Amplification device and method for digital droplet PCR (Polymerase Chain Reaction) and microfluidic system

Abstract

The invention provides an amplification device and method for digital droplet PCR and a microfluidic system, the amplification device is provided with two independent temperature control modules to cooperate with a driving structure to realize alternate contact of a microfluidic chip, and the problem that a single temperature control module needs to wait for temperature rise and fall in the amplification process can be effectively avoided; when one temperature control module carries out temperature treatment of the current stage on the micro-fluidic chip, the other temperature control module can synchronously complete temperature adjustment of the next stage, so that temperature changes of the two temperature control modules and alternate contact of the micro-fluidic chip form efficient cooperation, the overall time length of single amplification circulation is remarkably shortened, and the amplification efficiency of the micro-fluidic chip is improved. The PCR amplification efficiency is improved; meanwhile, the micro-fluidic chip can be stably fixed by the object placing table and the clamping assembly, and circulating temperature control of denaturation, annealing and extension can be completed without exporting liquid drops, so that the PCR amplification process is simplified, the risk of liquid drop breakage is reduced, and the detection accuracy is improved.

Description

Technical Field

[0001] This application relates to the field of microfluidic detection technology, specifically to an amplification device, method, and microfluidic system for digital droplet PCR. Background Technology

[0002] PCR is a rapid and economical DNA amplification technique. Before the reaction, template DNA, primers, DNA polymerase, deoxyribonucleotide triphosphates (DRPs), and a buffer solvent are combined in a mixture. After a series of thermal cycles, millions of copies of template DNA are generated. The denaturation phase involves heating to break the hydrogen bonds in the double-stranded DNA, resulting in two single-stranded DNA molecules. The annealing phase lowers the temperature to allow the primers to bind to the target sequence. In the extension phase, in the presence of free DTPs, the polymerase extends the incomplete DNA sequence, synthesizing new double-stranded DNA. By repeating the denaturation, annealing, and extension steps, typically for 25 to 40 cycles, the target DNA can be amplified to millions or even billions of copies, making it widely used in genetic engineering, genotyping, and sequencing.

[0003] The most widely used existing PCR amplification method involves placing centrifuge tubes containing samples into commercially available PCR amplification instruments. The amplification effect is achieved by the instrument actively controlling the temperature rise and fall. The amplification effect is closely related to the accuracy, uniformity, and heating / cooling rate of the amplification instrument. In particular, a high average heating / cooling rate is needed during amplification to shorten the reaction time, reduce the possibility of non-specific binding, and improve the specificity of the PCR reaction. Currently, the average temperature change rate of commercial instruments is generally between 1.5℃ / s and 4℃ / s. Furthermore, in existing technologies, droplets need to be exported from the microfluidic chip to a container before being placed into the amplification instrument for thermal cycling. During the export process, the droplets are prone to breakage due to pressure changes, affecting the accuracy of PCR detection. Summary of the Invention

[0004] The purpose of this application is to address the above-mentioned problems by providing an amplification device, method, and microfluidic system for digital droplet PCR.

[0005] In a first aspect, this application provides an amplification apparatus for digital droplet PCR, comprising: A base plate, with a top plate disposed above the base plate, the top plate being parallel to the base plate; an accommodating space is formed between the top plate and the base plate; A storage platform is provided within the accommodating space and is fixedly connected to the base plate. A clamping assembly is provided on the top of the storage platform for fixing the microfluidic chip. A heat dissipation module is fixedly installed on the top of the top plate. The top plate has a first through hole in the middle. Two temperature control modules are arranged along a first direction at the bottom of the heat dissipation module, and the two temperature control modules are located in the first through hole. The first direction is parallel to the length direction of the top plate. A driving structure is disposed within the accommodating space. The two ends of the driving structure are respectively connected to the bottom plate and the top plate. The driving structure is used to drive the top plate to reciprocate along the first direction, so that the two temperature control modules alternately move above the microfluidic chip, thereby cyclically applying denaturation temperature, annealing temperature and extension temperature to the microfluidic chip.

[0006] According to the technical solution provided in this application, the driving structure includes: two sets of driving components, which are respectively disposed on both sides of the platform along the first direction, and any one set of driving components is connected to a motor; The driving component includes: Two first bearing seats are provided, arranged along a second direction at the bottom of the top plate. A first bearing is fixedly installed in each of the two first bearing seats, and a first rotating shaft is provided between the two first bearings; the second direction is perpendicular to the first direction. Two second bearing seats are provided, arranged along the second direction on the top of the base plate. A second bearing is fixedly installed in each of the two second bearing seats, and a second rotating shaft is provided between the two second bearings. The motor is connected to the second rotating shaft and is used to drive the second rotating shaft to rotate. A first connecting rod, one end of which is fixedly connected to the first rotating shaft, and the other end of which is fixedly connected to the second rotating shaft.

