CN115477997A - Multi-temperature-zone PCR amplification device and amplification method thereof - Google Patents
Multi-temperature-zone PCR amplification device and amplification method thereof Download PDFInfo
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Abstract
The invention provides a multi-temperature-zone PCR amplification device, which comprises a PCR microfluidic chip, a motor and a lower heating plate, wherein the lower heating plate is fixed on a temperature-resistant bracket, a sample inlet of the PCR microfluidic chip is communicated with an amplification chamber, the PCR microfluidic chip is sleeved on a motor rotating bearing, the bottom surface of the PCR microfluidic chip is in contact with the lower heating plate, the lower heating plate consists of a plurality of lower heating plates made of semiconductor aluminum alloy, and a control unit controls the temperature of the heating plates and the rotation of the motor. The invention adopts multiple temperature zones for constant temperature heating, shortens the sample temperature changing time, accelerates the amplification speed and further increases the detection efficiency.
Description
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a multi-temperature-zone PCR amplification device and an amplification method thereof.
Background
Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying and amplifying specific DNA fragments, wherein PCR amplification utilizes the principle that DNA is changed into a single strand at high temperature of 95 ℃ in vitro, a primer is combined with the single strand at low temperature (usually about 60 ℃) according to the base complementary pairing principle, the temperature is adjusted to 72 ℃ which is the optimal reaction temperature of DNA polymerase, and the DNA polymerase synthesizes a complementary strand along the direction from phosphoric acid to pentose (5 '-3'). The key of the PCR amplification technology lies in a temperature control device which can well control the denaturation temperature, the renaturation temperature and the extension temperature.
At present, in the prior art, a PCR amplification device mostly adopts a fixed contact type variable temperature design, the temperature of a sample is kept consistent with that of a heating plate, the temperature rise and the temperature drop are carried out under the control of a program, one-time cyclic amplification is completed, and the PCR amplification is completed through multiple temperature cycles. This approach has the following disadvantages: 1) The temperature rising and falling speed is slow; 2) The requirement on the temperature control precision is high; 3) The heating device is wholly heated and cooled, the energy consumption is high, and the efficiency is low.
Therefore, there is a high necessity for a PCR amplification apparatus that can provide constant temperature heating, has a low requirement for temperature change accuracy, and effectively shortens the time for changing the temperature of a sample.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a multi-temperature-zone PCR amplification device and an amplification method thereof, which can provide constant-temperature heating, shorten the sample temperature changing time, accelerate the amplification speed and further increase the detection efficiency.
The invention provides the following technical scheme:
the utility model provides a multi-temperature-zone PCR amplification device, includes PCR micro-fluidic chip, motor and lower hot plate, its characterized in that, the hot plate is fixed on temperature resistant support down, PCR micro-fluidic chip's introduction port and amplification cavity intercommunication, PCR micro-fluidic chip cup joint on motor rolling bearing, and the hot plate is down contacted to the bottom surface, the hot plate comprises the lower heating plate of a plurality of semiconductor aluminum alloys down, and the control unit controls the temperature of heating plate and the rotation of motor.
Furthermore, the device also comprises an upper heating plate, wherein the upper heating plate consists of a plurality of upper heating plates made of semiconductor aluminum alloy, and the upper heating plate and the lower heating plate form a heating chamber for accommodating the microfluidic chip.
Furthermore, the number of the lower heating plates is 3, and the lower heating plates are surrounded by an included angle of 120 degrees; the number of the upper heating plates is 3, and the upper heating plates are enclosed by an included angle of 120 degrees.
Furthermore, at least one amplification chamber on the PCR microfluidic chip corresponds to the heating plate.
Furthermore, the upper heating plate is fixed on a fixing frame, the fixing frame is connected with a sliding block, a sliding rail is arranged in the Z-axis direction, and the sliding block moves on the sliding rail under the driving of a linear motor.
Furthermore, a chip clamping jaw is arranged on the motor rotating chuck and fixedly connected with the PCR microfluidic chip in a clamping mode.
Furthermore, a photoelectric separation blade is arranged between the chip clamping jaw and the motor and used for triggering the photoelectric switch.
Furthermore, a heat insulation baffle extends downwards between adjacent heating plates of the upper heating plate to divide the whole heating space into a plurality of independent heating bins.
Furthermore, each heating plate is connected with a temperature sensor, and the temperature sensors transmit temperature signals to the control unit.
Furthermore, the fixing frame is provided with a radiating aluminum fin and a radiating fan.
