Temperature control system for sample solution used for digital pcr, and method using system

Abstract

In the present invention, a device includes a first port, a second port, and a flow path connecting the plurality of chambers. The flow path includes: a first flow path that connects the first port to the plurality of chambers; and a second flow path that connects the second port to the plurality of chambers. The flow path resistance of the second flow path is greater than the flow path resistance of the first flow path. The temperature control system seals the plurality of chambers with a separation liquid, and subsequently causes a sample solution in the plurality of chambers to react by using a first temperature regulator while applying pressure on the first port.

Description

Temperature control system for sample solution used in digital PCR and method using same

[0001] The present invention relates to a temperature control system for a sample solution used in digital PCR and a method for using the temperature control system for a sample solution in digital PCR.

[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 is in minute amounts. To solve this problem, digital PCR technology has been attracting attention in recent years. In digital PCR, a sample containing the target nucleic acid is separated into multiple microscopic regions, and PCR is performed on each microscopic region. The type of nucleic acid present in each microscopic region is identified by distinguishing between compartments that contain the target nucleic acid and compartments that do not based on fluorescence intensity.

[0003] Digital PCR allows for highly accurate measurements. However, because digital PCR uses a single nucleic acid, the fluorescence intensity obtained is not as strong as that obtained by performing PCR using a large amount of nucleic acid in a PCR tube, as in conventional real-time PCR.

[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.

[0005] There are several configurations for controlling the sample temperature in digital PCR.

[0006] Patent Document 2 discloses a configuration for PCR temperature control in a cartridge-type device holding through-hole wells, in which the influence of air bubbles and the like is minimized by tilting a thermal cycler for temperature control.

[0007] Patent Document 3 discloses the configuration of a microdevice equipped with a valve, which opens and closes the valve in the process from sample introduction to PCR, while also ensuring the sealing of the solution and suppressing the effects of air bubbles.

[0008] Patent Document 4 discloses the configuration of a microdevice equipped with a reflux mechanism, which enables PCR using oil using a reflux device and also enables air bubbles generated within the device to be washed away within the reflux device.

[0009] JP 2018-108063 A JP 2016-518861 A Chinese Patent No. 107138193 Specification JP 2020-25537 A

[0010] Digital PCR requires a temperature control step to perform PCR. Furthermore, in the digital PCR that measures the DNA melting temperature described above, a step of measuring changes in fluorescence intensity while controlling the temperature is added to the PCR temperature control step. Therefore, the temperature control step is important when performing digital PCR.

[0011] Because temperature control is performed from room temperature to around 100°C, some of the sample solution trapped in the wells of the microdevice evaporates, generating bubbles. The bubbles generated inside the wells can push the solution inside the wells away from the covering oil and leak out, leading to contamination of surrounding wells.

[0012] Since digital PCR is a highly accurate measurement that allows absolute quantification, the occurrence of such contamination is a serious problem, and therefore, it is important to take measures against bubbles, which are the cause of contamination.

[0013] Therefore, existing technologies include tilting the temperature control mechanism, mounting valves, and introducing a reflux mechanism, as mentioned above. However, these mechanisms require bubble prevention mechanisms to be attached not only to the device but also to the equipment, which increases not only the cost but also the size of the equipment. Furthermore, opening and closing valves on the chip and the valves in the reflux device increases the number of steps the equipment must operate. Therefore, it is desirable to be able to implement bubble prevention measures during temperature control with a simple configuration.

[0014] An object of the present invention is to provide a method for suppressing the influence of bubbles in a sample solution temperature control system for digital PCR by generating flow path resistance within a device of the system.

[0015] Another object of the present invention is to provide a system having a simple and inexpensive configuration that is the same as the configuration of a device that introduces a sample solution and divides a sample, without implementing valves, a reflux device, or the like.

[0016] The above and other objects and novel features of the present invention will become apparent from the description of this specification and the accompanying drawings.

[0017] An example of a temperature control system according to the present invention is a temperature control system for a sample solution used in digital PCR, the temperature control system comprising a device, the device comprising: a first port that is pressurized; a second port that is open to the atmosphere; a plurality of chambers that store the sample solution; and a flow path connecting the first port, the second port, and the plurality of chambers, a separation liquid that divides the sample solution flows through the flow path, the flow path comprising: a first flow path connecting the first port and the plurality of chambers; and a second flow path connecting the second port and the plurality of chambers, the temperature control system further comprising: a pressurizing pump for pressurizing the device; and a first temperature regulator for regulating the temperature of the device, the flow path resistance of the second flow path being greater than the flow path resistance of the first flow path, and after sealing the plurality of chambers with the separation liquid, the temperature control system reacts the sample solution in the plurality of chambers using the first temperature regulator while pressurizing the first port.

[0018] An example of a method according to the present invention is a method of using a temperature control system for a sample solution in digital PCR, wherein the temperature control system comprises a device, the device comprising: a first port that is pressurized; a second port that is open to the atmosphere; a plurality of chambers that store the sample solution; and flow paths that connect the first port, the second port, and the plurality of chambers, a separation liquid that divides the sample solution flows through the flow paths, the flow paths comprising: a first flow path that connects the first port and the plurality of chambers; and a second flow path that connects the second port and the plurality of chambers, the temperature control system further comprising: a pressure pump that pressurizes the device; and a first temperature regulator that regulates the temperature of the device, wherein the flow path resistance of the second flow path is greater than the flow path resistance of the first flow path, and the method comprises the steps of: sealing the plurality of chambers with the separation liquid; and then reacting the sample solution in the plurality of chambers using the first temperature regulator while pressurizing the first port.