[0007] According to the technical solution provided in this application, the temperature control module is a semiconductor refrigeration chip, which is used to provide the microfluidic chip with the denaturation temperature, annealing temperature and extension temperature.

[0008] According to the technical solution provided in this application, the heat dissipation module includes heat dissipation fins, which are disposed on the top of the top plate, and the semiconductor cooling chip is disposed at the bottom of the heat dissipation fins. The heat dissipation fins are used to dissipate heat from the semiconductor cooling chip.

[0009] According to the technical solution provided in this application, the clamping assembly includes: a support plate and a drawer, wherein the support plate is made of metal; the support plate is disposed on the top of the platform, and the top of the support plate is used to contact the temperature control module; the drawer is disposed between the support plate and the platform, and the drawer is slidably connected to both the platform and the support plate, and the drawer is used to fix the microfluidic chip.

[0010] Secondly, this application provides an amplification method for digital droplet PCR, employing the amplification apparatus for digital droplet PCR as described in any one of the first aspects, characterized by comprising the following steps: S100: The microfluidic chip containing the droplets to be amplified is fixed on the stage using a clamping assembly; S200: A first temperature control region and a second temperature control region are constructed by the two temperature control modules and the heat dissipation module respectively. The temperature of the first temperature control region and the second temperature control region can be independently switched between the denaturation temperature, the annealing temperature and the extension temperature. S300: Set the temperature cycle logic of the first temperature control zone in the order of denaturation temperature, extension temperature, and annealing temperature, and configure the corresponding duration according to the temperature stage. S400: Set the temperature cycle logic of the second temperature control zone in the order of annealing temperature, denaturation temperature, and extension temperature, and configure the corresponding duration according to the temperature stage. S500: The control drive structure drives the top plate to reciprocate along the first direction, so that the microfluidic chip alternately contacts the first temperature control area and the second temperature control area; S600: Repeat step S500 until the preset number of amplification cycles is completed.

[0011] According to the technical solution provided in this application, the following steps are included before step S600: S510: Real-time monitoring of the contact duration between the first and second temperature control areas and the microfluidic chip, and determination of whether the contact duration exceeds the warning threshold. S511: If so, interrupt the current amplification cycle, and reset the temperature cycle logic of the first temperature region and the second temperature region according to the temperature control region and temperature stage of the microfluidic chip at the time of interruption, and continue to execute step S600.

[0012] According to the technical solution provided in this application, the temperature cycle logic of the first temperature region and the second temperature region is reset according to the temperature control region and temperature stage of the microfluidic chip at the time of interruption, including the following steps: When the interruption occurs during the denaturation temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of annealing temperature, denaturation temperature, and extension temperature; the temperature cycle logic of the other temperature control zone is set in the order of extension temperature, annealing temperature, and denaturation temperature. When the interruption occurs during the annealing temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of extension temperature, annealing temperature, and denaturation temperature; the temperature cycle logic of the other temperature control zone is set in the order of denaturation temperature, extension temperature, and annealing temperature. When the interruption occurs during the extension temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of denaturation temperature, extension temperature, and annealing temperature; the temperature cycle logic of the other temperature control zone is set in the order of annealing temperature, denaturation temperature, and extension temperature.

[0013] According to the technical solution provided in this application, the following steps are included after step S510: S512: If not, proceed to step S600.

[0014] Thirdly, this application provides a microfluidic system, including an amplification device for digital droplet PCR as described in any one of the first aspects, a microfluidic chip, and a detection device, wherein the microfluidic chip is used to generate droplets; and the detection device is used to perform optical detection on the droplets after amplification.

[0015] Compared with the prior art, the beneficial effects of this application are as follows: This application provides an amplification device, method, and microfluidic system for digital droplet PCR. The amplification device forms a receiving space through a bottom plate and a top plate arranged in parallel, and integrates a stage, clamping components, a heat dissipation module, a temperature control module, and a driving structure in an orderly manner. By setting two independent temperature control modules in conjunction with the driving structure to achieve alternating contact between the microfluidic chip and the microfluidic chip, the problem of waiting for temperature rise and fall during the amplification process can be effectively avoided by a single temperature control module. When one temperature control module performs the current stage of temperature treatment on the microfluidic chip, the other temperature control module can simultaneously complete the temperature adjustment for the next stage. This allows the temperature changes of the two temperature control modules to form an efficient coordination with the alternating contact of the microfluidic chip, significantly shortening the overall time of a single amplification cycle and improving PCR amplification efficiency. At the same time, the stage and clamping components can firmly fix the microfluidic chip, and the denaturation, annealing, and extension cycle temperature control can be completed without exporting the droplet, simplifying the PCR amplification process, reducing the risk of droplet breakage, and improving detection accuracy.

