Measuring Devices and Methods

Abstract

This measuring device comprises: a substrate having a plurality of through holes for introducing and dividing a nucleic acid solution; an oil that covers the through holes to block the through holes at a first surface of the substrate and a second surface opposite to the first surface of the substrate; a heat conduction plate provided on the second surface side for heating the substrate; and a reflection suppressing mechanism that suppresses the reflection of the excitation light radiated from the first surface side.

Description

[Technical Field]

[0001] The present invention relates to a measurement device and a measurement method, and in particular to digital PCR. [Background technology]

[0002] Traditionally, PCR and real-time PCR have been used for genetic testing. These technologies have had the problem of low measurement accuracy when the target (nucleic acid) to be measured is in trace amounts. In recent years, digital PCR technology has been attracting attention as a solution to this problem.

[0003] In digital PCR, a sample containing the target DNA is divided into many microscopic regions, and PCR is performed on each microscopic region. The type of DNA present in each microscopic region can be determined by distinguishing between regions that contain the target DNA and those that do not based on fluorescence intensity.

[0004] 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 DNA and a fluorescently labeled probe that hybridizes to the DNA. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2018-108063 Summary of the Invention [Problem to be solved by the invention]

[0006] In digital PCR combined with melting curve analysis, there are multiple types of genes to be measured, including wild-type and mutant genes. Because melting curves differ depending on the type of target gene and the fluorescently labeled probe, it is possible to distinguish the type of target gene by measuring the melting curve. When measuring multiple genes simultaneously, it is necessary to measure fluorescence intensity and melting curves with high precision and minimize measurement variability for each gene.

[0007] One of the factors that reduces the accuracy of measuring fluorescence intensity and melting curves is the presence of air bubbles. If air bubbles form near a microscopic region when the temperature rises, accurate measurement of fluorescence intensity becomes impossible, reducing the accuracy of melting curve measurement and the accuracy of gene type discrimination.

[0008] The problem with these bubbles is different from that of electrical noise, and it is difficult to remove them by signal processing or the like.

[0009] An object of the present invention is to provide a measurement device that can suppress unevenness in fluorescent images and fluctuations in fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene. The above and other objects and novel features of the present invention will become apparent from the description and accompanying drawings of this specification. [Means for solving the problem]

[0010] An example of a measuring device according to the present invention is a substrate having a plurality of through-holes for introducing and dividing a nucleic acid solution; oil covering the through holes on a first surface of the substrate and on a second surface of the substrate opposite to the first surface to seal the through holes; 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; The present invention is characterized by having the following.

[0011] The measurement method according to the present invention comprises: A measurement method using a measurement device, comprising: The measuring device is a substrate having a plurality of through holes; a thermally conductive plate for varying 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 thermal conduction plate side; and The measurement method includes: introducing a nucleic acid solution onto the substrate and fractionating it; introducing oil onto a first surface of the substrate and a second surface of the substrate opposite to the first surface to cover the through-holes into which the nucleic acid solution has been introduced; changing the temperature of the substrate by the thermally conductive plate; irradiating the substrate with excitation light from the first surface side while changing the temperature, detecting fluorescence from the first surface side, and measuring the nucleic acid solution; It has. [Effects of the Invention]

