PCR method, PCR apparatus, and PCR kit

Abstract

Its simple configuration, which does not require pressurization, suppresses the generation of air bubbles, enabling the separation of sample solutions and PCR. [Solution] The microdevice for digital PCR comprises a substrate having a plurality of recesses, a spacer provided between the substrate and a cover and positioned around the plurality of recesses, and a chamber formed by the spacer. The PCR method of this disclosure includes introducing a sample solution into the plurality of recesses, introducing a liquid photocurable resin having a glass transition temperature of 0°C or less and -120°C or higher into the chamber to cover the plurality of recesses and separate the plurality of recesses, irradiating the photocurable resin with light to gel the photocurable resin, and performing PCR by temperature-controlled the microdevice for digital PCR. The Young's modulus of the cover is 0.4 MPa or more and 4 Gpa or less, the distance from the substrate is variable during temperature control, and the area of ​​the cover on the chamber is 100 mm². 2 More than 2500mm 2 The following applies:

Description

[Technical Field]

[0001] This disclosure relates to a PCR method, a PCR device, and a PCR kit. [Background technology]

[0002] Traditionally, PCR or real-time PCR has been used for genetic testing. These techniques have the problem of low measurement accuracy when the amount of the target substance (nucleic acid) is minute. To solve this problem, digital PCR technology has attracted attention in recent years. In digital PCR, a sample containing the nucleic acid to be detected is separated into many minute regions, and PCR is performed on each minute region. By distinguishing between sections containing the target nucleic acid and sections that do not based on fluorescence intensity, the type of nucleic acid present in each minute region can be determined. Patent document 1 discloses a method for detecting DNA using digital PCR, in which the melting temperature of the DNA and the fluorescently labeled probe is measured in a droplet containing DNA and a fluorescently labeled probe that hybridizes to DNA.

[0003] UV-curing resins are often used as one of the components in microdevices for PCR. Patent Document 2 discloses a microfluidic chip that can easily dispense small amounts of sample. Patent Document 2 also states that UV-curing resins can be used as an example of an adhesive between the cover and substrate of a microdevice (see paragraph 0047). Patent Document 3 discloses a method for introducing a solution into a microfluidic device that can be used in digital PCR. Patent Document 3 also discloses that UV-curing resins can be used as a sealant material as an example of sealing wells.

[0004] In microfluidic devices used in digital PCR, oil is often used to separate samples into minute regions. Patent document 4 discloses a microarray device for separating minute amounts of samples containing nucleic acids. In this microarray device, after introducing the sample into a well, the well is covered with a hydrophobic substance such as mineral oil or silicone oil to perform solution separation. Since the sample in the well is hydrophilic and the oil is hydrophobic, the sample solution can be separated into each well without mixing due to the relationship between water and oil. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2018-108063 [Patent Document 2] Japanese Patent Publication No. 2011-163946 [Patent Document 3] International Publication No. 2014 / 193304 [Patent Document 4] U.S. Patent Publication No. 9518299 [Overview of the project] [Problems that the invention aims to solve]

[0006] When performing digital PCR using a microdevice for digital PCR, the sample solution confined in the well is a liquid in a minute volume of less than nanoliters. Resins are often used as materials for microdevices, but resins have water absorption properties. Therefore, even when using resin materials made of polypropylene (PP) or cycloolefin polymer (COP), which are known to be low water absorption materials of 0.01% or less, as the material for the microdevice, it has been found that the sample solution in the well is absorbed significantly into the resin by heating during PCR due to its minute volume. As a result, if the well is sealed with UV-curing resin, for example as in Patent Document 3, the sample solution in the well is absorbed into the microdevice during PCR. Consequently, the well becomes negatively pressurized by the volume change due to the loss of solution, and finally, bubbles are generated in the well to compensate for the lost volume. Bubbles become noise in fluorescence measurement in digital PCR. Therefore, the high-precision measurement, which is an advantage of digital PCR, becomes difficult due to the appearance of bubbles.

[0007] Furthermore, when performing PCR or digital PCR using a microdevice, it is crucial that the sample solution trapped in the well does not leak out due to the effects of heating during thermal cycling. As shown in Patent Document 4, hydrophobic substances such as oil are often used as sealing materials, but this alone is insufficient to prevent the sample solution from leaking out. Therefore, measures are taken to lower the vapor pressure of the sample solution by pressurizing the inside of the microdevice during PCR, thereby preventing the solution from evaporating and leaking out of the well. However, introducing a pressurization mechanism not only increases the size of the digital PCR device but also increases its cost.

[0008] To summarize, two features are necessary for a microdevice for digital PCR. First, a mechanism that prevents air bubbles from forming when the sample solution in the well is absorbed into the device, by having the sample separation solution replenish the volume absorbed. Second, a simple, non-pressurized mechanism to prevent the sample solution from leaking out of the well.

