A dual-temperature zone PCR microfluidic chip driven by dual-axis centrifugal

Abstract

The application discloses a double-temperature-zone PCR micro-fluidic chip driven by biaxial centrifugal force for space application, which can provide simulated gravity under microgravity conditions through centrifugal force, and can eliminate the problem of air bubbles generated in the qPCR process; meanwhile, a variable-temperature mode of fixed-temperature-zone driving liquid is adopted, so that the power consumption demand in the amplification process is reduced. X and Y axes which are perpendicular to each other are divided along the center of the chip, and four regions are formed; each region contains a pipeline system with the same structure. Each pipeline system is as follows: an amplification system buffer channel is connected to an amplification system quantitative pool through a first capillary valve; a side of the amplification system quantitative pool is provided with a channel connected to a waste liquid pool, and liquid more than the capacity of the amplification system flows into the waste liquid pool; the other side of the amplification system quantitative pool is connected to a dry powder preset channel through a second capillary valve; the dry powder preset channel is connected to an amplification pool; the direction extension line of the amplification pool and the x axis and the origin form an acute angle; the amplification pool is divided into low-temperature and high-temperature constant-temperature zones. A paraffin oil sampling port is located in the middle of the amplification pool.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field, and particularly relates to a double-temperature-zone PCR microfluidic chip driven by a double-shaft centrifugal. BACKGROUND

[0002] The qPCR real-time fluorescence quantitative polymerase chain reaction technology is an important tool for gene analysis in life science research. With the completion and operation of the space station in China, space life science research will be carried out in the space station. A large number of space experiments in the early stage adopt the mode of "space sampling-low temperature storage-downlink-ground analysis", and the downlink resources are tight and the experimental efficiency is low. A qPCR experiment system that can be carried out in a microgravity environment has important significance for reducing uplink and downlink resources and improving experimental efficiency.

[0003] Microgravity environment amplification cannot directly use the qPCR equipment commonly used in ground laboratories. The qPCR experiment reported in the Wetlab-2 system of the International Space Station mentioned that the bubbles generated in the tube will rise from the tube detection window under normal 1g conditions, while under microgravity conditions, bubbles will form, remain and expand in the tube optical window during the thermal cycling process, thereby producing noise in the resulting amplification curve. In the articles Microgravity validation of a novel system for RNAisolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station. PLoS ONE 12(9):e0183480 and Spaceflight validation of one-step Gene Sampling tool for genetic analysis on the International Space Station, Acta Astronautica, Volume 198, 2022, Pages 225-232, although the tube cover of the Wetlab-2 system was optimized to improve the sealing and reduce the generation of bubbles to a certain extent, but in the space station test in 2022, there are still reports of adverse effects of bubbles on the results, which affected the success rate and efficiency of the on-orbit test. It can be seen that solving the problem of bubbles in the amplification process in the microgravity environment is the most critical requirement for the development of qPCR space equipment.

[0004] Furthermore, traditional qPCR equipment uses a single metal heated stage for a three-step method (94℃-60℃-72℃) or a two-step method (94℃-60℃). Temperature variation can also be achieved by driving liquid flow within a constant temperature zone. Liquid-driven methods are diverse. For example, patent CN110872555A discloses a centrifugal reciprocating drive technology; patent CN110872557A discloses a chip-fixed rotating temperature zone technology; patents CN113574161A and CN103614294B disclose a pneumatic reciprocating drive technology; and patent CN110452803A discloses an external pump drive technology. While these patents provide technical solutions, they are all geared towards terrestrial applications, and their application in space experiments still presents challenges. The aforementioned methods for fixing the rotating temperature zone of the chip, like Wetlab-2, are all static liquid methods, which cannot avoid the influence of air bubbles. Centrifugal reciprocating drive and pneumatic reciprocating drive methods both have gas-liquid interfaces in actual liquid circuits. After the loss of gravity, the stability of the gas-liquid interface is insufficient, making it difficult to apply in the space environment. External pump drive methods, due to their connection between the liquid circuit interface and the flow path, have inconvenient consumable replacement and easy contamination of connectors, which bring great inconvenience to space astronauts.

[0005] Currently, there is no technical solution that can design a qPCR space device that can solve the bubble problem during amplification in a microgravity environment while achieving low power consumption optimization. Summary of the Invention

[0006] In view of this, the present invention provides a dual-temperature zone PCR microfluidic chip driven by biaxial centrifugation for space applications. It can provide simulated gravity under microgravity conditions through centrifugation, eliminating the bubble problem generated during qPCR. At the same time, it adopts a fixed temperature zone to drive the temperature change of the liquid, which greatly reduces the power consumption requirements during the amplification process.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a dual-temperature zone PCR microfluidic chip driven by biaxial centrifugation for space applications, wherein the chip is divided into mutually perpendicular X-axis and Y-axis along the center of the chip, and the X-axis and Y-axis divide the chip into four identical regions, namely the first quadrant to the fourth quadrant; each region contains a tubing system with the same structure.