[0012] According to 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 accuracy, and identify the type of gene with high accuracy. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 10 is a diagram illustrating the intensity measured when a bubble is generated below a through-hole well in a measurement device in a conventional example. [Figure 2] 10 is a reflection image illustrating that, in the conventional example, reflected light from the heat conduction plate below the measurement device is observed through the through-hole well. [Figure 3] FIG. 10 is a diagram illustrating that the influence of air bubbles on the measured intensity is eliminated by subjecting the heat conduction plate at the bottom of the measurement device to black surface treatment in Example 1 of the present invention. [Figure 4]10 is a reflection image illustrating that, in Example 1 of the present invention, the heat conduction plate below the measurement device is blackened, so that reflected light from the heat conduction plate is not observed through the through-hole wells. [Figure 5] FIG. 10 is a diagram illustrating that the measured intensity is not affected by bubbles by introducing particles into the lower part of the through-hole well in Example 2 of the present invention. [Figure 6] FIG. 10 is a diagram illustrating that the average reflected light intensity in the reflection image of the through-hole well is reduced by introducing particles into the lower part of the through-hole well in Example 2 of the present invention. [Figure 7] FIG. 10 is a diagram for explaining that the influence of air bubbles on the measured intensity is eliminated by making the heat conduction plate at the bottom of the measurement device transparent in Example 3 of the present invention. [Figure 8] FIG. 10 is a diagram for explaining that the influence of air bubbles on the measured intensity is prevented by providing a heat conduction plate at the bottom of the measurement device with an uneven structure in Example 4 of the present invention. [Figure 9] FIG. 10 is a diagram illustrating that the influence of air bubbles on the measured intensity is eliminated by coloring the oil in the measurement device black in Example 5 of the present invention. [Figure 10] FIG. 20 is a diagram illustrating that the measured intensity is not affected by air bubbles by introducing ink into the lower part of the through-hole well in Example 6 of the present invention. [Figure 11] FIG. 10 is a graph illustrating the change 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. [Figure 12] FIG. 10 is a diagram illustrating the change in fluorescence intensity observed when a measurement device having a reflection suppression mechanism 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. [Figure 13] FIG. 13 is a flow diagram of measuring fluorescence intensity with respect to temperature change in a measurement device incorporating a reflection suppression mechanism in Example 8 of the present invention. [Figure 14] 10A and 10B are diagrams 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. [Figure 15]FIG. 20 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. [Figure 16] FIG. 13 is a flow diagram of fluorescence intensity measurement and reflected image measurement in response to temperature changes in a measurement device incorporating a reflection suppression mechanism in Example 9 of the present invention. [Figure 17] 13A and 13B are diagrams illustrating changes in the coefficient of variation of reflected light intensity in a measurement device before and after the introduction of a reflection suppression mechanism in Example 9 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] A conventional example will be described with reference to FIGS. 1 and 2. FIG. Figure 1 is a schematic diagram of fluorescence measurement using a conventional measurement device holding through-hole wells 1. This measurement device is composed of a substrate 2 holding multiple through-hole wells 1, a mixed solution 3 of nucleic acid and fluorescently labeled probes injected into the wells, oil 4 covering the through-hole wells 1, and a heat-conducting plate 5.

[0015] In fluorescence measurement using a measurement device, excitation light 6 is irradiated onto through-hole well 1. A portion of excitation light 6 passes through through-hole well 1 as transmitted light 7. Transmitted light 7 is reflected by thermally conductive plate 5 and becomes reflected light 8. Reflected light 8 passes through through-hole well 1 again, and reflected light intensity 10, together with fluorescence intensity 9, is measured as the measured light intensity of the well.

[0016] If an air bubble 11 is present below the through-hole well 1, the transmitted light 7 through the through-hole well 1 is diffused by the air bubble 11 at the bottom of the through-hole well 1. Therefore, the light 12 that is not reflected by the thermal conduction plate 5 is not detected as strongly as the reflected light intensity 10. In other words, when an air bubble is present, the measured light intensity is the sum of the fluorescence intensity 9 and the reflected light intensity 13, which is attenuated from the original reflected light intensity 10.

[0017] From the above, it can be seen that the presence or absence of bubbles causes unevenness in the measured light intensity of the wells, and therefore, in the measurement image, the areas where bubbles exist are observed as dark measured intensities.

[0018] FIG. 2 shows reflection images of a substrate 101 holding a through-hole well 100, observed at low magnification (FIG. 2(a)) and high magnification (FIG. 2(b)). Focusing on the reflection image of the through-hole well 100, it is bright. Therefore, the excitation light 6 passes through the through-hole well 100 and is reflected by the thermally conductive plate 5. Furthermore, when comparing the through-hole well 100 and the through-hole well 102, there is a variation in brightness. This is due to the in-plane variation in the reflectivity of the thermally conductive plate 5.

[0019] As explained above, the presence of air bubbles 11 in a conventional measurement chip affects measurement. Below, we will explain how the air bubbles 11 do not affect measurement by applying a colored surface treatment to the heat conduction plate.

[0020] Hereinafter, an embodiment of the present invention will be described, and explanations of parts common to the above-mentioned conventional example may be omitted. [Example 1] Example 1 will be described below with reference to Figures 3 and 4. In Example 1, the heat conduction plate is blackened to make bubbles less observable. By using the measurement device according to this example, it is possible to measure the fluorescence intensity without being affected by bubbles.

[0021] 3 is a schematic diagram illustrating how the application of a black surface treatment film 201 to a heat conduction plate 200 at the bottom of the measurement device makes it difficult to observe the bubbles 202 observed in FIG. 1. 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 oil 210 covering the through-hole wells 203 on a first surface 209a of the substrate 209 and on a second surface 209b opposite the first surface of the substrate.

