WO2023218588A1 - 計測デバイスおよび測定方法 - Google Patents

計測デバイスおよび測定方法 Download PDF

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Publication number
WO2023218588A1
WO2023218588A1 PCT/JP2022/020026 JP2022020026W WO2023218588A1 WO 2023218588 A1 WO2023218588 A1 WO 2023218588A1 JP 2022020026 W JP2022020026 W JP 2022020026W WO 2023218588 A1 WO2023218588 A1 WO 2023218588A1
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Prior art keywords
substrate
reflection suppression
measuring
measurement
reflection
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PCT/JP2022/020026
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English (en)
French (fr)
Japanese (ja)
Inventor
惟夫 香村
淳子 田中
猛 石田
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Hitachi High Tech Corp
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Hitachi High Tech Corp
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Priority to US18/857,380 priority Critical patent/US20250264408A1/en
Priority to EP22941668.0A priority patent/EP4524227A4/en
Priority to PCT/JP2022/020026 priority patent/WO2023218588A1/ja
Priority to JP2024520169A priority patent/JP7810791B2/ja
Priority to CN202280094804.5A priority patent/CN119013386A/zh
Publication of WO2023218588A1 publication Critical patent/WO2023218588A1/ja
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/168—Specific optical properties, e.g. reflective coatings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention relates to a measuring device and a measuring method, and particularly relates to digital PCR.
  • PCR and real-time PCR have been used for genetic testing. These techniques have had the problem of low measurement accuracy when the amount of the target to be measured (nucleic acid) is minute. In order to solve this problem, digital PCR technology has attracted attention in recent years.
  • a sample containing DNA to be detected is divided into a large number of micro regions, and PCR is performed on each micro region.
  • the type of DNA present in each microregion is determined by distinguishing between sections that contain the DNA to be detected and sections that do not, based on the fluorescence intensity.
  • Patent Document 1 discloses a DNA detection method using digital PCR in which the melting temperature of DNA and a fluorescently labeled probe is measured in a droplet containing a fluorescently labeled probe that hybridizes to the DNA. ing.
  • melting curves differ depending on the type of target gene and fluorescently labeled probe, it is possible to determine the type of target gene by measuring the melting curve.
  • fluorescence intensity and melting curves When measuring a large number of genes simultaneously, it is necessary to measure fluorescence intensity and melting curves with high precision and to reduce measurement variations for each gene.
  • An object of the present invention is to provide a measurement device that can suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by bubbles in melting curve analysis of a target gene.
  • An example of the measurement device is a substrate having a plurality of through holes for introducing and dividing a nucleic acid solution; an oil that covers a first surface of the substrate and a second surface of the substrate opposite to the first surface to block the through hole; a heat conductive plate provided on the second surface side for heating the substrate; a reflection suppression mechanism that suppresses reflection of excitation light irradiated from the first surface side; It is characterized by having the following.
  • the measurement method includes: A measuring method using a measuring device,
  • the measurement device is a substrate having a plurality of through holes; a thermally conductive plate provided to change the temperature of the substrate; a reflection suppression mechanism that suppresses reflection of excitation light irradiated from a first surface side of the substrate opposite to the heat conduction plate side; has
  • the measurement method is a step of introducing a nucleic acid solution to the substrate and fractionating it; introducing oil to cover the through hole into which the nucleic acid solution has been introduced, on a first surface of the substrate and a second surface of the substrate opposite to the first surface; , changing the temperature of the substrate by the thermally conductive plate; irradiating excitation light from the first surface side of the substrate while changing the temperature, detecting fluorescence from the first surface side, and measuring the nucleic acid solution; has.
  • the present invention it is possible to provide a measurement device and a measurement method that can suppress the influence of bubbles, measure fluorescence intensity and melting curves with high precision, and discriminate the type of gene with high precision.
  • FIG. 3 is a diagram illustrating the intensity measured when air bubbles are generated at the bottom of a through-hole well in a measurement device in a conventional example.
  • a reflection image illustrating that in a conventional example, reflected light from a heat conduction plate at the bottom of a measurement device is observed through a through-hole well.