[0019] According to the present invention, it is possible to suppress air bubbles in digital PCR with a simple and inexpensive configuration, thereby making it possible to prevent contamination of adjacent wells.

[0020] Schematic diagram of the pressure gradient generated by installing a flow resistance within a device in a sample solution temperature control system. Several examples of configurations for generating flow resistance in a device within a system. A diagram explaining the shape of a digital PCR device composed of flow paths and wells. A diagram explaining the digital PCR device after the introduction of a sample solution, the digital PCR device after the introduction of a separation solution, and confirmation of digital PCR by temperature control of the thermal cycle, as well as their configuration diagrams. A diagram explaining the results of digital PCR measurements performed using this system. A diagram explaining the connection (contamination) between flow paths that occurs when flow path resistance is insufficient. A diagram explaining an example of further enhancing the influence of flow path resistance in a microfluidic device. A schematic diagram explaining the generation of flow path resistance using only high-viscosity oil in a sample solution temperature control system. A sample temperature control system combining two temperature controllers. A reference diagram explaining the generation of flow path connection between flow paths due to thermal expansion and contraction of the chip and sample solution in a sample temperature control system. A diagram explaining the temperature dependence of low-viscosity and high-viscosity oils at room temperature. A diagram explaining the presence or absence of mixing of low-viscosity and high-viscosity oils in an opening open to atmospheric pressure at room temperature. Schematic diagram of the device and a diagram explaining the shape of the port when introducing oil into the pressurization port. A diagram explaining an example of the configuration of the atmospheric pressure release port, where the sample solution is contained within the device without leaking out. A diagram explaining that melting temperature analysis can also be performed in the sample temperature control system.

[0021] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0022] First Embodiment A first embodiment will be described below with reference to FIGS. 1 to 5. FIG.

[0023] Example 1 shows an overview of the pressure gradient that occurs in a sample solution temperature control system by installing a flow path resistance in a device, and demonstrates that applying this to a microdevice enables digital PCR while suppressing the effects of air bubbles. The sample solution temperature control system of this example is used for digital PCR, and by using this, digital PCR can be performed with an inexpensive and simple device configuration.

[0024] 1(a) shows that by providing a flow path resistor in the device of the sample temperature control system, it is possible to control the temperature of the solution in the well while keeping it pressurized. The temperature control system includes this device.

[0025] The device has a pressurization port 6 (pressurized port or first port) and an atmospheric pressure release port 5 (port open to the atmosphere or second port). The temperature control system also has a pressurization pump 1 for pressurizing the device. During temperature control during PCR, separation solution 2 is continuously introduced through the pressurization port 6 by the pressurization pump 1.

[0026] At this time, a flow path resistance 4 is installed within the device. In Fig. 1, the flow path resistance 4 is indicated by a symbol. Opposite the pressurizing port 6 is an atmospheric pressure release port 5. In addition, a temperature controller 8 (first temperature controller) for controlling the temperature of the device is installed below the bottom surface 7 of the device.

[0027] Figure 1(b) shows a graph of pressure changes in the direction from port 6 to port 5 in the chip in this sample temperature control system. When there is no flow path resistance 4, the pressure from the pressurizing pump 1 decreases to atmospheric pressure toward port 5, which is open to atmospheric pressure, as shown by the dashed line. On the other hand, when there is flow path resistance 4, the pressure from the pressurizing pump 1 is maintained at approximately the same value up to the position where the flow path resistance exists, as shown by the solid line, and once it passes the position of the flow path resistance, the pressure decreases to atmospheric pressure. Therefore, the sample solution 3 confined in the well in the device continues to be pressurized by the pressurizing pump 1.

[0028] When the sample solution 3 confined in the well is subjected to pressure, its boiling point increases and its saturated vapor pressure rises. This is known from the Clausius-Clapeyron equation. Therefore, in this system, when the temperature is controlled to around 100°C during PCR, bubbles caused by evaporation are prevented from forming in the sample solution 3 confined in the well. As a result, the implementation of this system helps prevent the connection (contamination) of samples in multiple adjacent wells.

[0029] This system not only implements flow resistance in a microfluidic device with over 5,000 wells, but also suppresses air bubbles by using a system that controls temperature while applying pressure, which is a process unique to digital PCR, in the temperature control within each well. This does not require the implementation of valves, and can be realized with a simple configuration by simply leaving the atmospheric pressure release port 5 open to atmospheric pressure in relation to the pressurization port 6.

[0030] In this case, a syringe pump, a diaphragm pump, a compressor, etc. can be used as the pressurizing pump 1. The material of the device may be glass, resin (cycloolefin polymer, polypropylene, polymethyl methacrylate, polycarbonate, etc.), semiconductor (silicon, etc.), metal (copper, zinc, etc.), etc. A cartridge heater, a Peltier element, etc. can be used as the temperature regulator 8.

[0031] As shown in Figure 1(a), examples of the separating liquid 2 include photocurable resin and oil. In particular, liquids that are incompatible with water are suitable because they can avoid mixing with the sample solution 3. Oil is an example of a suitable separating liquid because it has low compatibility with water. Other examples of oil include silicone oil, mineral oil, and Fluorinert oil.

[0032] The flow path resistance has several elements. The flow path resistance will be explained using Equation 1. Δp=32 μLu / (d ２ ) ... (Equation 1) This equation is the flow path resistance (pressure loss) of laminar flow in a pipe. Δp is the flow path resistance, μ is the viscosity, L is the length, u is the average flow velocity, and d is the diameter of the pipe. According to Equation 1, in order to increase the flow path resistance, the viscosity, length, and average flow velocity parameters can be increased, or the diameter of the pipe can be reduced.