[0016] It should be understood that the descriptions of technical features, technical solutions, beneficial effects, or similar language in this application do not imply that all features and advantages can be achieved in any single embodiment. Rather, it is understood that the description of a feature or beneficial effect means that a specific technical feature, technical solution, or beneficial effect is included in at least one embodiment. Therefore, the descriptions of technical features, technical solutions, or beneficial effects in this specification do not necessarily refer to the same embodiment. Furthermore, the technical features, technical solutions, and beneficial effects described in this embodiment can be combined in any suitable manner. Those skilled in the art will understand that embodiments can be implemented without one or more specific technical features, technical solutions, or beneficial effects of a particular embodiment. In other embodiments, additional technical features and beneficial effects may be identified in specific embodiments that do not embody all embodiments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this embodiment, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A schematic diagram of an amplification device for digital droplet PCR provided in Embodiment 1 of this application; Figure 2 This is a schematic diagram showing the connection between the heat dissipation module and the temperature control module and the top plate provided in Embodiment 1 of this application; Figure 3 This is a schematic diagram showing the connection between the storage platform and the clamping assembly provided in Embodiment 1 of this application; Figure 4 This is an internal top view of the microfluidic chip provided in Embodiment 3 of this application; Figure 5 This is an internal front view of the microfluidic chip provided in Embodiment 3 of this application; Figure 6 This is a schematic diagram of the detection device provided in Embodiment 3 of this application; Figure 7 This is a flowchart of an amplification method for digital droplet PCR provided in Embodiment 2 of this application.

[0019] In the diagram: 1. Base plate; 2. Top plate; 3. Display platform; 4. First bearing seat; 5. First bearing; 6. First rotating shaft; 7. Second bearing seat; 8. Second bearing; 9. Second rotating shaft; 10. Motor; 11. First connecting rod; 12. Temperature control module; 13. Heat dissipation module; 14. Support plate; 15. Drawer; 16. LED light source; 17. Concentrator; 18. Microfluidic chip; 19. Photomultiplier tube; 20. Optical fiber; 21. Data processing system; 22. Shallow channel; 23. Deep channel; 24. Stepped structure; 25. Placement platform. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. The descriptions in this section are merely illustrative and explanatory, and should not be construed as limiting the scope of protection of this application. Specifically, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort should fall within the scope of protection of this application.

[0021] It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such process, method, product, or apparatus.

[0022] To make the technical solution of this application clearer and easier to understand, the following describes an amplification device, method and microfluidic system for digital droplet PCR provided in the embodiments of this application.

[0023] Example 1 like Figure 1 As shown, this embodiment provides an amplification device for digital droplet PCR, comprising: A base plate 1 is provided, and a top plate 2 is provided above the base plate 1. The top plate 2 is arranged parallel to the base plate 1; an accommodating space is formed between the top plate 2 and the base plate 1. The platform 3 is set in the accommodating space and is fixedly connected to the base plate 1. The top of the platform 3 is provided with a clamping component for fixing the microfluidic chip 18. The heat dissipation module 13 is fixedly installed on the top of the top plate 2. The top plate 2 has a first through hole in the middle. Two temperature control modules 12 are arranged along the bottom of the heat dissipation module 13 in a first direction, and the two temperature control modules 12 are located in the first through hole. The first direction is parallel to the length direction of the top plate 2. The driving structure is set in the accommodating space. The two ends of the driving structure are connected to the bottom plate 1 and the top plate 2 respectively. The driving structure is used to drive the top plate 2 to reciprocate along the first direction so that the two temperature control modules 12 alternately move above the microfluidic chip 18, thereby cyclically applying the denaturation temperature, annealing temperature and extension temperature to the microfluidic chip 18.