[0022] The mixed solution 208 comprises a nucleic acid solution. In the present specification and drawings, references to a mixed solution can be construed as references to a nucleic acid solution.

[0023] The measurement device has a heat conduction 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 heat conduction plate 200 side.

[0024] The material of the heat conduction plate 200 is, for example, metal or resin, but it may also be glass. By using such a material, it is possible to configure a heat conduction plate that meets the requirements of the measurement device. However, other materials may also be used.

[0025] In conventional configurations, the intensity of reflected light varies depending on the presence or absence of bubbles 202 below through-hole wells 203. However, the measurement device according to this embodiment has a reflection suppression mechanism that suppresses reflection of excitation light 207 irradiated from the first surface 209a side (e.g., reflection toward the first surface 209a side). In this embodiment, the reflection suppression mechanism is a colored surface treatment structure of thermal conduction plate 200.

[0026] As a specific example, the reflection suppression mechanism includes a black surface treatment film 201 as an anti-reflection film. The surface treatment film 201 is disposed on the heat conduction plate 200, and the surface treatment film 201 absorbs the excitation light (or light having the same wavelength as the excitation light). By providing the heat conduction plate 200 with the black surface treatment film 201, the reflected light 204 from the heat conduction plate 200 generates an attenuated reflected light intensity 205. Therefore, the measured light intensity of each through-hole well 203 is the sum of the fluorescent light intensity 206 and the attenuated reflected light intensity 205. This prevents air bubbles from being observed as unevenness on the measurement image.

[0027] The structure and method of forming the surface treatment film 201 can be designed as appropriate by those skilled in the art, and the thickness varies depending on the surface treatment process. To give some examples, an anodic oxide coating such as anodizing is about 5 to 40 μm thick, and a film treated by electroplating is about 2 to 20 μm thick. Furthermore, a film formed by spray painting is about 15 to 30 μm thick. In this embodiment, the film thickness is preferably 40 μm or less.

[0028] FIG. 4 shows reflection images of a substrate 300 after a black surface treatment film 201 has been applied to a thermally conductive plate, observed at low magnification (FIG. 4(a)) and high magnification (FIG. 4(b)). Focusing on the reflection image within through-hole well 301, it can be seen that it is dark. Furthermore, a comparison of the reflected light intensity between through-hole well 301 and through-hole well 302 reveals that there is no variation in the reflected light intensity, and that the reflected light intensity is generally low. This reflection image indicates that reflection from the thermally conductive plate 200 is suppressed.

[0029] In this embodiment, a black surface treatment film 201 is used as the colored surface treatment structure, and its reflectance is important for suppressing reflection from the heat conduction plate 200. In this embodiment, "colored" means that the reflectance is 10% or less over a wavelength range of 400 nm to 700 nm, specifically black. Such a colored surface treatment structure suppresses reflection from the heat conduction plate 200.

[0030] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0031] [Example 2] Example 2 will be described below with reference to Figures 5 and 6. In Example 2, microparticles 401 are injected into the lower part of through-hole well 400, making bubbles 402 less observable. By using the measurement device according to this example, it becomes possible to measure fluorescence intensity without being affected by bubbles 402. Hereinafter, explanations of parts common to Example 1 may be omitted.

[0032] 5 is a schematic diagram showing fluorescence measurement when microparticles 401 are introduced below 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 microparticles 401 introduced into through-hole well 400. It is preferable that microparticles 401 do not transmit excitation light 404.

[0033] If the particles 401 were not present, the same phenomenon as in Figure 1 would occur. However, the presence of the particles 401 prevents the excitation light 404 from passing through the well, and the reflected light 405 is suppressed by the heat conduction plate. Therefore, the measured light intensity is the sum of the fluorescence intensity 406 and the attenuated reflected light intensity 407. This makes it difficult to observe the bubbles 402 in the measurement image.

[0034] Figure 6 shows a comparison of the reflected images of the measurement devices shown in Figure 1 and Figure 5. Figure 6(a) is a reflected image obtained by observing a conventional measurement device, and Figure 6(b) is a reflected image obtained by observing the measurement device according to this embodiment into which particles have been injected.