[0033] 2(a) to 2(c) show examples in which the above-mentioned elements are applied to the flow path resistance 4 shown in FIG. 1. The device includes multiple wells (multiple chambers) for storing sample solutions. The device also includes flow paths 9, which connect the pressurization port 6, the atmospheric pressure release port 5, and the multiple wells. In particular, the flow paths 9 include a first flow path 9a connecting the pressurization port 6 and the multiple wells, and a second flow path 9b connecting the atmospheric pressure release port 5 and the multiple wells.

[0034] The flow path resistance of the second flow path 9b is greater than the flow path resistance of the first flow path 9a. Therefore, when the pressurizing port 6 is pressurized, the pressure applied to the first flow path 9a is greater than the pressure applied to the second flow path 9b. This suppresses the generation of bubbles in the first flow path 9a.

[0035] 2A, a highly viscous separation liquid 100 is placed at the port 5 that is open to atmospheric pressure. Therefore, the flow path resistance is increased by the viscosity parameter μ.

[0036] 2B, the length 102 between the atmospheric pressure release port 5 and the well 101 closest to the atmospheric pressure release port 5 is increased. Therefore, the flow path resistance is increased by the length parameter L.

[0037] 2(c), the flow path between the atmospheric pressure release port 5 and the well 101 closest to the atmospheric pressure release port 5, i.e., the flow path diameter 103 of the second flow path 9b, is narrowed. Therefore, the flow path resistance is increased by the piping diameter parameter d.

[0038] The Reynolds number is an indicator of a highly viscous separation liquid 100. This will be explained using Equation 2. Re=ρvl / μ (Equation 2) Re is the Reynolds number, ρ is density, v is representative flow velocity, l is length, and μ is viscosity. In this case, l corresponds to the diameter of the flow path in the device or the diameter of the port 5 that is open to atmospheric pressure.

[0039] The Reynolds number is a measure of the effect of viscosity on a flow. Therefore, a large Reynolds number for a separating liquid means that it moves easily, and a small Reynolds number means that it moves less easily. In other words, taking into account the flow path resistance mentioned above, the smaller the Reynolds number, the greater the flow path resistance.

[0040] Generally, a Reynolds number below 2300 indicates laminar flow, and a Reynolds number above 4000 indicates turbulent flow. Values ​​in between are called transitional flows. A guideline for the highly viscous separation liquid 100 in this system is that it should be laminar, with a viscosity that results in a Reynolds number below 2300.

[0041] It is also effective to use a photocurable resin for the separation liquid 2. After introducing the photocurable resin into the flow path, it is possible to change the viscosity by photocuring. Therefore, the photocurable resin can be introduced in a low viscosity state, and can be photocured after introduction to increase the viscosity.

[0042] The degree of viscosity correlates with the glass transition temperature (Tg) of the photocurable resin. When a photocurable resin with a Tg above 0°C is photocured, it becomes as hard as a resin plate in a normal dPCR environment (around room temperature). On the other hand, when a photocurable resin with a Tg below 0°C is photocured, it becomes gel-like in a normal dPCR environment, as shown in Figure 1(a). Thus, a more suitable viscosity can be achieved by using a photocurable resin with a Tg below 0°C.

[0043] Here is an example of using these photocurable resins. When a photocurable resin is introduced regardless of Tg, only the photocurable resin in the same position as the high-viscosity separating liquid 100 shown in Figure 2(a) is photocured. This makes it possible to generate flow path resistance in the vicinity of the atmospheric pressure release port 5 and in the flow path up to the well.

[0044] As described above, it is possible to increase the flow path resistance by several factors. The above are only examples, and it is also possible to increase the flow path resistance by other factors such as bending loss, branching loss, exit loss, and unevenness on the flow path surface, and devices using these can also be constructed.

[0045] Figure 3 shows the shape of a digital PCR device composed of channels and wells. Figures 3(a) and 3(b) show a portion of the device at low and high magnification, respectively.

[0046] The microchannel actually used has an inlet portion 200 (first port), a channel 201, and a plurality of wells 202 (a plurality of chambers). Note that the outlet portion is not specifically shown in FIG.

[0047] A sample solution is introduced from an inlet 200, passes through a flow path 201, and is introduced into a well 202. The size of the well 202 may range from the nanometer scale to the micrometer scale, and the shape may be various, such as a square, a circle, or a hexagon. Furthermore, the number of wells 202 is at least one, and at most 3 million.

[0048] In this device, flow resistance can be generated by the branching configuration to each well, the width of the microchannel itself, the configuration of the curved section, etc. This allows the sample solution in each well to be pressurized, making it possible to suppress bubbles.

[0049] In Figure 4, Figure 4(a) shows the digital PCR device after the sample solution has been introduced, Figure 4(b) shows the digital PCR device after the separation solution has been introduced, and Figure 4(c) shows that digital PCR is confirmed by temperature control of the thermal cycle.

[0050] As shown in Figure 4(a), when a sample is introduced, the multiple wells 300 (multiple chambers) and flow channel 301 of the digital PCR device are filled with sample solution 3. Then, as shown in Figure 4(b), a separation liquid 2 is introduced from a pressurizing port 6 using a pressurizing pump 1. At that time, the sample solution present in the flow channel 301 in the device is replaced with the separation liquid 2. In this way, the separation liquid 2 that divides the sample solution flows through the flow channel 301.

[0051] The sample solution is then divided into individual wells 300 by the separation liquid 2. After sealing the wells 300 with the separation liquid, the temperature control system photo-cures the photocurable resin before reacting the sample solution. In this way, sealing and pressurization can be performed appropriately. Note that the configuration for photo-curing the photocurable resin (such as a light irradiation means and its control means) can be designed appropriately by those skilled in the art.