[0009] Therefore, this disclosure provides a technology that suppresses the generation of bubbles with a simple configuration that does not require pressurization, enabling the separation of sample solutions and PCR. [Means for solving the problem]

[0010] To solve the above problems, this disclosure provides a PCR method for a digital PCR microdevice, the digital PCR microdevice comprising: a substrate having a plurality of recesses capable of holding a sample solution to be introduced; a cover disposed above the substrate; a spacer provided between the substrate and the cover and disposed around the plurality of recesses; and a chamber formed by the spacer. The PCR method includes introducing the sample solution into the plurality of recesses; introducing a liquid photocurable resin having a glass transition temperature of 0°C or less and -120°C or more into the chamber to cover the plurality of recesses with the photocurable resin and separate the plurality of recesses; irradiating the photocurable resin introduced into the chamber with light to gel the photocurable resin; and performing PCR by temperature-controlled the digital PCR microdevice, wherein the cover has a Young's modulus of 0.4 MPa or more and 4 Gpa or less, the distance from the substrate is variable during temperature control, and the area of ​​the cover on the chamber is 100 mm². 2 More than 2500mm 2 The following applies:

[0011] Further features relating to this disclosure will become apparent from the description herein and the accompanying drawings. Furthermore, aspects of this disclosure are achieved and realized through elements and various combinations of elements and the modes of the claims described herein and the accompanying claims. The descriptions herein are merely illustrative and do not limit in any way the claims or applications of this disclosure. [Effects of the Invention]

[0012] According to this disclosure, it is possible to suppress the generation of bubbles with a simple configuration that does not require pressurization, and to perform separation of sample solutions and PCR.

[0013] Problems, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

Brief Description of the Drawings

[0014] [Figure 1A] It is a schematic cross-sectional view showing a configuration example of a microdevice for digital PCR according to the first embodiment. [Figure 1B] It is a bright-field microscope image of the wells formed on the substrate. [Figure 2] It is a schematic cross-sectional view for explaining a method of introducing a sample solution and a photocurable resin into a microdevice and a change in the properties of the photocurable resin due to light irradiation after the introduction. [Figure 3] It is a fluorescence image of the microdevice after performing PCR after performing a separation flow of the sample solution using the microdevice. [Figure 4] It is a diagram showing the relationship between the glass transition temperature (Tg) and the melting temperature (Tm) of the photocurable resin and the hardness of the resin material. [Figure 5A] It is a diagram showing a bright-field observation image after performing PCR using a photocurable resin having a glass transition temperature below the freezing point. [Figure 5B] It is a diagram showing a bright-field observation image after performing PCR using a photocurable resin having a glass transition temperature near 100°C. [Figure 6] It is a table summarizing the conditions of various resins and the photocurable resin. [Figure 7A] It is a schematic cross-sectional view of the microdevice for explaining that the cover deflects during PCR. [Figure 7B] It is a top view of the microdevice for explaining that the cover deflects during PCR. [Figure 8] It is a diagram showing the Young's modulus of various materials. [Figure 9] It is a schematic cross-sectional view for explaining the movement of the photocurable resin and the deflection of the cover during PCR. [Figure 10] It is a schematic diagram of a PCR device according to the second embodiment. [Figure 11] This is a schematic diagram of a kit consisting of a microdevice for digital PCR and a photocurable resin. [Modes for carrying out the invention]

[0015] [First Embodiment] In the first embodiment, it is explained that robust sample solution separation is possible and digital PCR can be performed by introducing a photocurable resin having a glass transition temperature (Tg) of 0°C or lower that satisfies specific conditions into a microdevice for digital PCR. In this specification, a photocurable resin having a glass transition temperature (Tg) of 0°C or lower that satisfies specific conditions may be simply referred to as "photocurable resin".

[0016] <Example of a microdevice configuration> Figure 1A is a schematic cross-sectional view showing an example configuration of a microdevice 100 for digital PCR according to the first embodiment. The microdevice 100 comprises a substrate 1, a cover 2, a spacer 3, and a port 4. The substrate 1 has multiple wells 5 which are recesses. The material of the substrate 1 can be a resin with low water absorption and low cost. Examples of materials include resins such as cycloolefin polymer and polypropylene.

[0017] Cover 2 is provided above substrate 1. Spacers 3 are provided between substrate 1 and cover 2, and around multiple wells 5. The chamber 6 is defined by the space between substrate 1, cover 2, and spacers 3. The specific elements of cover 2 will be described later. Cover 2 has an opening that connects to a port 4, and port 4 is provided at a position corresponding to the opening in cover 2. A sample solution and a photocurable resin that satisfies the conditions described later are introduced through port 4. Two ports 4 are not necessarily required; one is sufficient.

[0018] Figure 1B is a bright-field microscope image of the wells 5 formed in the substrate 1. It can be seen that the square wells 5 are aligned vertically and horizontally. The wells 5 do not necessarily have to be aligned regularly. When maximizing the number of wells 5 on the substrate 1, they can be arranged in a hexagonal or square arrangement. There are no limitations on the shape of the wells 5; for example, they may be square, hexagonal, or cylindrical.