[0035] In the example of Figure 6(a), the average reflected light intensity averaged over 50 through-hole wells was 133 (arbitrary units), and in the example of Figure 6(b), the average reflected light intensity was 99. Focusing on the average reflected light intensity of multiple through-hole wells, the average reflected light intensity of multiple wells was reduced by injecting particles into the through-hole wells. Therefore, the excitation light is prevented from passing through the through-hole wells and reflecting on the thermal conduction plate.

[0036] The conditions for the injected microparticles include size, specific gravity, surface treatment, and number. The size (e.g., diameter) is preferably 30 nm or more. When microparticles scatter light in the visible to near-infrared range, the scattering phenomenon is based on Mie scattering theory. Since Mie scattering shows significant scattering intensity from about 30 nm, this value can be used as the lower limit. On the other hand, it is preferable that the size of the microparticles be smaller than the through-hole well into which the microparticles are injected. Therefore, when the well size is 60 μm, the upper limit of the microparticle size can be set to 60 μm.

[0037] In terms of specific gravity, it is preferable that the microparticles settle to the bottom of the through-hole well. Therefore, it is preferable that the specific gravity of the microparticles is higher than that of the mixed solution of nucleic acid and fluorescently labeled probe injected into the through-hole well. In other words, it is preferable that the specific gravity of the microparticles is higher than that of the mixed solution.

[0038] As an example, polymer particles (polystyrene, specific gravity: 1.04 to 1.07 g / cm) are used. 3 ), magnetic fine particles (iron, specific gravity: 7.85 g / cm 3 ), metal fine particles (silver, specific gravity: 10.49g / cm 3 ) or other particles with a specific gravity greater than 1 (i.e., the specific gravity of the particles is 1 g / cm 3 That's it.) The microparticles then settle within the through-hole wells.

[0039] In terms of surface treatment of microparticles, it is useful to consider the affinity of the microparticles with the mixed solution of nucleic acid and fluorescently labeled probe. Because the mixed solution of nucleic acid and fluorescently labeled probe is an aqueous solution, it is preferable to treat the microparticles with hydrophilicity (e.g., hydrophilic coating). Furthermore, to prevent the nucleic acid from adsorbing to the microparticles, it is preferable to negatively charge the microparticle surfaces.

[0040] In one example, the particles are made hydrophilic by being treated to place carboxyl groups on their surfaces. In another example, a magnet is placed at the bottom of the measurement device, and the hydrophilic magnetic particles can be sedimented to the bottom of the through-hole wells by magnetic force. Because the through-hole wells are surrounded by oil, the hydrophilic magnetic particles remain at the bottom of the through-hole wells. Therefore, using this method, it is possible to control the position of the magnetic particles.

[0041] The condition required from the viewpoint of number is that the cross-sectional area of ​​the microparticles multiplied by the number of injected particles must be equal to or less than the bottom area of ​​the through-hole well: (Cross-sectional area of ​​particle) × (number of particles to be injected) ≦ (bottom area of ​​through-hole well) (Formula 1) It is preferable to satisfy the following condition. Note that it is not necessary to cover the entire bottom area of ​​the through-hole well with the particles. In one example, it was confirmed that the effect of air bubbles was reduced by using the condition that about 10% of the bottom area of ​​the well is covered with the particles.

[0042] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0043] [Example 3] Example 3 will be described below with reference to Fig. 7. In Example 3, a transparent heat conduction plate 500 is used as a reflection suppression mechanism at the bottom of the measurement device, making it difficult to observe bubbles 501. By using the measurement device according to this example, it becomes possible to measure fluorescence intensity without being affected by bubbles 501. Hereinafter, explanations of parts common to Examples 1 and 2 may be omitted.

[0044] 7 is a schematic diagram of fluorescence measurement when the lower part of the measurement device is a transparent heat conduction plate 500. Excitation light 502 passes through through-hole wells 503 and reaches the transparent heat conduction plate 500. At this time, the excitation light 502 becomes light 504 that passes through the heat conduction plate 500 due to the transparent heat conduction plate 500. This makes it possible to suppress reflection of the excitation light.

[0045] Therefore, the measured light intensity is the sum of the fluorescent light intensity 505 and the attenuated reflected light intensity 506. This makes it difficult to observe the air bubbles 501 in the measurement image. Examples of materials for the transparent heat-conducting plate 500 include plastics such as polycarbonate, glass, etc. Alternatively, the transparent heat-conducting plate 500 may be a transparent conductive substrate formed by doping a glass substrate with indium tin oxide or the like.