[0052] 4(c) shows a state in which the PCR temperature is controlled by applying pressure through the pressure port 6 using a pressure pump 1 in the same device configuration as in FIG. 4(b). After sealing the well 300 with separation liquid 2, the temperature control system reacts the sample solution in the well 300 using a temperature controller 8 while applying pressure through the pressure port 6.

[0053] Thus, this embodiment relates to a method of using a temperature control system for a sample solution in digital PCR, and this method includes a step of sealing the well 300 with a separation liquid 2, and then a step of reacting the sample solution in the well 300 using a temperature controller 8 while applying pressure to the pressure port 6.

[0054] The pressurizing pump 1 can pressurize the device when introducing the separation liquid 2 into the device and when reacting the sample solution. In this way, the pressurizing pump 1 used for introducing the separation liquid 2 can also be used for suppressing air bubbles, which is efficient.

[0055] At this time, the presence of flow path resistance 4 within the device keeps the sample solution in the wells bubble-suppressed, so the sample solution does not flow into separation liquid 2 present in flow path 301.

[0056] In the evaluation of digital PCR, fluorescence observation is performed after the PCR temperature is adjusted. During PCR, PCR is promoted in wells where nucleic acids are present, and PCR does not occur in wells where nucleic acids are not present. By introducing a fluorescently labeled probe into the sample solution in advance, only wells where PCR has occurred emit light during fluorescence measurement.

[0057] 4(c), glowing (positive) wells 302 and non-glowing (negative) wells 303 are observed in the device during fluorescence observation. From this result, it is determined that digital PCR was performed without any problems.

[0058] In this way, in this system, the system configuration after sample division is the same as that during PCR, and there is no need to add a new device for PCR, etc. Therefore, the system configuration is inexpensive and can be said to be simple in terms of the system configuration and the process of operating the system.

[0059] Figure 5 shows the results of digital PCR observed using this system. Figure 5(a) shows a schematic cross-sectional view similar to Figure 4. The fluorescent image shown in Figure 5(b) shows that illuminated (positive) wells 401 and non-illuminated (negative) wells 402 were observed in the actual measurement. This shows that digital PCR was performed without any problems, proving the effectiveness of this system.

[0060] Example 2 Example 2 will be described below with reference to Figures 6 and 7. Example 2 shows the measurement results of digital PCR observed when the flow path resistance is insufficient and measures to be taken to reduce the flow path resistance.

[0061] For reference, Figure 6 shows the results of digital PCR observed when the flow path resistance is insufficient. Figure 6(a) shows a schematic cross-sectional view similar to Figure 4, and Figure 6(b) shows an example of a fluorescent image.

[0062] When the flow path resistance 500 is insufficient during PCR temperature control, contaminated wells 501 occur in wells close to the port 5 on the atmospheric pressure release side. This occurs because pressurization is not properly applied to the wells due to insufficient flow path resistance 500, and bubbles are not suppressed, causing the sample solution to leak into the flow path. In fact, a region 502 where contamination has occurred can be confirmed in the digital PCR image.

[0063] In this way, if the wells are connected to each other, the advantage of highly accurate digital PCR, which allows absolute quantification, is lost. That is, insufficient flow path resistance 500 results in a lack of high accuracy of digital PCR.

[0064] 7(a) and (b) show examples of a device according to this embodiment that does not introduce insufficient flow path resistance. In Fig. 7(a), a flow path 602 (second flow path) from an outlet portion 600 (second port) on the opposite side of the inlet portion 200 (first port) to the well 601 closest to that port is made longer. In Fig. 7(b), a flow path 603 (second flow path) from an outlet portion 600 on the opposite side of the inlet portion 200 to the well 601 closest to that port has a narrower flow path diameter.

[0065] From the viewpoint of molding a microchannel chip, it is easy to form the channel 602 in a tournament or serpentine shape. Therefore, from the viewpoint of channel resistance, it is preferable to form the channel 602 in a tournament or serpentine shape with a total length longer than 0.1 mm, and from the viewpoint of device size, it can be said that it is easy to use if the channel 602 is shorter than 30 mm.

[0066] On the other hand, in terms of processing accuracy, the limit of the diameter of the flow channel 603 is several hundred nanometers to several tens of micrometers. This is a numerical value in terms of width, but the limit is also several hundred nanometers to several tens of micrometers in terms of depth.

[0067] In this system, the diameter and depth of the channel of the microdevice are preferably smaller than 100 μm. Furthermore, when comparing the diameters and depths of the channel 201 and the channel 603, it is preferable that the diameter and depth of the channel 603 be equal to or smaller than the diameter and depth of the channel 201. Similarly, when comparing the overall lengths of the channel 201 and the channel 602, it is preferable that the overall length of the channel 602 be equal to or greater than that of the channel 201.

[0068] Example 3 Example 3 will be described below with reference to Fig. 8. Fig. 8 is a diagram illustrating that it is possible to generate local flow path resistance simply by using high viscosity oil, without devising the shape of the flow path or the like.

[0069] The main differences from the system shown in Figure 1 are that the separation liquid flowing through the flow path is separation liquid 701 whose viscosity is temperature dependent, and that the temperature regulator does not heat the area around the atmospheric pressure release port 5, but rather regulates the temperature of only the pressurization port 6 and the well, being temperature regulator 700 (first temperature regulator).

[0070] An overview of this system will be explained using Figure 8(a). A pressure pump 1 is used to introduce a separation liquid 701, whose viscosity is temperature-dependent, through a pressure inlet 6. At this time, to facilitate the introduction of the temperature-dependent viscosity 701, the pressure inlet 6 and the well may be heated to approximately 30 to 80°C by a temperature regulator 700 that regulates the temperature of only the pressure inlet 6 and the well. That is, in one example, when the separation liquid 701 is introduced into the device, the temperature regulator 700 heats the separation liquid 701 to 30 to 80°C.