[0024] Specifically, in this embodiment, the first direction is the horizontal direction; Specifically, in this application, the top plate 2 and the bottom plate 1 are always in a parallel state. When the amplification device is placed on the platform, the contact surface between the temperature control module 12 and the microfluidic chip 18 is ensured to be horizontal, and the contact area is also maximized, thereby improving the heat transfer efficiency. Specifically, such as Figure 2 As shown, in practical application scenarios, a first temperature control region and a second temperature control region are constructed by two temperature control modules 12 and a heat dissipation module 13, respectively. The temperatures of the first temperature control region and the second temperature control region can be switched independently between the denaturation temperature, annealing temperature, and extension temperature. The temperature control module 12 is connected to the control system, and the temperature switching of the temperature control module 12 is controlled by the control system. For specific methods, please refer to Embodiment 2.

[0025] Working principle: This application achieves alternating contact with the microfluidic chip 18 by setting two independent temperature control modules 12 in conjunction with the driving structure. This effectively avoids the problem of waiting for temperature rise and fall during the amplification process when only one temperature control module 12 is used. When one temperature control module 12 performs the current stage of temperature treatment on the microfluidic chip 18, the other temperature control module 12 can simultaneously complete the next stage of temperature adjustment. This allows the temperature changes of the two temperature control modules 12 and the alternating contact with the microfluidic chip 18 to form an efficient coordination, significantly shortening the overall time of a single amplification cycle and improving PCR amplification efficiency. At the same time, the stage 3 and the clamping components can firmly fix the microfluidic chip 18, and the denaturation, annealing, and extension cycle temperature control can be completed without exporting droplets. This simplifies the PCR amplification process, reduces the risk of droplet breakage, and improves detection accuracy.

[0026] In some embodiments, the drive structure includes two sets of drive components, which are respectively disposed on both sides of the platform 3 along the first direction, and any one set of drive components is connected to the motor 10. The driver components include: There are two first bearing seats 4, arranged along the second direction at the bottom of the top plate 2. A first bearing 5 is fixedly installed in each of the two first bearing seats 4, and a first rotating shaft 6 is arranged between the two first bearings 5; the second direction is perpendicular to the first direction. There are two second bearing seats 7, which are arranged along the second direction on the top of the base plate 1. A second bearing 8 is fixedly installed in each of the two second bearing seats 7, and a second rotating shaft 9 is arranged between the two second bearings 8. The motor 10 is connected to the second rotating shaft 9 and is used to drive the second rotating shaft 9 to rotate. The first connecting rod 11 has one end fixedly connected to the first rotating shaft 6 and the other end fixedly connected to the second rotating shaft 9.

[0027] In this embodiment, the second direction is horizontal and perpendicular to the first direction; Specifically, the drive structure consists of two sets of symmetrically arranged drive components. The two sets of drive components are respectively set on both sides of the platform 3 along the first direction. One set of drive components is connected to the motor 10, so that the two sets of drive components can be driven collaboratively by the same motor 10. Specifically, each drive assembly includes two first bearing seats 4 arranged along the second direction at the bottom of the top plate 2 and two second bearing seats 7 arranged along the second direction at the top of the bottom plate 1. A first bearing 5 is fixed inside each of the two first bearing seats 4, and a first rotating shaft 6 is disposed between the two first bearings 5. A second bearing 8 is fixed inside each of the two second bearing seats 7, and a second rotating shaft 9 is disposed between the two second bearings 8. A first connecting rod 11 serves as the core transmission component, with one end fixedly connected to the first rotating shaft 6 and the other end fixedly connected to the second rotating shaft 9. When the motor 10 drives the second rotating shaft 9 to rotate, it synchronously pushes the top plate 2 to reciprocate horizontally along the first direction via the first connecting rods 11 on both sides. This design ensures that the top plate 2 remains horizontal throughout its movement while significantly reducing system complexity. To ensure stability, each drive assembly includes two first connecting rods 11. Specifically, in this embodiment, the motor 10 is a stepper motor. Based on the swing angle of the first link 11, the angle is used as a parameter input into the stepper motor control program to realize that the two temperature control modules 12 alternately move above the microfluidic chip 18.

[0028] In some implementations, the temperature control module 12 is a thermoelectric cooler used to provide the microfluidic chip 18 with the denaturation temperature, annealing temperature and extension temperature.

[0029] In this embodiment, the temperature control module 12 is a thermoelectric cooler, which can flexibly perform heating or cooling operations by switching the direction of the DC current. When the current flows in the forward direction, the thermoelectric cooler actively absorbs heat and cools the lower surface of the microfluidic chip 18 to precisely maintain the temperature required for annealing. When the current flows in the reverse direction, the lower surface releases heat and heats up, efficiently providing high-temperature conditions for the denaturation stage. For extended temperatures, the heating or cooling mode is intelligently selected based on the difference between the target temperature and the current state. This bidirectional temperature control capability allows a single device to cover the temperature requirements of the entire PCR process. Combined with the spatial displacement brought by the drive structure, it completely avoids the thermal inertia delay in the heating and cooling process of traditional equipment.