[0046] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0047] [Example 4] Example 4 will be described below with reference to Fig. 8. Example 4 uses a heat conduction plate 600 with an uneven structure to make bubbles 601 less observable. By using the measurement device according to this example, it becomes possible to measure the fluorescence intensity without being affected by bubbles 601. Hereinafter, explanations of parts common to Examples 1 to 3 may be omitted.

[0048] 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. As described above, the reflection suppression mechanism according to this embodiment has an uneven structure, and the uneven structure is placed on the heat conduction plate 600 with an uneven structure.

[0049] Excitation light 602 passes through through-hole wells 603 and reaches a thermally conductive plate 600 with a concave-convex structure. At this time, the concave-convex structure can suppress reflected light 604 of the excitation light. Therefore, the measured light intensity is the sum of the fluorescent light intensity 605 and the attenuated reflected light intensity 606. This makes it difficult to observe bubbles 601 in the measurement image. In order to reduce reflection by the concave-convex structure, it is preferable that the structure be periodic, and it is even more preferable that the pitch (spatial repeat period) of the concave-convex structure be 1 μm or less.

[0050] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0051] [Example 5] Example 5 will be described below with reference to Fig. 9. In Example 5, the oil covering the through-hole is colored to make the bubbles 700 less observable. By using the measurement device according to this example, it becomes possible to measure the fluorescence intensity without being affected by the bubbles 700. Hereinafter, explanations of parts common to Examples 1 to 4 may be omitted.

[0052] 9 is a schematic diagram showing fluorescence measurement when through-hole wells 702 are covered with colored oil 701 in a measurement device. In this example, oil 701 is colored black, but colored oil 701 is not necessarily limited to oil that has been colored, and the color is not limited to black.

[0053] The excitation light 703 is absorbed by the colored oil 701. Therefore, it is possible to suppress the reflected light 705 from the heat conduction plate 704. This makes it difficult to observe the bubbles 700 in the measurement image. What is important in coloring the oil is the reflectance of the coloring dye. In this embodiment, "colored" means that the reflectance is 10% or less over the wavelength range of 400 nm to 700 nm, for example. With such a reflectance, the reflection of the excitation light can be efficiently suppressed.

[0054] In reality, oil 701 may also be present above through-hole well 702, in which case it may also block fluorescence. Here, assuming that the reflectance of the excitation light and fluorescence in oil 701 is 10% and the transmittance is 10%, 10% of the total amount of excitation light (100%) enters through-hole well 702, 1% is reflected at the lower end of through-hole well 702, and 0.1% is transmitted to the upper side of through-hole well 702 and detected. On the other hand, of the total amount of fluorescence (100%), 10% is transmitted to the upper side of through-hole well 702 and detected. In this way, the excitation light becomes negligible compared to the fluorescence above through-hole well 702, and it can be said that reflection of the excitation light is efficiently suppressed.

[0055] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0056] [Example 6] Example 6 will be described below with reference to Figure 10. In Example 6, bubbles 802 are made less observable by introducing ink 801 into the lower part of a through-hole well 800. By using the measurement device according to this example, it becomes possible to measure fluorescence intensity without being affected by bubbles 802. Hereinafter, explanations of parts common to Examples 1 to 5 may be omitted.

[0057] 10 is a schematic diagram of fluorescence measurement when ink 801 is injected into the bottom of through-hole well 800. In this example, the reflection suppression mechanism includes ink 801 introduced into through-hole well 800. After transmitting through through-hole well 800, excitation light 803 is absorbed by ink 801. This makes it difficult to observe bubbles 802 in the measurement image.

[0058] In this embodiment, the ink 801 is preferably a pigment ink. In terms of specific gravity, the pigment ink preferably has a specific gravity greater than that of the mixed solution of nucleic acid and fluorescently labeled probe. In other words, it is preferable that the ink 801 has a specific gravity greater than that of the mixed solution 805. By satisfying this condition, it is possible to cause the ink to settle at the bottom of the through-hole well. As a specific example, the specific gravity of the ink 801 is 1 g / cm 3 It is preferable that the ink density is equal to or higher than 1.7 g / cm. In addition, carbon black (specific gravity: 1.7 to 1.8 g / cm) is used as the pigment ink. 3 ) etc. may also be used.

[0059] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0060] [Example 7] Example 7 will be described below with reference to Figures 11 and 12. Example 7 shows the effect that by using the measuring device according to Example 1, bubbles are actually no longer observed.