[0071] After the separation liquid has advanced through the flow path to the atmospheric pressure release port 5, PCR temperature control begins using the pressurization port 6 and the temperature regulator 700, which controls the temperature of only the wells. At this time, the pressurization pump 1 continues to apply pressure in the same manner as in Example 1.

[0072] The separation liquid 701, which is present in the region from the pressurization port 6 to the well and has a temperature-dependent viscosity, has a low viscosity due to this temperature control, making it easier to apply pressure from the pressurization pump 1. Therefore, the generation of bubbles in the sample solution 3 in the well is suppressed during PCR temperature control.

[0073] On the other hand, the temperature of the second flow path 9b (see FIG. 2) is lower than the temperature of the temperature regulator 700. Therefore, in the vicinity of the atmospheric pressure release port 5, which is not heated by the temperature regulator, the separation liquid 701, whose viscosity is temperature-dependent, is close to room temperature because the temperature is not regulated, and becomes a highly viscous separation liquid 702. Therefore, in this region, the pressure of the pressurizing pump 1 is suddenly subjected to flow path resistance and changes to atmospheric pressure.

[0074] In fact, it was confirmed that digital PCR can be performed normally using this system, and the results are shown in FIG. 8(b).

[0075] Unlike Example 1, the advantage of this example is that there is no need to generate flow path resistance by narrowing or lengthening the flow path. In digital PCR, lengthening the flow path leads to the waste of more valuable sample solution (increased dead volume). Furthermore, narrowing the flow path has the disadvantage of taking longer to introduce the sample and separation liquid. On the other hand, this example can be achieved simply by using a separation liquid whose viscosity is temperature-dependent and by using a temperature controller.

[0076] The separation liquid, whose viscosity is temperature-dependent, can be any liquid that is immiscible (compatible) with water, such as oil or photocurable resin. A good viscosity guideline is between 50 mPa·s and 500 mPa·s at room temperature. As shown in Figure 8(a), by setting the viscosity of the separation liquid within the range of 50 mPa·s to 500 mPa·s, appropriate fluidity can be ensured. The upper limit guideline is calculated from the time for which the separation liquid is introduced.

[0077] Here, the relationship between flow rate, pressure, and viscosity will be explained using Equation 3 and Equation 4. (Flow rate) ∝ (Differential pressure) １／２ ... (Formula 3) (Flow rate) ∝ 1 / Viscosity ... (Formula 4)

[0078] Suppose that separation liquid can be introduced into a device under certain conditions. If the separation liquid is changed to one with four times the viscosity and the same flow rate must be applied to the device, the pressure on the device and separation liquid will be 16 times higher than normal. In other words, the pressure required for introduction is proportional to the square of the viscosity. It can also be understood that as the viscosity increases, the flow rate decreases in inverse proportion to the viscosity. In other words, the higher the viscosity, the longer it takes to introduce the liquid, or it may not be possible to introduce it at all.

[0079] If there is no upper limit to the time, the separating liquid may be introduced while being heated up to about 2000 mPa·s. On the other hand, the lower limit is a condition for ensuring a sufficient pressure gradient.

[0080] 8, no temperature regulator is provided at the atmospheric pressure release port 5 or the second flow path 9b, which simplifies the configuration of the temperature regulation system.

[0081] Figure 9 shows a sample temperature control system that combines two temperature controllers, which is a derivative of the temperature controller in Figure 8. In order to partially turn the temperature-dependent viscosity of the separation liquid 701 into a high-viscosity separation liquid 702, a partial heating system was used in Figure 8, but in Figure 9, a new temperature controller 800 (second temperature controller) is installed to control the temperature of the device.

[0082] The temperature regulator 700 is installed in the first flow path 9a (see FIG. 2(a)) (and in the example of FIG. 9, the port 6 for pressurization and the well), and the temperature regulator 800 is installed in the second flow path 9b (and in the example of FIG. 9, the port 5 for atmospheric pressure release). The temperature regulator 800 makes it possible to cool to a temperature below room temperature, thereby enabling a lower temperature and higher viscosity, and improving the degree of flow path resistance. In other words, the temperature regulator 700 functions as a heater, and the temperature regulator 800 functions as a cooler.

[0083] It is also effective to use a photocurable resin for the separation liquid 701. After introducing the photocurable resin into the flow path, the viscosity can be changed by photocuring. Therefore, the photocurable resin can be introduced in a low viscosity state, and can be photocured after introduction to increase the viscosity.

[0084] The degree of viscosity correlates with the glass transition point (Tg) of the photocurable resin; photocurable resins with a Tg above 0°C become hard like a resin plate, while photocurable resins with a Tg below 0°C become gel-like.

[0085] An example of using these photocurable resins is as follows. When a photocurable resin is introduced regardless of Tg, it is possible to photocur only the photocurable resin at the same position as the highly viscous separation liquid 702. This makes it possible to generate flow path resistance in the second flow path 9b.

[0086] Of course, Example 3 is merely an example, and various modifications are possible. For example, taking into consideration Example 1, etc., the accuracy of the flow channel resistance may be improved by combining the shape of the flow channel, a separation liquid whose viscosity is temperature-dependent, a temperature regulator that regulates the temperature of only the pressurizing port and the well, etc.

[0087] Example 4 will be described below with reference to Figures 10, 11, and 12. These figures explain the possibility that the sample solution may flow back from the port that is released to atmospheric pressure depending on the viscosity of the separation liquid used.