[0019] Figure 2 is a schematic cross-sectional view illustrating the method of introducing the sample solution and photocurable resin into the microdevice, and the change in the properties of the photocurable resin due to light irradiation after introduction. In step (i), the sample solution 101 is introduced into the microdevice 100 from port 4. The wells 5 and chambers 6 in the microdevice 100 are filled with the sample solution 101.

[0020] In step (ii), a liquid photocurable resin 102 with a glass transition temperature (Tg) of 0°C or lower is introduced from port 4. The liquid photocurable resin 102 displaces the sample solution 101 present in chamber 6. As a result, the sample solution 101 is sealed within well 5. In this state, the sample solution 101 in well 5 is sealed by the liquid photocurable resin 102. Therefore, the airtightness is not sufficiently high.

[0021] Therefore, in step (iii), the light source 104 is used to convert the liquid photocurable resin 102 into a gel-like photocurable resin 103. As a result, each sample solution 101 in well 5 changes from being sealed with a liquid material to being sealed with a gel material, thus ensuring airtightness. At this time, the light source 104 can accelerate the photopolymerization reaction by irradiating with light near the absorption wavelength of the liquid photocurable resin 102. Therefore, for example, light with a wavelength of 250 to 500 nm can be irradiated. However, wavelengths in the ultraviolet region may destroy the nucleic acids being measured. Therefore, by using light with a wavelength of 350 to 500 nm, for example, the destruction of nucleic acids can be avoided.

[0022] Examples of light sources 104 include lasers, LEDs, mercury lamps, deuterium lamps, tungsten lamps, xenon lamps, and halogen lamps. Specific conditions for the liquid photocurable resin 102 will be described later.

[0023] Figure 3 shows the fluorescence image of microdevice 100 after performing PCR on the sample solution shown in Figure 2 using the microdevice 100 shown in Figure 1B. In digital PCR, PCR is performed using a heating medium such as a thermal cycler, so that PCR is promoted in wells where nucleic acids are present, and does not occur in wells where nucleic acids are not present. A fluorescently labeled probe is introduced into the sample solution beforehand, and only the wells where PCR occurred emit fluorescence during fluorescence measurement. By examining the fluorescence image, it can be confirmed from the contrast that there are wells that are glowing and wells that are not glowing. Therefore, from this fluorescence observation result, it can be seen that digital PCR can be performed without the influence of air bubbles, etc., in a microdevice made by gelling a photocurable resin with a glass transition temperature (Tg) of 0°C or lower.

[0024] Incidentally, when a liquid medium such as oil was introduced instead of the liquid photocurable resin 102 and PCR was performed in the same way, no digital PCR results were obtained, and connections between adjacent wells and dispersion of the solution were observed (not shown). Therefore, it can be seen that sealing with a gelling photocurable resin improves the airtightness of the wells.

[0025] In fluorescence observation, light from sources other than the sample solution in the well becomes noise. Therefore, the photocurable resin 102 and cover 2 can suppress noise by exhibiting low autofluorescence in the wavelength range of 400-700 nm. Furthermore, noise can be suppressed by using materials with high transparency, such as a transmittance of 60% or more for the photocurable resin and cover.

[0026] <Conditions for photocuring resin> -Regarding the glass transition temperature- This document describes the conditions for a photocurable resin with a glass transition temperature (Tg) of 0°C or lower, which is introduced into a microdevice. By using the photocurable resin under the conditions described below, it is possible to perform highly sealed sample solution separation and PCR without generating air bubbles. Furthermore, a pressurization mechanism is not required during PCR, enabling the realization of a digital PCR device with a simple configuration.

[0027] Figure 4 shows the relationship between the glass transition temperature (Tg) and melting temperature (Tm) of a photocurable resin and the hardness of the resin material. Every resin has a glass transition temperature (Tg) and a melting temperature (Tm), which serve as indicators of the resin's state (hardness). For example, when the melting temperature (Tm) of a resin is room temperature (~20°C), the resin is in an intermediate state between gel and liquid, where the two are mixed together. Similarly, the glass transition temperature (Tg) is the temperature at which the intermediate state between glass and gel is observed, where the two are mixed together. The glass transition temperature (Tg) varies greatly depending on the resin; some have a Tg of around -100°C, while others have a Tg of around 200°C. Photocurable resins always exist in a liquid state before light irradiation, but after light irradiation, they become the state (hardness) determined by the resin's glass transition temperature (Tg) and melting temperature (Tm).

[0028] The photocurable resin disclosed herein is preferably in a gel-like state rather than a hard, glass-like state after light irradiation. Therefore, the glass transition temperature should be lower than room temperature. Furthermore, since the glass transition temperature is an intermediate state between gel and glass, it must be at least 0°C or below. In addition, in DSC analysis to measure the glass transition temperature, many resins undergo a gradual change to a gel-like state starting from the glass transition temperature. A change of about 50°C from the glass transition temperature causes a change from an intermediate state between glass and gel to a single component of the gel state. Based on the above, a photocurable resin with a glass transition temperature of 0°C or below, particularly a photocurable resin with a glass transition temperature of -30°C or below, can be used. On the other hand, the lower limit of the glass transition temperature can be, for example, -120°C or above, and in particular, -80°C or above, which is the glass transition temperature (Tg) of fluorosilicone rubber, but is not limited to this.