[0061] In this measurement, the measurement device is heated to 85°C for a certain period of time, and then visible excitation light is irradiated onto the measurement device to acquire a fluorescent image. The exposure time for acquiring the fluorescent image is 1300 ms, and 80 or more fluorescent images are taken continuously to evaluate the presence or absence of the influence of air bubbles.

[0062] As a comparative example, Figure 11 shows the change in fluorescence intensity 901 of a single well 900 in the measurement device shown in Figure 1, which does not have a reflection suppression mechanism. Also, a portion of the fluorescence image of the through-hole well is shown in the upper right corner of the graph (only the single well 900 has been corrected to white for visibility). In the graph, the horizontal axis represents the number of fluorescence images, and the vertical axis represents fluorescence intensity. The image number indicates the order in which the images were acquired, and therefore the horizontal axis corresponds to time.

[0063] The data in the graph is the change in fluorescence intensity of a single well 900 in each fluorescence image. In the graph, the fluorescence intensity does not always show a constant value, and a steep change in fluorescence intensity 902 occurs. When this steep change in fluorescence intensity 902 occurs, an air bubble is present at the bottom of the well. Therefore, the presence of an air bubble can be confirmed from the steep change in fluorescence intensity 902.

[0064] Fig. 12 shows the change in fluorescence intensity 1000 of a single well in the measurement device according to Example 1, which is equipped with a reflection suppression mechanism. Compared to the case of Fig. 11, it can be seen that no steep change in fluorescence intensity is observed. Therefore, by using Example 1, it is possible to suppress the influence of bubbles. It is believed that the same effect can be obtained with the measurement devices according to Examples 2 to 6.

[0065] As described above, the measurement device according to this embodiment makes it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0066] [Example 8] Example 8 will be described below with reference to Figures 13 to 15. Example 8 illustrates a method for melting curve analysis using the measurement devices of Examples 1 to 6. Use of the measurement device according to this example enables highly accurate measurement of melting curve analysis.

[0067] FIG. 13 shows the measurement flow from injecting a mixed solution of nucleic acid and fluorescently labeled probe into the measurement device according to this embodiment to performing melting curve analysis.

[0068] First, a mixed solution of nucleic acid and fluorescently labeled probe is introduced into the measurement device, and the solution is fractionated into each of the through-hole wells, thereby fractionating the nucleic acid into the through-hole wells (S1100).

[0069] Next, oil is introduced to cover the periphery of the through-hole well (S1101). In the example of Fig. 3, oil is introduced to cover the first surface 209a of the substrate 209 and the second surface 209b opposite to the first surface 209a of the substrate 209 to block the through-hole well 203 into which the mixed solution 208 has been introduced.

[0070] After the oil is introduced, PCR is performed to amplify the nucleic acid in the through-hole well (S1102). In the example of Fig. 3, the temperature of the measurement device (particularly the substrate 209) is changed by the thermally conductive plate 200.

[0071] Finally, while changing the temperature of the measurement device, excitation light is irradiated and the fluorescence intensity from the mixed solution of nucleic acid and fluorescently labeled probe in the through-hole well is detected (S1103). In the example of Fig. 3, excitation light 207 is irradiated from the side of first surface 209a of substrate 209, and fluorescence is detected from the side of first surface 209a, thereby measuring mixed solution 208.

[0072] As a comparative example, Figure 14 shows an example of the melting curve analysis results for a conventional measurement device without a reflection suppression mechanism. Figure 14(a) is a graph plotting the change in fluorescence intensity versus temperature in a single well in the measurement device. A decrease in fluorescence intensity can be confirmed. If air bubbles are observed during this decrease, a steep decrease in fluorescence intensity of 1200 is observed.

[0073] Figure 14(b) shows the temperature derivative of the melting curve in Figure 14(a), and the melting temperature is calculated from the peak of the derivative curve. In the derivative curve where bubbles are observed, not only the true melting temperature 1201 but also a melting temperature artifact 1202 is observed. This artifact is caused by a steep decrease in fluorescence intensity 1200. Therefore, the influence of bubbles significantly affects the melting curve analysis.

[0074] Figure 15 shows an example of the melting curve analysis results in this example. Figure 15(a) is a plot of the change in fluorescence intensity versus temperature in a single well in the measurement device. A monotonic decrease in fluorescence intensity can be confirmed.