[0088] Figure 10 is a reference schematic diagram showing the process of sample solution backflow. Temperature control during PCR causes expansion and contraction of the device and expansion and contraction of air bubbles present in the atmospheric pressure release port. These expansions and contractions are the main cause of the generation of a mixed solution 900 (see Figure 10(a)) of the sample solution and separation solution present in the atmospheric pressure release port 5, which is drawn into the device. As a result, wells 901 in which the sample solutions are connected to each other are created, as shown in Figure 10(b).

[0089] One solution to prevent this sample backflow is to prevent the generation of a mixed solution 900 of the sample solution and the separation liquid. In order to prevent the generation of a mixed solution, the Reynolds number shown in Equation 2 above can be used. The Reynolds number is a measure that represents the effect of viscosity on a flow. Therefore, a large Reynolds number for the separation liquid means that it moves easily, and a small Reynolds number means that it moves less easily. In other words, the smaller the Reynolds number, the more likely the separation liquid is to form a laminar flow and the more difficult it is to mix.

[0090] Generally, a Reynolds number lower than 2300 indicates laminar flow, and a Reynolds number higher than 4000 indicates turbulent flow. Values ​​between these are called transitional flows. In this embodiment, it is desirable for the separation liquid 2 to be a laminar flow, and therefore a viscosity that results in a Reynolds number lower than 2300 is desirable.

[0091] It is also effective to use a photocurable resin for the separation liquid 2. After introducing the photocurable resin into the flow path, it is possible to change the viscosity by photocuring. Therefore, the photocurable resin can be introduced in a low viscosity state, and can be photocured after introduction to increase the viscosity.

[0092] The degree of viscosity correlates with the glass transition point (Tg) of the photocurable resin; photocurable resins with a Tg above 0°C become hard like a resin plate, while photocurable resins with a Tg below 0°C become gel-like. Therefore, a similar effect can be achieved by using a photocurable resin that becomes highly viscous upon photocuring. For example, photocurable resins with a Tg below 0°C have a viscosity close to that of a gel. Therefore, the Reynolds number is lower than 2300, resulting in a laminar flow.

[0093] Figure 11 shows a graph of the change in viscosity of the separated liquid with temperature, where (a) and (b) show separated liquids with low and high viscosities at room temperature, respectively.

[0094] As shown in Figure 10, the viscosity of the separation liquid present at the atmospheric pressure release port 5 decreases as it is heated during PCR temperature control. Therefore, the lower viscosity increases the Reynolds number, making it more likely to mix with the sample solution. Therefore, to prevent mixing with the sample solution, it is preferable for the viscosity of the separation liquid to be high at room temperature, as shown in Figure 11(b), not only at room temperature but also during PCR temperature control. In the example of Figure 11(b), the viscosity of the separation liquid at room temperature (e.g., 25°C) is 300 cst, and the viscosity of the separation liquid during PCR is 60 to 100 cst.

[0095] 12(a) and 12(b) are schematic diagrams showing the state of the sample solution and the separated liquid present at the atmospheric pressure release port 5 when the separation liquids with low and high viscosities at room temperature shown in FIGS. 11(a) and 11(b) are used. When the separation liquid shown in FIG. 11(b) is used, the separated liquid forms a laminar flow at the atmospheric pressure release port 5 as shown in FIG. 12(b).

[0096] To summarize the above, to prevent the sample solution and the separation liquid from mixing, it is desirable for the separation liquid to be in a laminar flow state, and it is preferable that this condition also be met when heated during PCR. This makes it possible to prevent backflow of the sample solution. Furthermore, by adjusting the temperature control cycle conditions during PCR in consideration of the viscosity of the separation liquid that results in laminar flow, it is expected that the effectiveness of preventing backflow can be enhanced. For example, this could involve slowing down the temperature cycle during the temperature rise and / or fall of PCR overall (<10°C / sec), or slowing down the temperature cycle during temperature fall compared to the temperature rise. These measures contribute to lowering (slowing down) the degree (speed) of expansion and contraction of bubbles, etc., thereby reducing the amount of the mixed solution 900 of sample solution and separation liquid drawn into the device.

[0097] [Embodiment 5] Hereinafter, embodiment 5 will be described with reference to Figures 13 and 14. These figures are diagrams for explaining the configuration of the atmospheric pressure release port and the pressurization port.

[0098] Figure 13 shows an example of the shape of the pressurizing port 6. Before pressurizing the pressurizing port 6, the separated liquid 2 is poured into the port. At this time, it is desirable to prevent air from getting in when the separated liquid is poured. Therefore, it is preferable to provide the pressurizing port 6 with a tapered portion 6a so that the shape makes it difficult for air to get in.

[0099] Furthermore, the pressurizing port 6 may be surface-treated in advance so as to reduce the contact angle between the separation liquid 2 and the wall surface of the pressurizing port 6. Measures to prevent air contamination can also be taken by the method of injecting the separation liquid 2, rather than by the structure of the pressurizing port 6. For example, the separation liquid dispenser 1000 may be designed so that the separation liquid 2 runs down the wall surface of the pressurizing port 6 when being injected.

[0100] FIG. 14 is a diagram illustrating a configuration for preventing the sample solution from eluting out of the device at the atmospheric pressure release port 5.

[0101] In this embodiment, the system is designed to continuously apply pressure during temperature control during PCR. As a result, the separation solution and the sample solution present inside it are constantly being pressed. If at any moment the sample solution adheres (contaminates) to the outside of the device, for example, the experimental environment or the inside of the equipment, this can lead to carryover. In other words, if a similar measurement is performed using a different device after such contamination, the measurement data will be inaccurate. Therefore, it is desirable to avoid exposing the sample solution as much as possible at the atmospheric pressure release port.

