A multi-temperature zone PCR amplification device and amplification method thereof

Abstract

The present invention proposes a multi-temperature zone PCR amplification device, comprising a PCR microfluidic chip, a motor, and a lower heating plate. The lower heating plate is fixed to a heat-resistant bracket. The sample inlet of the PCR microfluidic chip is connected to the amplification chamber. The PCR microfluidic chip is mounted on the motor's rotating bearing, with its bottom surface contacting the lower heating plate. The lower heating plate is composed of several lower heating plates made of semiconductor aluminum alloy. A control unit controls the temperature of the heating plates and the rotation of the motor. The present invention utilizes multiple temperature zones for constant temperature heating, shortening sample temperature change time, accelerating amplification, and thus increasing detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, and in particular relates to a multi-temperature zone PCR amplification device and an amplification method thereof. Background Art

[0002] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. PCR amplification utilizes the fact that DNA denatures into single strands at 95°C in vitro. At lower temperatures (usually around 60°C), primers bind to the single strands based on complementary base pairing. The temperature is then adjusted to the optimal reaction temperature of DNA polymerase, 72°C, where the enzyme synthesizes complementary strands along the phosphate-to-pentose (5'-3') chain. The key to PCR amplification technology lies in temperature control equipment, which allows for precise control between denaturation, annealing, and extension temperatures.

[0003] Currently, most PCR amplification devices use a fixed-contact variable temperature design, where the sample and the heating plate are kept at the same temperature. The temperature is raised and lowered under program control to complete a single amplification cycle. PCR amplification is completed after multiple temperature cycles. This approach has the following drawbacks: 1) slow ramping speeds; 2) high temperature control precision requirements; and 3) high energy consumption and low efficiency due to the overall heating and cooling of the heating element.

[0004] Therefore, there is an urgent need for a PCR amplification device that can provide constant temperature heating, has low requirements for temperature change accuracy, and effectively shortens the sample temperature change time. Summary of the Invention

[0005] In order to overcome the problems of the prior art, the present invention provides a multi-temperature zone PCR amplification device and an amplification method thereof, which can provide constant temperature heating, shorten the sample temperature change time, accelerate the amplification speed and thus increase the detection efficiency.

[0006] The present invention provides the following technical solutions:

[0007] A multi-temperature zone PCR amplification device includes a PCR microfluidic chip, a motor and a lower heating plate, characterized in that the lower heating plate is fixed on a temperature-resistant bracket, the sample inlet of the PCR microfluidic chip is connected to the amplification chamber, the PCR microfluidic chip is sleeved on the motor rotating bearing, and the bottom surface contacts the lower heating plate. The lower heating plate is composed of several lower heating disks made of semiconductor aluminum alloy, and a control unit controls the temperature of the heating disks and the rotation of the motor.

[0008] Furthermore, it also includes an upper heating plate, which is composed of several upper heating disks of semiconductor aluminum alloy. The upper and lower heating plates form a heating chamber to accommodate the microfluidic chip.

[0009] Furthermore, there are three lower heating discs, which form a lower heating plate at an angle of 120°; there are three upper heating discs, which form an upper heating plate at an angle of 120°.

[0010] Furthermore, at least one amplification chamber on the PCR microfluidic chip corresponds to the heating plate.

[0011] Furthermore, the upper heating plate is fixed on a fixing frame, the fixing frame is connected to a slider, and a slide rail is provided in the Z-axis direction. Driven by a linear motor, the slider moves on the slide rail.

[0012] Furthermore, a chip clamp is installed on the motor rotating chuck, and the clamp clamps and fixes the PCR microfluidic chip.

[0013] Furthermore, a photoelectric barrier is provided between the chip clamp and the motor for triggering a photoelectric switch.

[0014] Furthermore, a heat-insulating baffle extends downward between adjacent heating discs of the upper heating plate to divide the entire heating space into several independent heating chambers.

[0015] Furthermore, each heating plate is connected to a temperature sensor, and the temperature sensor transmits a temperature signal to the control unit.

[0016] Furthermore, heat dissipation aluminum fins and a heat dissipation fan are provided on the fixing frame.