[0008] The piping system includes an amplification system buffer channel, a first capillary valve, an amplification system quantitative cell, a waste liquid cell, a second capillary valve, a dry powder pre-set channel, an amplification cell, and a paraffin oil inlet.

[0009] The amplification system buffer channels in the first and fourth quadrants are both open at their ends, serving as the injection port and exhaust port, respectively; the injection port is used to insert a conventional pipette tip for sample injection.

[0010] The amplification system buffer channel is connected to the amplification system quantitative pool through the first capillary valve; the amplification system buffer channel is not parallel to the x-axis in the connection direction of the first capillary valve.

[0011] A channel is arranged on the side of the amplification system quantitative pool to communicate with the waste liquid pool, and liquid flows into the waste liquid pool when the liquid is more than the capacity of the amplification system; the other side of the amplification system quantitative pool is connected to the dry powder pre-setting channel through the second capillary valve; the second capillary valve is perpendicular to the Y-axis in the direction.

[0012] The dry powder pre-setting channel is connected to the amplification pool, and the total volume of the amplification pool is greater than twice the volume of the amplification system quantitative pool; the extension line of the amplification pool forms an acute angle with the x-axis and the origin; the amplification pool is divided into a low-temperature constant-temperature zone and a high-temperature constant-temperature zone; the temperature of the low-temperature constant-temperature zone is set to be between 50-70℃, and the temperature of the high-temperature constant-temperature zone is set to be between 90-100℃.

[0013] The paraffin oil sampling port is connected to the amplification pool at a position in the middle of the amplification pool.

[0014] Further, the total capacity of the four amplification system buffer channels is greater than the total volume of the four amplification system quantitative pools.

[0015] Further, a sharp corner is arranged on the side of the amplification system quantitative pool connected to the waste liquid pool to isolate the amplification system quantitative pool and the waste liquid pool, and the capacity of the range below the dashed line formed along the direction parallel to the X-axis from the vertex of the sharp corner to the amplification system capacity does not differ by more than 5%.

[0016] Beneficial effects:

[0017] 1. The application provides a double-temperature-zone PCR microfluidic chip with double-axis centrifugal driving for space application, which provides simulated gravity under microgravity conditions through centrifugation, and eliminates the problem of bubbles generated in the qPCR process; when the centrifugal force simulates gravity and is provided to be approximately equal to the gravity on the ground (about 1g), the bubbles generated in the amplification pool float above the oil layer as on the ground, thereby reducing the influence of the bubbles on the amplification process and obtaining good amplification results. Meanwhile, the variable-temperature mode of driving liquid by using a fixed temperature zone greatly reduces the power consumption requirement in the amplification process, and the double-axis centrifugal mode has the application advantages of stable driving, independent consumables and no external interface compared with the existing pneumatic, single-axis centrifugal and external pump driving modes.

[0018] 2. The application provides a double-temperature-zone PCR microfluidic chip with double-axis centrifugal driving for space application, which adopts multi-axis centrifugation, and can generate centrifugal forces in multiple directions on the chip in turn during use to drive the liquid in the chip to flow in different regions.

[0019] 3. The application provides a double-temperature zone PCR microfluidic chip driven by double-axis centrifugation for space application, which is designed without external pipeline, is fully closed after sample addition, is in the form of independent consumables, is simple to operate and has no connector pollution. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the chip structure;

[0021] Figure 2 is a schematic diagram of the low-temperature and high-temperature constant temperature zones. DETAILED DESCRIPTION

[0022] The application will be described in detail below with reference to the drawings and examples.

[0023] The application provides a double-temperature zone PCR microfluidic chip driven by double-axis centrifugation for space application, which is designed without external pipeline, is fully closed after sample addition, is in the form of independent consumables, is simple to operate and has no connector pollution. Figure 1 As shown in the figure, the chip as a whole can be circular, and other shapes are also possible. The X-axis and Y-axis perpendicular to each other are divided along the center of the chip, and the X-axis and Y-axis divide the chip into four identical regions, which are the first quadrant to the fourth quadrant region; each region contains the same structure of pipeline system.