[0102] 14(a) to 14(e) are possible configurations. These show how a separation liquid 1100 and a sample solution 1101 are pooled in the port 5 that is released to atmospheric pressure after PCR. By taking measures above the sample solution while it is released to atmospheric pressure, it is possible to prevent it from leaking out of the device.

[0103] 14(a) shows an example in which oil 1102 is placed above a sample solution 1101. This oil may be the same as the separation liquid 1100. It is preferable that the oil be lighter than the sample solution, and it is particularly preferable that the oil has a lower density than the sample solution. It is also preferable that the oil be hydrophobic to avoid mixing with the sample solution.

[0104] 14(b) shows a case in which a photocurable resin 1103 is placed on top of a sample solution 1101. It is preferable that this photocurable resin 1103 also has a lower density than the sample solution 1101. It is also preferable that the photocurable resin is hydrophobic and does not mix with the sample solution. The photocurable resin 1103 can be hardened after PCR and used as a solid lid.

[0105] 14C shows the installation of a gapped lid 1104. Pressure release caps are commercially available, so a lid with such a configuration may be installed.

[0106] 14(d) shows the installation of a breathable film 1105. Examples of breathable films include PTFE and PDMS, and it is desirable that the breathable film has water repellency.

[0107] 14(e) shows the installation of an absorbent material 1106. The absorbent material may be absorbent cotton or the like, and absorbs excess sample solution 1101 while ensuring breathability.

[0108] Ideally, the lid, breathable film, absorbent material, oil, and photocurable resin should be installed before the start of PCR. There are various methods for timing of installation.

[0109] In the first example, a sample solution is introduced through the atmospheric pressure release port 5. Oil or photocurable resin may be placed on the sample solution in advance. In this state, a vacuum may be drawn through the pressurization port 6, and the sample may be introduced from the atmospheric pressure release port 5 toward the pressurization port 6. Alternatively, a pressurization pump may be connected to the atmospheric pressure release port 5, and the sample may be introduced from the atmospheric pressure release port 5 toward the pressurization port 6. After the sample solution is introduced, a separation liquid may be introduced through the pressurization port 6, thereby achieving the configurations shown in FIGS. 14( a) and 14(b).

[0110] It is also possible to introduce the sample solution through the pressurizing port 6. In this case, oil or photocurable resin, sample solution, and separation liquid are placed in the pressurizing port 6 from the bottom, and the sample is introduced through the pressurizing port 6 using a pressurizing pump, and then the sample is divided, thereby achieving the configurations shown in Figures 14(a) and 14(b).

[0111] 14(a) and 14(b), the temperature control system can place photocurable resin 1103 or oil 1102 in the atmospheric pressure release port 5 after sealing the well with separation liquid 1100 and before reacting with sample solution 1101. This allows pressure control to be performed at the appropriate timing.

[0112] The temperature control system can be configured to automatically place the photocurable resin 1103 or the oil 1102. Specific means for placing the photocurable resin 1103 or the oil 1102 (such as a fluid supply means and its control means) can be designed appropriately by a person skilled in the art.

[0113] 14(c) to (e), the temperature control system can install a lid 1104 (pressure release cap), a water-repellent breathable film 1105, or an absorbent material 1106 on the atmospheric pressure release port 5 after sealing the well with the separation liquid 1100 and before reacting the sample solution 1101. This allows pressure control to be performed at an appropriate timing.

[0114] The temperature control system can be configured to automatically install the lid 1104, the breathable film 1105, or the absorbent material 1106. Specific means for installing these members in the atmospheric pressure release port 5 (such as a gripping means, a transporting means, and a control means for these) can be appropriately designed by a person skilled in the art.

[0115] Example 6 Example 6 will be described below with reference to Fig. 15. Fig. 15 is a diagram illustrating that the melting temperature of a sample solution can be analyzed using this system.

[0116] One DNA detection method using digital PCR involves measuring the melting temperature of a droplet containing DNA and a fluorescently labeled probe that hybridizes to the DNA. Figure 15(a) shows the configuration of an apparatus for performing melting temperature analysis.

[0117] An optical system for fluorescence measurement is prepared above the temperature control system. For fluorescence measurement, light of the required wavelength is extracted from excitation light 1200 by an excitation light filter 1201, and the extracted light is focused onto a sample by a lens 1202. A dichroic half mirror 1203 transmits the light extracted from the excitation light 1200 and reflects the fluorescence emitted from the sample. The fluorescence from the sample passes through a fluorescence filter 1204 and is then imaged onto a photodetector 1206 by an imaging lens 1205.

[0118] In this fluorescence measurement, the melting temperature of DNA can be calculated by acquiring a fluorescence image while the temperature regulator 8 is heating or cooling at a constant ramp rate.

[0119] FIG. 15(b) shows an example of a graph of temperature and change in fluorescence intensity in melting temperature analysis. When the change in fluorescence intensity is acquired while heating, a monotonic sigmoid curve is obtained. The inflection point of the sigmoid curve corresponds to the melting temperature of DNA. Therefore, by differentiating the fluorescence intensity with respect to temperature based on the sigmoid curve (FIG. 15(c)), the melting temperature 1207 can be calculated as the peak value.

[0120] In this way, this sample temperature control system can also be used in combination with an optical system. Temperature control in melting temperature analysis differs from that in PCR in that the sample is not heated to nearly 100°C. Therefore, the pump mechanism for pressurization, etc., can be removed during fluorescence measurement.

[0121] In this way, the temperature control system can perform fluorescence measurement (for example, melting temperature measurement) accompanied by temperature control using the temperature controller 8. This allows the fluorescence measurement to be performed appropriately.

[0122] The present invention is not limited to the above-described embodiments, but 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.