[0030] In some embodiments, the heat dissipation module 13 includes heat dissipation fins disposed on the top of the top plate 2, and a thermoelectric cooler is disposed at the bottom of the heat dissipation fins, the heat dissipation fins being used to dissipate heat from the thermoelectric cooler.

[0031] Specifically, in this embodiment, a fan is installed on the heat dissipation fins, which are fixedly covered on the top of the top plate 2, and their bottom is in close contact with the upper surface of the thermoelectric cooler. By utilizing the structural characteristics of the heat dissipation fins themselves, an efficient heat conduction channel is constructed, which can promptly dissipate the heat generated by the thermoelectric cooler during operation, providing support for maintaining a stable operating temperature environment for the thermoelectric cooler, ensuring that its cooling function can be continuously and reliably performed, and thus helping the temperature control system of the entire amplification device to operate stably, so as to meet the requirements of precise temperature control in the digital droplet PCR amplification process.

[0032] In some embodiments, the clamping assembly includes a support plate 14 and a drawer 15. The support plate 14 is made of metal. The support plate 14 is disposed on the top of the stage 3, and the top of the support plate 14 is used to contact the temperature control module 12. The drawer 15 is disposed between the support plate 14 and the stage 3, and the drawer 15 is slidably connected to both the stage 3 and the support plate 14. The drawer 15 is used to fix the microfluidic chip 18.

[0033] Specifically, such as Figure 3As shown, the clamping assembly consists of a support plate 14 and a drawer 15. The support plate 14 is made of metal, possessing good structural stability and thermal conductivity. It is located on the top of the stage 3. The top of the support plate 14 is used to contact the temperature control module 12, and the metal material can efficiently transfer heat, enabling precise temperature control in conjunction with the temperature control module 12. The drawer 15 is installed in the space between the support plate 14 and the stage 3, and it is slidably connected to both the stage 3 and the support plate 14. The operator can easily place the microfluidic chip 18 into the drawer 15 by pulling it out. The drawer 15 can stably clamp and position the microfluidic chip 18, ensuring that the chip position remains stable during PCR amplification. This provides a reliable support for the droplet amplification reaction, allowing the heat from the temperature control module 12 to effectively act on the droplets on the chip, ensuring the smooth progress of the amplification process.

[0034] Example 2 like Figure 7 As shown, this embodiment provides an amplification method for digital droplet PCR, using the amplification device for digital droplet PCR provided in Example 1, and includes the following steps: S100: The microfluidic chip containing the droplets to be amplified is fixed on the stage using a clamping assembly; S200: The first temperature control area and the second temperature control area are constructed by two temperature control modules and a heat dissipation module, respectively. The temperature of the first temperature control area and the second temperature control area can be independently switched between the denaturation temperature, annealing temperature and extension temperature. S300: Set the temperature cycle logic of the first temperature control zone in the order of denaturation temperature, extension temperature, and annealing temperature, and configure the corresponding duration according to the temperature stage. S400: Set the temperature cycle logic of the second temperature control zone in the order of annealing temperature, denaturation temperature, and extension temperature, and configure the corresponding duration according to the temperature stage. S500: The control drive structure drives the top plate to reciprocate along the first direction, so that the microfluidic chip alternately contacts the first temperature control area and the second temperature control area; S600: Repeat step S500 until the preset number of amplification cycles is completed.

[0035] Specifically, in practical application scenarios, a first temperature control area and a second temperature control area are constructed by two temperature control modules 12 and a heat dissipation module 13, respectively. The temperatures of the first temperature control area and the second temperature control area can be switched independently between the denaturation temperature, the annealing temperature, and the extension temperature. The PCR reaction needs to be carried out at denaturation temperature T1 for a seconds, annealing temperature T2 for b seconds, and extension temperature T3 for c seconds; let a+b+c=n, and 2n is a complete cycle; in this embodiment, denaturation temperature T1 is 95℃, annealing temperature T2 is 60℃, and extension temperature T3 is 72℃. The temperature cycle logic of the first temperature control zone is as follows: after maintaining the denaturation temperature T1 for a seconds in the time period (0, a+b), the temperature is lowered to the extension temperature T3; after maintaining the extension temperature T3 for c seconds in the time period (a+b, n+a), the temperature is lowered to the annealing temperature T2; after maintaining the annealing temperature T2 for b seconds in the time period (n+a, 2n), the temperature is raised to the denaturation temperature T1, thus completing one sub-cycle. The temperature cycle logic of the second temperature control zone is as follows: after maintaining the annealing temperature T2 for b seconds in the time period (a, n), the temperature is increased to the denaturation temperature T1; after maintaining the denaturation temperature T1 for a seconds in the time period (n, n+a+b), the temperature is decreased to the extension temperature T3; after maintaining the extension temperature T3 for c seconds in the time period (n+a+b, 2n+a), the temperature is decreased to the annealing temperature T2, thus completing one sub-cycle; Specifically, the temperature control strategies for the first and second temperature control zones are shown in Table 1 below: Table 1 Temperature Variable Strategies for the First and Second Temperature Control Zones