[0017] A method for performing PCR amplification using a multi-temperature zone PCR amplification device comprises the following steps:

[0018] Step 1: Sleeve the PCR microfluidic chip onto the motor rotating bearing, and clamp the chip to fix the PCR microfluidic chip;

[0019] Step 2: Add reaction buffer, primers, Taq enzyme, deoxynucleoside triphosphate substrate, and DNA template reagent from the PCR microfluidic chip inlet. The motor drives the microfluidic chip to rotate, and the reagents enter the amplification chamber and are mixed evenly.

[0020] Step 3: The linear motor drives the upper heating plate to move downward to form several independent heating chambers, and the amplification chamber of the PCR microfluidic chip rotates into the heating chambers;

[0021] Step 4: The control unit controls the heating of the semiconductor chip on the heating plate. The different heating chambers rotate in sequence to maintain the temperatures at the denaturation reaction temperature, annealing reaction temperature, and amplification temperature of the PCR experiment. The upper and lower heating plates in the same heating chamber are kept at the same temperature.

[0022] In step 5, the motor drives the PCR microfluidic chip to rotate 120 degrees each time, and the amplification chamber enters the heating chambers of denaturation temperature, annealing temperature, and amplification temperature in sequence. The heating is continued for 30 seconds to 2 minutes in each heating chamber, and the microfluidic chip is rotated for amplification cycles.

[0023] Furthermore, in step 2, when the motor rotates, the photoelectric barrier rotates to the action point of the photoelectric switch, the photoelectric switch sends a signal, and the control unit determines the motor origin;

[0024] Furthermore, in step 4, the temperature sensor detects the temperature of the heating plate, and calculates the chip temperature through temperature compensation. After reaching the target temperature, the control unit reduces the power output to keep the temperature at a constant value.

[0025] By adopting the above technical solution, the present invention has the following beneficial effects:

[0026] (1) Using multiple temperature zones for constant temperature heating shortens the sample temperature change time, accelerates the amplification speed and thus increases the detection efficiency;

[0027] (2) It uses upper and lower double heating plates for heating, with fast heating and cooling speed. The low temperature zone is heated to 65°C within 1 minute, and the high temperature zone is heated to 95°C within 2 minutes.

[0028] (3) The traditional variable temperature mode is changed to several independent constant temperature heating units, which reduces the requirements for temperature control accuracy; the heating plate does not need to cycle the temperature, and the energy consumption is small when maintaining a fixed temperature;

[0029] (4) Cyclic detection mode to increase detection reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the structure of the PCR microfluidic chip in Example 1 of the present invention;

[0031] Figure 2 It is a structural schematic diagram of the microfluidic chip, motor, and lower heating plate of the present invention;

[0032] Figure 3 1 is a side view of the multi-temperature zone PCR amplification device in Example 1 of the present invention;

[0033] Figure 4 Schematic diagram of the structure of the PCR microfluidic chip in Example 2 of the present invention;

[0034] Figure 5 Schematic diagram of the structure of the multi-temperature zone PCR amplification device in Example 2 of the present invention;

[0035] Figure 6 is a structural cross-sectional view of the multi-temperature zone PCR amplification device in Example 2 of the present invention;

[0036] Figure 7 1 is a schematic structural diagram of a heating chamber of a multi-temperature zone PCR amplification device in Example 2 of the present invention;

[0037] Figure 8 Schematic diagram of the structure of the lower heating plate of the multi-temperature zone PCR amplification device in Example 2 of the present invention.

[0038] Figure 9 Schematic diagram of the structure of the PCR microfluidic chip in Example 3 of the present invention.

[0039] Among them: 1. PCR microfluidic chip, 101. injection port, 102. amplification chamber, 103. waste liquid port, 2. motor, 3. lower heating plate, 301. lower heating plate, 302. thermal insulation strip, 4. temperature-resistant bracket, 5. upper heating plate, 501. upper heating plate, 502. thermal insulation strip, 6. heating chamber, 7. fixing frame, 8. slider, 9. slide rail, 10. linear motor, 11. clamp, 12. photoelectric baffle, 13. photoelectric switch, 14. thermal insulation baffle, 15. temperature sensor, 16. heat dissipation aluminum fins, 17. cooling fan. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] Example 1

[0042] like Figure 1-2 As shown, the present invention provides a multi-temperature zone PCR amplification device, including a PCR microfluidic chip 1, a motor 2 and a lower heating plate 3. The lower heating plate is fixed on a temperature-resistant bracket 4. The lower heating plate is a hollow structure and is mounted on the motor rotating shaft. It is fixed relative to the motor rotating shaft. An insulating mica plate can be provided between the lower heating plate and the temperature-resistant bracket for thermal insulation.