[0029] Figure 5A shows a bright-field image after PCR using a photocurable resin with a glass transition temperature below freezing. In Figure 5A, isooctyl acrylate with a glass transition temperature (Tg) of -54°C is used as the photocurable resin. From Figure 5A, it can be seen that when a photocurable resin with a glass transition temperature (Tg) of -54°C is used, no air bubbles are observed in the wells after PCR.

[0030] Figure 5B shows bright-field images after PCR using a photocurable resin with a glass transition temperature (Tg) of approximately 100°C. In Figure 5B, tricyclodecanedimethanol diacrylate with a glass transition temperature (Tg) of 97°C is used as the photocurable resin. From Figure 5B, it can be seen that when a photocurable resin with a glass transition temperature (Tg) of 97°C is used, bubbles are observed in some wells. As mentioned above, this is because the sample solution in the wells is absorbed into the device during PCR, resulting in a negative pressure state in the wells due to the volume change caused by the loss of solution. Ultimately, bubbles are generated in the wells to compensate for the lost volume. On the other hand, with a photocurable resin with a glass transition temperature (Tg) of -54°C, the photocurable resin, which becomes gel-like upon exposure to light, can enter the wells to compensate for the volume lost, so the wells do not become negatively pressured and bubbles do not form. Therefore, it can be seen that the glass transition temperature (Tg) of the photocurable resin is significantly involved in bubble generation.

[0031] This section explains the guidelines for the hardness of gel-like photocurable resin. When the penetration degree of gel-like photocurable resin is 20 or higher, the resin is easily pulled into the well, and a good sealing effect on the well can be achieved. On the other hand, if the penetration degree exceeds 150, the gel is highly fluid, resulting in insufficient sealing of the well and a risk of contamination between adjacent wells. Therefore, the penetration degree of gel-like photocurable resin can be set, for example, between 20 and 150.

[0032] The wells in microdevices are spread out on a plane, and compared to digital PCR devices composed of microchannels, the solution in adjacent wells is more likely to connect. Therefore, it is important to ensure as much adhesion as possible between the substrate containing the wells and the photocurable resin material, so that the sample solution does not penetrate the walls between wells and the sample solution does not easily connect between adjacent wells. To achieve this, it is desirable to select a photocurable resin material that has a molecular skeleton similar to that of the substrate, or a material with a composition similar to rubber, in order to improve adhesion with the substrate.

[0033] -Regarding viscosity- In addition to the glass transition temperature (Tg), viscosity and hydrophobicity are important conditions for the photocurable resin to be introduced into the microdevice 100. Viscosity is an important indicator when introducing the resin into the chamber 6 inside the microdevice 100. If the viscosity of the photocurable resin is too high, introduction will be difficult. Furthermore, if high pressure is applied to introduce a highly viscous photocurable resin, there is a possibility that the microdevice 100 may be damaged. Therefore, the viscosity of the liquid photocurable resin 102 before introduction into the microdevice 100 can be kept below 2000 mPa·s.

[0034] Since one side of well 5 of the microdevice 100 is open to the chamber 6, care must be taken when introducing the photocurable resin 102, which has a glass transition temperature (Tg) of 0°C or lower. If the introduction rate of the photocurable resin 102 is too fast, the sample solution 101 in well 5 will escape. As a result, the sample solution 101 will be depleted from well 5 and replaced by the photocurable resin 102. To prevent this, the viscosity of the photocurable resin 102 can be slightly increased, or the introduction rate of the photocurable resin 102 can be adjusted to be slower. When increasing the viscosity of the photocurable resin 102, for example, the viscosity can be increased to 100 mPa·s or higher.

[0035] -Regarding hydrophobicity- The hydrophobicity of the photocurable resin 102 is a necessary indicator in PCR. The sample solution 101 is basically an aqueous solution. Therefore, if the photocurable resin 102 is miscible or compatible with water, components in the sample solution 101 may elute into the photocurable resin 102. As a result, PCR may not occur (i.e., PCR may be inhibited). Therefore, the photocurable resin 102 must be insoluble in water. As a material for hydrophobic photocurable resins, materials with a normal alkane or isoalkane molecular skeleton can strongly exhibit hydrophobic function. Similarly, materials with a silicone molecular skeleton also exhibit hydrophobic function. Furthermore, if the solubility of the photocurable resin 102 in water is 100 g / L or less, it is expected that the effect of PCR inhibition will be drastically reduced.