[0024] The pipeline system includes an amplification system buffer channel 3, a first capillary valve 4, an amplification system quantitative pool 5, a waste pool 6, a second capillary valve 7, a dry powder pre-setting channel 8, an amplification pool 9, and a paraffin oil sample inlet 10; the ends of the amplification system buffer channel 3 in the first quadrant and the fourth quadrant region are both opened and are respectively provided as a sample inlet 1 and an exhaust port 2; the sample inlet 1 hole is inserted into a conventional pipette tip for sample injection.

[0025] The amplification system buffer channel 3 is connected to the amplification system quantitative pool 5 through the first capillary valve 4; the connection direction of the amplification system buffer channel 3 and the first capillary valve 4 is not parallel to the x-axis.

[0026] A channel is provided on the side of the amplification system quantitative pool 5 to communicate with the waste pool 6, and the liquid flows into the waste pool 6 when the liquid is more than the capacity of the amplification system; the other side of the amplification system quantitative pool 5 is connected to the dry powder pre-setting channel 8 through the second capillary valve 7; the direction of the second capillary valve 7 is perpendicular to the Y-axis.

[0027] The dry powder pre-setting channel 8 communicates with the amplification pool 9, and the total volume of the amplification pool 9 is greater than 2 times the volume of the amplification system quantitative pool 5; the direction extension line of the amplification pool 9 and the x-axis and the origin form an acute angle; the amplification pool 9 is divided into a low-temperature constant temperature zone 9-1 and a high-temperature constant temperature zone 9-2; the temperature of the low-temperature constant temperature zone 9-1 is set to be between 50-70℃, and the temperature of the high-temperature constant temperature zone 9-2 is set to be between 90-100℃; in the embodiment of the application, the low-temperature constant temperature zone 9-1 and the high-temperature constant temperature zone 9-2 are realized by setting the temperature-controllable copper block.

[0028] The paraffin oil injection port 10 is connected with the amplification pool 9 at a position in the middle of the amplification pool 9.

[0029] In the embodiment of the present application, the total capacity of the four amplification system buffer channels 3 is greater than the total volume of the four amplification system quantitative pools 5.

[0030] In the embodiment of the present application, one sharp corner 5-1 is arranged on one side of the amplification system quantitative pool 5 connected with the waste liquid pool 6, which is used to isolate the amplification system quantitative pool 5 and the waste liquid pool 6. The capacity of the range below the dashed area starting from the vertex of the sharp corner 5-1 and along the direction parallel to the X axis is equal to the capacity of the amplification system.

[0031] The working process of the space application double-shaft centrifugal driving double-temperature zone PCR microfluidic chip provided in the embodiment of the present application comprises the following steps:

[0032] Step 1: The primers are prepositioned on the position of the dry powder prepositioning channel 8 in the chip before the chip is bonded, and then the primers are added through the paraffin oil injection port 10 after the chip is bonded.

[0033] Step 2: The nucleic acid sample is added from the injection port 1 by using a pipetting tool meeting the specification shape requirement, so that the nucleic acid sample fills the amplification system buffer channel 3.

[0034] Step 3: The X axis is taken as the rotation axis to rotate, under the action of the centrifugal force, the amplification system in the amplification system buffer channel 3 enters the amplification system quantitative pool 5 through the first capillary valve 4, and then the excess amplification system overflowed enters the waste liquid pool 6; when the nucleic acid sample in the amplification system buffer channel 3 all enters the amplification system quantitative pool 5, step 4 is entered.

[0035] Step 4: The Y axis is taken as the rotation axis to rotate, under the action of the centrifugal force, the nucleic acid sample in the amplification system quantitative pool 5 enters the dry powder prepositioning channel 8 through the second capillary valve 7, dissolves the prepositioned primer dry powder, and then continues to enter the amplification pool 9 and stays in the high-temperature constant temperature zone 9-2 of the amplification pool 9.

[0036] Step 5: The nucleic acid sample stays in the high-temperature constant temperature zone 9-2 for a time, the X axis is taken as the rotation axis to rotate, under the action of the centrifugal force, the low-temperature constant temperature zone 9-1 is moved, and the rotation is maintained; the amplification system stays in the low-temperature constant temperature zone 9-1 for a time, the Y axis is taken as the rotation axis to rotate, under the action of the centrifugal force, the high-temperature constant temperature zone 9-2 is moved, and the rotation is maintained, and the circulation is repeated until the experiment is finished.

[0037] In the present application, the capillary valve can be replaced by other commonly used valves of the microfluidic chip, such as a pressure valve made of soft material, a paraffin valve, etc.

[0038] In the embodiment of the present application, the capillary valve parameters and the rotation speed are one of the feasible combinations, and different capillary valve parameters and matching rotation speeds can be designed.

[0039] The primers in the embodiment of the application can also not be pre-arranged in the chip, but directly loaded in the system for four same parallel amplifications.