  • FIG. 3 is a diagram illustrating that, in Example 1 of the present invention, by subjecting the heat conductive plate at the bottom of the measurement device to a black surface treatment, the influence of air bubbles does not increase on the measured intensity.
  • 2 is a reflection image illustrating that, in Example 1 of the present invention, by blackening the heat conduction plate at the bottom of the measurement device, reflected light from the heat conduction plate is not observed through the through-hole well.
  • FIG. 7 is a diagram illustrating that, in Example 2 of the present invention, by introducing fine particles into the lower part of the through-hole well, the measured intensity is not influenced by air bubbles.
  • FIG. 7 is a diagram illustrating that the average reflected light intensity in the reflected image of the through-hole well is reduced by introducing fine particles into the lower part of the through-hole well in Example 2 of the present invention.
  • FIG. 7 is a diagram illustrating that, in Example 3 of the present invention, by making the heat conductive plate at the bottom of the measurement device transparent, the influence of air bubbles is not added to the measured intensity.
  • FIG. 7 is a diagram illustrating that the influence of air bubbles does not increase the measured intensity by making the heat conductive plate at the bottom of the measurement device have an uneven structure in Example 4 of the present invention.
  • FIG. 7 is a diagram illustrating that the influence of air bubbles is not added to the measured intensity by coloring the oil in the measuring device black in Example 5 of the present invention.
  • FIG. 7 is a diagram illustrating that the influence of air bubbles is not increased on the measured intensity by introducing ink into the lower part of the through-hole well in Example 6 of the present invention.
  • FIG. 3 is a diagram illustrating changes in fluorescence intensity observed when a measurement device without a reflection suppression mechanism is heated at 85° C. for a certain period of time in a comparative example.
  • FIG. 7 is a diagram illustrating changes in fluorescence intensity observed when a measurement device in which a reflection suppression mechanism is introduced between a through-hole well and a heat conduction plate is heated at 85° C. for a certain period of time in Example 7 of the present invention.
  • FIG. 8 is a flow diagram of fluorescence intensity measurement with respect to temperature change in a measurement device incorporating a reflection suppression mechanism in Example 8 of the present invention.
  • FIG. 7 is a diagram illustrating changes in fluorescence intensity with respect to temperature changes in a measurement device that does not incorporate a reflection suppression mechanism in a comparative example.
  • FIG. 8 is a diagram illustrating changes in fluorescence intensity with respect to temperature changes in a measurement device incorporating a reflection suppression mechanism in Example 8 of the present invention.
  • FIG. 9 is a flowchart of fluorescence intensity measurement and reflection image measurement with respect to temperature changes in a measurement device incorporating a reflection suppression mechanism in Example 9 of the present invention.
  • FIG. 9 is a diagram illustrating changes in the coefficient of variation of reflected light intensity in a measurement device before and after introducing a reflection suppression mechanism in Example 9 of the present invention.
  • FIG. 1 is a schematic diagram when fluorescence measurement is performed using a conventional measurement device holding a through-hole well 1.
  • This measurement device is composed of a substrate 2 holding a plurality of through-hole wells 1, a mixed solution 3 of nucleic acid and fluorescently labeled probe injected into the wells, oil 4 covering the through-hole wells 1, and a thermally conductive plate 5.
  • excitation light 6 is irradiated into the through-hole well 1.
  • a part of the excitation light 6 becomes transmitted light 7 that passes through the through-hole well 1 .
  • the transmitted light 7 is reflected by the heat conduction plate 5 and becomes reflected light 8.
  • the reflected light 8 passes through the through-hole well 1 again, and the reflected light intensity 10 as well as the fluorescence intensity 9 are measured as the measurement light intensity of the well.
  • the transmitted light 7 of the through-hole well 1 is diffused at the lower part of the through-hole well 1 by the air bubbles 11. Therefore, the light 12 that is not reflected by the thermally conductive plate 5 is not detected as strongly as the reflected light intensity 10. That is, when bubbles exist, the measured light intensity is the sum of the fluorescence intensity 9 and the reflected light intensity 13 which is attenuated compared to the original reflected light intensity 10.