A method for carrying out PCR amplification by a multi-temperature-zone PCR amplification device comprises the following steps:
step 1, sleeving the PCR microfluidic chip on a motor rotating bearing, and clamping and fixing the PCR microfluidic chip by a chip clamping jaw;
and 5, driving the PCR microfluidic chip to rotate 120 degrees each time by the motor, sequentially allowing the amplification chamber to enter heating bins with denaturation temperature, annealing temperature and amplification temperature, continuously heating in each heating bin for 30s-2min, and rotating the microfluidic chip for amplification circulation.
Further, in step 2, when the motor rotates, after the photoelectric barrier rotates to the action point of the photoelectric switch, the photoelectric switch sends a signal, and the control unit determines the origin of the motor;
further, in step 4, the temperature sensor detects the temperature of the heating plate, the temperature of the chip is estimated through temperature compensation, and after the target temperature is reached, the control unit reduces the power output to keep the temperature at a constant value.
By adopting the technical scheme, the invention has the following beneficial effects:
(1) The multi-temperature zone is adopted for constant-temperature heating, so that the sample temperature change time is shortened, the amplification speed is accelerated, and the detection efficiency is increased;
(2) Heating by adopting an upper heating plate and a lower heating plate, wherein the heating and cooling speed is high, the low-temperature area is heated to 65 ℃ within 1 minute, and the high-temperature area is heated to 95 ℃ within 2 minutes;
(3) The traditional variable temperature mode is changed into a plurality of independent constant temperature heating units, so that the requirement on the temperature control precision is reduced; the heating plate does not need circulating temperature change, and the energy consumption is low when the fixed temperature is maintained;
(4) And the detection mode is circulated, so that the detection reliability is improved.
Drawings
FIG. 1 is a schematic diagram of the structure of a PCR microfluidic chip in example 1 of the present invention;
FIG. 2 is a schematic diagram of the structure of the microfluidic chip, the motor and the lower heating plate of the present invention;
FIG. 3 is a side view of a multi-temperature-zone PCR amplification apparatus in example 1 of the present invention;
FIG. 4 is a schematic structural diagram of a PCR microfluidic chip in example 2 of the present invention;
FIG. 5 is a schematic view of the construction of a multi-temperature-zone PCR amplification apparatus in example 2 of the present invention;
FIG. 6 is a sectional view showing the structure of a multi-temperature zone PCR amplification apparatus in example 2 of the present invention;
FIG. 7 is a schematic structural view of a heating chamber of a multi-temperature-zone PCR amplification apparatus in example 2 of the present invention;
FIG. 8 is a schematic view of the structure of the lower heating plate of the multi-temperature-zone PCR amplification apparatus in example 2 of the present invention.
FIG. 9 is a schematic diagram of the structure of the PCR microfluidic chip in example 3 of the present invention.
Wherein: 1. PCR micro-fluidic chip, 101, a sample inlet, 102, an amplification chamber, 103, a waste liquid port, 2, a motor, 3, a lower heating plate, 301, a lower heating plate, 302, a heat insulation strip, 4, a temperature-resistant support, 5, an upper heating plate, 501, an upper heating plate, 502, a heat insulation strip, 6, a heating bin, 7, a fixing frame, 8, a sliding block, 9, a sliding rail, 10, a linear motor, 11, a clamping jaw, 12, a photoelectric blocking piece, 13, a photoelectric switch, 14, a heat insulation blocking piece, 15, a temperature sensor, 16, a heat-dissipation aluminum fin, 17 and a heat-dissipation fan.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the block diagrams and specific examples are set forth only for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Example 1
As shown in FIGS. 1-2, the invention provides a multi-temperature-zone PCR amplification device, which comprises a PCR microfluidic chip 1, a motor 2 and a lower heating plate 3, wherein the lower heating plate is fixed on a temperature-resistant support 4, is of a hollow structure, is sleeved on a motor rotating shaft and is fixed relative to the motor rotating shaft, and a heat-insulating mica plate can be arranged between the lower heating plate and the temperature-resistant support for heat insulation.
The sample inlet 101 of the PCR microfluidic chip is communicated with the amplification chambers 102, the sample inlet can be connected with a plurality of amplification chambers, the amplification chambers are connected with the waste liquid port 103, the PCR microfluidic chip is sleeved on the motor rotating bearing 201 through a middle through hole, the bottom surface of the chip is contacted with the lower heating plate, and the plane is driven by the motor rotating bearing to rotate.