[0043] The sample injection port 101 of the PCR microfluidic chip is connected to the amplification chamber 102. The sample injection port can be connected to multiple amplification chambers. The amplification chamber is connected to the waste liquid port 103. The PCR microfluidic chip is mounted on the motor rotating bearing 201 through the middle through-hole. The bottom surface of the chip contacts the lower heating plate, and the plane rotates driven by the motor rotating bearing.

[0044] The lower heating plate consists of several aluminum alloy lower heating plates 301. These plates are bonded to the semiconductor chips. Forward power to the heating plates heats the chips, while reverse power to the heating plates cools them, enabling rapid cooling. The semiconductor chips have low thermal resistance and rapid temperature increases and decreases. The low-temperature zone can be heated to 65°C in one minute, and the high-temperature zone can reach 95°C in two minutes. Adjacent heating plates are insulated by insulation strips, and a control unit controls the temperature of the heating plates and the rotation of the motor.

[0045] like Figure 3 As shown, the multi-temperature zone PCR amplification device also includes an upper heating plate 5, which is composed of several upper heating plates 501 made of a semiconductor aluminum alloy. The heating plates are made of aluminum alloy plates bonded to the semiconductor chip. Forward power is applied to the heating plates to heat them, while reverse power is applied to them to cool them, enabling rapid cooling. The upper and lower heating plates form a heating chamber 6, which houses the microfluidic chip and ensures a constant temperature within the amplification chamber.

[0046] During use, the temperatures of the left and right heating chambers can be set to the denaturation temperature of 95°C and the annealing temperature of 60°C for the PCR reaction respectively. The temperatures of the upper and lower heating plates in the same heating chamber are the same.

[0047] The motor controls the microfluidic chip to rotate on the lower heating plate. The amplification chamber first completes the denaturation reaction in a 95°C heating chamber, rotates the chip 180 degrees, and reaches a 60°C heating chamber to complete the annealing reaction. The control unit controls the 60°C heating chamber to heat up to the amplification temperature of 72°C. Each reaction time is 30s-1min, completing an amplification cycle. The microfluidic chip continues to rotate, and the control unit controls the temperature change of the lower heating plate to perform continuous amplification. The temperature of the two heating chambers can be changed according to the temperature change requirements of the amplification experiment. The multi-temperature zone PCR amplification device of this embodiment can reduce the time for heating and cooling in the experiment and improve detection efficiency.

[0048] Example 2

[0049] like Figure 4 As shown, the present invention provides a multi-temperature zone PCR amplification device, including a PCR microfluidic chip 1, a motor 2, and a lower heating plate 3, which is fixed to a heat-resistant bracket 4. The lower heating plate is a hollow structure, mounted on the motor shaft and fixed relative to the motor shaft. An insulating mica plate can be provided between the lower heating plate and the heat-resistant bracket for thermal insulation.

[0050] The sample inlet of the PCR microfluidic chip is connected to several amplification chambers. The microfluidic chip can reduce the amount of sample used. Multiple amplification chambers are set up to conduct multiple parallel experiments at the same time. There are three lower heating trays, which form a lower heating plate at an angle of 120 degrees. Insulation strips 302 are installed between adjacent lower heating trays 301 to prevent temperature loss. Each heating tray corresponds to at least one amplification chamber. Figure 4The PCR microfluidic chip shown in FIG3 corresponds to one heating plate with three amplification chambers.

[0051] The PCR microfluidic chip is mounted on the motor rotating bearing 201, and its bottom surface contacts the lower heating plate. The lower heating plate is composed of several lower heating disks 301 made of semiconductor aluminum alloy. The control unit controls the temperature of the heating disks and the rotation of the motor.