  • FIG. 2 is a reflection image of the substrate 101 holding the through-hole well 100 observed at low magnification (FIG. 2(a)) and high magnification (FIG. 2(b)). If we pay attention to the reflected image of the through-hole well 100, it is bright. Therefore, the excitation light 6 passes through the through-hole well 1 and is reflected by the thermally conductive plate 5. Further, when comparing the through-hole well 100 and the through-hole well 102, there are variations in brightness. This is due to in-plane variations in the reflectance of the heat conductive plate 5.
  • Example 1 will be described below with reference to FIGS. 3 and 4.
  • the thermally conductive plate is treated with black color to make it difficult to observe bubbles.
  • FIG. 3 is a schematic diagram illustrating that by applying a black surface treatment film 201 to the heat conduction plate 200 at the bottom of the measurement device, the bubbles 202 observed in FIG. 1 become difficult to observe.
  • the measurement device has a substrate 209 having a plurality of through-hole wells 203 (through-holes) for introducing and dividing a mixed solution 208.
  • the measurement device has an oil 210 covering the through-hole well 203 on a first side 209a of the substrate 209 and on a second side 209b of the substrate opposite to the first side.
  • the mixed solution 208 contains a nucleic acid solution.
  • references to mixed solutions can be interpreted as references to nucleic acid solutions.
  • the measurement device has a heat conductive plate 200 provided on the second surface 209b side in order to change the temperature of the substrate 209 (for example, to heat the substrate 209).
  • the second surface 209b can be said to be the surface of the substrate 209 on the thermally conductive plate 200 side.
  • the material of the heat conductive plate 200 is, for example, metal or resin, but may also be glass. By using such materials, it is possible to construct a thermally conductive plate suitable for the requirements of the measurement device. Note that materials other than these may also be used.
  • the measurement device has a reflection suppression mechanism that suppresses reflection of the excitation light 207 irradiated from the first surface 209a side (for example, reflection toward the first surface 209a side).
  • the reflection suppression mechanism is a colored surface treatment structure of the thermally conductive plate 200.
  • the reflection suppression mechanism includes a black surface treatment film 201 as a reflection suppression film.
  • the surface treatment film 201 is placed on the heat conduction plate 200, and the surface treatment film 201 absorbs excitation light (or light having the same wavelength as the excitation light).
  • the black surface treatment film 201 absorbs excitation light (or light having the same wavelength as the excitation light).
  • the reflected light 204 from the heat conduction plate 200 produces an attenuated reflected light intensity 205. Therefore, the measured light intensity of each through-hole well 203 is the sum of the fluorescence intensity 206 and the attenuated reflected light intensity 205. Therefore, bubbles are prevented from being observed as unevenness on the measurement image.
  • the structure and formation method of the surface treatment film 201 can be appropriately designed by those skilled in the art, and the thickness varies depending on the surface treatment process.
  • an anodized film such as alumite treatment has a thickness of about 5 to 40 ⁇ m
  • a film treated by electroplating has a thickness of about 2 to 20 ⁇ m.
  • the film formed by spray coating is approximately 15 to 30 ⁇ m. In the case of this example, the film thickness is preferably 40 ⁇ m or less.
  • FIG. 4 is a reflection image observed at low magnification (FIG. 4(a)) and high magnification (FIG. 4(b)) of the substrate 300 after the black surface treatment film 201 has been applied to the heat conductive plate. . If we pay attention to the reflected image inside the through-hole well 301, we can see that it is dark. Further, in comparing the reflected light intensity between the through-hole well 301 and the through-hole well 302, it can be seen that there is no variation in the reflected light intensity, and the overall reflected light intensity is small. From this reflected image, it can be said that the reflection from the heat conductive plate 200 is suppressed.
  • the black surface treatment film 201 was used as the colored surface treatment structure, but its reflectance is important in order to suppress reflection from the heat conduction plate 200.
  • "colored" means that the reflectance is 10% or less over a wavelength of 400 nm to 700 nm, and specifically means black.
  • Such a colored surface treatment structure suppresses reflection from the thermally conductive plate 200.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 2 will be described below with reference to FIGS. 5 and 6.
  • fine particles 401 are injected into the lower part of the through-hole well 400 to make it difficult for bubbles 402 to be observed.
  • fluorescence intensity measurement can be performed without being affected by the bubbles 402.