[0075] Figure 15(b) shows the temperature derivative of the fluorescence intensity change in Figure 15(a). Because bubbles are difficult to observe with the measurement device of this example, a steep decrease in fluorescence intensity is difficult to observe in the melting curve. Therefore, only the true melting temperature of 1300 is observed in the differential curve of the melting curve. Therefore, the measurement device of this example can suppress unnecessary artifacts, enabling highly accurate melting curve analysis.

[0076] As described above, the measurement device and measurement method according to this embodiment make it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0077] [Example 9] Example 9 will be described below with reference to Figures 16 and 17. Example 9 shows a method for confirming that the black surface treatment of the heat conduction plate in Example 1 and the injection of fine particles into the through-hole wells in Example 2 have the effect of making air bubbles less observable.

[0078] Fig. 16 shows a measurement flow in which operations for measuring a reflected image are added to the measurement flow diagram shown in Fig. 13 of Example 8. S1400 to S1402 and S1404 in Fig. 16 can be similar to S1100 to S1102 and S1103 in Fig. 13, respectively.

[0079] After S1402, the reflected image of the measurement device is measured (S1403). In the example of Fig. 3, excitation light 207 or white light is irradiated from the first surface 209a side of substrate 209, and the reflected image is measured.

[0080] The above is merely an example, and the operation of measuring the reflected image is performed after PCR (S1402), but it may also be performed after the oil introduction step (S1401) that covers the through-hole. The step of S1403 can be performed after S1401 and before the end of S1404.

[0081] Figure 17 shows the reflected images actually observed by each measurement device. As the reflected images, images focused on the heat-conducting plate are acquired.

[0082] In the case of a conventional measurement device (Figure 17(a)) that does not have a reflection suppression mechanism or other treatment, unevenness in the reflected light is observed. Furthermore, the coefficient of variation of the reflected light intensity is 10%. Here, the coefficient of variation is the value obtained by dividing the standard deviation of the reflected light intensity for multiple through-hole wells by the average reflected light intensity.

[0083] On the other hand, when reflection suppression mechanisms such as the injection of magnetic microparticles (Example 2, Figure 17(b)) or black surface treatment of the thermal conduction plate (Example 1, Figure 17(c)) are implemented, the coefficients of variation of the reflected light intensity are 3% and 4%, respectively.

[0084] These values ​​are merely examples. However, compared to conventional measurement devices, the coefficient of variation of reflected light intensity is reduced in the measurement devices according to the examples of the present invention. Therefore, the performance of the measurement devices according to the examples can be evaluated by evaluating the reflected image before performing melting curve analysis.

[0085] As described above, the measurement device and measurement method according to this embodiment make it possible to suppress unevenness in the fluorescent image and fluctuations in the fluorescent intensity caused by the influence of air bubbles in melting curve analysis of a target gene.

[0086] The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations. [Explanation of symbols]

[0087] 200...Heat conduction plate 201...Surface treatment film (reflection suppression mechanism, reflection suppression film) 202...bubbles 203...Through hole well (through hole) 204...Reflected light 205...Attenuated reflected light intensity 206...Fluorescence intensity 207...Excitation light 208…Mixed solution (nucleic acid solution) 209... Circuit board 209a...First side 209b...Second side 210...Oil 300...Substrate 301...Through hole well (through hole) 302...Through hole well (through hole) 400...Through hole well (through hole) 401...Fine particles (reflection suppression mechanism) 402...bubbles 403…Mixed solution (nucleic acid solution) 404...Excitation light 405...Reflected light 406...Fluorescence intensity 407...Attenuated reflected light intensity 500...Transparent heat conduction plate (anti-reflection mechanism) 501...bubbles 502...Excitation light 503...Through hole well (through hole) 504...Light passing through the heat conduction plate 505...Fluorescence intensity 506...Attenuated reflected light intensity 600...Concave and concave heat conduction plate (reflection suppression mechanism) 601...bubbles 602...Excitation light 603...Through hole well (through hole) 604…Reflected light 605...Fluorescence intensity 606...Attenuated reflected light intensity 700...bubbles 701...Colored oil (anti-reflection 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...Change in fluorescence intensity 902...Sudden change in fluorescence intensity 1000...Change in fluorescence intensity 1200: Steep decrease in fluorescence intensity 1201...True melting temperature 1202...Melting temperature artifact 1300...true melting temperature

Claims

1. A measurement device configured as a digital PCR device, a substrate having a plurality of through-holes for introducing and dividing a nucleic acid solution; oil covering the through-holes on a first surface of the substrate and a second surface of the substrate opposite to the first surface to seal the through-holes; 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; A measuring device comprising:

2. The measuring device according to claim 1 , wherein the anti-reflection mechanism comprises microparticles introduced into the through-hole, the microparticles being hydrophilically coated and having a specific gravity greater than that of the nucleic acid solution.