[0036] -Examples of resins that meet the conditions- Figure 6 is a table summarizing the conditions for various resins and photocurable resins. The viscosity requirement is 2000 mPa·s or less, which is sufficient for introduction into microdevices. The hydrophobicity requirement is that it does not inhibit PCR. The glass transition temperature (Tg) requirement is 0°C or less, so that it is liquid when introduced into microdevices and becomes gel-like after photocuring. For eight types of resins—polyethylene glycol (600) diacrylate, dimethylacrylamide, aliphatic polybutadiene-based urethane acrylate, isobornyl acrylate, tricyclodecanedimethanol diacrylate, phenoxyethyl acrylate, isooctyl acrylate, and fluorosilicone rubber—whether each of the above conditions is met is indicated with ○ or ×.

[0037] As shown in Figure 6, isooctyl acrylate and fluorosilicone rubber have a glass transition temperature (Tg) of 0°C or lower, are hydrophobic, and have a viscosity of 2000 mPa·s or lower. Note that, as shown in Figure 5B, bubbles were observed in a photocurable resin (phenoxyethyl acrylate) with a glass transition temperature (Tg) of 2°C, therefore, the glass transition temperature (Tg) is set to at least below freezing point, 0°C or lower.

[0038] As described in this embodiment, the advantage of this technology is that solution separation and PCR can be performed using only one type of photocurable resin. If a mixture of multiple types of photocurable resins, or a mixture of photocurable resin and oils other than photocurable resin, is used, the compatibility of the mixture may be disrupted over time, potentially leading to separation. Furthermore, if a mixture of photocurable resin and oil, or a mixture of photocurable resin and emulsifier is prepared and cured, only the photocurable resin may harden or gel, while other substances remain on the surface of the photocurable resin. In addition, since the photocurable resin will contain impurities unrelated to photocrosslinking, the degree of solidification and gelation may be poor. If a mixture of photocurable resin and oil / emulsifier is used, and solution separation and PCR are performed using the microdevice 100 shown in the first embodiment, in the mixture of photocurable resin and oil / emulsifier in chamber 6, only the photocurable resin will gel, and the mixture other than the resin will remain on the surface of the gelled resin. In that case, the well is sealed by a mixture of gelled photocurable resin and other liquids, so the airtightness is lower than that of a gel made from a single type of photocurable resin. Therefore, a pressurizing mechanism or other device may be required. Thus, the separation of sample solutions using a single type of photocurable resin in this embodiment not only simplifies the materials but also demonstrates good well airtightness. In addition, any additive can be added to the photocurable resin 102 as long as it does not hinder the effects of this disclosure. The amount of such additive added can be, for example, 1% by weight or less of the total amount of the photocurable resin 102.

[0039] <Cover Conditions> The following describes the conditions of the cover 2 of the microdevice 100. First, we will explain the bending of the cover in the microdevice. By using the cover 2 of the microdevice 100 of this disclosure, when the sample solution 101 in well 5 is absorbed into the microdevice 100 during PCR, not only the photocurable resin but also the cover 2 of the microdevice 100 bends by the amount of the change in water absorption volume. As a result, highly sealed sample solution separation and PCR become possible without the generation of air bubbles.

[0040] Figure 7A is a schematic cross-sectional view of the microdevice 100 to illustrate the bending of cover 2 during PCR. Figure 7B is a top view of the microdevice 100 to illustrate the bending of cover 2 during PCR. During PCR, the sample solution 101 is absorbed into the substrate 1 in the microdevice 100, causing the photocurable resin 103 to be pulled toward well 5, and consequently, the cover 2 of the microdevice 100 to bend. As mentioned above, the gel-like photocurable resin 103 is pulled toward well 5 as the sample solution 101 is absorbed into the substrate 1. As a result, the pulled gel-like photocurable resin 103 is present in well 5, as well as the bent cover 2. By combining a photocurable resin with a glass transition temperature (Tg) of 0°C or lower with a bent cover 2, not only are no bubbles generated in well 5 of the microdevice 100, but no bubbles are generated in the chamber 6 either. As a result, PCR can be performed stably. The area 202 of the flexible cover 2 corresponds to the area in the base material 1 where the well 5 exists, and is the area shown by the dashed line in Figures 7A and 7B.

[0041] Let us illustrate how much of the sample solution 101 is absorbed by the microdevice 100. Assuming the substrate 1 of the microdevice 100 is polypropylene (water absorption rate 0.01%), and the depth of well 5 is approximately 30 μm and the width of well 5 is approximately 20 μm, about 10% of the sample solution 101 in well 5 will be absorbed. Therefore, in the case of the illustrated well 5, the amount of water absorbed per well is 1.2 pL / well. As mentioned above, since well 5 is a minute volume consisting of a size and depth of approximately 100 μm or less, even an absorption amount of about 1.2 pL per well has a significant impact on digital PCR measurement. Furthermore, if there are 500,000 of these wells, about 0.6 μL of sample solution will be lost in all wells combined. Therefore, the photocurable resin and the cover 2 of the microdevice in this disclosure need to change in volume by the amount of water absorbed. The amount by which the cover 2 needs to move from the volume change can be explained by the following formula (1).