[0040] The double-axis centrifugation is not limited to the x-axis and the y-axis in the embodiment of the application, but can be any double-axis combination mode of the x-axis and the z-axis or other different angles.

[0041] To sum up, the above is only a preferred embodiment of the application, and is not used to limit the protection scope of the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A dual-temperature zone PCR microfluidic chip driven by biaxial centrifugation for space applications, characterized in that, The chip is divided into four identical regions by mutually perpendicular X-axis and Y-axis along its center: the first quadrant to the fourth quadrant; each region contains a piping system with the same structure. The pipeline system includes an amplification system buffer channel (3), a first capillary valve (4), an amplification system quantitative cell (5), a waste liquid cell (6), a second capillary valve (7), a dry powder pre-positioning channel (8), an amplification cell (9), and a paraffin oil inlet (10). The amplification system buffer channel (3) in the first and fourth quadrants is open at the end, and is set as the injection port (1) and the exhaust port (2) respectively; the injection port (1) hole is inserted into a conventional pipette tip for sample injection; The amplification system buffer channel (3) is connected to the amplification system quantitative cell (5) through the first capillary valve (4); the connection direction between the amplification system buffer channel (3) and the first capillary valve (4) is not parallel to the x-axis; The amplification system quantitative cell (5) has a channel on its side that connects to the waste liquid pool (6). When the liquid exceeds the capacity of the amplification system, it flows into the waste liquid pool (6). The other side of the amplification system quantitative cell (5) is connected to the dry powder pre-positioned channel (8) through a second capillary valve (7). The direction of the second capillary valve (7) is perpendicular to the Y-axis. The dry powder pre-placed channel (8) is introduced into the amplification pool (9), and the total volume of the amplification pool (9) is more than twice the volume of the quantitative amplification system pool (5); the directional extension line of the amplification pool (9) forms an acute angle with the x-axis and the origin; the amplification pool (9) is divided into a low temperature constant temperature zone (9-1) and a high temperature constant temperature zone (9-2); the temperature of the low temperature constant temperature zone (9-1) is set between 50-70℃, and the temperature of the high temperature constant temperature zone (9-2) is set between 90-100℃; The connection point between the paraffin oil inlet (10) and the amplification cell (9) is located in the middle of the amplification cell (9); Its workflow includes the following steps: Step 1: Before chip bonding, the primers are pre-placed in the dry powder pre-placement channel (8) of the chip, and after bonding, they are added through the paraffin oil injection port (10); Step 2: Use a pipette that meets the required specifications and shape to add the nucleic acid sample from the injection port (1) to fill the amplification system buffer channel (3) with the nucleic acid sample; Step 3: Rotate with the X-axis as the rotation axis. Under the action of centrifugal force, the amplification system in the amplification system buffer channel (3) enters the amplification system quantitative pool (5) through the first capillary valve (4), and then the excess amplification system that overflows enters the waste liquid pool (6); after all the nucleic acid samples in the amplification system buffer channel (3) have entered the amplification system quantitative pool (5), proceed to step (4); Step 4: Rotate with the Y-axis as the rotation axis. Under the action of centrifugal force, the nucleic acid sample in the quantitative cell (5) of the amplification system enters the dry powder pre-placement channel (8) through the second capillary valve (7), dissolves the pre-placed primer dry powder, and continues to enter the amplification cell (9) and stays in the high temperature constant temperature zone (9-2) of the amplification cell (9). Step 5: After the nucleic acid sample has been in the high-temperature isothermal zone (9-2) for the specified time, it is rotated around the X-axis and moved to the low-temperature isothermal zone (9-1) under centrifugal force, while maintaining the rotation. After the amplification system has been in the low-temperature isothermal zone (9-1) for the specified time, it is rotated around the Y-axis and moved to the high-temperature isothermal zone (9-2) under centrifugal force, while maintaining the rotation. This process is repeated until the experiment is completed.

2. The dual-temperature zone PCR microfluidic chip driven by biaxial centrifugation for space applications as described in claim 1, characterized in that, The total capacity of the four amplification system buffer channels (3) is greater than the total volume of the four amplification system quantitative pools (5).

3. The dual-temperature zone PCR microfluidic chip driven by biaxial centrifugation for space applications as described in claim 1, characterized in that, A sharp corner (5-1) is provided on one side of the amplification system quantitative cell (5) connected to the waste liquid cell (6) to isolate the amplification system quantitative cell (5) and the waste liquid cell (6). Starting from the vertex of the sharp corner (5-1), along the direction parallel to the X-axis, the capacity of the area below the dashed line is no more than 5% different from the capacity of the amplification system.

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