and size of the individual lid 74 and the shape of the well 13, the position of the individual lid 74 in the well 13 can be adjusted.
  • FIG. 9 is a diagram for explaining a third modification of the embodiment to which the present invention is applied. In this modification, culture and measurement are performed simultaneously.
  • the sample measurement method according to this modification includes a step of injecting the sample S into the well 12 (step S50).
  • the sample S is a mixed solution of a culture object (for example, microorganisms, animal cells, plant cells, etc.) and a medium. Depending on the purpose of measurement, it is also possible to mix reagents for the culture object.
  • the sample measurement method according to the present modification includes a step of changing the sample S over time with the lid 20 on the multi-well plate 10 (step S60).
  • this process is performed on the conditions suitable for a culture target.
  • this step can be realized by leaving it in a CO2 incubator with a temperature control function for a predetermined period.
  • the sample measurement method according to the present embodiment includes a step (step S70) of measuring the state of the sample S.
  • the phosphor 30 is irradiated with excitation light from the lower side of the multi-well plate 10, and the fluorescence of the phosphor 30 is received from the lower side of the multi-well plate 10, and a predetermined calculation is performed. Measure the state.
  • this process may be performed for every predetermined period and may be performed continuously.
  • the multi-well plate 10 can be arranged on the measuring table 50 of the sample measuring device 2 at a predetermined timing, and the state of the sample S can be measured intermittently.
  • the step of measuring the state of the sample S can be realized by measuring the concentration of the specific component contained in the sample S. Specifically, it can be measured as information on the dissolved oxygen concentration contained in the sample S, but it can also be measured as pH or dissolved carbon dioxide concentration.
  • the state of the sample S can be measured as turbidity information.
  • the success or failure of the culture of the object can be confirmed for each well 12, so that it is possible to confirm whether or not the medium or reagent has an influence on the object.
  • a method of measuring the state of the sample based on the disappearance time of the fluorescence may be applied in the step of measuring the state of the sample S (step S70). It is also possible to apply a method for measuring the state. For example, when the culture of the object progresses and the growth in the well 12 proceeds, the measured fluorescence intensity decreases. Therefore, the progress of culture can be confirmed by using the fluorescence intensity.
  • the phosphor 30 is irradiated with excitation light and fluorescent light. Can be received.
  • the phosphor 30 may be irradiated with excitation light using a surface emitting device.
  • the light emitting / receiving module (measuring base) can also be configured to include an optical integrated device 80 as shown in FIG.
  • the optical integrated device 80 is a device disposed between the light emitting element 54 and the light receiving element 56 and the measurement table.
  • the optical integrated device 80 includes an optical fiber 82 and a light shielding tube 84 that covers the side surface of the optical fiber 82.
  • the optical integrated device 80 is disposed such that the first end 86 of the optical fiber 82 faces the light emitting element 54 and the light receiving element 56 and the second end 88 faces the sample measurement kit 1 (phosphor 30). According to this, irradiation of excitation light and reception of fluorescence are performed through the optical fiber 82, and the excitation light and fluorescence are integrated without diffusing, so that the detection sensitivity can be increased.
  • the detection sensitivity can be further increased. Furthermore, according to the present embodiment, it is possible to prevent light (fluorescence) from other than the measurement target from entering, and thus it is possible to measure the state of the sample more accurately.
  • the present invention is not limited to this, and although not particularly illustrated, the present invention can be used for a system having only one test tank.
  • a technique for measuring the state of a sample in a test tank is used for the purpose of culturing cells and microorganisms, toxicity tests, and the like, and one that uses a phosphor is known.
  • it is possible to accurately measure the state of a sample by using a lid with a simple structure by irradiating the phosphor with excitation light and receiving the fluorescence from the lower side of the multi-well plate. Become. Further, by measuring the amount of dissolved oxygen in the sample, it is possible to detect whether or not the reagent is toxic to cells and to what extent.

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Abstract

La présente invention vise à fournir un procédé de mesure d'échantillon avec lequel un instrument ayant une structure simple peut être utilisé, et qui peut être appliqué à des cellules dépendantes de l'adhérence ou planctoniques, par exemple, et pour fournir un couvercle de plaque à puits multiples, un kit de mesure d'échantillon et un dispositif de mesure d'échantillon. Ce procédé de mesure de l'état d'un échantillon liquide à l'aide d'une plaque à puits multiples comprend : une étape de préparation d'une plaque à puits multiples (10) ayant un échantillon liquide S injecté dans chaque puits (12); une étape de placement d'un couvercle (20) sur la plaque à puits multiples; et une étape de mesure de l'état de l'échantillon. Le couvercle comprend une pluralité de parties en saillie (22) correspondant à chaque puits dans la plaque à puits multiples, les parties en saillie étant conçues de telle sorte que, lorsque le couvercle est fixé à la plaque à puits multiples, une extrémité distale de chaque partie en saillie vient en contact avec l'échantillon tout en étant séparée du fond du puits. L'étape de mesure de l'état de l'échantillon comprend une étape de rayonnement de lumière d'excitation depuis le dessous de la plaque puits multiples sur un matériau fluorescent (30) disposé au niveau des extrémités distales des parties saillantes, et de réception de fluorescence à partir de celle-ci.
PCT/JP2019/004318 2018-02-05 2019-01-31 Procédé de mesure d'échantillon, couvercle de plaque à puits multiples, kit de mesure d'échantillon et dispositif de mesure d'échantillon WO2019151542A1 (fr)

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WO2021054325A1 (fr) * 2019-09-17 2021-03-25 ウシオ電機株式会社 Dispositif de mesure de lumière et lecteur de microplaque
JP7380053B2 (ja) 2019-10-09 2023-11-15 株式会社リコー 蓋付き容器
US11874221B2 (en) 2021-01-26 2024-01-16 Yokogawa Electric Corporation Measurement of dissolved oxygen using optical radiation induced luminescence

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