The lower heating plate consists of a plurality of aluminum alloy lower heating plates 301, the heating plates are attached to semiconductor chips through aluminum alloy plates, the forward electrified heating plates heat, and the reverse electrified heating plates refrigerate to enable the heating plates to be rapidly cooled. The semiconductor chip has small thermal resistance and high temperature rise and fall speed, the low-temperature region can be heated to 65 ℃ within 1 minute, and the high-temperature region can be heated to 95 ℃ within 2 minutes. The adjacent heating plates are insulated by heat insulating strips, and the control unit controls the temperature of the heating plates and the rotation of the motor.
As shown in FIG. 3, the PCR amplification device with multiple temperature zones further comprises an upper heating plate 5, wherein the upper heating plate is composed of a plurality of upper heating plates 501 made of semiconductor aluminum alloy, each heating plate is attached to a semiconductor chip by an aluminum alloy plate, the heating plates are powered on positively for heating, and the heating plates are powered on reversely for refrigerating, so that the heating plates can be cooled rapidly. The upper heating plate and the lower heating plate form a heating chamber 6, the microfluidic chip is accommodated in the heating chamber, and the constant temperature of an amplification chamber in the heating chamber can be guaranteed.
When the PCR reaction device is used, the temperatures of the left heating bin and the right heating bin can be respectively set to be 95 ℃ of denaturation temperature and 60 ℃ of annealing temperature of PCR reaction, and the temperatures of the upper heating plate and the lower heating plate of the same heating bin are the same.
The motor controls the micro-fluidic chip to rotate on the lower heating plate, the amplification chamber finishes denaturation reaction in a heating chamber at 95 ℃, the chip is rotated by 180 ℃, annealing reaction is finished in the heating chamber at 60 ℃, the control unit controls the heating chamber at 60 ℃ to be heated to the amplification temperature of 72 ℃, each reaction time is 30s-1min, and an amplification cycle is finished. The micro-fluidic chip is continuously rotated, and the control unit controls the temperature change of the lower heating plate to carry out continuous amplification. The temperature of the two heating chambers can be varied as required by the temperature change of the amplification experiment. By adopting the multi-temperature-zone PCR amplification device of the embodiment, the temperature rise and fall time in an experiment can be reduced, and the detection efficiency is improved.
Example 2
As shown in FIG. 4, the invention provides a multi-temperature-zone PCR amplification device, which comprises a PCR microfluidic chip 1, a motor 2 and a lower heating plate 3, wherein the lower heating plate is fixed on a temperature-resistant bracket 4. The lower heating plate is of a hollow structure, is sleeved on the motor rotating shaft and is fixed relative to the motor rotating shaft, and a heat insulation mica plate can be arranged between the lower heating plate and the temperature-resistant support and used for heat insulation.
The sample inlet of the PCR microfluidic chip is communicated with a plurality of amplification chambers, the microfluidic chip can reduce the use amount of samples, the amplification chambers are arranged in a plurality, and a plurality of parallel experiments are carried out simultaneously. The lower heating plates are 3 in number, the lower heating plates are surrounded by an included angle of 120 degrees, and heat insulation strips 302 are arranged between the adjacent lower heating plates 301, so that the temperature loss is avoided. Each heating plate corresponds to at least one amplification chamber, and the PCR microfluidic chip shown in fig. 4 corresponds 3 amplification chambers to one heating plate.
The PCR microfluidic chip is sleeved on the motor rotating bearing 201, the bottom surface of the PCR microfluidic chip is in contact with the lower heating plate, the lower heating plate is composed of a plurality of lower heating plates 301 made of semiconductor aluminum alloy, and the control unit controls the temperature of the heating plates and the rotation of the motor.
The micro-fluidic chip heating device further comprises an upper heating plate 5, wherein the upper heating plate is composed of a plurality of aluminum alloy upper heating plates 501, the upper heating plate and the lower heating plate form a heating chamber 6, and the micro-fluidic chip is accommodated in the heating chamber. The number of the upper heating plates is 3, the upper heating plates are surrounded by an included angle of 120 degrees, and heat insulation strips 502 are arranged between the adjacent upper heating plates, so that temperature loss is avoided. Thus, the amplification chamber corresponds to the upper and lower heating plates, and the control unit adjusts the temperature of the heating plate to make the temperatures of the upper and lower surfaces of the amplification chamber consistent.