[0042] (Change in cover deflection due to volume change) < (well depth) × (water absorption rate of substrate) × (coefficient (≧350)) (1)

[0043] The right-hand side of equation (1) shows the volume of water absorbed per well relative to the depth of the well. For example, when the water absorption is about 10%, as in the example above, this means a volume of 3 μm relative to a well depth of 30 μm. In this case, the change in deflection of cover 2 due to the volume change can be less than 3 μm. This is because, for each well, not only the area of ​​cover 2 directly above well 5 deflects, but the entire cover 2 over the area where well 5 exists deflects. The coefficient is the water absorption coefficient that actually takes into account the shape of well 5 (fine irregularities, presence or absence of surface coating, circular or square shape, etc.). The example above shows one example where well 5 does not absorb water easily (well shape: square, water absorption rate: 0.01%), and the water absorption coefficient in that case is 350, so that value was used as the minimum value. Depending on the actual application, the above coefficient may be greater than 350.

[0044] The flexibility of cover 2 is largely influenced by Young's modulus and the size of cover 2. Young's modulus is closely related to the stiffness of a material; the larger the Young's modulus, the harder the material, requiring greater force to deform. In this disclosure, the Young's modulus of the material used for cover 2 can be set to, for example, 0.4 MPa to 4 GPa, thereby making it more flexible.

[0045] Figure 8 shows the Young's modulus for various materials. From Figure 8, it can be seen that materials such as glass and metal are difficult to apply to cover 2. In addition to polystyrene, polyethylene, and rubber shown in Figure 8, resins with a Young's modulus of 0.4 MPa to 4 GPa, such as polypropylene, can be used as cover 2.

[0046] The size of cover 2 can be explained by the following formula (2).

[0047] δ∝F·L3 / E·I (2)

[0048] The amount of deflection δ is proportional to the external force F and the length L of the cover, and inversely proportional to the elastic modulus F and the second moment of area I. From equation (2), it can be seen that the length L is the most dominant value because it affects the amount of deflection in powers of 3. Therefore, it can be said that the larger the length of the cover, i.e., the area, the easier it is to deflect. In this disclosure, the deflecting region 202 of the cover 2 shown in Figures 7A and 7B is, for example, 100 mm. 2 More than 2500mm 2 The following is possible:

[0049] The key feature of this technology is that, instead of the cover area directly above each well bending in response to the well, the entire cover over the area where the well exists bends. In the fabrication of microdevices, there are always walls between wells on the substrate. Therefore, within the area where the well exists, there is a well area where the resin is stretched and volume changes occur during PCR, and a well wall area that does not participate in volume changes. In other words, the cover over the area where the well exists can compensate only for the area of ​​the well wall that does not participate in volume changes, thereby reducing the amount of bending. This allows for the aforementioned 100mm 2 More than 2500mm 2 Within the following range, the cover will flex by the amount of volume change due to water absorption by the substrate, thereby suppressing the generation of air bubbles.

[0050] Figure 9 is a schematic cross-sectional view illustrating the movement of the photocurable resin 103 and the bending of the cover 2 during PCR. During PCR, the microdevice 100 is positioned in the temperature control unit 105 such that the temperature control unit 105 is in contact with the bottom surface of the substrate 1. The temperature control unit 105 is, for example, a thermal cycler, and its temperature is controlled to perform the thermal cycle of PCR. After sample separation by the photocurable resin, during the heating to 95°C and cooling to 60°C cycle in PCR, the sample solution 101 in the well 5 is absorbed by the substrate 1, and the photocurable resin 103 is pulled into the well 5. In addition, the cover 2 of the microdevice 100 bends up and down during heating and cooling. The cover 2 of the microdevice 100 remains bent even after the completion of PCR. This prevents the generation of bubbles caused by the absorption of the sample solution 101 into the substrate 1, and allows for the construction of a simple device configuration without the need for a pressurization mechanism. The chamber 6 between the substrate 1 and the cover 2 requires space to allow for the deflection of the cover 2, and the thickness of the chamber 6 can be, for example, between 2 μm and 1 mm. The thickness of the cover 2 affects the degree of deflection and interference during fluorescence measurement, so it can be, for example, between 100 μm and 2 mm. Furthermore, since the configuration of this microdevice 100 is characterized by the deflection of the cover 2, it is difficult to control the temperature of PCR from the cover 2 side. If temperature control is performed from the cover 2 side, there will be parts that do not come into contact with the temperature control unit 105 due to the effect of deflection, resulting in significant in-plane temperature unevenness within the microdevice 100. Therefore, heating by the temperature control unit 105 must be performed from the substrate 1 side, which is the opposite direction from the cover 2.