3. The specific gravity of the fine particles is 1 g / cm ３ The measuring device according to claim 2, characterized in that:

4. The measurement device according to claim 1 , wherein the anti-reflection mechanism comprises ink introduced into the through-hole, the ink having a specific gravity greater than that of the nucleic acid solution.

5. The specific gravity of the ink is 1 g / cm ３ The measuring device according to claim 4, characterized in that:

6. The measurement device according to claim 1 , wherein the reflection suppressing mechanism includes an anti-reflection film, the anti-reflection film being disposed on the thermally conductive plate, and the anti-reflection film absorbing the excitation light.

7. The measurement device according to claim 1 , wherein the reflection suppression mechanism comprises a concave-convex structure, the concave-convex structure is disposed on the thermal conduction plate, and the pitch of the concave-convex structure is 1 μm or less.

8. the anti-reflection mechanism is a colored surface treatment structure of the heat conduction plate; The term "colored" means that the reflectance is 10% or less over the wavelength range of 400 nm to 700 nm.

2. The measuring device according to claim 1, characterized in that it comprises:

9. 2. The measuring device according to claim 1, wherein the material of the heat-conducting plate is any one of metal, resin, and glass.

10. the oil is colored, The term "colored" means that the reflectance is 10% or less over the wavelength range of 400 nm to 700 nm.

2. The measuring device according to claim 1, characterized in that it comprises:

11. A measurement method using a measurement device configured as a digital PCR device, The measuring device is a substrate having a plurality of through holes; a thermally conductive plate for varying 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 thermal conduction plate side; and The measurement method includes: introducing a nucleic acid solution onto the substrate and fractionating it; introducing oil onto a first surface of the substrate and a second surface of the substrate opposite to the first surface to cover the through-holes into which the nucleic acid solution has been introduced, to seal the through-holes; changing the temperature of the substrate by the thermally conductive plate; irradiating the substrate with excitation light from the first surface side while changing the temperature, detecting fluorescence from the first surface side, and measuring the nucleic acid solution; A measurement method comprising:

12. 12. The measurement method according to claim 11, wherein the anti-reflection mechanism comprises microparticles, the microparticles are hydrophilically coated, and the microparticles have a specific gravity greater than that of the nucleic acid solution.

13. The specific gravity of the fine particles is 1 g / cm ３ The measuring method according to claim 12, characterized in that:

14. The measurement method according to claim 11 , wherein the reflection suppression mechanism comprises ink introduced into the through-hole, the ink having a specific gravity greater than that of the nucleic acid solution.

15. The specific gravity of the ink is 1 g / cm ３ The measuring method according to claim 14, wherein the above is true.

16. The measurement method according to any one of claims 12 to 15, further comprising the step of irradiating the substrate with the excitation light or white light from the side of the first surface and measuring a reflected image after the step of introducing the oil and before the step of measuring the nucleic acid solution is completed.

17. 12. The measurement method according to claim 11, wherein the reflection suppressing mechanism includes an anti-reflection film, the anti-reflection film being disposed on the thermally conductive plate, and the anti-reflection film absorbing the excitation light.

18. 12. The measurement method according to claim 11, wherein the anti-reflection mechanism comprises a concave-convex structure, the concave-convex structure is disposed on the thermal conduction plate, and the pitch of the concave-convex structure is 1 μm or less.

19. the anti-reflection mechanism is a colored surface treatment structure of the heat conduction plate; The term "colored" means that the reflectance is 10% or less over the wavelength range of 400 nm to 700 nm. The measuring method according to claim 11 .

20. 12. The measuring method according to claim 11, wherein the material of the heat-conducting plate is any one of metal, resin, and glass.

21. the oil is colored, The term "colored" means that the reflectance is 10% or less over the wavelength range of 400 nm to 700 nm. The measuring method according to claim 11 .

22. The measurement method according to any one of claims 17 to 21, further comprising the step of irradiating excitation light or white light from the side of the first surface of the substrate and measuring a reflected image after the step of introducing the oil and before the step of measuring the nucleic acid solution is completed.

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