  • descriptions of parts common to Example 1 may be omitted.
  • FIG. 5 is a schematic diagram of fluorescence measurement when microparticles 401 are introduced at the bottom of a mixed solution 403 of nucleic acid and fluorescently labeled probe injected into a through-hole well 400 in a measurement device.
  • the reflection suppression mechanism according to this embodiment includes fine particles 401 introduced into a through-hole well 400. Preferably, the fine particles 401 do not allow the excitation light 404 to pass therethrough.
  • the measurement light intensity is the sum of the fluorescence intensity 406 and the attenuated reflected light intensity 407. This makes it difficult for bubbles 402 to be observed in the measurement image.
  • FIG. 6 shows a comparison of the reflected images of the measurement devices shown in FIGS. 1 and 5.
  • FIG. 6(a) is a reflected image observed of a conventional measuring device
  • FIG. 6(b) is a reflected image observed of a measuring device according to this embodiment in which fine particles are injected.
  • the average reflected light intensity averaged over 50 through-hole wells was 133 (arbitrary units), and in the example of FIG. 6(b), the average reflected light intensity was 99. . Focusing on the average reflected light intensity of the plurality of through-hole wells, the average reflected light intensity of the plurality of wells is reduced by injecting fine particles into the through-hole well. Therefore, the excitation light is prevented from passing through the through-hole well and being reflected on the heat conductive plate.
  • Conditions for the fine particles to be injected include size, specific gravity, surface treatment, and number.
  • the size (for example, diameter) is desirably 30 nm or more.
  • the scattering phenomenon is based on Mie's scattering theory. Since Mie scattering shows significant scattering intensity from about 30 nm, this value can be set as the lower limit.
  • the size of the microparticles is preferably smaller than the through-hole well into which the microparticles are injected. Therefore, when the well size is 60 ⁇ m, the upper limit of the fine particles can be 60 ⁇ m.
  • the fine particles settle at the bottom of the through-hole well. Therefore, it is preferable to increase the specific gravity of the microparticles relative to the mixed solution of nucleic acid and fluorescently labeled probe injected into the through-hole well. That is, it is preferable that the fine particles have a higher specific gravity than the mixed solution.
  • fine particles examples include polymer fine particles (polystyrene, specific gravity: 1.04 to 1.07 g/cm 3 ), magnetic fine particles (iron, specific gravity: 7.85 g/cm 3 ), and metal fine particles (silver, specific gravity: 10.49 g/cm 3 ). 3 ), etc., with a specific gravity greater than 1 are used (that is, the specific gravity of the particles is 1 g/cm 3 or more).
  • the microparticles then settle within the through-hole wells.
  • a mixed solution of nucleic acids and fluorescently labeled probe is an aqueous solution
  • hydrophilic treatment for example, by hydrophilic coating
  • the surface of the fine particles be negatively charged.
  • the fine particles are made to exhibit hydrophilicity by performing a treatment that places carboxy groups on the surface of the fine particles.
  • the condition required from the viewpoint of the number of particles is that the cross-sectional area of microparticles x the number of particles injected is equal to or less than the bottom area of the through-hole well: (Particle cross-sectional area) x (Number of particles to be injected) ⁇ (Bottom area of through-hole well)... (Formula 1) It is preferable to satisfy the following. Note that it is not necessarily necessary to cover the entire bottom area of the through-hole well with the particles. In one example, it has been confirmed that the effect of air bubbles is reduced by using a condition in which about 10% of the bottom area of the well is covered with fine particles.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 3 will be described below with reference to FIG.
  • a transparent heat conductive plate 500 is used at the bottom of the measurement device to make it difficult for bubbles 501 to be observed.
  • fluorescence intensity measurement can be performed without being affected by the bubbles 501.
  • descriptions of parts common to Embodiment 1 or 2 may be omitted.
  • FIG. 7 is a schematic diagram of fluorescence measurement when the lower part of the measurement device is made of a transparent heat conduction plate 500.
  • Excitation light 502 passes through through-hole well 503 and reaches transparent thermally conductive plate 500 . At this time, the excitation light 502 becomes light 504 that passes through the transparent heat conduction plate 500. This makes it possible to suppress reflection of excitation light.