[0123] DESCRIPTION OF SYMBOLS 1...Pressure pump 2...Separation liquid 3...Sample solution 4...Flow path resistance 5...Atmospheric pressure release port (second port) 6...Pressure port (first port) 6a...Tapered portion 7...Device bottom 8...Temperature controller (first temperature controller) 9...Flow path 9a...First flow path 9b...Second flow path 100...Highly viscous separation liquid 101...Well closest to the atmospheric pressure release port 102...Length between the atmospheric pressure release port and the well closest to that port 103...Flow path diameter between the atmospheric pressure release port and the well closest to that port 200...Inlet portion (first port) 201...Flow path 202...Well (multiple chambers) 300...Well (multiple chambers) 301...Flow path 302...Glowing (positive) well 303...Negative well 401...Glowing (positive) well in actual measurement 402... Well that does not glow in actual measurement (negative) 500... Insufficient flow path resistance 501... Well in which contamination has occurred 502... Area in which contamination has occurred 600... Outlet portion (second port) 601... Well closest to outlet portion 602... Flow path from outlet portion to well closest to outlet portion (second flow path) 603... Flow path from outlet portion to well closest to outlet portion (second flow path) 700... Temperature controller (first temperature controller) 701... Separating liquid whose viscosity is temperature-dependent 702... Highly viscous separating liquid 800... Temperature controller (second temperature controller) 900... Mixed solution of sample solution and separating liquid 901... Well in which sample solutions are connected to each other 1000... Dispenser 1100... Separating liquid 1101... Sample solution 1102... Oil 1103... Photocurable resin 1104: Lid (pressure release cap) 1105: Breathable film 1106: Absorbent material 1200: Excitation light 1201: Excitation light filter 1202: Lens 1203: Dichroic half mirror 1204: Fluorescence filter 1205: Imaging lens 1206: Photodetector 1207: Melting temperature

Claims

1. A temperature control system for a sample solution used in digital PCR, wherein the temperature control system comprises a device, the device comprising: a first port to be pressurized; a second port in an open state to the atmosphere; a plurality of chambers for storing the sample solution; and a flow path connecting the first port, the second port, and the plurality of chambers, wherein a separation liquid for dividing the sample solution flows through the flow path, the flow path comprising: a first flow path connecting the first port and the plurality of chambers; and a second flow path connecting the second port and the plurality of chambers, the temperature control system further comprising: a pressurizing pump for pressurizing the device; and a first temperature controller for controlling the temperature of the device, wherein the flow path resistance of the second flow path is greater than that of the first flow path, and the temperature control system reacts the sample solution in the plurality of chambers using the first temperature controller while pressurizing the first port after sealing the plurality of chambers with the separation liquid. A temperature control system characterized by the above.

2. The temperature control system according to claim 1, wherein the separation liquid has a temperature-dependent viscosity.

3. The temperature control system according to claim 2, wherein when the separation liquid is introduced into the device, the first temperature controller heats the separation liquid to 30°C to 80°C.

4. The temperature control system according to claim 1, wherein the pressure applied to the first flow path is greater than the pressure applied to the second flow path.

5. The temperature control system according to claim 1, wherein the temperature of the second flow path is lower than the temperature of the first temperature controller.

6. The temperature control system according to claim 5, wherein no temperature controller is installed in the second port and the second flow path.

7. The temperature control system according to claim 5, wherein the temperature control system comprises a second temperature controller for controlling the temperature of the device, the first temperature controller is installed in the first flow path, and the second temperature controller is installed in the second flow path.

8. The temperature control system according to claim 1, wherein the pressurizing pump is capable of pressurizing the device when introducing the separation liquid into the device and when reacting the sample solution.

9. The temperature control system according to claim 1, wherein the separation liquid becomes laminar flow at the second port.

10. The temperature control system according to claim 9, wherein the viscosity of the separation liquid is in the range of 50 mPa·s to 500 mPa·s.

11. The temperature control system according to claim 1, wherein the separation liquid is immiscible with water.

12. The temperature control system according to claim 11, wherein the separation liquid is oil.

13. The temperature control system according to claim 11, wherein the separation liquid is a photocurable resin.

14. The temperature control system according to claim 13, wherein the photocurable resin has a glass transition point of 0 °C or lower.

15. The temperature control system according to claim 13, wherein the photocurable resin is photocured before the sample solution is reacted.

16. The temperature control system according to claim 1, wherein a photocurable resin or oil is installed at the second port before the sample solution is reacted.

17. The temperature control system according to claim 16, wherein the photocurable resin or oil has a density lower than that of the sample solution and is hydrophobic.

18. The temperature control system according to claim 1, wherein a pressure release cap, a water-repellent breathable film, or an absorbent is installed at the second port before the sample solution is reacted.

19. The temperature control system according to claim 1, wherein it is possible to perform fluorescence measurement with temperature control using the temperature controller.

20. A method of using a temperature control system for a sample solution in digital PCR, wherein the temperature control system comprises a device, the device comprising: a first port to be pressurized; a second port in an open state to the atmosphere; a plurality of chambers for storing the sample solution; and a flow path connecting the first port, the second port, and the plurality of chambers, wherein a separation liquid for dividing the sample solution flows through the flow path, the flow path comprising: a first flow path connecting the first port and the plurality of chambers; and a second flow path connecting the second port and the plurality of chambers, the temperature control system further comprising: a pressurizing pump for pressurizing the device; and a first temperature controller for controlling the temperature of the device, wherein the flow path resistance of the second flow path is greater than that of the first flow path, the method comprising: sealing the plurality of chambers with the separation liquid; and then reacting the sample solution in the plurality of chambers using the first temperature controller while pressurizing the first port.

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