[0036] When performing digital droplet PCR amplification, starting from the first temperature-controlled region, the microfluidic chip 18 is driven by a driving structure to reciprocate between the first and second temperature-controlled regions, causing the droplets within the microfluidic chip 18 to alternately contact the two temperature-controlled regions. The timing of this movement is strictly matched to the temperature cycling logic of the two temperature-controlled regions, specifically: During the time period (0, a), the microfluidic chip 18 remains stationary in the first temperature control region, and the droplet is heated for a seconds at the denaturation temperature T1. During the time period (a, a+b), the second temperature control region moves to the microfluidic chip 18, and the droplet is heated for b seconds at the annealing temperature T2. During the time period (a+b, n), the first temperature control region moves to the microfluidic chip 18, and the droplet is heated for c seconds at the extended temperature T3. During the time period (n, n+a), the second temperature control region moves to the microfluidic chip 18, and the droplet is heated for a seconds at the denaturation temperature T1. During the time interval (n+a, n+a+b), the first temperature control region moves to the microfluidic chip 18, and the droplet is heated at the annealing temperature T2 for b seconds. During the time period (n+a+b, 2n), the second temperature control region moves to the microfluidic chip 18, and the droplet is heated for c seconds at the extended temperature T3. Completing the above-mentioned actions constitutes a cycle. Repeat the above steps until the preset number of expansion cycles is completed.

[0037] In some implementations, the following steps are included before step S600: S510: Real-time monitoring of the contact duration between the first and second temperature control areas and the microfluidic chip, and determination of whether the contact duration exceeds the warning threshold. S511: If so, interrupt the current amplification cycle, and reset the temperature cycle logic of the first temperature region and the second temperature region according to the temperature control region and temperature stage of the microfluidic chip at the time of interruption, and continue to execute step S600.

[0038] Specifically, before step S600 is executed, the contact time between the first temperature region and the second temperature control region and the microfluidic chip 18 is monitored in real time, and it is continuously determined whether the duration of each contact process exceeds the preset warning threshold (this warning threshold is determined based on the longest duration of each temperature stage during PCR amplification, usually 1.5 times the longest duration of the denaturation, annealing, and extension stages). Once it is detected that the contact time exceeds the limit, such as due to the response delay of the motor 10, the amplification device fails to rotate as expected, causing the microfluidic chip 18 to remain in a certain temperature control region for too long (it should have rotated to the next position but there was a delay / error). If the temperature control zone cannot be switched normally, and the warning time is exceeded, the current amplification cycle is immediately interrupted. Then, based on the temperature control zone where the microfluidic chip 18 is located at the moment of interruption, and the temperature stage of that zone at that time (such as denaturation stage, annealing stage, extension stage), the temperature cycle logic of the first temperature control zone and the second temperature control zone is re-planned and set, and the cycle strategy is adjusted, including redetermining the starting state of the new cycle. After the adjustment is completed, step S600 is continued to ensure that the PCR amplification process can still conform to the preset reaction conditions as much as possible after the abnormality is recovered, and maintain the reliability of the amplification results. In some implementations, the following steps are included after step S510: S512: If not, proceed to step S600.

[0039] Specifically, when the contact duration is detected to be within the limit, step S500 is repeated until the preset number of amplification cycles is completed.