[0051] <Summary of the First Embodiment> According to the first embodiment, the microdevice 100 for digital PCR includes a substrate 1 having a plurality of wells 5 (recesses), a spacer 3 provided between the substrate 1 and the cover 2 and disposed around the plurality of wells 5, and a chamber 6 formed by the spacer 3. The PCR method of the present disclosure includes introducing a sample solution 101 into the plurality of wells 5, introducing a liquid photocurable resin 102 having a glass transition point of 0°C or lower and -120°C or higher into the chamber 6 to cover the plurality of wells 5 and separate the plurality of wells 5, irradiating the photocurable resin 102 with light to gelify the photocurable resin, and performing PCR by temperature control of the microdevice 100 for digital PCR. The Young's modulus of the cover 2 is 0.4 MPa or more and 4 GPa or less, and the distance from the substrate 1 is variable during temperature control. The area on the chamber 6 in the cover 2 is 100 mm 2 or more and 2500 mm 2 or less.

[0052] Since the volume of the sample solution 101 in the well 5 absorbed during PCR is complemented by the photocurable resin having a glass transition point (Tg) of 0°C or lower and -120°C or higher and the cover 2 of the microdevice 100, no bubbles are generated. Further, since the photocurable resin 102 having a glass transition point (Tg) of 0°C or lower becomes gel-like when irradiated with light, highly robust separation can be achieved without leakage of the sample solution 101 in the well 5 and without the need for pressurization. Also, since pressurization is not required, digital PCR with a simple configuration is possible.

[0053] [Second Embodiment] In the second embodiment, an overview of a PCR apparatus that performs sample solution separation and PCR using a microdevice for digital PCR will be described.

[0054] [Configuration Example of PCR Apparatus] Figure 10 is a schematic diagram of a PCR apparatus 300 according to a second embodiment. The PCR apparatus 300 comprises a temperature control unit 301, a pressure pump 302, a pressure / depressurization pump 303, a photocurable resin storage unit 304, and a light source 305. The operation of these components is controlled by a control device (not shown). The microdevice 100 is placed on the temperature control unit 301. The photocurable resin storage unit 304 contains liquid photocurable resin 102. The photocurable resin storage unit 304 is connected to port 4 of the microdevice 100. The pressure pump 302 is connected to the photocurable resin storage unit 304 and is used to pressurize the photocurable resin storage unit 304 and introduce the photocurable resin 102 into the microdevice 100.

[0055] The pressurizing / depressurizing pump 303 is configured to perform at least one of either pressurizing or depressurizing. The pressurizing / depressurizing pump 303 is used when introducing the sample solution 101 into the microdevice 100. When introducing the sample solution 101 under pressure, the pressurizing / depressurizing pump 303 does not need to be installed inside the PCR apparatus 300. In this case, the pressurizing pump 302 can be used to introduce the sample solution 101 and the photocurable resin 102, and the pressure line can be controlled by a valve or the like.

[0056] After at least one well 5 in the microdevice 100 is sealed with liquid photocurable resin 102, light is irradiated from the light source 305. This light causes all of the liquid photocurable resin 102 in the microdevice 100 to gel, enabling the separation of the sample solution 101. Since the PCR device 300 needs to accumulate the photocurable resin inside the PCR device 300, the PCR device 300 needs an environment that shields it from external light. If a light-shielding environment is difficult, the photocurable resin accumulation section 304 may be shielded from light by making it a black container.

[0057] <Summary of the second embodiment> By using the PCR apparatus 300 of this disclosure, sample solution separation using photocurable resin and PCR without generating bubbles become possible. Furthermore, a large pressurization mechanism capable of pressurizing the entire PCR apparatus 300 is not required. The PCR apparatus 300 can be used in various derivative devices. For example, by adding a multi-wavelength excitation light source, optical filter, focusing lens, and a photodetector such as a CCD or CMOS camera to the light source 305, fluorescence measurement of the microdevice 100 after PCR becomes possible.

[0058] [Third Embodiment] In the third embodiment, a kit of a microdevice for digital PCR and a photocurable resin (PCR kit) will be described.

[0059] <Example of kit contents> Figure 11 is a schematic diagram of a kit 400 containing a microdevice for digital PCR and a photocurable resin. The kit 400 includes the microdevice 100 described in the first embodiment (Figure 1), a container 401 containing liquid photocurable resin 102, and a package 402 that encloses these. By shielding the kit 400 from light, it is possible to prevent the photocurable resin 102 from hardening during transport. Either the container 401 containing the photocurable resin 102 or the package 402 of the kit 400 can be shielded from light.

[0060] <Summary of the second embodiment> By using Kit 400 of this disclosure, users no longer need to separately prepare a photocurable resin for separating sample solutions. Sample solution separation and PCR can be performed using a photocurable resin simply by preparing a pressure application device such as a pump and a temperature controller such as a thermal cycler.