  • the measurement light intensity is the sum of the fluorescence intensity 505 and the attenuated reflected light intensity 506. This makes it difficult for bubbles 501 to be observed in the measurement image.
  • the material of the transparent heat conductive plate 500 include plastic such as polycarbonate, glass, and the like. Further, the transparent heat conductive plate 500 may be a transparent conductive substrate made of a glass substrate doped with indium tin oxide or the like.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 4 will be described below using FIG. 8.
  • bubbles 601 are made difficult to be observed by using a heat conductive plate 600 having an uneven structure.
  • the measurement device By using the measurement device according to this embodiment, it is possible to measure fluorescence intensity without being affected by the bubbles 601.
  • descriptions of parts common to Examples 1 to 3 may be omitted.
  • FIG. 8 is a schematic diagram of fluorescence measurement when the lower part of the measurement device is a heat conduction plate 600 with an uneven structure.
  • the reflection suppressing mechanism according to this embodiment has a concave-convex structure, and the concave-convex structure is arranged on the heat conductive plate 600 having the concave-convex structure.
  • the excitation light 602 passes through the through-hole well 603 and reaches the heat conductive plate 600 having an uneven structure. At this time, the reflected light 604 of the excitation light can be suppressed by the uneven structure. Therefore, the measurement light intensity is the sum of the fluorescence intensity 605 and the attenuated reflected light intensity 606. This makes it difficult for bubbles 601 to be observed in the measurement image.
  • the structure be periodic, and it is more preferable that the pitch (spatial repetition period) of the uneven structure be 1 ⁇ m or less.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 5 will be described below with reference to FIG.
  • the oil covering the through holes is colored to make it difficult to observe the bubbles 700.
  • fluorescence intensity measurement can be performed without being affected by the bubbles 700.
  • descriptions of parts common to Examples 1 to 4 may be omitted.
  • FIG. 9 is a schematic diagram of fluorescence measurement when the through-hole well 702 is covered with colored oil 701 in the measurement device.
  • the oil 701 is colored black, but the colored oil 701 is not necessarily colored, and the color is not limited to black.
  • the excitation light 703 is absorbed by the colored oil 701. Therefore, it is possible to suppress the reflected light 705 by the heat conductive plate 704. This makes it difficult for the bubbles 700 to be observed in the measurement image.
  • What is important in oil coloring is the reflectance of the coloring pigment.
  • "colored" means that the reflectance is 10% or less over a wavelength of 400 nm to 700 nm, for example. With such a reflectance, reflection of excitation light can be efficiently suppressed.
  • the oil 701 may actually exist above the through-hole well 702, and in that case, the fluorescence may also be blocked.
  • the reflectance of the excitation light and fluorescence in the oil 701 is 10% and the transmittance is 10%
  • 10% of the total amount of excitation light is 100%
  • enters the through-hole well 702 1% is reflected at the lower end of the through-hole well 702, and 0.1% is transmitted to the upper side of the through-hole well 702 and detected.
  • the total amount of fluorescence is 100%, 10% is transmitted to the upper side of the through-hole well 702 and detected. In this way, above the through-hole well 702, the excitation light is negligible compared to the fluorescence, and it can be said that reflection of the excitation light is effectively suppressed.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 6 will be described below with reference to FIG.
  • ink 801 is introduced into the lower part of through-hole well 800, thereby making it difficult for bubbles 802 to be observed.
  • the measurement device according to this embodiment it is possible to measure fluorescence intensity without being affected by the bubbles 802.
  • descriptions of parts common to Examples 1 to 5 may be omitted.
  • FIG. 10 is a schematic diagram of fluorescence measurement when ink 801 is injected into the lower part of through-hole well 800.
  • the reflection suppression mechanism includes ink 801 introduced into a through-hole well 800. After the excitation light 803 passes through the through-hole well 800, it is absorbed by the ink 801. This makes it difficult for bubbles 802 to be observed in the measurement image.
  • the ink 801 is preferably a pigment ink.
  • the specific gravity is larger than that of a mixed solution of a nucleic acid and a fluorescently labeled probe. That is, it is preferable that the ink 801 has a higher specific gravity than the mixed solution 805. By satisfying this condition, it is possible to cause the ink to settle in the lower part of the through-hole well.