Preferably, the upper heating plate is fixed on a fixing frame 7, the fixing frame is connected with a sliding block 8, a sliding rail 9 is arranged in the Z-axis direction, and the sliding block moves on the sliding rail under the driving of a linear motor 10, so that the upper heating plate is driven to move up and down, the upper heating plate is contacted with or separated from the microfluidic chip, the chip is conveniently placed manually, and meanwhile, temperature conduction and isolation are carried out. The fixing frame is also provided with radiating aluminum fins 16 and a radiating fan 17, so that the radiating effect is enhanced, and the performance of the semiconductor chip is improved.
The chip clamping jaw 11 is arranged on the motor rotating chuck 22, the clamping jaw corresponds to the hollow shape of the chip, the clamping jaw and the chip can be meshed and fixed, and the clamping jaw and the PCR microfluidic chip are clamped and fixed. A photoelectric blocking sheet 12 is arranged between the chip clamping jaw and the motor and used for triggering a photoelectric switch 13 and determining the initial chip detection position.
Extend thermal-insulated baffle 14 downwards between the adjacent heating plate of last hot plate, the baffle adopts heat preservation thermal insulation material such as aerogel, cuts apart into 3 heated warehouses with whole heating space, and thermal-insulated baffle reduces the air convection, effectively reduces heat loss speed, makes not take place the heat exchange between the heated warehouses, and the amplification cavity is heated evenly in the heated warehouses.
Each heating plate is connected with a temperature sensor 15, the temperature sensors transmit temperature signals to the control unit, the temperature of the chip is calculated through temperature compensation, and after the target temperature is reached, the control unit reduces power output to keep the temperature at a constant value.
Example 3
As shown in fig. 9, the PCR microfluidic chip can be configured as a set of amplification chambers, including a sample inlet, a main liquid channel, an amplification chamber, and a waste liquid storage chamber. The sample inlet is communicated with the waste liquid storage cavity through the main liquid channel, the main liquid channel is communicated with the amplification cavity through the liquid path, an online degassing channel is arranged between the sample inlet and the main liquid channel, and the top layer of the online degassing channel is covered by a semipermeable membrane. The main liquid channel is connected to the lower part of the amplification chamber through the liquid inlet branch channel, the upper part of the amplification chamber is connected to the liquid outlet branch channel, and the top of the outlet position of the liquid outlet branch channel is sealed by a semipermeable membrane.
The distribution of the amplification chamber corresponds to the heating plate, so that the motor drives the chip to rotate, the chip can sequentially pass through the heating plates with different temperature settings, the temperature and the heating time of each heating bin can be different according to the requirement of a PCR amplification experiment, and the chip sequentially rotates to complete the cyclic amplification.
Example 4
According to the structure of embodiment 2, the method for performing PCR amplification by using the multi-temperature-zone PCR amplification device provided by the invention comprises the following steps:
step 1, sleeving a PCR microfluidic chip on a motor rotating bearing, and clamping and fixing the PCR microfluidic chip by a chip clamping jaw;
and 5, driving the PCR microfluidic chip to rotate 120 degrees each time by a motor, sequentially enabling the amplification chamber to enter heating bins with denaturation temperature, annealing temperature and amplification temperature, continuously heating for 30s-2min, preferably 1min, in each heating bin, and rotating the microfluidic chip to perform amplification circulation.
Preferably, in step 2, when the motor rotates, after the photoelectric barrier rotates to the action point of the photoelectric switch, the photoelectric switch sends a signal, and the control unit determines the origin of the motor;
in step 4, the temperature sensor detects the temperature of the heating plate, the temperature of the chip is calculated through temperature compensation, and after the target temperature is reached, the control unit reduces the power output to keep the temperature at a constant value.
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (13)
1. The utility model provides a multi-temperature-zone PCR amplification device, includes PCR micro-fluidic chip, motor and lower hot plate, its characterized in that, the hot plate is fixed on the temperature resistant support down, PCR micro-fluidic chip's introduction port and amplification cavity intercommunication, PCR micro-fluidic chip cup joints on motor rolling bearing, and the bottom surface contacts hot plate down, the hot plate comprises the lower heating plate of a plurality of semiconductor aluminum alloys down, and the control unit control heating plate's temperature and the rotation of motor.
2. The multi-temperature-zone PCR amplification device according to claim 1, further comprising an upper heating plate, wherein the upper heating plate is composed of a plurality of upper heating plates made of semiconductor aluminum alloy, and the upper and lower heating plates form a heating chamber for accommodating the microfluidic chip therein.