[0040] In some implementations, the temperature cycling logic of the first and second temperature regions is reset according to the temperature control region and temperature stage of the microfluidic chip at the time of the interruption, including the following steps: When the interruption occurs during the denaturation temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of annealing temperature, denaturation temperature, and extension temperature; the temperature cycle logic of the other temperature control zone is set in the order of extension temperature, annealing temperature, and denaturation temperature. Taking the denaturation temperature stage in the first temperature control zone during interruption as an example, the temperature cycle logic of the first and second temperature control zones is shown in Table 2. Table 2 Temperature Cycle Logic of First and Second Temperature Control Zones

[0041] When the interruption occurs during the annealing temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of extension temperature, annealing temperature, and denaturation temperature; the temperature cycle logic of the other temperature control zone is set in the order of denaturation temperature, extension temperature, and annealing temperature. Taking the annealing temperature stage in the first temperature control zone during interruption as an example, the temperature cycle logic of the first and second temperature control zones is shown in Table 3. Table 3 Temperature Cycle Logic of First and Second Temperature Control Zones

[0042] When the interruption occurs during the extension temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of denaturation temperature, extension temperature, and annealing temperature; the temperature cycle logic of the other temperature control zone is set in the order of annealing temperature, denaturation temperature, and extension temperature.

[0043] Taking the annealing temperature stage in the first temperature control zone during interruption as an example, the temperature cycle logic of the first and second temperature control zones is shown in Table 4. Table 4 Temperature Cycle Logic of the First and Second Temperature Control Zones

[0044] Example 3 This embodiment provides a microfluidic system, including the expansion device for digital droplet PCR provided in Embodiment 1, the microfluidic chip 18, and the detection device. The microfluidic chip 18 is used to generate droplets; the detection device is used to perform optical detection on the droplets after amplification.

[0045] Specifically, such as Figures 4-5As shown, the microfluidic chip 18 includes: a shallow channel 22 and a deep channel 23 connected together, the height difference between the shallow channel 22 and the deep channel 23 forming a stepped structure 24; the shallow channel 22 is connected to an air pump, and the shallow channel 22 has a first inlet and a second inlet. Water is first delivered to the first inlet under the action of the air pump, and then oil is delivered to the second inlet under the action of the air pump. Multiple water-in-oil droplets are formed at the stepped structure 24 and enter the deep channel 23 for storage. Specifically, such as Figure 6 As shown, the detection device includes a placement platform 25 for placing a microfluidic chip 18. An LED light source 16 is provided on the top of the placement platform 25, and a condenser 17 is provided at the output end of the LED light source 16. The microfluidic chip 18 and the photomultiplier tube 19 are connected by an optical fiber 20, and the photomultiplier tube 19 is connected to the data processing system 21 for signal analysis. The microfluidic chip 18 in this application is a transparent chip made of PDMS material. The LED light source 16 can irradiate the microfluidic chip 18 through the condenser 17 to excite the fluorescent probe. When a droplet containing the fluorescent probe flows through the detection area, fluorescence is generated by the excitation of the light source. The fluorescence is transmitted to the photomultiplier tube 19 through the optical fiber 20. The photomultiplier tube 19 receives the fluorescence signal and converts it into a photoelectric signal. The photoelectric signal is received and processed by the data processing system 21 and transmitted to the computer for waveform analysis. Through processing, droplets that emit fluorescence and droplets that do not emit fluorescence can be distinguished, that is, positive droplets and negative droplets can be distinguished.

[0046] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. An amplification device for digital droplet PCR, characterized in that, include: A base plate (1) is provided above the base plate (1), and the top plate (2) is provided parallel to the base plate (1); an accommodating space is formed between the top plate (2) and the base plate (1); A platform (3) is provided in the accommodating space and is fixedly connected to the base plate (1). A clamping assembly is provided on the top of the platform (3) for fixing the microfluidic chip (18). A heat dissipation module (13) is fixedly installed on the top of the top plate (2). The top plate (2) has a first through hole in the middle. Two temperature control modules (12) are arranged along the bottom of the heat dissipation module (13) in a first direction, and the two temperature control modules (12) are located in the first through hole. The first direction is parallel to the length direction of the top plate (2). The driving structure is disposed in the accommodating space. The two ends of the driving structure are respectively connected to the bottom plate (1) and the top plate (2). The driving structure is used to drive the top plate (2) to reciprocate along the first direction so that the two temperature control modules (12) move alternately above the microfluidic chip (18) to realize the cyclic application of denaturation temperature, annealing temperature and extension temperature to the microfluidic chip (18).