[0052] The system also includes an upper heating plate 5, which is composed of several aluminum alloy upper heating plates 501. The upper and lower heating plates form a heating chamber 6, which houses the microfluidic chip. Three upper heating plates are arranged at a 120-degree angle to form the upper heating plate. Insulation strips 502 are installed between adjacent upper heating plates to prevent temperature loss. The amplification chamber is thus aligned with the upper and lower heating plates. The control unit adjusts the temperature of the heating plates to ensure that the upper and lower surfaces of the amplification chamber are at the same temperature.

[0053] Preferably, the upper heating plate is fixed to a fixed frame 7, which is connected to a slider 8. A slide rail 9 is provided in the Z-axis direction. Driven by a linear motor 10, the slider moves on the slide rail, thereby driving the upper heating plate up and down. The upper heating plate contacts or separates from the microfluidic chip, facilitating manual chip placement while providing temperature conduction and isolation. The fixed frame is also equipped with heat dissipation aluminum fins 16 and a cooling fan 17 to enhance heat dissipation and improve semiconductor chip performance.

[0054] A chip clamp 11 is mounted on a motor-driven chuck 22. The clamp 11 corresponds to the hollow shape of the chip, allowing the two to engage and secure the PCR microfluidic chip. A photoelectric barrier 12 is located between the chip clamp and the motor to trigger a photoelectric switch 13, determining the initial position of the chip for detection.

[0055] An insulating baffle 14 extends downward between adjacent heating disks of the upper heating plate. The baffle is made of thermal insulation materials such as aerogel, dividing the entire heating space into three heating chambers. The insulating baffle reduces air convection, effectively lowers the rate of heat loss, prevents heat exchange between the heating chambers, and ensures that the amplification chamber is heated evenly in the heating chamber.

[0056] Each heating plate is connected to a temperature sensor 15, which transmits the temperature signal to the control unit. The chip temperature is calculated through temperature compensation. When the target temperature is reached, the control unit reduces the power output to keep the temperature at a constant value.

[0057] Example 3

[0058] like Figure 9As shown, a PCR microfluidic chip can be configured as a set of amplification chambers, including an inlet, a main liquid channel, an amplification chamber, and a waste liquid storage chamber. The inlet is connected to the waste liquid storage chamber via the main liquid channel, which in turn is connected to the amplification chamber via a fluid path. An online degassing channel is provided between the inlet and the main liquid channel, and the top layer of the online degassing channel is capped with a semipermeable membrane. The main liquid channel is connected to the lower portion of the amplification chamber via an inlet branch channel, and the upper portion of the amplification chamber is connected to the outlet branch channel, whose outlet is capped at the top by a semipermeable membrane.

[0059] The distribution of the amplification chambers corresponds to the heating plates, so that the motor drives the chip to rotate and it can pass through the heating plates with different temperature settings in sequence. According to the needs of the PCR amplification experiment, the temperature and heating time of each heating chamber can be different, and the chip rotates in sequence to complete the cyclic amplification.

[0060] Example 4

[0061] According to the structure of Example 2, the present invention provides a method for performing PCR amplification using a multi-temperature zone PCR amplification device, comprising the following steps:

[0062] Step 1: Sleeve the PCR microfluidic chip onto the motor rotating bearing, and clamp the PCR microfluidic chip with the chip clamp;

[0063] Step 2: Add reaction buffer, primers, Taq enzyme, deoxynucleoside triphosphate substrate, and DNA template reagent from the PCR microfluidic chip inlet. The motor drives the microfluidic chip to rotate, and the reagents enter the amplification chamber and are mixed evenly.

[0064] Step 3: The linear motor drives the upper heating plate to move downward to form three independent heating chambers, and the amplification chamber of the PCR microfluidic chip rotates into the heating chamber;

[0065] Step 4: The control unit controls the heating of the semiconductor chip on the heating plate. The temperatures of the different heating chambers are maintained in sequence according to the order in which the chips rotate, such as the denaturation reaction temperature of the PCR experiment, for example, 95°C, the annealing reaction temperature, for example, 60°C, and the amplification temperature, for example, 72°C. The upper and lower heating plates in the same heating chamber are kept at the same temperature.

[0066] In step 5, the motor drives the PCR microfluidic chip to rotate 120 degrees each time, and the amplification chamber enters the heating chambers of denaturation temperature, annealing temperature, and amplification temperature in sequence. The heating is continued in each heating chamber for 30 seconds to 2 minutes, preferably 1 minute, and the microfluidic chip is rotated for amplification cycle.