[0061] [Differentiation] This disclosure is not limited to the embodiments described above, but includes various modifications. For example, the embodiments described above are described in detail for the purpose of illustrating this disclosure, and do not necessarily have to include all the configurations described. Furthermore, parts of one embodiment can be replaced with the configurations of another embodiment. Furthermore, configurations of other embodiments can be added to the configuration of one embodiment. Furthermore, parts of the configuration of each embodiment can be added, deleted, or replaced with parts of the configurations of other embodiments. [Explanation of Symbols]

[0062] 1: Base material 2: Cover 3: Spacer 4: Port 5: Well 6: Chamber 100: Microdevices 101: Sample Solution 102: Liquid photocurable resin 103: Gel-like photocurable resin 104: Light source 300: PCR device 301: Temperature control section 302: Pressure pump 303: Pressurizing / Depressurizing Pumps 304: Photocuring resin storage section 305: Light source 400: Kit 401: Container 402: Package

Claims

1. A PCR method for a digital PCR microdevice, The aforementioned microdevice for digital PCR is A substrate having multiple recesses capable of holding the sample solution to be introduced, A cover positioned above the substrate, A spacer is provided between the substrate and the cover and is arranged around the plurality of recesses, The chamber formed by the spacer, The PCR method described above is Introducing the sample solution into the plurality of recesses, A liquid photocurable resin having a glass transition temperature of 0°C or less and -120°C or higher is introduced into the chamber, the photocurable resin covers the plurality of recesses, and the plurality of recesses are separated. The photocurable resin introduced into the chamber is irradiated with light to gel the photocurable resin, This includes performing PCR while temperature-controlled the aforementioned digital PCR microdevice, The cover has a Young's modulus of 0.4 MPa or more and 4 GPa or less, and its distance from the substrate is variable during temperature control. The area on the chamber in the cover is 100 mm ２ 2500mm or more ２ The PCR method is as follows.

2. The aforementioned cover is Let X be the water absorption rate of the substrate. The PCR method according to claim 1, characterized in that, during the temperature control, the distance moved is less than the depth of the plurality of recesses × X% × coefficient (≥ 350).

3. The PCR method according to claim 1, characterized in that, when performing PCR with temperature control, the heating is performed from the substrate side, which is in the opposite direction to the cover.

4. The PCR method according to claim 1, characterized in that the thickness of the cover is 100 μm or more and 2 mm or less.

5. The PCR method according to claim 1, characterized in that the thickness of the spacer is 2 μm or more and 1 mm or less.

6. The PCR method according to claim 1, characterized in that the penetration degree of the gelled photocurable resin is 20 or more and 150 or less.

7. The PCR method according to claim 1, characterized in that the viscosity of the liquid photocurable resin is 100 mPa·s or more and 2000 mPa·s or less.

8. The PCR method according to claim 1, characterized in that the photocurable resin is hydrophobic.

9. The PCR method according to claim 8, characterized in that the photocurable resin has a n-alkane, isoalkane, or silicone-based molecular skeleton.

10. The PCR method according to claim 8, characterized in that the photocurable resin has a solubility in water of 100 g / L or less.

11. The PCR method according to claim 1, wherein the photocurable resin is isooctyl acrylate or fluorosilicone rubber.

12. The PCR method according to claim 1, characterized in that the photocurable resin and the cover are transparent.

13. The PCR method according to claim 12, characterized in that the transparency of the photocurable resin and the cover is such that the transmittance is 60% or more at a wavelength of 400 to 700 nm.

14. The PCR method according to claim 1, characterized in that the photocurable resin and the cover exhibit low autofluorescence in the wavelength range of 400 to 700 nm.

15. The PCR method according to claim 1, characterized in that the light has a wavelength in the range of 250 to 500 nm.

16. The PCR method according to claim 1, characterized in that the cover flexes during temperature control.

17. A PCR device using a digital PCR microdevice, The aforementioned microdevice for digital PCR is A substrate having multiple recesses capable of holding the sample solution to be introduced, A cover positioned above the substrate, A spacer is provided between the substrate and the cover and is arranged around the plurality of recesses, The chamber formed by the spacer, The PCR device is A pressure application mechanism that applies pressure to the chamber to introduce the sample solution into the plurality of recesses, A pressurizing mechanism for introducing a liquid photocurable resin having a glass transition temperature of 0°C or lower and -120°C or higher into the chamber, A light source that irradiates the photocurable resin introduced into the chamber with light to gel the photocurable resin, The system includes a temperature control unit that adjusts the temperature of the aforementioned microdevice for digital PCR to perform PCR, The cover has a Young's modulus of 0.4 MPa or more and 4 GPa or less, and its distance from the substrate is variable during temperature control by the temperature control unit. The area on the chamber in the cover is 100 mm ２ 2500mm or more ２ The following is the PCR device.

18. A PCR kit comprising a microdevice for digital PCR and a liquid photocurable resin contained in a container, The aforementioned microdevice for digital PCR is A substrate having multiple recesses capable of holding the sample solution to be introduced, A cover positioned above the substrate, A spacer is provided between the substrate and the cover and is arranged around the plurality of recesses, The chamber formed by the spacer, The aforementioned photocurable resin has a glass transition temperature of 0°C or lower and -120°C or higher, and gels upon irradiation with light. The cover has a Young's modulus of 0.4 MPa or more and 4 GPa or less, and its distance from the substrate is variable during temperature control. The area on the chamber in the cover is 100 mm ２ 2500mm or more ２ The following is the PCR kit.

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