  • the specific gravity of the ink 801 is preferably 1 g/cm 3 or more.
  • carbon black (specific gravity: 1.7 to 1.8 g/cm 3 ) or the like may be used as the pigment ink.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 7 will be described below with reference to FIGS. 11 and 12.
  • Example 7 shows the effect of using the measurement device according to Example 1, in which bubbles are actually no longer observed.
  • the measurement device is heated for a certain period of time to maintain it at 85°C, and the measurement device is irradiated with excitation light in the visible range to obtain a fluorescence image.
  • the exposure time when acquiring a fluorescence image is 1300 ms, and 80 or more fluorescence images are continuously photographed to evaluate the presence or absence of the influence of air bubbles.
  • FIG. 11 shows a fluorescence intensity change 901 of a single well 900 in the measurement device shown in FIG. 1 without a reflection suppression mechanism.
  • a portion of the fluorescence image of the through-hole well is shown in the upper right corner of the graph (for visibility, only the single well 900 has been corrected to white).
  • the horizontal axis represents the number of fluorescence images
  • the vertical axis represents the fluorescence intensity.
  • the image number represents the order in which the images were acquired, so the horizontal axis corresponds to time.
  • the data in the graph is the fluorescence intensity change of a single well 900 in each fluorescence image.
  • the fluorescence intensity does not always show a constant value, and a steep fluorescence intensity change 902 occurs.
  • this steep fluorescence intensity change 902 occurs, air bubbles are present at the bottom of the well. Therefore, the presence of bubbles can be confirmed from the steep fluorescence intensity change 902.
  • FIG. 12 shows a fluorescence intensity change 1000 of a single well in the measurement device according to Example 1 which is provided with a reflection suppression mechanism. It can be seen that, compared to the case in FIG. 11, no steep fluorescence intensity change was observed. Therefore, by using Example 1, it is possible to suppress the influence of bubbles. It is thought that the measurement devices according to Examples 2 to 6 have similar effects.
  • the measurement device According to the measurement device according to the present example, it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of air bubbles in melting curve analysis of a target gene.
  • Example 8 will be described below with reference to FIGS. 13 to 15.
  • Example 8 shows a measurement method for melting curve analysis using the measurement devices of Examples 1 to 6. By using the measurement device according to this embodiment, highly accurate measurement of melting curve analysis is possible.
  • FIG. 13 shows a measurement flow from injecting a mixed solution of nucleic acids and fluorescently labeled probes into the measurement device according to this example to performing melting curve analysis.
  • a mixed solution of nucleic acids and fluorescently labeled probes is introduced into the measurement device and fractionated into each of the through-hole wells, thereby fractionating the nucleic acids into the through-hole wells (S1100).
  • oil is introduced so as to cover the periphery of the through-hole well (S1101).
  • the through-hole well 203 into which the mixed solution 208 has been introduced is blocked on the first surface 209a of the substrate 209 and the second surface 209b of the substrate 209 on the opposite side to the first surface 209a. Introduce covering oil.
  • PCR is performed to amplify the nucleic acid in the through-hole well (S1102).
  • the temperature of the measurement device is changed by the heat conduction plate 200.
  • excitation light is irradiated while changing the temperature of the measurement device, and the fluorescence intensity from the mixed solution of nucleic acid and fluorescently labeled probe in the through-hole well is detected (S1103).
  • excitation light 207 is irradiated from the first surface 209a side of the substrate 209, fluorescence is detected from the first surface 209a side, and the mixed solution 208 is thereby measured.
  • FIG. 14 shows an example of melting curve analysis results for a conventional measurement device without a reflection suppression mechanism.
  • FIG. 14(a) is a graph plotting changes in fluorescence intensity against changes in temperature in a single well within the measurement device. A decrease in fluorescence intensity can be confirmed. If bubbles are observed during this decrease, a steep decrease in fluorescence intensity 1200 is observed.
  • FIG. 14(b) shows the temperature differential of the fluorescence intensity change in FIG. 14(a), and the differential curve of the melting curve is calculated.
  • the melting temperature is calculated from the peak of the differential curve.