3. The multi-temperature-zone PCR amplification device according to claim 1 or 2, wherein the number of the lower heating plates is 3, and the lower heating plates are surrounded by an included angle of 120 degrees; the number of the upper heating plates is 3, and the upper heating plates are surrounded by an included angle of 120 degrees.
4. The multi-temperature-zone PCR amplification device of claim 3, wherein at least one amplification chamber on the PCR microfluidic chip corresponds to the heating plate.
5. The multi-temperature-zone PCR amplification device of claim 3, wherein the upper heating plate is fixed on a fixing frame, the fixing frame is connected with a slide block, a slide rail is arranged in the Z-axis direction, and the slide block moves on the slide rail under the driving of a linear motor.
6. The multi-temperature-zone PCR amplification device of claim 5, wherein a chip clamping jaw is mounted on the motor rotating chuck and clamped to fix the PCR microfluidic chip.
7. The multi-temperature-zone PCR amplification device of claim 6, wherein a photoelectric barrier is disposed between the chip clamping jaw and the motor for triggering the photoelectric switch.
8. The multi-temperature-zone PCR amplification device of claim 7, wherein a heat insulation baffle extends downwards between adjacent heating plates of the upper heating plate to divide the whole heating space into a plurality of independent heating chambers.
9. The multi-temperature-zone PCR amplification apparatus according to claim 8, wherein each heating plate is connected to a temperature sensor, and the temperature sensor transmits a temperature signal to the control unit.
10. The multi-temperature-zone PCR amplification device of claim 5, wherein a heat-dissipating aluminum fin and a heat-dissipating fan are disposed on the fixing frame.
11. The method for PCR amplification by the multi-temperature-zone PCR amplification device according to claim 8, comprising the following steps:
step 1, sleeving the PCR microfluidic chip on a motor rotating bearing, and clamping and fixing the PCR microfluidic chip by a chip clamping jaw;
step 2, adding reaction buffer solution, primers, taq enzyme, deoxynucleoside triphosphate substrates and DNA template reagents from a sample inlet of the PCR microfluidic chip, driving the microfluidic chip to rotate by a motor, and uniformly mixing the reagents after the reagents enter an amplification chamber;
step 3, the linear motor drives the upper heating plate to move downwards to form a plurality of independent heating bins, and the amplification chamber of the PCR microfluidic chip rotates into the heating bins;
step 4, the control unit controls the semiconductor chip of the heating plate to be heated, different heating chambers sequentially keep the temperature at the denaturation reaction temperature, the annealing reaction temperature and the amplification temperature of the PCR experiment according to the rotation sequence, and the temperatures of an upper heating plate and a lower heating plate in the same heating chamber are the same;
and 5, driving the PCR microfluidic chip to rotate 120 degrees each time by the motor, sequentially allowing the amplification chamber to enter heating bins with denaturation temperature, annealing temperature and amplification temperature, continuously heating in each heating bin for 30s-2min, and rotating the microfluidic chip for amplification circulation.
12. The method of claim 11, wherein in step 2, when the motor rotates, the photoelectric switch sends a signal after the photoelectric barrier rotates to the action point of the photoelectric switch, and the control unit determines the origin of the motor.
13. The method of PCR amplification of claim 11, wherein in step 4, the temperature sensor detects the temperature of the heating plate, the temperature of the chip is estimated by temperature compensation, and the control unit reduces the power output to maintain the temperature at a constant value after the target temperature is reached.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116515621A (en) * | 2023-05-06 | 2023-08-01 | 南方医科大学南方医院 | Multi-temperature-zone PCR amplification system, method and equipment |
| CN116574603A (en) * | 2023-07-14 | 2023-08-11 | 潍坊安普未来生物科技有限公司 | Isothermal amplification detection device and detection method |
-
2022
- 2022-10-27 CN CN202211323220.9A patent/CN115477997A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116515621A (en) * | 2023-05-06 | 2023-08-01 | 南方医科大学南方医院 | Multi-temperature-zone PCR amplification system, method and equipment |
| CN116515621B (en) * | 2023-05-06 | 2023-10-17 | 南方医科大学南方医院 | Multi-temperature-zone PCR amplification system, method and equipment |
| CN116574603A (en) * | 2023-07-14 | 2023-08-11 | 潍坊安普未来生物科技有限公司 | Isothermal amplification detection device and detection method |
| CN116574603B (en) * | 2023-07-14 | 2023-09-19 | 潍坊安普未来生物科技有限公司 | Isothermal amplification detection device and detection method |
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