[0067] Preferably, in step 2, when the motor rotates, the photoelectric baffle rotates to the action point of the photoelectric switch, the photoelectric switch sends a signal, and the control unit determines the motor origin;

[0068] In step 4, the temperature sensor detects the temperature of the heating plate and calculates the chip temperature through temperature compensation. After reaching the target temperature, the control unit reduces the power output to keep the temperature at a constant value.

[0069] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A multi-temperature zone PCR amplification device, comprising a PCR microfluidic chip, a motor and a lower heating plate, characterized in that: The lower heating plate is fixed on a temperature-resistant bracket, the sample inlet of the PCR microfluidic chip is connected to the amplification chamber, the PCR microfluidic chip is sleeved on the motor rotating bearing, and the bottom surface contacts the lower heating plate. The lower heating plate is composed of a plurality of lower heating disks of semiconductor aluminum alloy. The control unit controls the temperature of the heating disk and the rotation of the motor; and also includes an upper heating plate, which is composed of a plurality of upper heating disks of semiconductor aluminum alloy. The upper and lower heating plates form a heating chamber to accommodate the microfluidic chip. There are three lower heating disks, which form a lower heating plate at an angle of 120°; there are three upper heating disks, which form an upper heating plate at an angle of 120°; a heat-insulating baffle extends downward between adjacent heating disks on the upper heating plate, dividing the entire heating space into several independent heating chambers; there is at least one amplification chamber on the PCR microfluidic chip corresponding to the heating disk; the upper heating plate is fixed on a fixed frame, which is connected to a slider, and a slide rail is provided in the Z-axis direction. Driven by a linear motor, the slider moves on the slide rail; each heating disk is connected to a temperature sensor, which transmits a temperature signal to a control unit.

2. The multi-temperature zone PCR amplification device according to claim 1, characterized in that: A chip clamp is installed on the motor rotating chuck, and the clamp clamps and fixes the PCR microfluidic chip.

3. The multi-temperature zone PCR amplification device according to claim 2, characterized in that: A photoelectric baffle is provided between the chip clamp and the motor to trigger the photoelectric switch.

4. The multi-temperature zone PCR amplification device according to claim 1, characterized in that: The fixing frame is provided with heat dissipation aluminum fins and a heat dissipation fan.

5. A method for performing PCR amplification using the multi-temperature zone PCR amplification device according to claim 4, characterized in that: The following steps are involved: Step 1: Sleeve the PCR microfluidic chip onto the motor rotating bearing, and clamp the chip to fix the PCR microfluidic chip; Step 2: Add reaction buffer, primers, Taq enzyme, deoxynucleoside triphosphate substrate, and DNA template reagent from the PCR microfluidic chip inlet. The motor drives the microfluidic chip to rotate, and the reagents enter the amplification chamber and are mixed evenly. Step 3: The linear motor drives the upper heating plate to move downward to form several independent heating chambers, and the amplification chamber of the PCR microfluidic chip rotates into the heating chambers; Step 4: The control unit controls the heating of the semiconductor chip on the heating plate. The different heating chambers rotate in sequence to maintain the temperatures at the denaturation reaction temperature, annealing reaction temperature, and amplification temperature of the PCR experiment. The upper and lower heating plates in the same heating chamber are kept at the same temperature. In step 5, the motor drives the PCR microfluidic chip to rotate 120 degrees each time, and the amplification chamber enters the heating chambers of denaturation temperature, annealing temperature, and amplification temperature in sequence. The heating is continued for 30 seconds to 2 minutes in each heating chamber, and the microfluidic chip is rotated for amplification cycles.

6. The method for PCR amplification according to claim 5, characterized in that In step 2, when the motor rotates, the photoelectric barrier rotates to the action point of the photoelectric switch, the photoelectric switch sends a signal, and the control unit determines the motor origin.

7. The method for PCR amplification according to claim 6, characterized in that In step 4, the temperature sensor detects the temperature of the heating plate and calculates the chip temperature through temperature compensation. After reaching the target temperature, the control unit reduces the power output to keep the temperature at a constant value.

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