  • a melting temperature artifact 1202 is observed in the differential curve where bubbles are observed. This artifact is caused by a steep decrease in fluorescence intensity 1200. Therefore, the effect of air bubbles greatly affects the melting curve analysis.
  • FIG. 15 shows an example of the melting curve analysis results in this example.
  • FIG. 15(a) is a plot of fluorescence intensity changes with respect to temperature changes in a single well within the measurement device. A monotonous decrease in fluorescence intensity can be confirmed.
  • FIG. 15(b) shows the temperature differential of the fluorescence intensity change in FIG. 15(a). Since bubbles are difficult to observe in the measurement device of this example, it is difficult to observe a steep decrease in fluorescence intensity in the melting curve. Therefore, only the true melting temperature 1300 is observed in the differential curve of the melting curve. Therefore, the measurement device according to this embodiment can suppress unnecessary artifacts and enable highly accurate melting curve analysis.
  • the measurement device and measurement method according to the present example it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of bubbles in melting curve analysis of a target gene.
  • Example 9 will be described below with reference to FIGS. 16 and 17.
  • Example 9 shows a method for confirming that the black surface treatment of the thermally conductive plate of Example 1 and the injection of fine particles into the through-hole wells of Example 2 have the effect of making it difficult to observe bubbles. be.
  • FIG. 16 shows a measurement flow in which the reflection image measurement operation is added to the measurement flow diagram shown in FIG. 13 of Example 8.
  • S1400 to S1402 and S1404 in FIG. 16 can be the same as S1100 to S1102 and S1103 in FIG. 13, respectively.
  • the reflected image of the measurement device is measured (S1403).
  • the excitation light 207 or white light is irradiated from the first surface 209a side of the substrate 209, and the reflected image is measured.
  • the reflection image measurement operation is performed after PCR (S1402), but it may also be performed after the oil introduction step (S1401) to cover the through hole.
  • S1403 can be executed after S1401 and before the end of S1404.
  • FIG. 17 shows reflected images actually observed in each measurement device. An image focused on the thermally conductive plate is obtained as a reflected image.
  • the variation coefficient of the reflected light intensity is 10%.
  • the coefficient of variation is a value obtained by dividing the standard deviation of the reflected light intensity by the average of the reflected light intensity for a plurality of through-hole wells.
  • the measurement device and measurement method according to the present example it is possible to suppress unevenness in fluorescence images and fluctuations in fluorescence intensity caused by the influence of bubbles in melting curve analysis of a target gene.
  • the present invention is not limited to the above-described embodiments, and includes various modifications.
  • the embodiments described above are described in detail to explain the present invention in an easy-to-understand manner, and the present invention is not necessarily limited to having all the configurations described.
  • it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment and it is also possible to add the configuration of another embodiment to the configuration of one embodiment.
  • Reflected light 605 Fluorescence intensity 606... Attenuated reflected light intensity 700... Bubbles 701... Colored oil (reflection suppression mechanism) 702...Through hole well (through hole) 703...Excitation light 704...Heat conduction plate 705...Reflected light 800...Through hole well (through hole) 801...Ink (reflection suppression mechanism) 802...Bubbles 803...Excitation light 805...Mixed solution (nucleic acid solution) 900...Single well (through hole) 901... Fluorescence intensity change 902... Steep fluorescence intensity change 1000... Fluorescence intensity change 1200... Steep fluorescence intensity decrease 1201... True melting temperature 1202... Melting temperature artifact 1300... True melting temperature

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WO2016072416A1 (ja) * 2014-11-04 2016-05-12 凸版印刷株式会社 核酸導入方法、核酸検出方法、生体成分解析方法、生体成分定量用アレイデバイス、及び生体成分解析キット
WO2019239805A1 (ja) * 2018-06-14 2019-12-19 株式会社日立製作所 デジタルｐｃｒの測定方法および測定装置
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WO2016072416A1 (ja) * 2014-11-04 2016-05-12 凸版印刷株式会社 核酸導入方法、核酸検出方法、生体成分解析方法、生体成分定量用アレイデバイス、及び生体成分解析キット
WO2019239805A1 (ja) * 2018-06-14 2019-12-19 株式会社日立製作所 デジタルｐｃｒの測定方法および測定装置
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