2. The amplification device for digital droplet PCR according to claim 1, characterized in that, The drive structure includes two sets of drive components, which are respectively arranged on both sides of the platform (3) along the first direction, and any one set of drive components is connected to the motor (10). The driving component includes: Two first bearing seats (4) are provided, and the two first bearing seats (4) are arranged along the second direction at the bottom of the top plate (2). A first bearing (5) is fixedly provided in each of the two first bearing seats (4), and a first rotating shaft (6) is provided between the two first bearings (5); the second direction is perpendicular to the first direction. Two second bearing seats (7) are provided, and the two second bearing seats (7) are arranged along the second direction on the top of the base plate (1). A second bearing (8) is fixedly provided in each of the two second bearing seats (7), and a second rotating shaft (9) is provided between the two second bearings (8). The motor (10) is connected to the second rotating shaft (9), and the motor (10) is used to drive the second rotating shaft (9) to rotate. The first connecting rod (11) has one end fixedly connected to the first rotating shaft (6) and the other end fixedly connected to the second rotating shaft (9).

3. The amplification device for digital droplet PCR according to claim 1, characterized in that, The temperature control module (12) is a semiconductor refrigeration chip, which is used to provide the microfluidic chip (18) with the denaturation temperature, annealing temperature and extension temperature.

4. The amplification device for digital droplet PCR according to claim 3, characterized in that, The heat dissipation module (13) includes heat dissipation fins, which are disposed on the top of the top plate (2). The bottom of the heat dissipation fins is provided with the semiconductor cooling chip, and the heat dissipation fins are used to dissipate heat from the semiconductor cooling chip.

5. The amplification device for digital droplet PCR according to claim 1, characterized in that, The clamping assembly includes a support plate (14) and a drawer (15). The support plate (14) is made of metal. The support plate (14) is located on the top of the platform (3), and the top of the support plate (14) is used to contact the temperature control module (12). The drawer (15) is located between the support plate (14) and the platform (3). The drawer (15) is slidably connected to both the platform (3) and the support plate (14). The drawer (15) is used to fix the microfluidic chip (18).

6. An amplification method for digital droplet PCR, employing the amplification apparatus for digital droplet PCR as described in any one of claims 1-5, characterized in that, Includes the following steps: S100: The microfluidic chip containing the droplets to be amplified is fixed on the stage using a clamping assembly; S200: A first temperature control region and a second temperature control region are constructed by the two temperature control modules and the heat dissipation module respectively. The temperature of the first temperature control region and the second temperature control region can be independently switched between the denaturation temperature, the annealing temperature and the extension temperature. S300: Set the temperature cycle logic of the first temperature control zone in the order of denaturation temperature, extension temperature, and annealing temperature, and configure the corresponding duration according to the temperature stage. S400: Set the temperature cycle logic of the second temperature control zone in the order of annealing temperature, denaturation temperature, and extension temperature, and configure the corresponding duration according to the temperature stage. S5 00: The control drive structure drives the top plate to reciprocate along the first direction, so that the microfluidic chip alternately contacts the first temperature control area and the second temperature control area; S600: Repeat step S500 until the preset number of amplification cycles is completed.

7. The amplification method for digital droplet PCR according to claim 6, characterized in that, The following steps are included before step S600: S510: Real-time monitoring of the contact duration between the first and second temperature control areas and the microfluidic chip, and determination of whether the contact duration exceeds the warning threshold. S511: If so, interrupt the current amplification cycle, and reset the temperature cycle logic of the first temperature region and the second temperature region according to the temperature control region and temperature stage of the microfluidic chip at the time of interruption, and continue to execute step S600.

8. The amplification method for digital droplet PCR according to claim 7, characterized in that, Based on the temperature control region and temperature stage of the microfluidic chip at the time of the interruption, the temperature cycle logic of the first and second temperature regions is reset, including the following steps: When the interruption occurs during the denaturation temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of annealing temperature, denaturation temperature, and extension temperature; the temperature cycle logic of the other temperature control zone is set in the order of extension temperature, annealing temperature, and denaturation temperature. When the interruption occurs during the annealing temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of extension temperature, annealing temperature, and denaturation temperature; the temperature cycle logic of the other temperature control zone is set in the order of denaturation temperature, extension temperature, and annealing temperature. When the interruption occurs during the extension temperature stage, the temperature cycle logic of the current temperature control zone is set in the order of denaturation temperature, extension temperature, and annealing temperature; the temperature cycle logic of the other temperature control zone is set in the order of annealing temperature, denaturation temperature, and extension temperature.

9. The amplification method for digital droplet PCR according to claim 8, characterized in that, Step S510 is followed by the following steps: S512: If not, proceed to step S600.

10. A microfluidic system, characterized in that, include: The amplification device, microfluidic chip (18), and detection device for digital droplet PCR according to any one of claims 1-5, wherein the microfluidic chip (18) is used to generate droplets; and the detection device is used to perform optical detection on the droplets after amplification.

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